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Cystatin C is a disease‐associated protein subject to multiple regulation

Cystatin C is a disease‐associated protein subject to multiple regulation Immunology and Cell Biology (2015) 93, 442–451 & 2015 Australasian Society for Immunology Inc. All rights reserved 0818-9641/15 www.nature.com/icb REVIEW Cystatin C is a disease-associated protein subject to multiple regulation Yuekang Xu, Ying Ding, Xinchen Li and Xiaobing Wu A protease inhibitor, cystatin C (Cst C), is a secreted cysteine protease inhibitor abundantly expressed in body fluids. Clinically, it is mostly used to measure glomerular filtration rate as a marker for kidney function due to its relatively small molecular weight and easy detection. However, recent findings suggest that Cst C is regulated at both transcriptional and post-translational levels, and Cst C production from haematopoietic cell lineages contributes significantly to the systematic pools of Cst C. Furthermore, Cst C is directly linked to many pathologic processes through various mechanisms. Thus fluctuation of Cst C levels might have serious clinical implications rather than a mere reflection of kidney functions. Here, we summarize the pathophysiological roles of Cst C dependent and independent on its inhibition of proteases, outline its change of expression by various stimuli, and elucidate the regulatory mechanisms to control this disease-related protease inhibitor. Finally, we discuss the clinical implications of these findings for translational gains. Immunology and Cell Biology (2015) 93, 442–451; doi:10.1038/icb.2014.121; published online 3 February 2015 Cystatin C (Cst C) is a potent extracellular inhibitor of cysteine alteration in patients with cancer, HIV infection, cardiovascular 11–13 protease, and has been generally considered a ubiquitously expressed diseases and neurological disorders. Moreover, new studies protein as no obvious regulatory elements were found in its gene reported direct involvement of Cst C in many pathogenic processes 1 14,15 promoter. Clinically, Cst C is mostly used as a biomarker of kidney other than renal disorders. Thus, the oscillation of blood Cst C function for its relatively lower molecular weight (~13.3 kDa) and easy levels could actually reflect the change of Cst C production, detection compared to the injection of compounds, radioisotopes or consumption, inactivation or fibrillation (mentioned later) rather than radiocontrast agent to measure glomerular filtration rate (GFR), an its filtration in the kidney, and the readout of plasma Cst C index of kidney health, because Cst C is removed from the blood concentration might have different clinical implications. In this review, stream by glomerular filtration, whose decline as a result of failed we first summarize the major pathophysiological roles of Cst C to kidney function will lead to increased serum Cst C concentration. Cst illustrate its importance as a functional protein in the body rather than C serum levels were claimed to be a more precise index of kidney an ubiquitously expressed measuring substance. Second, we outline function than that of creatinine under the assumption that serum the impact of different stimuli on its expression and elucidate some of input of Cst C is constant, and the main determinant of blood Cst C the latest developments on the regulatory mechanisms to explain Cst levels is the rate at which it is filtered at the glomerulus. Furthermore, C variation under certain circumstances. In the end, the clinical recent advances have facilitated the use of Cst C as a clinical measure implications derived from these studies are also discussed. of kidney function (the update on Cst C in recent kidney disease guidelines has been reviewed elsewhere ). Although, the bulk of PATHOPHYSIOLOGICAL ROLES OF CST C literature report the use of Cst C for GFR estimation, precaution Functions dependent of its inhibition of proteases should be taken when the outcomes of this measurement is Cysteine cathepsins play fundamental roles in multiple biological interpreted because recent studies indicated that both genetic processes such as protein turnover, pro-protein processing, bone 5,6 7,8 polymorphisms and clinical interference could make Cst C an 16 remodeling, antigen presentation and apoptosis. They are also involved unreliable index of GFR. Indeed, accumulating reports have docu- in numerous pathological processes such as cardiovascular disease and mented that subjected to the influence of many factors, serum Cst C inflammation. The activities of these enzymes both inside and outside levels do vary independent of renal functions, and the synthesis and of cells thus need to be tightly controlled by their endogenous inhibitors, secretion of Cst C seem to be tightly regulated under different of which Cst C is the most abundant and potent member. pathophysiological conditions. For example, body composition, thyroid function, glucocorticoid and C-reactive protein levels or even Intracellular roles cigarette smoking and pregnancy status of the candidates examined Apoptosis. Intracellular lysosomes undergo membrane permeabili- 9,10 have been found to affect Cst C blood concentration, let alone its zation in response to extra- or intra-lysosomal stimuli, and the Anhui Provincial Key Laboratory for Conservation and Exploitation of Biological Resources, School of Life Sciences, Anhui Normal University, Anhui, P.R. China Correspondence: Professor Y Xu, School of Life Science, Anhui Normal University, 1 Beijing East Road, Wuhu, Anhui Province 241000, P.R. China. E-mail: yuekang.xu@hotmail.com Received 28 October 2014; revised 7 December 2014; accepted 8 December 2014; published online 3 February 2015 Regulation of cystatin C in diseases YXu et al involvement of lysosomal cysteine proteases in apoptosis has been immature and mature DCs suggests that the regulatory potential of Cst 17,18 confirmed in several systems. However, the roles of the endogen- C toward these cathepsins inside DCs is limited, which could explain ous lysosomal cysteine protease inhibitor Cst C in apoptosis remain the inconsistent findings related to the intracellular roles of Cst C. controversial. Upregulation of Cst C expression was found to correlate Instead, large secretion of Cst C over cathepsins S, L and H was 19 30 with oxidative stress-induced apoptosis in cultured rat neurons, and observed in the culture media, indicating that the extracellular Cst C injection into rat hippocampus led to neuronal cell death in the compartment is the primary site for these interactions. granule cell layer of dentate gyrus in vivo, indicating a possible Extracellular roles. The structure of Cst C predisposes this protease functional role of Cst C in apoptosis induction. Since, the Cst inhibitor to extracellular functions as a secreted protein. Accordingly, C-induced neuronal cell death could be inhibited by simultaneous Cst C is found in all body fluids at significant concentrations, which co-application of cathepsin B, the inhibitory activity of Cst C on a makes it a major regulator of cysteine protease activity in the cysteine protease was proposed to be involved in the process of extracellular medium. apoptotic neuronal cell death. Recent studies indicated that the roles of Cst C in neuronal cell apoptosis induction include decreasing B-cell Atherosclerosis. Atherosclerosis-based vascular disease is an inflam- leukemia-2 (Bcl-2) and increasing active caspase-9 protein levels via matory disease characterized by extensive remodeling of the extra- the Jun N-terminal kinase (JNK)-dependent pathway, and upregula- cellular matrix of the arterial walls. Apart from the well-known matrix tion and accumulation of the insoluble α-synuclein in oligodendro- metalloproteinases and serine proteases, lysosomal cysteine proteases cytes and neurons. Interestingly, apart from these documented pro- 32 were also found to be involved. Present in substantial amounts in the apoptotic roles, Cst C was also found to have anti-apoptotic effects on normal vessel walls, the expression of Cst C was found to be severely neuronal cells. For example, expression of Cst C in PC12 cells derived reduced in both atherosclerostic and aneurysmal lesions, and increased from a pheochromocytoma of rat adrenal medulla prevents oxidative abdominal aortic diameter correlated with lower serum Cst C levels in stress-induced death in vitro. In accordance with this finding, the 13 humans. Furthermore, the pathogenic roles of Cst C were tested in a anti-apoptotic function of Cst C was further demonstrated in vivo in a model of atherosclerosis-prone mice with apolipoprotein E deficiency mouse model of the inherited neurodegenerative disorder, progressive − / − (apoE ) where the elevated cathepsins were associated with the myoclonic epilepsy type 1, where loss of function in cystatin B and 33 atherosclerostic process. In two independent studies, Cst C- and enhanced cathepsin B and D activities are the underlying pathologies. apoE-double-deficient mice were generated, both of them confirmed Crossbreeding of the mice with either Cst C-overexpressing transgenic − / − an anti-atherosclerostic function of Cst C in the apoE mice, or Cst C-deficient mice revealed that Cst C levels in vivo can affect although differences regarding lesion size and composition were neuron degeneration caused by the proteases, indicating that Cst C 34,35 found. These differences are most probably caused by differences partially prevents neural cell death in vivo through inhibition of in the duration of high-fat diet of the mice, sex of the mice and cathepsins activity. (Described in the section of Neuroprotective anatomic site of analyzed lesions. Interestingly, in line with the anti- roles.) atherosclerostic role of Cst C in this animal model, polymorphisms in the promoter regions of the Cst C gene were found to influence the Antigen presentation. Proteolysis of antigen by cysteine cathepsins to plasma Cst C concentration in human coronary artery disease, generate immunogenic peptides and guide the transit of both major indicating the production of this protease inhibitor could be subject histocompatibility complex class II (MHCII) and MHC-like molecules to regulation under the diseased conditions. through the endocytic compartments are important intracellular activities in antigen-presenting cells. As a potent inhibitor of cysteine Tumor metastasis. Metastatic tumor cells invade host tissues through cathepsins, Cst C had initially been implicated in playing a regulatory a series of steps that require proteolytic enzymes including cysteine role in the developmental control of MHCII presentation in dendritic cathepsins to degrade components of the extracellular matrix. As a cells (DCs) by inhibiting cathepsin S in invariant chain cleavage. most abundant endogenous cysteine proteinase inhibitor, Cst C is Consistent with the role of Cst C in antigen presentation, a cystatin believed to prevent tumor progression by inhibiting the activities of a homolog produced by the filarial nematode parasite was found to family of lysosomal cysteine cathepsins. Results from the first Cst inhibit lysosomal cysteine protease activities, which subsequently C-deficient animals indicated that Cst C concentration in vivo might impeded the generation of human B cells. Further experiments influence tumor metastasis in some tissues. In agreement, the using bone marrow-derived DCs indicate that interleukin 6 (IL-6)- reduced Cst C levels correlate with increased metastasis of different mediated signal transducer and activator of transcription 3 (STAT3) tumors in human tissues or patients. Furthermore, local over- activation decreased Cst C expression and MHCII αβ dimer levels. expression of Cst C in the host tissue microenvironment could lead to However, a separate study using mouse primary DCs isolated from Cst successful reduction of metastasis via cysteine cathepsin inhibition in C-deficient mice demonstrated that Cst C is neither necessary nor 40 an experimental tumor model. This inverse correlation between sufficient to control MHCII expression and antigen presentation in Cst C and tumor aggressiveness, however, may not always involve DCs. These discrepant results obtained from different laboratories inhibition of cysteine proteases. For example, elevated matrix metal- could be due to the different cell types investigated, and/or to the loproteinases 2 and crosstalk between Cst C and androgen receptor- compensatory roles played by other cysteine protease inhibitors in the mediated pathways were reportedly implicated in prostate cancer absence of Cst C. In support of this view, cell-specificregulation of invasion and metastasis, indicating multiple roles of Cst C in tumor cathepsin activity by Cst C was identified in two similar antigen- metastasis. presenting cells in the brain: Cst C was found to inhibit cathepsin L activity in astrocytes, but does not regulate cathepsins L and S in Pathogen invasion. Although a secreted protein, Cst C was also microglia. reported to be up-taken by cells of both foreign and endogenous When intracellular localization of the Cst C and its target proteases origins in various tissues to regulate both intracellular and extracellular were examined in human DC differentiated from monocytes in vitro, cysteine protease activities. One of the major roles of cystatins is to the different compartmentation of Cst C and cathepsins S, L and H in protect the host against invading microorganisms and parasites that Immunology and Cell Biology Regulation of cystatin C in diseases YXu et al use cysteine proteases to enter the body. Chicken cystatin was first amyloidogenesis begin with dimerization by a process known as reported to partially block poliovirus replication in infected human ‘three-dimentional domain swapping’, in which two parts of the cells. Further study demonstrated that a small peptide derivative that cystatin structure become separated from each other and next mimics part of the proteinase-binding center of human Cst C could exchanged between two molecules. Interestingly, with their inhibi- inhibit a cysteine protease specific for the growth of group A tory region hidden within the dimer interface, Cst C dimers cannot streptococci, blocking the growth of these bacteria both in vivo and inhibit cysteine proteases. This none-inhibitory Cst C dimer is in vitro. Similarly, recombinant human Cst C was also proven required for the formation of the Cst C oligomers, intermediates in fibrillogenesis, because variants of monomeric Cst C, stabilized against to inhibit the growth of herpes simplex virus and human coronaviruses. Moreover, a family of cathepsin L- and B-like cysteine domain swapping to block the inhibitory site, fail to produce proteases, found in all species of Leishmania examined, are required oligomers, indicating that Cst C fibrils are formed by propagated 48 61 for the parasite growth and virulence. Since, these parasite cysteine domain swapping. proteases may not only digest the host extracellular matrix to facilitate Domain swapping must be preceded by at least partial unfolding of their invasion, but also help to ensure a Th -like response led to the molecule, therefore, any factors affecting the stabilization of the parasite proliferation, cystatin treatment in combination with inter- molecule could initiate the domain swapping process. For instance, the feron γ (IFNγ) that leads to reduced parasite numbers, successful Th point mutation with substitution of native leucine in position 68 by to Th conversion, and NO generation, which finally resulted in glutamine (L68Q) disrupts a network of the hydrophobic interactions, abrogation of parasite infection in a mouse model of leishmaniasis. which leads to increased tendency for dimerization and aggregation in vitro. The L68Q replacement is also a naturally occurring point Functions independent of its inhibition of proteases mutation in the Cst C protein sequence with autosomal dominant Modulating roles. Most abundantly expressed in tissues, Cst C also hereditary. This Cst C variants form fibrils in vivo in the brain serves numerous functions independent of protease inhibition includ- vasculature, which cause hemorrhage, dementia and eventually death ing affecting signaling properties of other molecules. For example, Cst in people carrying this mutation, a condition known as hereditary C antagonizes the binding of transforming growth factor-beta (TGF-β) cystatin C amyloid angiopathy, also called hereditary cerebral to its cell receptors by physically interacting with TGF-β type II hemorrhage with amyloidosis, icelandic type. The cellular transport receptor independent of its protease inhibitory activity, as over- of Cst C is impeded by the mutated L68Q variants, resulting in 66,67 expression of Cst C mutant that is impaired in its ability to inhibit diminished Cst C levels in cerebrospinal fluid and retained Cst C cathepsin activity blocked TGF-β-dependent invasion of 3T3-L1 mutants in blood monocytes from patients. Consistently, in vitro fibroblasts. This novel function of signaling modulation allows Cst studies demonstrated that clones expressing the gene encoding L68Q C to inhibit the oncogenic activities of TGF-β through stimulation of Cst C secreted either lower amounts of Cst C, or unstable protein 52 69 mammary epithelial-mesenchymal transition. Apart from blocking susceptible to a serine protease, contributing to the reduced interaction between TGF-β and its receptor, Cst C was also found to extracellular Cst C levels. prevent TGF-β signaling partly by reducing the extent of mothers Cst C amyloid fibrils not only affect brain vasculature but also may against decapentaplegic homolog 2 (Smad2), p38 mitogen-activated lead to toxicity in other tissues. As the most abundant protein, Cst C protein kinases (p38 MAPKs) and extracellular signal-regulated was found in tissues outside of the brain including the testis, and so kinases 1/2 (Erk1/2) phosphorylation in murine 4T1 breast cancer was the L68Q variant. A recent study reports that heterozygous cells. In addition to affecting TGF-β signaling, CstCis involved in transgenic mice that express this pathogenic variant were unable to the IFNγ signal transduction pathway. It activates nuclear factor generate offspring, indicating the L68Q Cst C amyloid affects sperm kappa-light-chain-enhancer of activated B cells (NF-κB) p65, induces function. Further analysis of the L68Q mice demonstrated that their NO synthase, but downregulates IL-10 in macrophages. epididymal spermatozoa were unable to fertilize oocytes and exhibited Through interacting with other proteins with effects on their poor sperm motility in the presence of Cst C amyloid that were not signaling properties, Cst C can also affect biological functions of cells. found in the wild-type mice. The L68Q epididymal fluid, when During brain development, Cst C was identified as a factor to depleted of the Cst C amyloids, however, did not impair the motility upregulate the glial fibrilllary acidic protein promoter. As a result, of wild-type spermatozoa, suggesting that amyloids in the epididymal addition of human Cst C to the culture medium of primary brain cells fluid can be cytotoxic to the maturing spermatozoa resulting in male increased the number of glial fibrilllary acidic protein-positive cells infertility. However, two other groups in earlier studies have also and neurospheres formed from the embryonic brain. Again, the generated the transgenic lines expressing this variant form of Cst C, 72,73 promotion of astrocyte development by Cst C appears to be unrelated but they were live and fertile, questioning whether the pathology to its protease inhibitor activity, as another cysteine protease inhibitor described is really brought about by the Cst C variants or the site did not have this effect. Along the same line, a recent study identified where the transgene was inserted. a novel function of Cst C in mediating amyloid β (Aβ) precursor Neuroprotective roles. Although mutant Cst C is toxic by amyloidosis, protein-induced proliferation of neural stem/progenitor cells. wild-type Cst C has multiple neuroprotective roles (comprehensively Although the mechanism of Aβ precursor protein in stimulating reviewed by Gauthier et al. ). An amyloidogenic protein itself, Cst C neural stem/progenitor cells to secrete Cst C needs further character- has an anti-toxic role against another amyloid protein, Aβ.Studies ization, this study contributes to a better understanding of multi- in vitro demonstrated that Cst C binds to Aβ and inhibits its functional roles of Cst C in the pathogenesis of Alzheimer’sdisease. 75 76 oligomerization and amyloid fibril formation. Furthermore, in Amyloidogenic roles. In addition to cysteine protease inhibition, Cst C Aβ precursor protein transgenic mice, Cst C was found to physically is also one of the few amyloidogenic proteins that form a fibrillary associate with the soluble, non-pathological form of Aβ in vivo, which structure deposited in the vascular walls, affecting the health of blood inhibited the aggregation and deposition of Aβ plaques in the 73,77 vessels (angiopathy). The proteins identified in cerebral amyloid brain. Moreover, Cst C can also directly protect neuronal cells angiopathy include beta/A4, transthyretin and Cst C. Cst C from amyloid toxicity, as extracellular addition of human Cst C Immunology and Cell Biology Regulation of cystatin C in diseases YXu et al promoted the survival of cultured neuronal cells against the preformed TGF-β1 has been reported to upregulate Cst C secretion from vascular oligomeric or fibrillar Aβ. smooth muscle cells, murine embryo cells, cultured differentiated Autophagy is important for the survival and homeostasis of neurons podocytes, 3T3-L1 fibroblasts, and more recently human lung 13,51,90 as they cannot dilute accumulating detrimental substances or damaged fibroblasts. Clinically, a significant influence of circulating Cst organelles by cell division (see latest review for details). Cst C was Clevelsby TGF-β1 were also observed in patients with thyroid first reported to induce a fully functional autophagy to protect dysfunction, and both Cst C mRNA and protein levels were increased neuronal cells against various stress via the mammalian target of by TGF-β in cultured human hepatoblastoma cells. rapamycin (mTOR) pathway in vitro independent of its inhibitor Dexamethasone is a potent synthetic member of glucocorticoid class activity. Consistently, following experimental subarachnoid hemor- commonly used in the clinic to treat many inflammatory and rhage, exogenous Cst C administration was recently found to activate autoimmune conditions. Interestingly, this steroid drug can drive autophagy pathway ex vivo, which plays a beneficial role in early brain promoter-mediated upregulation of Cst C gene transcription, which 81,82 injury in a rat model. In addition to these apoptotic factors like leads to a significant and dose-dependent increase in the Cst C nutritional deprivation, oxidative stress and hemorrhage, another production by up to 80%. Recently, dexamethasone induced neurotoxic element that can be counterbalanced by Cst C partly secretions of Cst C from human cancer cells were found to be through autophagy is mutant Cu/Zn superoxide dismutase, a frequent enhanced by co-application of cisplatin and 5-fluorouracil, two agents 83 93 cause of inherited amyotrophic lateral sclerosis. commonly used in esophageal cancer chemotherapy, adding further factors to the repertoire of Cst C-altering elements. REGULATION OF CST C BY DIFFERENT STIMULI The numerous pathophysiological roles of Cst C foretell that the Physicochemical damages original application of this small molecular weight (MW) protein as a Rich in cerebrospinal fluid and brain tissue, Cst C is susceptible to measurement of the GFR for kidney function might no longer be physical and chemical insults that may occur in the central nervous appropriate, as participation in various pathophysiological processes system. For example, enhanced Cst C expression was observed in could lead to consumption and subsequent regulation of this multi- response to all sorts of neurological injuries, including transient 94 95 functional protease inhibitor. Indeed, Cst C levels could be altered by forebrain ischemia and seizure. Consistently, the severity of many common stimuli under both physiological and diseased neuronal damage in the CA1 subfield of the hippocampus correlates conditions. with enhanced Cst C immunostaining in microglia, the major Cst C-expressing cell type in normal brain tissues. Indeed electrical Inflammatory cytokines and pathogens induction of a status epilepticus causes upregulation of Cst C Inflammation is a quite common condition caused by various expression in rat neurons and glia. In addition, persistent pathogens that elicit the burst of inflammatory cytokines by the host environmental toxicants, like dieldrin, and neurotoxin MPP as a first line of defense. The influence of inflammatory cytokines on (1-methy1-4phenyl-1,2,3,6-tetrahydropyridine), were also reported to the production of Cst C was documented almost 30 years ago. injure dopaminergic neurons and stimulate their secretion of Cst C for Treatment of resident mouse peritoneal macrophages in vitro with the microglia activation and neurotoxicity. bacterial compound lipopolysaccharide (LPS) or pro-inflammatory Another Cst C-sensitive tissue that is also vulnerable to physical IFN-γ downregulates Cst C secretion. Likewise, inflammatory impairment comes from blood vasculature where proteolytic activity cytokine IL-6 signaling in vivo was found to decrease Cst C expression of cysteine proteases requires strict regulation by their endogenous in DCs. Moreover, human immunodeficiency virus infection could inhibitor. Balloon injury was recently reported to increase serum Cst C either inhibit Cst C expression in DCs or reduce the reactivity of Cst levels, which correlated with proliferating cell nuclear antigen in 85 99 C with its target enzyme cathepsin B in macrophages. Along the smooth muscle cells. same line, we found that in an inflammatory mouse model created by intravenous injection of CpG oligodeoxynucleotides, mimics of Oxidative stress bacterial and viral DNA responsible for immune stimulation, the Interestingly, the two tissues vulnerable to Cst C levels and damages as synthesis of Cst C in DCs as well as the circulating pools of Cst C in mentioned above are also sensitive to the disturbances in their normal blood were greatly reduced. In non-hematopoietic cells, however, redox state. Oxidative stress initiates pathological progression through addition of periodontal pathogens and pro-inflammatory cytokines to the production of peroxides and free radicals that damage all human gingival fibroblasts was found to enhance their Cst C components of the cells, including proteins, lipids and DNA. Although expression, and upregulated levels of Cst C was observed in the the exact mechanisms by which Cst C is possibly involved in the ethmoid sinus mucosa of patients with chronic sinusitis, indicating disease development remain to be clarified, oxidative stress was different roles and/or regulatory mechanisms of Cst C might exist in repeatedly reported to upregulate Cst C expression in neurological different cell types and tissues. However, the substantial impact of and cardiovascular systems. For example, oxidative stress causes an pathogens on immune cells, and our recent finding that serum levels increase in Cst C expression in cultured rat primary neurons of Cst C could be significantly affected by the replacement of bone and cerebral microvascular smooth muscle cells. Moreover, marrows highlights the important impact of altered Cst C produc- 6-hydroxydopamine induced a temporal and concentration- tion from hematopoietic cells on the systematic pools of Cst C during dependent increase in Cst C secretion from pheochromocytoma inflammation. cells. The release of Cst C in response to H O as one of the 2 2 cytoprotective, anti-apoptotic factors from the embryonic stem cells Growth factors and hormones were believed to be the mechanism to explain why transplanted Growth factors or hormones are naturally occurring substance capable embryonic stem cells subsequent to myocardial infarction differentiate of regulating a variety of cellular processes by stimulating cellular into the major cell types in the heart and improve cardiac function. growth, proliferation, healing and differentiation. They are also Interestingly, this H O induced release of Cst C was not only 2 2 frequently used in the clinic to adjust the imbalanced cellular process. observed in embryonic stem cells but was also observed later in Immunology and Cell Biology Regulation of cystatin C in diseases YXu et al Figure 1 Regulation of cystatin C. Multiple regulatory mechanisms exist to modulate the production and activity of cystatin C inside and outside of cells, at transcriptional and post-translational levels. JAK, Janus kinase; MyD88, myeloid differentiation primary response gene (88); ROSs, reactive oxygen species; STAT, signal transducer and activator of transcription; TLR, Toll-like receptor. A full color version of this figure is available at the Immunology and Cell Biology journal online. cardiomyocytes isolated from rat hearts because coronary artery bacterial and viral infection. However, the transcriptional regulation blocking-induced myocardial ischemia causes an increase in the levels of Cst C was not extensively explored probably due to the ubiquitous of Cst C protein in the plasma. expression pattern of the protein. With the increasing recognition of the important roles of Cst C in cardiovascular diseases, the association Others of this elastolytic cysteine protease inhibitor with human coronary Other factors affecting Cst C production include the lung toxicants artery disease began to be examined at genetic levels. Two common crystalline silica and arsenic trioxide, which were reported to stimulate promoter polymorphisms, a G-to-C substitution at position − 82 and Cst C release from rat alveolar macrophages. In cancer patients a T-to-G substitution at position − 78, were found to influence the during malignant progression-like melanoma and colorectal cancer, binding of nuclear factors and affect the basal rate of gene transcrip- high serum concentrations of the cysteine proteases cathepsins B and tion in an allele-specific manner, which are also associated with the H induced by the tumors stimulate the production of Cst C for plasma concentration of Cst C in healthy individuals and patients with counterbalance. In addition, common factors such as cigarette recent myocardial infarction. In another study, the major haplotype smoking or C-reactive protein, an acute-phase protein in response − 82G/− 5G/+4A of the cst3 gene was found to determine plasma to inflammation, were also among the lists reported to increase serum levels of Cst C as the respondents with homozygous genotypes have Cst C levels independent of renal functions. the highest plasma levels. Collectively, these data suggest that an altered promoter activity of the Cst C gene could be a causal factor MECHANISMS OF CST C REGULATION for the association between Cst C genotype and plasma Cst C The dynamic changes of Cst C caused by different stimuli herald the concentration. existence of, and prompt people to search for, the regulatory Consistent with the potential regulatory mechanisms of human Cst mechanisms of Cst C to keep this multifunctional and disease- C at genetic levels, we found that Cst C is differentially expressed associated protein under check. Like many other proteins, the among mouse cells of the immune system. Specifically, cells of the production of Cst C is subject to both transcriptional and post- monocyte/macrophage and DC lineages express it at much higher translational regulation (Figure 1). 28,89 levels than lymphocytes. Mouse DC can be divided into two + − subsets by their surface expression of CD8α (CD8 and CD8 DC). Transcriptional regulation Further analysis of the DC subsets directly isolated from the spleen Although several polymorphisms and sequence variations were 1,105 demonstrated that the CD8 DC were the major producer of Cst detected in the promoter region of cst3, the gene coding Cst C, 89,109 C. This was also verified with DCs that had been generated in it generally shares common features with those of housekeeping culture from bone marrow precursors supplemented with Flt3L, in genes. Interestingly, the promoter region of murine cst3 gene was + hi later found to contain a core sequence of the androgen-responsive which only the CD8 DC equivalent, the CD24 DC subset, contained high levels of Cst C. The differential expression pattern of Cst C element and two potential binding sites for activator protein 1, a among cells of common lineages suggests that its gene could be transcription factor that regulates gene expression in response to a variety of stimuli including cytokines, growth factors, stress and controlled by cell-specifictranscription factors. Immunology and Cell Biology Regulation of cystatin C in diseases YXu et al This preferential expression pattern of Cst C in monocyte/macro- Therefore, the factors that cause this conformational change of Cst C phage and DCs is of clinical relevance in view of the recent reports by dimerization can be regarded as a way to regulate the activity of this showing that these cell types are present in tissues where Cst C plays protease inhibitor. It is intriguing to think that Cst C adopts the pathological roles such as atherosclerosis and angiopathy. Vascular DCs unreactive dimer conformation as storage form, which quickly (aortic DCs), which increase in number in atherosclerotic lesions as monomerizes in response to the stimulation of its target enzymes. + 111 disease progresses, were reported to be related to CD8 DC, and the This post-translational regulation of Cst C by dimerization was first + − + Flt3L-signaling-dependent CD103 /CD11b (CD8 equivalent) DCs described in transfected Chinese hamster ovary cells, where Cst C is protect against atherosclerosis. Consistently, our analysis of DC inactivated during the early part of its trafficking through the secretory purified from aortas indicates that these cells indeed express Cst C pathway and then reactivated prior to secretion. Interestingly, we (unpublished observation). Similarly, microglia, also called the mono- found that steady-state (immature)CD8 DCs isolated from primary cyte/microphage in the brain, is the major cell type that express Cst C in spleens constitutively contain Cst C homo-dimers, which can be 96 64 the brain, where deposition of Cst C fibrils produces angiopathy. separated from monomers by size-exclusion chromatography of cell To uncover the transcriptional mechanisms underlying the prefer- lysates and their non-reactivity with the Cst C target enzyme papain, ential expression of disease-related Cst C in these cells, a novel cis supporting the notion that they are domain-swapped dimers. element for transcription factors, denoted as IRF (interferon regulatory Furthermore, when CD8 DC underwent maturation by incubation factor)-Ets composite sequence (IECS), in the promoter region of the in vitro,they no longer producedCst Cdimers. Disappearance of cst3 gene was identified in a reporter assay system employing a self- dimer could be the strategy employed by healthy primary cells to inactivating retrovirus. This element consists of a core IRF-binding process this potentially pathogenic form on their way toward motif for IRF8, and an Ets-binding motif for PU.1, an Ets transcrip- maturation, which might then be disrupted in diseased conditions. tion factor and binding partner that facilitates a more stable binding of To identify the mechanistic factors leading to Cst C dimerization IRF8 to chromatin. Notably, both IRF8 and PU.1 are essential for either as post-translational regulation of its activity, or as amyloid 115,116 the development of DC subsets, of which IRF8 is required for precursor protein, the intracellular accumulation of reactive oxygen + + 115 the development of CD8 CD103 DC, the major producers of species in the immature and mature states were compared. A strong Cst C. This could imply that Cst C expression by these cells might be correlation between reactive oxygen species levels and Cst C dimer was the direct consequence of the presence of IRF8-binding motif in the observed not only in same cell type at different developmental stages, Cst C promoter region in cells that depend on IRF8 for their but also in different cell types at the same developmental stages. development. However, this cannot be the only explanation because Furthermore, artificial enhancement of the intracellular oxidative IRF8 is also required in plasmacytoid DC development, yet this DC status resulted in a time-dependent Cst C dimer enrichment, which type does not express Cst C. Thus, the molecular mechanisms could be prevented by inhibiting mitochondrial activity, indicating the responsible for this phenomenon remain to be revealed. reactive oxygen species released from mitochondria are responsible for To investigate the involvement of these two transcription factors in the observed constitutive Cst C dimer formation. Although, the Cst C expression in primary DC in vivo, the physical interaction of exact process by which oxidative stress exerts the conformational IRF8 and PU.1 with the cis element IECS was examined by chromatin changes of Cst C is not fully understood, a recently published report immunoprecipitation in three different DC subsets freshly isolated on recombinant human Cst C stabilized by genetically introduced from splenic cells. Consistent with their Cst C expression profile disulfide linkage demonstrated a disappearance of a dithiothreitol- + − (CD8 DC express Cst C, both CD8 DC and plasmacytoid DC do not induced dimer if the concentration of this reducing agent was further express Cst C), the binding of IRF8 to the IECS sequence was detected increased, suggesting a direct impact of redox environment on the + − in CD8 DC, but not in CD8 DC and plasmacytoid DC. Interestingly, conformational changes of Cst C protein. in the plasmacytoid DC where PU.1 expression is low but IRF8 Different from type I cystain family members, the type II cystatin expression is the same as CD8 DC, the binding of IRF8 to the IECS Cst C is synthesized with a signal peptide, hence being secreted and sequence is still not detected, indicating a quantitative requirement of consequently found in body fluids. Although its intracellular roles in PU.1 in IRF8 binding to the chromatin, a result consistent with processing MHCII molecules via lysosomal proteinase in DCs were previous findings with fluorescence recovery after filling to mathema- debatable, the uptake of Cst C in amounts sufficient to affect the tical models. Collectively, the chromatin immunoprecipitation data activities of intracellular cysteine proteases was described in the 120 121 suggest that IRF8 binding to the IECS of the Cst C promoter in the eyes, proximal tubule cells and several human cancer cell 122,123 presence of sufficient PU.1 could drive cst 3 expression. To finally lines. Thus, trafficking inside and outside of the cell membrane confirm the role of IRF8 in Cst C expression, a CD8 DC line 1940, of Cst C can regulate or fine tune its division of labor between where both IRF8 and PU.1 were amply expressed, were transfected intracellular compartments and the extracellular matrix. However, the with the retrovirus vector LMP encoding shRNAs for irf8 to silence the structural determinants on the internalized inhibitor required for gene. When the synthesis of IRF8 was reduced in the cells, that of Cst efficient uptake were not characterized until recently 12 variants of Cst C, but not MHC I, was also compromised. This causal impact of IRF8 C with substitutions of selected amino acids were generated. Uptake on Cst C production, combined with the diminished Cst C expression of Cst C in human breast adenocarcinoma cells is dependent on both − /− in bone marrow (BM)-derived DC from irf8 mice compared with charged amino acids of the N-terminal segment and on a hydrophobic wild-type control, strongly suggest that IRF8 is the key transcription amino acid in domains involved in the inhibition of cysteine factor regulating Cst C expression. cathepsins. Furthermore, natural arginine (Arg) residues in positions 24 and 25 are of importance for the uptake process. Post-translational regulation Another mechanism for post-translational regulation of Cst C could The translational expression of Cst C in quantity does not necessarily come from digestion of this cysteine protease inhibitor by proteases of lead to a functional protein product. The activity of Cst C can still be another family. For example, earlier proteomic studies have shown further modulated after its transcription. As discussed before, the that Cst C is a substrate of matrix metalloproteinase 2 with specific reactivity of Cst C with its target protease is lost if it dimerizes. inactivation upon cleavage. In addition, human aspartic Immunology and Cell Biology Regulation of cystatin C in diseases YXu et al family members for early prevention, or selectively remove Cst C dimers from biological fluids containing both dimers and monomers in patients with hereditary cystatin C amyloid angiopathy. In addition, the mechanistic factors affecting the inhibitive activity of Cst C can also be harnessed for therapeutic gains. Drugs to improve the intracellular redox environment by removal of reactive oxygen species and compounds may be beneficial in not only reducing the Cst C amyloidogenesis, but also regulating the elastic proteolysis in the diseased locus as elevated protease activity in the local body fluids is partly responsible for the tissue destruction in the disease associated with Cst C amyloid. Last but not least, identification of transplantable cellular sources for major Cst C production will also be of great clinical value. Since Cst C is involved in inflammatory diseases, in which immune cells accumulate and play important roles, bone marrow-derived cells are an important cellular source for Cst C manipulation. Along this line, we found that hematopoietic cells contribute significantly to the systematic pools of Cst C. Therefore, bone marrow transplantation Figure 2 Multiple factors are involved in the progression of cystatin C-related would be an applicable approach in clinic to treat patients with Cst C diseases. A scheme summarizing factors leading to the fluctuation of amyloidogenesis or reduction. cystatin C levels, which triggles or participates in the pathogenic processes of several diseases. CRP, C-reactive protein. A full color version of this figure CONCLUSION is available at the Immunology and Cell Biology journal online. With the recent identification of regulatory elements in the promoter region of Cst C and increasing reports of factors affecting its production endoproteinase cathepsin D was also able to inactivate human Cst C and/or activities, precaution should be taken when Cst C is used as an by cutting hydrophobic amino acid residues into several fragments index of GFR because its blood concentration is subject to changes in vitro. Along the same line, the amount of Cst C in the caused by many factors independent of kidney function. These factors extracellular environment is reduced in the secretome of mouse include cigarette smoking, body composition, viral infection, tumor embryonic fibroblasts stably transfected with human cathepsin D, malignancy or gene mutations. Ultimately, the altered Cst C levels or and the tumor-derived cathepsin D assists breast cancer progression 126 activity could lead to pathological processes in cardiovascular diseases, by inhibiting Cst C activity. Importantly, the relevance of this neurological disorders or even mortality (Figure 2). Such an important mechanism was also found in other biological process in which Cst C disease-associated protein thus should be effectively regulated for was shown to be a proteolytic target of cathepsin D, affecting the 127 therapeutic gains. Further characterization of the signaling pathways differentiation of DCs from hematopoietic stem cells. leading to Cst C expression will help to develop antagonists and/or synagonists for medical intervention or regulation of the abnormal CLINICAL IMPLICATIONS production and/or activity of this disease-associated enzyme inhibitor. Given the increasingly discovered roles of Cst C in various pathophy- Likewise, identification of the post-translational modifications in the siological processes and identification of its regulatory mechanisms at microheterogeneity of the pathogenic species will also assist in under- both transcriptional and post-transcriptional levels, manipulation of standing the mechanisms or direct factors triggering Cst C dimerization. Cst C expression either locally or systematically may have many The knowledge should provide the information for a better under- clinical implications. Characterization of the factors that control Cst C standing of the mysterious mechanisms underlying the diseases expression at the transcriptional level could provide valuable clues for associated with Cst C abnormalities. the treatment of pathologies associated with insufficient control of extracellular proteases. For example, signaling molecules that upregu- CONFLICT OF INTEREST late Cst C expression could be used to promote Cst C secretion in the The authors declare no conflict of interest. case that requires slightly higher Cst C concentration to protect neuronal cells from cell death in Alzheimer’s disease, or at sites of ACKNOWLEDGEMENTS inflammation in which excessive protease activity causes tissue This work was funded by a Project Grant from National Health and Medical damage, as has been suggested to occur in atherosclerosis and aortic Research Council (NHMRC) of Australia (no. 1006428), and a start-up fund aneurysm. 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FEBS J 2008; 275: 4571–4582. 127 Martino S, Tiribuzi R, Ciraci E, Makrypidi G, D'Angelo F, di Girolamo I et al. 123 Wallin H, Bjarnadottir M, Vogel LK, Wasselius J, Ekstrom U, Abrahamson M. Cystatins Coordinated involvement of cathepsins S, D and cystatin C in the commitment of —extra- and intracellular cysteine protease inhibitors: high-level secretion and uptake hematopoietic stem cells to dendritic cells. Int J Biochem Cell Biol 2011; 43: of cystatin C in human neuroblastoma cells. Biochimie 2010; 92:1625–1634. 775–783. Immunology and Cell Biology http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Immunology and Cell Biology Pubmed Central

Cystatin C is a disease‐associated protein subject to multiple regulation

Immunology and Cell Biology , Volume 93 (5) – Feb 3, 2015

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Abstract

Immunology and Cell Biology (2015) 93, 442–451 & 2015 Australasian Society for Immunology Inc. All rights reserved 0818-9641/15 www.nature.com/icb REVIEW Cystatin C is a disease-associated protein subject to multiple regulation Yuekang Xu, Ying Ding, Xinchen Li and Xiaobing Wu A protease inhibitor, cystatin C (Cst C), is a secreted cysteine protease inhibitor abundantly expressed in body fluids. Clinically, it is mostly used to measure glomerular filtration rate as a marker for kidney function due to its relatively small molecular weight and easy detection. However, recent findings suggest that Cst C is regulated at both transcriptional and post-translational levels, and Cst C production from haematopoietic cell lineages contributes significantly to the systematic pools of Cst C. Furthermore, Cst C is directly linked to many pathologic processes through various mechanisms. Thus fluctuation of Cst C levels might have serious clinical implications rather than a mere reflection of kidney functions. Here, we summarize the pathophysiological roles of Cst C dependent and independent on its inhibition of proteases, outline its change of expression by various stimuli, and elucidate the regulatory mechanisms to control this disease-related protease inhibitor. Finally, we discuss the clinical implications of these findings for translational gains. Immunology and Cell Biology (2015) 93, 442–451; doi:10.1038/icb.2014.121; published online 3 February 2015 Cystatin C (Cst C) is a potent extracellular inhibitor of cysteine alteration in patients with cancer, HIV infection, cardiovascular 11–13 protease, and has been generally considered a ubiquitously expressed diseases and neurological disorders. Moreover, new studies protein as no obvious regulatory elements were found in its gene reported direct involvement of Cst C in many pathogenic processes 1 14,15 promoter. Clinically, Cst C is mostly used as a biomarker of kidney other than renal disorders. Thus, the oscillation of blood Cst C function for its relatively lower molecular weight (~13.3 kDa) and easy levels could actually reflect the change of Cst C production, detection compared to the injection of compounds, radioisotopes or consumption, inactivation or fibrillation (mentioned later) rather than radiocontrast agent to measure glomerular filtration rate (GFR), an its filtration in the kidney, and the readout of plasma Cst C index of kidney health, because Cst C is removed from the blood concentration might have different clinical implications. In this review, stream by glomerular filtration, whose decline as a result of failed we first summarize the major pathophysiological roles of Cst C to kidney function will lead to increased serum Cst C concentration. Cst illustrate its importance as a functional protein in the body rather than C serum levels were claimed to be a more precise index of kidney an ubiquitously expressed measuring substance. Second, we outline function than that of creatinine under the assumption that serum the impact of different stimuli on its expression and elucidate some of input of Cst C is constant, and the main determinant of blood Cst C the latest developments on the regulatory mechanisms to explain Cst levels is the rate at which it is filtered at the glomerulus. Furthermore, C variation under certain circumstances. In the end, the clinical recent advances have facilitated the use of Cst C as a clinical measure implications derived from these studies are also discussed. of kidney function (the update on Cst C in recent kidney disease guidelines has been reviewed elsewhere ). Although, the bulk of PATHOPHYSIOLOGICAL ROLES OF CST C literature report the use of Cst C for GFR estimation, precaution Functions dependent of its inhibition of proteases should be taken when the outcomes of this measurement is Cysteine cathepsins play fundamental roles in multiple biological interpreted because recent studies indicated that both genetic processes such as protein turnover, pro-protein processing, bone 5,6 7,8 polymorphisms and clinical interference could make Cst C an 16 remodeling, antigen presentation and apoptosis. They are also involved unreliable index of GFR. Indeed, accumulating reports have docu- in numerous pathological processes such as cardiovascular disease and mented that subjected to the influence of many factors, serum Cst C inflammation. The activities of these enzymes both inside and outside levels do vary independent of renal functions, and the synthesis and of cells thus need to be tightly controlled by their endogenous inhibitors, secretion of Cst C seem to be tightly regulated under different of which Cst C is the most abundant and potent member. pathophysiological conditions. For example, body composition, thyroid function, glucocorticoid and C-reactive protein levels or even Intracellular roles cigarette smoking and pregnancy status of the candidates examined Apoptosis. Intracellular lysosomes undergo membrane permeabili- 9,10 have been found to affect Cst C blood concentration, let alone its zation in response to extra- or intra-lysosomal stimuli, and the Anhui Provincial Key Laboratory for Conservation and Exploitation of Biological Resources, School of Life Sciences, Anhui Normal University, Anhui, P.R. China Correspondence: Professor Y Xu, School of Life Science, Anhui Normal University, 1 Beijing East Road, Wuhu, Anhui Province 241000, P.R. China. E-mail: yuekang.xu@hotmail.com Received 28 October 2014; revised 7 December 2014; accepted 8 December 2014; published online 3 February 2015 Regulation of cystatin C in diseases YXu et al involvement of lysosomal cysteine proteases in apoptosis has been immature and mature DCs suggests that the regulatory potential of Cst 17,18 confirmed in several systems. However, the roles of the endogen- C toward these cathepsins inside DCs is limited, which could explain ous lysosomal cysteine protease inhibitor Cst C in apoptosis remain the inconsistent findings related to the intracellular roles of Cst C. controversial. Upregulation of Cst C expression was found to correlate Instead, large secretion of Cst C over cathepsins S, L and H was 19 30 with oxidative stress-induced apoptosis in cultured rat neurons, and observed in the culture media, indicating that the extracellular Cst C injection into rat hippocampus led to neuronal cell death in the compartment is the primary site for these interactions. granule cell layer of dentate gyrus in vivo, indicating a possible Extracellular roles. The structure of Cst C predisposes this protease functional role of Cst C in apoptosis induction. Since, the Cst inhibitor to extracellular functions as a secreted protein. Accordingly, C-induced neuronal cell death could be inhibited by simultaneous Cst C is found in all body fluids at significant concentrations, which co-application of cathepsin B, the inhibitory activity of Cst C on a makes it a major regulator of cysteine protease activity in the cysteine protease was proposed to be involved in the process of extracellular medium. apoptotic neuronal cell death. Recent studies indicated that the roles of Cst C in neuronal cell apoptosis induction include decreasing B-cell Atherosclerosis. Atherosclerosis-based vascular disease is an inflam- leukemia-2 (Bcl-2) and increasing active caspase-9 protein levels via matory disease characterized by extensive remodeling of the extra- the Jun N-terminal kinase (JNK)-dependent pathway, and upregula- cellular matrix of the arterial walls. Apart from the well-known matrix tion and accumulation of the insoluble α-synuclein in oligodendro- metalloproteinases and serine proteases, lysosomal cysteine proteases cytes and neurons. Interestingly, apart from these documented pro- 32 were also found to be involved. Present in substantial amounts in the apoptotic roles, Cst C was also found to have anti-apoptotic effects on normal vessel walls, the expression of Cst C was found to be severely neuronal cells. For example, expression of Cst C in PC12 cells derived reduced in both atherosclerostic and aneurysmal lesions, and increased from a pheochromocytoma of rat adrenal medulla prevents oxidative abdominal aortic diameter correlated with lower serum Cst C levels in stress-induced death in vitro. In accordance with this finding, the 13 humans. Furthermore, the pathogenic roles of Cst C were tested in a anti-apoptotic function of Cst C was further demonstrated in vivo in a model of atherosclerosis-prone mice with apolipoprotein E deficiency mouse model of the inherited neurodegenerative disorder, progressive − / − (apoE ) where the elevated cathepsins were associated with the myoclonic epilepsy type 1, where loss of function in cystatin B and 33 atherosclerostic process. In two independent studies, Cst C- and enhanced cathepsin B and D activities are the underlying pathologies. apoE-double-deficient mice were generated, both of them confirmed Crossbreeding of the mice with either Cst C-overexpressing transgenic − / − an anti-atherosclerostic function of Cst C in the apoE mice, or Cst C-deficient mice revealed that Cst C levels in vivo can affect although differences regarding lesion size and composition were neuron degeneration caused by the proteases, indicating that Cst C 34,35 found. These differences are most probably caused by differences partially prevents neural cell death in vivo through inhibition of in the duration of high-fat diet of the mice, sex of the mice and cathepsins activity. (Described in the section of Neuroprotective anatomic site of analyzed lesions. Interestingly, in line with the anti- roles.) atherosclerostic role of Cst C in this animal model, polymorphisms in the promoter regions of the Cst C gene were found to influence the Antigen presentation. Proteolysis of antigen by cysteine cathepsins to plasma Cst C concentration in human coronary artery disease, generate immunogenic peptides and guide the transit of both major indicating the production of this protease inhibitor could be subject histocompatibility complex class II (MHCII) and MHC-like molecules to regulation under the diseased conditions. through the endocytic compartments are important intracellular activities in antigen-presenting cells. As a potent inhibitor of cysteine Tumor metastasis. Metastatic tumor cells invade host tissues through cathepsins, Cst C had initially been implicated in playing a regulatory a series of steps that require proteolytic enzymes including cysteine role in the developmental control of MHCII presentation in dendritic cathepsins to degrade components of the extracellular matrix. As a cells (DCs) by inhibiting cathepsin S in invariant chain cleavage. most abundant endogenous cysteine proteinase inhibitor, Cst C is Consistent with the role of Cst C in antigen presentation, a cystatin believed to prevent tumor progression by inhibiting the activities of a homolog produced by the filarial nematode parasite was found to family of lysosomal cysteine cathepsins. Results from the first Cst inhibit lysosomal cysteine protease activities, which subsequently C-deficient animals indicated that Cst C concentration in vivo might impeded the generation of human B cells. Further experiments influence tumor metastasis in some tissues. In agreement, the using bone marrow-derived DCs indicate that interleukin 6 (IL-6)- reduced Cst C levels correlate with increased metastasis of different mediated signal transducer and activator of transcription 3 (STAT3) tumors in human tissues or patients. Furthermore, local over- activation decreased Cst C expression and MHCII αβ dimer levels. expression of Cst C in the host tissue microenvironment could lead to However, a separate study using mouse primary DCs isolated from Cst successful reduction of metastasis via cysteine cathepsin inhibition in C-deficient mice demonstrated that Cst C is neither necessary nor 40 an experimental tumor model. This inverse correlation between sufficient to control MHCII expression and antigen presentation in Cst C and tumor aggressiveness, however, may not always involve DCs. These discrepant results obtained from different laboratories inhibition of cysteine proteases. For example, elevated matrix metal- could be due to the different cell types investigated, and/or to the loproteinases 2 and crosstalk between Cst C and androgen receptor- compensatory roles played by other cysteine protease inhibitors in the mediated pathways were reportedly implicated in prostate cancer absence of Cst C. In support of this view, cell-specificregulation of invasion and metastasis, indicating multiple roles of Cst C in tumor cathepsin activity by Cst C was identified in two similar antigen- metastasis. presenting cells in the brain: Cst C was found to inhibit cathepsin L activity in astrocytes, but does not regulate cathepsins L and S in Pathogen invasion. Although a secreted protein, Cst C was also microglia. reported to be up-taken by cells of both foreign and endogenous When intracellular localization of the Cst C and its target proteases origins in various tissues to regulate both intracellular and extracellular were examined in human DC differentiated from monocytes in vitro, cysteine protease activities. One of the major roles of cystatins is to the different compartmentation of Cst C and cathepsins S, L and H in protect the host against invading microorganisms and parasites that Immunology and Cell Biology Regulation of cystatin C in diseases YXu et al use cysteine proteases to enter the body. Chicken cystatin was first amyloidogenesis begin with dimerization by a process known as reported to partially block poliovirus replication in infected human ‘three-dimentional domain swapping’, in which two parts of the cells. Further study demonstrated that a small peptide derivative that cystatin structure become separated from each other and next mimics part of the proteinase-binding center of human Cst C could exchanged between two molecules. Interestingly, with their inhibi- inhibit a cysteine protease specific for the growth of group A tory region hidden within the dimer interface, Cst C dimers cannot streptococci, blocking the growth of these bacteria both in vivo and inhibit cysteine proteases. This none-inhibitory Cst C dimer is in vitro. Similarly, recombinant human Cst C was also proven required for the formation of the Cst C oligomers, intermediates in fibrillogenesis, because variants of monomeric Cst C, stabilized against to inhibit the growth of herpes simplex virus and human coronaviruses. Moreover, a family of cathepsin L- and B-like cysteine domain swapping to block the inhibitory site, fail to produce proteases, found in all species of Leishmania examined, are required oligomers, indicating that Cst C fibrils are formed by propagated 48 61 for the parasite growth and virulence. Since, these parasite cysteine domain swapping. proteases may not only digest the host extracellular matrix to facilitate Domain swapping must be preceded by at least partial unfolding of their invasion, but also help to ensure a Th -like response led to the molecule, therefore, any factors affecting the stabilization of the parasite proliferation, cystatin treatment in combination with inter- molecule could initiate the domain swapping process. For instance, the feron γ (IFNγ) that leads to reduced parasite numbers, successful Th point mutation with substitution of native leucine in position 68 by to Th conversion, and NO generation, which finally resulted in glutamine (L68Q) disrupts a network of the hydrophobic interactions, abrogation of parasite infection in a mouse model of leishmaniasis. which leads to increased tendency for dimerization and aggregation in vitro. The L68Q replacement is also a naturally occurring point Functions independent of its inhibition of proteases mutation in the Cst C protein sequence with autosomal dominant Modulating roles. Most abundantly expressed in tissues, Cst C also hereditary. This Cst C variants form fibrils in vivo in the brain serves numerous functions independent of protease inhibition includ- vasculature, which cause hemorrhage, dementia and eventually death ing affecting signaling properties of other molecules. For example, Cst in people carrying this mutation, a condition known as hereditary C antagonizes the binding of transforming growth factor-beta (TGF-β) cystatin C amyloid angiopathy, also called hereditary cerebral to its cell receptors by physically interacting with TGF-β type II hemorrhage with amyloidosis, icelandic type. The cellular transport receptor independent of its protease inhibitory activity, as over- of Cst C is impeded by the mutated L68Q variants, resulting in 66,67 expression of Cst C mutant that is impaired in its ability to inhibit diminished Cst C levels in cerebrospinal fluid and retained Cst C cathepsin activity blocked TGF-β-dependent invasion of 3T3-L1 mutants in blood monocytes from patients. Consistently, in vitro fibroblasts. This novel function of signaling modulation allows Cst studies demonstrated that clones expressing the gene encoding L68Q C to inhibit the oncogenic activities of TGF-β through stimulation of Cst C secreted either lower amounts of Cst C, or unstable protein 52 69 mammary epithelial-mesenchymal transition. Apart from blocking susceptible to a serine protease, contributing to the reduced interaction between TGF-β and its receptor, Cst C was also found to extracellular Cst C levels. prevent TGF-β signaling partly by reducing the extent of mothers Cst C amyloid fibrils not only affect brain vasculature but also may against decapentaplegic homolog 2 (Smad2), p38 mitogen-activated lead to toxicity in other tissues. As the most abundant protein, Cst C protein kinases (p38 MAPKs) and extracellular signal-regulated was found in tissues outside of the brain including the testis, and so kinases 1/2 (Erk1/2) phosphorylation in murine 4T1 breast cancer was the L68Q variant. A recent study reports that heterozygous cells. In addition to affecting TGF-β signaling, CstCis involved in transgenic mice that express this pathogenic variant were unable to the IFNγ signal transduction pathway. It activates nuclear factor generate offspring, indicating the L68Q Cst C amyloid affects sperm kappa-light-chain-enhancer of activated B cells (NF-κB) p65, induces function. Further analysis of the L68Q mice demonstrated that their NO synthase, but downregulates IL-10 in macrophages. epididymal spermatozoa were unable to fertilize oocytes and exhibited Through interacting with other proteins with effects on their poor sperm motility in the presence of Cst C amyloid that were not signaling properties, Cst C can also affect biological functions of cells. found in the wild-type mice. The L68Q epididymal fluid, when During brain development, Cst C was identified as a factor to depleted of the Cst C amyloids, however, did not impair the motility upregulate the glial fibrilllary acidic protein promoter. As a result, of wild-type spermatozoa, suggesting that amyloids in the epididymal addition of human Cst C to the culture medium of primary brain cells fluid can be cytotoxic to the maturing spermatozoa resulting in male increased the number of glial fibrilllary acidic protein-positive cells infertility. However, two other groups in earlier studies have also and neurospheres formed from the embryonic brain. Again, the generated the transgenic lines expressing this variant form of Cst C, 72,73 promotion of astrocyte development by Cst C appears to be unrelated but they were live and fertile, questioning whether the pathology to its protease inhibitor activity, as another cysteine protease inhibitor described is really brought about by the Cst C variants or the site did not have this effect. Along the same line, a recent study identified where the transgene was inserted. a novel function of Cst C in mediating amyloid β (Aβ) precursor Neuroprotective roles. Although mutant Cst C is toxic by amyloidosis, protein-induced proliferation of neural stem/progenitor cells. wild-type Cst C has multiple neuroprotective roles (comprehensively Although the mechanism of Aβ precursor protein in stimulating reviewed by Gauthier et al. ). An amyloidogenic protein itself, Cst C neural stem/progenitor cells to secrete Cst C needs further character- has an anti-toxic role against another amyloid protein, Aβ.Studies ization, this study contributes to a better understanding of multi- in vitro demonstrated that Cst C binds to Aβ and inhibits its functional roles of Cst C in the pathogenesis of Alzheimer’sdisease. 75 76 oligomerization and amyloid fibril formation. Furthermore, in Amyloidogenic roles. In addition to cysteine protease inhibition, Cst C Aβ precursor protein transgenic mice, Cst C was found to physically is also one of the few amyloidogenic proteins that form a fibrillary associate with the soluble, non-pathological form of Aβ in vivo, which structure deposited in the vascular walls, affecting the health of blood inhibited the aggregation and deposition of Aβ plaques in the 73,77 vessels (angiopathy). The proteins identified in cerebral amyloid brain. Moreover, Cst C can also directly protect neuronal cells angiopathy include beta/A4, transthyretin and Cst C. Cst C from amyloid toxicity, as extracellular addition of human Cst C Immunology and Cell Biology Regulation of cystatin C in diseases YXu et al promoted the survival of cultured neuronal cells against the preformed TGF-β1 has been reported to upregulate Cst C secretion from vascular oligomeric or fibrillar Aβ. smooth muscle cells, murine embryo cells, cultured differentiated Autophagy is important for the survival and homeostasis of neurons podocytes, 3T3-L1 fibroblasts, and more recently human lung 13,51,90 as they cannot dilute accumulating detrimental substances or damaged fibroblasts. Clinically, a significant influence of circulating Cst organelles by cell division (see latest review for details). Cst C was Clevelsby TGF-β1 were also observed in patients with thyroid first reported to induce a fully functional autophagy to protect dysfunction, and both Cst C mRNA and protein levels were increased neuronal cells against various stress via the mammalian target of by TGF-β in cultured human hepatoblastoma cells. rapamycin (mTOR) pathway in vitro independent of its inhibitor Dexamethasone is a potent synthetic member of glucocorticoid class activity. Consistently, following experimental subarachnoid hemor- commonly used in the clinic to treat many inflammatory and rhage, exogenous Cst C administration was recently found to activate autoimmune conditions. Interestingly, this steroid drug can drive autophagy pathway ex vivo, which plays a beneficial role in early brain promoter-mediated upregulation of Cst C gene transcription, which 81,82 injury in a rat model. In addition to these apoptotic factors like leads to a significant and dose-dependent increase in the Cst C nutritional deprivation, oxidative stress and hemorrhage, another production by up to 80%. Recently, dexamethasone induced neurotoxic element that can be counterbalanced by Cst C partly secretions of Cst C from human cancer cells were found to be through autophagy is mutant Cu/Zn superoxide dismutase, a frequent enhanced by co-application of cisplatin and 5-fluorouracil, two agents 83 93 cause of inherited amyotrophic lateral sclerosis. commonly used in esophageal cancer chemotherapy, adding further factors to the repertoire of Cst C-altering elements. REGULATION OF CST C BY DIFFERENT STIMULI The numerous pathophysiological roles of Cst C foretell that the Physicochemical damages original application of this small molecular weight (MW) protein as a Rich in cerebrospinal fluid and brain tissue, Cst C is susceptible to measurement of the GFR for kidney function might no longer be physical and chemical insults that may occur in the central nervous appropriate, as participation in various pathophysiological processes system. For example, enhanced Cst C expression was observed in could lead to consumption and subsequent regulation of this multi- response to all sorts of neurological injuries, including transient 94 95 functional protease inhibitor. Indeed, Cst C levels could be altered by forebrain ischemia and seizure. Consistently, the severity of many common stimuli under both physiological and diseased neuronal damage in the CA1 subfield of the hippocampus correlates conditions. with enhanced Cst C immunostaining in microglia, the major Cst C-expressing cell type in normal brain tissues. Indeed electrical Inflammatory cytokines and pathogens induction of a status epilepticus causes upregulation of Cst C Inflammation is a quite common condition caused by various expression in rat neurons and glia. In addition, persistent pathogens that elicit the burst of inflammatory cytokines by the host environmental toxicants, like dieldrin, and neurotoxin MPP as a first line of defense. The influence of inflammatory cytokines on (1-methy1-4phenyl-1,2,3,6-tetrahydropyridine), were also reported to the production of Cst C was documented almost 30 years ago. injure dopaminergic neurons and stimulate their secretion of Cst C for Treatment of resident mouse peritoneal macrophages in vitro with the microglia activation and neurotoxicity. bacterial compound lipopolysaccharide (LPS) or pro-inflammatory Another Cst C-sensitive tissue that is also vulnerable to physical IFN-γ downregulates Cst C secretion. Likewise, inflammatory impairment comes from blood vasculature where proteolytic activity cytokine IL-6 signaling in vivo was found to decrease Cst C expression of cysteine proteases requires strict regulation by their endogenous in DCs. Moreover, human immunodeficiency virus infection could inhibitor. Balloon injury was recently reported to increase serum Cst C either inhibit Cst C expression in DCs or reduce the reactivity of Cst levels, which correlated with proliferating cell nuclear antigen in 85 99 C with its target enzyme cathepsin B in macrophages. Along the smooth muscle cells. same line, we found that in an inflammatory mouse model created by intravenous injection of CpG oligodeoxynucleotides, mimics of Oxidative stress bacterial and viral DNA responsible for immune stimulation, the Interestingly, the two tissues vulnerable to Cst C levels and damages as synthesis of Cst C in DCs as well as the circulating pools of Cst C in mentioned above are also sensitive to the disturbances in their normal blood were greatly reduced. In non-hematopoietic cells, however, redox state. Oxidative stress initiates pathological progression through addition of periodontal pathogens and pro-inflammatory cytokines to the production of peroxides and free radicals that damage all human gingival fibroblasts was found to enhance their Cst C components of the cells, including proteins, lipids and DNA. Although expression, and upregulated levels of Cst C was observed in the the exact mechanisms by which Cst C is possibly involved in the ethmoid sinus mucosa of patients with chronic sinusitis, indicating disease development remain to be clarified, oxidative stress was different roles and/or regulatory mechanisms of Cst C might exist in repeatedly reported to upregulate Cst C expression in neurological different cell types and tissues. However, the substantial impact of and cardiovascular systems. For example, oxidative stress causes an pathogens on immune cells, and our recent finding that serum levels increase in Cst C expression in cultured rat primary neurons of Cst C could be significantly affected by the replacement of bone and cerebral microvascular smooth muscle cells. Moreover, marrows highlights the important impact of altered Cst C produc- 6-hydroxydopamine induced a temporal and concentration- tion from hematopoietic cells on the systematic pools of Cst C during dependent increase in Cst C secretion from pheochromocytoma inflammation. cells. The release of Cst C in response to H O as one of the 2 2 cytoprotective, anti-apoptotic factors from the embryonic stem cells Growth factors and hormones were believed to be the mechanism to explain why transplanted Growth factors or hormones are naturally occurring substance capable embryonic stem cells subsequent to myocardial infarction differentiate of regulating a variety of cellular processes by stimulating cellular into the major cell types in the heart and improve cardiac function. growth, proliferation, healing and differentiation. They are also Interestingly, this H O induced release of Cst C was not only 2 2 frequently used in the clinic to adjust the imbalanced cellular process. observed in embryonic stem cells but was also observed later in Immunology and Cell Biology Regulation of cystatin C in diseases YXu et al Figure 1 Regulation of cystatin C. Multiple regulatory mechanisms exist to modulate the production and activity of cystatin C inside and outside of cells, at transcriptional and post-translational levels. JAK, Janus kinase; MyD88, myeloid differentiation primary response gene (88); ROSs, reactive oxygen species; STAT, signal transducer and activator of transcription; TLR, Toll-like receptor. A full color version of this figure is available at the Immunology and Cell Biology journal online. cardiomyocytes isolated from rat hearts because coronary artery bacterial and viral infection. However, the transcriptional regulation blocking-induced myocardial ischemia causes an increase in the levels of Cst C was not extensively explored probably due to the ubiquitous of Cst C protein in the plasma. expression pattern of the protein. With the increasing recognition of the important roles of Cst C in cardiovascular diseases, the association Others of this elastolytic cysteine protease inhibitor with human coronary Other factors affecting Cst C production include the lung toxicants artery disease began to be examined at genetic levels. Two common crystalline silica and arsenic trioxide, which were reported to stimulate promoter polymorphisms, a G-to-C substitution at position − 82 and Cst C release from rat alveolar macrophages. In cancer patients a T-to-G substitution at position − 78, were found to influence the during malignant progression-like melanoma and colorectal cancer, binding of nuclear factors and affect the basal rate of gene transcrip- high serum concentrations of the cysteine proteases cathepsins B and tion in an allele-specific manner, which are also associated with the H induced by the tumors stimulate the production of Cst C for plasma concentration of Cst C in healthy individuals and patients with counterbalance. In addition, common factors such as cigarette recent myocardial infarction. In another study, the major haplotype smoking or C-reactive protein, an acute-phase protein in response − 82G/− 5G/+4A of the cst3 gene was found to determine plasma to inflammation, were also among the lists reported to increase serum levels of Cst C as the respondents with homozygous genotypes have Cst C levels independent of renal functions. the highest plasma levels. Collectively, these data suggest that an altered promoter activity of the Cst C gene could be a causal factor MECHANISMS OF CST C REGULATION for the association between Cst C genotype and plasma Cst C The dynamic changes of Cst C caused by different stimuli herald the concentration. existence of, and prompt people to search for, the regulatory Consistent with the potential regulatory mechanisms of human Cst mechanisms of Cst C to keep this multifunctional and disease- C at genetic levels, we found that Cst C is differentially expressed associated protein under check. Like many other proteins, the among mouse cells of the immune system. Specifically, cells of the production of Cst C is subject to both transcriptional and post- monocyte/macrophage and DC lineages express it at much higher translational regulation (Figure 1). 28,89 levels than lymphocytes. Mouse DC can be divided into two + − subsets by their surface expression of CD8α (CD8 and CD8 DC). Transcriptional regulation Further analysis of the DC subsets directly isolated from the spleen Although several polymorphisms and sequence variations were 1,105 demonstrated that the CD8 DC were the major producer of Cst detected in the promoter region of cst3, the gene coding Cst C, 89,109 C. This was also verified with DCs that had been generated in it generally shares common features with those of housekeeping culture from bone marrow precursors supplemented with Flt3L, in genes. Interestingly, the promoter region of murine cst3 gene was + hi later found to contain a core sequence of the androgen-responsive which only the CD8 DC equivalent, the CD24 DC subset, contained high levels of Cst C. The differential expression pattern of Cst C element and two potential binding sites for activator protein 1, a among cells of common lineages suggests that its gene could be transcription factor that regulates gene expression in response to a variety of stimuli including cytokines, growth factors, stress and controlled by cell-specifictranscription factors. Immunology and Cell Biology Regulation of cystatin C in diseases YXu et al This preferential expression pattern of Cst C in monocyte/macro- Therefore, the factors that cause this conformational change of Cst C phage and DCs is of clinical relevance in view of the recent reports by dimerization can be regarded as a way to regulate the activity of this showing that these cell types are present in tissues where Cst C plays protease inhibitor. It is intriguing to think that Cst C adopts the pathological roles such as atherosclerosis and angiopathy. Vascular DCs unreactive dimer conformation as storage form, which quickly (aortic DCs), which increase in number in atherosclerotic lesions as monomerizes in response to the stimulation of its target enzymes. + 111 disease progresses, were reported to be related to CD8 DC, and the This post-translational regulation of Cst C by dimerization was first + − + Flt3L-signaling-dependent CD103 /CD11b (CD8 equivalent) DCs described in transfected Chinese hamster ovary cells, where Cst C is protect against atherosclerosis. Consistently, our analysis of DC inactivated during the early part of its trafficking through the secretory purified from aortas indicates that these cells indeed express Cst C pathway and then reactivated prior to secretion. Interestingly, we (unpublished observation). Similarly, microglia, also called the mono- found that steady-state (immature)CD8 DCs isolated from primary cyte/microphage in the brain, is the major cell type that express Cst C in spleens constitutively contain Cst C homo-dimers, which can be 96 64 the brain, where deposition of Cst C fibrils produces angiopathy. separated from monomers by size-exclusion chromatography of cell To uncover the transcriptional mechanisms underlying the prefer- lysates and their non-reactivity with the Cst C target enzyme papain, ential expression of disease-related Cst C in these cells, a novel cis supporting the notion that they are domain-swapped dimers. element for transcription factors, denoted as IRF (interferon regulatory Furthermore, when CD8 DC underwent maturation by incubation factor)-Ets composite sequence (IECS), in the promoter region of the in vitro,they no longer producedCst Cdimers. Disappearance of cst3 gene was identified in a reporter assay system employing a self- dimer could be the strategy employed by healthy primary cells to inactivating retrovirus. This element consists of a core IRF-binding process this potentially pathogenic form on their way toward motif for IRF8, and an Ets-binding motif for PU.1, an Ets transcrip- maturation, which might then be disrupted in diseased conditions. tion factor and binding partner that facilitates a more stable binding of To identify the mechanistic factors leading to Cst C dimerization IRF8 to chromatin. Notably, both IRF8 and PU.1 are essential for either as post-translational regulation of its activity, or as amyloid 115,116 the development of DC subsets, of which IRF8 is required for precursor protein, the intracellular accumulation of reactive oxygen + + 115 the development of CD8 CD103 DC, the major producers of species in the immature and mature states were compared. A strong Cst C. This could imply that Cst C expression by these cells might be correlation between reactive oxygen species levels and Cst C dimer was the direct consequence of the presence of IRF8-binding motif in the observed not only in same cell type at different developmental stages, Cst C promoter region in cells that depend on IRF8 for their but also in different cell types at the same developmental stages. development. However, this cannot be the only explanation because Furthermore, artificial enhancement of the intracellular oxidative IRF8 is also required in plasmacytoid DC development, yet this DC status resulted in a time-dependent Cst C dimer enrichment, which type does not express Cst C. Thus, the molecular mechanisms could be prevented by inhibiting mitochondrial activity, indicating the responsible for this phenomenon remain to be revealed. reactive oxygen species released from mitochondria are responsible for To investigate the involvement of these two transcription factors in the observed constitutive Cst C dimer formation. Although, the Cst C expression in primary DC in vivo, the physical interaction of exact process by which oxidative stress exerts the conformational IRF8 and PU.1 with the cis element IECS was examined by chromatin changes of Cst C is not fully understood, a recently published report immunoprecipitation in three different DC subsets freshly isolated on recombinant human Cst C stabilized by genetically introduced from splenic cells. Consistent with their Cst C expression profile disulfide linkage demonstrated a disappearance of a dithiothreitol- + − (CD8 DC express Cst C, both CD8 DC and plasmacytoid DC do not induced dimer if the concentration of this reducing agent was further express Cst C), the binding of IRF8 to the IECS sequence was detected increased, suggesting a direct impact of redox environment on the + − in CD8 DC, but not in CD8 DC and plasmacytoid DC. Interestingly, conformational changes of Cst C protein. in the plasmacytoid DC where PU.1 expression is low but IRF8 Different from type I cystain family members, the type II cystatin expression is the same as CD8 DC, the binding of IRF8 to the IECS Cst C is synthesized with a signal peptide, hence being secreted and sequence is still not detected, indicating a quantitative requirement of consequently found in body fluids. Although its intracellular roles in PU.1 in IRF8 binding to the chromatin, a result consistent with processing MHCII molecules via lysosomal proteinase in DCs were previous findings with fluorescence recovery after filling to mathema- debatable, the uptake of Cst C in amounts sufficient to affect the tical models. Collectively, the chromatin immunoprecipitation data activities of intracellular cysteine proteases was described in the 120 121 suggest that IRF8 binding to the IECS of the Cst C promoter in the eyes, proximal tubule cells and several human cancer cell 122,123 presence of sufficient PU.1 could drive cst 3 expression. To finally lines. Thus, trafficking inside and outside of the cell membrane confirm the role of IRF8 in Cst C expression, a CD8 DC line 1940, of Cst C can regulate or fine tune its division of labor between where both IRF8 and PU.1 were amply expressed, were transfected intracellular compartments and the extracellular matrix. However, the with the retrovirus vector LMP encoding shRNAs for irf8 to silence the structural determinants on the internalized inhibitor required for gene. When the synthesis of IRF8 was reduced in the cells, that of Cst efficient uptake were not characterized until recently 12 variants of Cst C, but not MHC I, was also compromised. This causal impact of IRF8 C with substitutions of selected amino acids were generated. Uptake on Cst C production, combined with the diminished Cst C expression of Cst C in human breast adenocarcinoma cells is dependent on both − /− in bone marrow (BM)-derived DC from irf8 mice compared with charged amino acids of the N-terminal segment and on a hydrophobic wild-type control, strongly suggest that IRF8 is the key transcription amino acid in domains involved in the inhibition of cysteine factor regulating Cst C expression. cathepsins. Furthermore, natural arginine (Arg) residues in positions 24 and 25 are of importance for the uptake process. Post-translational regulation Another mechanism for post-translational regulation of Cst C could The translational expression of Cst C in quantity does not necessarily come from digestion of this cysteine protease inhibitor by proteases of lead to a functional protein product. The activity of Cst C can still be another family. For example, earlier proteomic studies have shown further modulated after its transcription. As discussed before, the that Cst C is a substrate of matrix metalloproteinase 2 with specific reactivity of Cst C with its target protease is lost if it dimerizes. inactivation upon cleavage. In addition, human aspartic Immunology and Cell Biology Regulation of cystatin C in diseases YXu et al family members for early prevention, or selectively remove Cst C dimers from biological fluids containing both dimers and monomers in patients with hereditary cystatin C amyloid angiopathy. In addition, the mechanistic factors affecting the inhibitive activity of Cst C can also be harnessed for therapeutic gains. Drugs to improve the intracellular redox environment by removal of reactive oxygen species and compounds may be beneficial in not only reducing the Cst C amyloidogenesis, but also regulating the elastic proteolysis in the diseased locus as elevated protease activity in the local body fluids is partly responsible for the tissue destruction in the disease associated with Cst C amyloid. Last but not least, identification of transplantable cellular sources for major Cst C production will also be of great clinical value. Since Cst C is involved in inflammatory diseases, in which immune cells accumulate and play important roles, bone marrow-derived cells are an important cellular source for Cst C manipulation. Along this line, we found that hematopoietic cells contribute significantly to the systematic pools of Cst C. Therefore, bone marrow transplantation Figure 2 Multiple factors are involved in the progression of cystatin C-related would be an applicable approach in clinic to treat patients with Cst C diseases. A scheme summarizing factors leading to the fluctuation of amyloidogenesis or reduction. cystatin C levels, which triggles or participates in the pathogenic processes of several diseases. CRP, C-reactive protein. A full color version of this figure CONCLUSION is available at the Immunology and Cell Biology journal online. With the recent identification of regulatory elements in the promoter region of Cst C and increasing reports of factors affecting its production endoproteinase cathepsin D was also able to inactivate human Cst C and/or activities, precaution should be taken when Cst C is used as an by cutting hydrophobic amino acid residues into several fragments index of GFR because its blood concentration is subject to changes in vitro. Along the same line, the amount of Cst C in the caused by many factors independent of kidney function. These factors extracellular environment is reduced in the secretome of mouse include cigarette smoking, body composition, viral infection, tumor embryonic fibroblasts stably transfected with human cathepsin D, malignancy or gene mutations. Ultimately, the altered Cst C levels or and the tumor-derived cathepsin D assists breast cancer progression 126 activity could lead to pathological processes in cardiovascular diseases, by inhibiting Cst C activity. Importantly, the relevance of this neurological disorders or even mortality (Figure 2). Such an important mechanism was also found in other biological process in which Cst C disease-associated protein thus should be effectively regulated for was shown to be a proteolytic target of cathepsin D, affecting the 127 therapeutic gains. Further characterization of the signaling pathways differentiation of DCs from hematopoietic stem cells. leading to Cst C expression will help to develop antagonists and/or synagonists for medical intervention or regulation of the abnormal CLINICAL IMPLICATIONS production and/or activity of this disease-associated enzyme inhibitor. Given the increasingly discovered roles of Cst C in various pathophy- Likewise, identification of the post-translational modifications in the siological processes and identification of its regulatory mechanisms at microheterogeneity of the pathogenic species will also assist in under- both transcriptional and post-transcriptional levels, manipulation of standing the mechanisms or direct factors triggering Cst C dimerization. Cst C expression either locally or systematically may have many The knowledge should provide the information for a better under- clinical implications. Characterization of the factors that control Cst C standing of the mysterious mechanisms underlying the diseases expression at the transcriptional level could provide valuable clues for associated with Cst C abnormalities. the treatment of pathologies associated with insufficient control of extracellular proteases. For example, signaling molecules that upregu- CONFLICT OF INTEREST late Cst C expression could be used to promote Cst C secretion in the The authors declare no conflict of interest. case that requires slightly higher Cst C concentration to protect neuronal cells from cell death in Alzheimer’s disease, or at sites of ACKNOWLEDGEMENTS inflammation in which excessive protease activity causes tissue This work was funded by a Project Grant from National Health and Medical damage, as has been suggested to occur in atherosclerosis and aortic Research Council (NHMRC) of Australia (no. 1006428), and a start-up fund aneurysm. 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Published: Feb 3, 2015

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