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Opinion

TRENDS in Molecular Medicine

Vol.13 No.2

The collagen-specific molecular chaperone HSP47: is there a role in fibrosis? Takashi Taguchi1 and M. Shawkat Razzaque1,2 1

Department of Pathology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan 2 Department of Developmental Biology, Research and Educational Building, Room 304, Harvard School of Dental Medicine, 190 Longwood Avenue, Boston, MA 02115, USA

Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone that is required for molecular maturation of various types of collagens. Recent studies have shown a close association between increased expression of HSP47 and excessive accumulation of collagens in scar tissues of various human and experimental fibrotic diseases. It is presumed that the increased levels of HSP47 in fibrotic diseases assist in excessive assembly and intracellular processing of procollagen molecules and, thereby, contribute to the formation of fibrotic lesions. Studies have also shown that suppression of HSP47 expression can reduce accumulation of collagens to delay the progression of fibrotic diseases in experimental animal models. Because HSP47 is a specific chaperone for collagen synthesis, it provides a selective target to manipulate collagen production, a phenomenon that might have enormous clinical impact in controlling a wide range of fibrotic diseases. Here, we outline the fibrogenic role of HSP47 and discuss the potential usefulness of HSP47 as an anti-fibrotic therapeutic target. Heat shock proteins Heat shock proteins (HSPs) are a distinctive class of molecules that protects cells against a wide range of injuries. The primary structures of stress proteins are highly conserved [1]. HSPs assist in the recovery from stress either by repairing damaged proteins (protein refolding) or by removing them through degradation to restore protein homeostasis and to promote overall cell survival [2]. Some HSPs are strictly stress-inducible, whereas others are constitutively expressed or developmentally regulated. Several HSPs are constitutively expressed and actively involved in maintaining cellular homeostasis by acting as molecular chaperones [3–5]. The stress responses in mammalian cells are transcriptionally regulated by the heat shock transcription factor (HSF), which can selectively bind to the heat shock promoter element (HSE) [6,7]. In normal, unstressed cells, HSF is present in the cytoplasm but, in stressful microenvironments, HSF is Corresponding authors: Razzaque, M.S. (mrazzaque@hms.harvard.edu), (razzaquems@yahoo.com). Available online 13 December 2006. www.sciencedirect.com

converted from an inactive monomeric form into an active trimeric DNA-binding form, which then translocates into the nucleus and interacts with HSE to induce transcription of HSP genes [8,9]. Oligomerization of the HSF and its interaction with the HSE are the hallmark of an active transcriptional response to a wide range of stresses that include but are not limited to physical, environmental and chemical stresses. Both in vivo and in vitro studies have documented the important roles of HSPs in the pathogenesis of various diseases, ranging from autoimmune to fibrotic diseases [10–14]. Furthermore, inhibitors of the HSPs affect normal cellular activities by preventing proper folding and assembly of key signaling proteins, including tyrosine kinases and steroid receptors [15]. For example, HSP90

Glossary Collagen: it is the major components of the extracellular matrix and the most abundant protein in mammals, making up to 25% of the total protein to support and maintain cell and tissue structures. It is the main component of cartilage, ligaments, tendons, bone and teeth, and is responsible for maintaining strength and elasticity of many soft tissues, including skin and blood vessels. Collagen has unique amino acid compositions with large amounts of proline, and glycine being present at almost every third residue, along with two other uncommon amino acid derivatives in the form of hydroxyproline and hydroxylysine. There are as many as 28 different types of collagen that have been identified. Defect or mutations in the collagen gene is associated with some well-characterized human diseases, including osteogenesis imperfecta (type I collagen), inherited osteonecrosis (type II collagen), Ehlers–Danlos syndrome (type III collagen) and Alport syndrome (type IV collagen), whereas increased synthesis and accumulation of mainly interstitial collages form fibrotic mass. Fibrosis: when a particular tissue or organ is damaged, the natural response of the body is to heal the injury by producing granulation tissue, which is mostly composed of interstitial type I and type III collagens. However, in some diseases the granulation or fibrous tissue continue to grow even after covering the original wound, causing abnormal pathological fibrosis or scarring. Fibrous tissue is thicker than the surrounding tissue with a limited blood supply, and has restricted movement and sensation. Although procedures are available to remove excessive fibrotic tissues, any surgery will always induce a new scar and, most importantly, aggravate the risk of recurrence and worsening of fibrosis. There is no specific or effective therapy available to treat fibrotic diseases beyond supportive care. Heat shock protein 47 (HSP47): it is a collagen-binding protein that resides in the ER of collagen-producing cells. Available information suggests that HSP47 is involved in the correct folding of triple-helical procollagen and is believe to assist in transporting procollagen from the ER into the Golgi complex. The expression of HSP47 always increases in fibrotic diseases. Unlike most molecular chaperones that recognize several target proteins, collagen is the only substrate protein for HSP47.

1471-4914/$ – see front matter ß 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.molmed.2006.12.001


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inhibitors can inactivate, destabilize and degrade HSP90 client proteins to exert anti-tumor activity. Currently, a small-molecule inhibitor of HSP90 (17-allylaminogeldanamycin) is in Phase II clinical trials as an anticancer agent [16–18]. Thus, it seems that selective manipulation of HSPs might be clinically useful. A comprehensive review of all the aspects of HSPs is beyond the scope of this article; rather, we briefly present our views on the possible role of HSP47 in fibrosis (see Glossary), and its potentials as a novel anti-fibrotic target with possible limitations. Heat shock protein 47 HSP47 was first characterized by Kurkinen et al. [19] in murine parietal endoderm cells. Subsequently, collagenbinding proteins were identified and characterized in various species, including J6 in the mouse [20], gp46 in human and rat [21] and HSP47 in the chick and rabbit [22,23]. All these proteins were then found to be the same group of molecules with common collagen-binding properties. HSP47 is a 47-kDa glycoprotein that is mostly present in the endoplasmic reticulum (ER) of collagen-producing cells, and is involved in the molecular maturation of various types of collagens by assisting correct folding of the procollagens [14,24–26] (Figure 1). Collagen biosynthesis is a multi-step complex process that includes both intracellular and extracellular events. The synthesis of a-polypeptide chains, their hydroxylation and the formation of stable triple-helical procollagen molecules are intracellular processes, mostly regulated by several specific enzymes (e.g. prolyl 4-hydroxylase,

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prolyl 3-hydroxylase, lysyl hydroxylase, hydroxylysyl galactosyltransferase and galactosyl-hydroxylysyl glucosyltransferase), whereas the extracellular processes involve a different set of enzymes (e.g. procollagen Nproteinase, procollagen C-proteinase and lysyl oxidase) [24,27–32]. Furthermore, several chaperones and/or folding proteins such as protein disulphide isomerase (PDI) and HSP47 are also involved in collagen synthesis [14,24,26]. PDI binds to the C-propeptides of procollagen to facilitate trimer formation at the C-terminus by catalyzing disulfide-bond formation, which then proceeds towards the N-terminus [33], whereas HSP47 assists in the correct folding and stabilization of triple-helical procollagen molecules [14,34], which are eventually transported to the extracellular space across the Golgi complex, where N- and C-propeptides are cleaved by procollagen N- and Cproteinases to assemble into collagen fibrils [30,35] (Figure 2). HSP47 resides in the ER; it has a signal sequence at the N-terminus that targets the molecule to the ER, whereas it has an ER retention signal (RDEL) at the Cterminus [23]. HSP47 binds to nascent procollagen chains in the ER and, once the RDEL signal is removed from the C-terminus, HSP47 seems to dissociate from the procollagens, which is then rapidly secreted from the cell [36]. The collagen-binding ability of HSP47 has been demonstrated by co-immunoprecipitation studies [37]. Both native and synthetic HSP47 bind to various types of collagens, as determined by in vitro pull-down studies, using surface plasmon resonance [38,39]. In vitro binding analysis using a synthetic peptide model of collagen has identified a

Figure 1. Possible regulation of HSP47. HSFs are normally bound to HSPs as inactive molecules in the cytosol. Upon exposure to stressors, HSFs are phosphorylated (P) by protein kinases, rapidly form trimers and translocate into the nucleus where they interact with HSE to induce the transcription of HSPs, which are then transcribed and relocate to the cytosol. In the cytosol, HSP47 is involved in correct assembly of procollagen molecules [6–9,54,55]. Correctly folded procollagen is transported to the extracellular space and cleaved by procollagen N- and C-proteinases, and proceeds to assemble into collagen fibrils. Note that this is only an oversimplified outline, and further studies are needed to clarify exact molecular interactions. www.sciencedirect.com


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Figure 2. Multi-step intracellular and extracellular events of fibrillar collagen synthesis. Step 1. Transcription of collagen gene. Step 2. Synthesis of a chains of pre-procollagen on ribosomes and ER. Step 3. Hydroxylation of proline residues to hydroxyproline and lysine residues to hydroxylysine. Step 4. Glycosylation of some of the hydroxylysine residues. Step 5. Assembly of three a chains to form procollagen (HSP47 is involved in correct folding and assembly of procollagen molecules). Step 6. Procollagen is transported to the extracellular space across the Golgi complex by exocytosis. Step 7. Cleavage of registration peptides by procollagen N- and C-proteinases. Step 8. Self assembly and polymerization into collagen fibrils. Step 9. Cross-linkage between fibrils to form collagen fibers. Note that this is only a simplified diagram where only the essential steps of collagen synthesis have been included.

specific HSP47-binding sequence. It is suggested that the binding of HSP47 to typical collagen model peptides (Pro– Pro–Gly)n occurs when n> 8 [40], suggesting that HSP47 binding to the triplet repeats increases with higher numbers of repeats. Interestingly, HSP47 cannot bind to these peptides when the proline residue is hydroxylated in the second position. Hydroxylation, therefore, might be a regulatory step for association and dissociation of HSP47 during the maturation process of collagen [40]. Similar to the synthetic peptides, using cyanogen bromide (CNBr) fragments of naturally occurring a chains of type I and type II collagens, Thomson et al. [41] have shown HSP47– collagen interactions with the strongest affinities in the www.sciencedirect.com

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N-terminal region. Furthermore, arginine residues at Yaa position of collagenous Gly–Xaa–Yaa repeats have been suggested to be crucial for HSP47 interactions [42–44]. Recently, employing computational HSP47-binding protein search program, multiple HSP47-binding sites have been shown to be present in a chains of collagens I–XXVII [45]. Interestingly, besides collagen molecules, several collagen-related proteins, including pulmonary-surfactant proteins and C1q-related proteins, were selected as possible clients of HSP47 by such database search [45]. Additional studies are needed to determine the relevance of HSP47 in regulating biological activities of collagenrelated proteins. The essential role of HSP47 in collagen maturation is further evidenced by in vivo deletion studies. Genomic ablation of Hsp47 gene causes embryonic lethality in homozygous mice at embryonic day 10.5 [46]. Embryos that are lacking the Hsp47 gene have maturation defects in both type I and type IV collagens. Also, triple helices formed in the absence of Hsp47 were shown to be more susceptible to protease digestion [46]. Moreover, using Hsp47 / embryonic stem cells, it has been shown that type IV collagen secreted from these null cells cannot correctly assemble the triple helical structures, and this results in the failure to form basement membrane in the embryoid bodies of these null cells [47]. Embryoid bodies are formed by the aggregation of embryonic stem cells containing differentiated cells from all germ layers and are useful reproducible models to study various morphogenic events and, therefore, provide a dynamic tool to study the role of a particular protein during early development. Impaired basement-membrane formation was also documented in Hsp47 / mouse embryos [46]. These studies clearly suggest that, when HSP47 is absent, there is abnormal molecular maturation of its substrate protein collagen. Interstitial type I and type III collagens are the major component of fibrotic mass, and experiments have demonstrated physical interactions of HSP47 with both these collagens [48–51]. Studies have shown the essential roles of HSP47 not only in correct folding but also in the prevention of abnormal aggregation of type I collagen in the ER. Such effects of HSP47 are also crucial for subsequent secretion, processing and fibril formation of type I collagen; using a gene-deletion approach, the importance of HSP47 in the molecular maturation of type I collagen has been demonstrated in Hsp47 / cells [52]. The fibrils of type I collagen that are produced by Hsp47 / cells are abnormally thin and have frequently branched features. Furthermore, ablation of Hsp47 activities results in abnormal retention of type I collagen in the ER of Hsp47 / cells. The rate of procollagen secretion from Hsp47 / cells is slower than that of wild-type cells, and this results in the accumulation of the insoluble type I collagen aggregates within Hsp47 / cells [52]. Similarly, using in vitro transfection studies, a possible role for HSP47 has been suggested in type III collagen modification and secretion [53]. These observations demonstrate that HSP47 influences molecular maturations of both type I and type III collagens, the two most-important matrix proteins, the abnormal accumulation of which results in fibrosis.


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Studies have also identified the cis-acting DNA elements for the transcriptional activation of HSP47 and the transacting factor that binds to this element to induce the transcription of HSP47 in collagen-synthesizing cells. Putative SP1-binding site (at position 210 bp in the promoter region) has been suggested to be required for the basal expression of HSP47, whereas the other binding site (a 500-bp region located within the first introns) has been suggested to be necessary for cell-type specific expression of HSP47 [54]. In human embryonic lung fibroblasts, transforming growth factor-b1 (TGF-b1) and interleukin (IL)-b1 could induce trimer formation of HSF1, which then binds to HSE, located in the promoter region, to induce the synthesis of HSP47 [55] (Figure 1). A better understanding of the biology of HSP47 requires the investigation of other functional aspects and therapeutic prospects of this protein. As mentioned before, Hsp47 / mice have abnormal collagen maturation in the embryos compared with that of wild-type littermates [46,52]; however, early embryonic lethality of Hsp47 / mice did not provide enough information regarding Hsp47-ablation effect in postnatal collagen synthesis. Additional in vivo studies that selectively ablate the Hsp47 gene from a specific tissue or organ, perhaps by using the Cre–loxP system [56], will provide a better understanding of the essential roles of HSP47 in postnatal tissue growth and development. Such inducible approach will also help to determine controlled reduction and/or ablation of HSP47 activity in diseases that are associated with increased synthesis and accumulation of collagens. Several studies have suggested possible molecular interactions between known fibrogenic factors and HSP47. For example, treatment of MC3T3-E1 mouse osteoblasts with TGF-b1 induces the expression of both HSP47 and type I collagen in a dose-dependent manner [57]. TGF-b1 also transcriptionally induces the expression of HSP47 in various human cells, including conjunctival fibroblasts, dermal fibroblasts and aortic smooth muscle cells [58–60]. In addition to TGF-b1, certain other known profibrogenic factors, including IL-4, IL-13 and connectivetissue growth factor (CTGF) at various concentrations can induce the expression of both HSP47 and collagens in human conjunctival fibroblasts [61–63]. It is therefore plausible that one might be able to modulate the expression of HSP47, and perhaps its activity, by manipulating known fibrogenic factors such as TGF-b1, CTGF or cytokines in fibrotic diseases, although there are certain limitations in organ-specific targeting of circulating factors. Induction of the expression of HSP47 is also reported in mesangial cells following exposure of advanced glycation end-products [64], which has an important role in the pathogenesis of diabetic complications including nephropathy. Similarly, a correlation between increased expression of HSP47 and excessive accumulation of collagens has been reported in both human and experimental diabetic nephrosclerosis [65,66]. From the above-mentioned studies, it is apparent that HSP47 is not only physiologically important in prenatal organogenesis, but also exerts a pathological role in postnatal fibrogenesis. www.sciencedirect.com

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Molecular events in fibrotic diseases Why is fibrosis a harmful process? Most fibrotic diseases are progressive in nature, and excessive accumulation of matrix proteins due to their uncontrolled synthesis and/or degradation leads to gradual expansion of fibrotic mass, which destroys the normal structure of the tissues, resulting in organ dysfunction and, ultimately, end-stage organ failure. Although the exact molecular mechanisms of fibroproliferative diseases are not fully understood, increased synthesis and deposition of collagens are consistently observed in most fibrotic diseases, irrespective of their origin [48–50]. It is becoming clear that a final common pathway for fibrosis exists in most affected organs, despite differences in the primary causes of the diseases [48–50,67]. The process and progression of fibrosis can be broadly divided into three phases or events, although they sometimes overlap: initial triggering events, inflammatory events and fibrotic events. The ideal approach to control fibrosis would be to identify the primary cause and to treat the causative factors. However, due to the diversity of the primary causes of fibrosis, and the long latency between commencement of the fibrotic process and appearance of clinical symptoms, it is not always easy to treat and eliminate the initial triggering events. Alcohol consumption, viral infections, toxic vapors, inorganic dusts, systemic diseases, metabolic diseases and immunological diseases can induce the process of fibrosis in the liver, lung, heart, kidney, eye and skin [48,50,51,60,68–70]. These diverse etiological factors of cellular activation and/or injury subsequently trigger inflammatory responses that induce numerous mitogenic and fibrogenic molecules such as epidermal growth factor (EGF), plateletderived growth factor (PDGF), TGF-b1, CTGF, fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF). Each of these molecules enhances the process of fibrosis. Thus, EGF is mitogenic for epithelial cells and fibroblasts, whereas PDGF is involved in the proliferation and migration of fibroblasts and myofibroblasts. FGF-2 and VEGF are involved in neovascularisation, whereas CTGF and TGF-b1 induce overproduction and reduced degradation of matrix proteins to promote fibrogenesis [50,51,69,71–73]. Accumulation of collagen is the main component of the fibrotic mass, and the understanding of collagen synthesis and processing has provided the potential molecular targets to manipulate its production selectively. HSP47 is a collagen-specific molecular chaperone that contributes to molecular maturation of collagen. HSP47 was shown to be highly upregulated in all the studied human and experimental fibrotic diseases [14,74]. Because no effective anti-fibrotic drug is currently available, and targeting circulating fibrogenic factors or blocking their receptors has not provided the expected results, the search for a novel therapeutic target to control progression of fibrosis is currently a field of intense research. The profibrotic effects of HSP47 make it a potential target for developing anti-fibrotic therapy. Because collagen is the only substrate for HSP47, it provides a selective target to manipulate collagen production, a phenomenon that might have huge impact on controlling fibrotic diseases.


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Moreover, the recent identification of small-molecule chemical inhibitors of HSP47 has increased its therapeutic applicability [75]. Here, we briefly discuss our opinion, based on experimental evidences, on the prospect of HSP47 as a novel anti-fibrotic therapeutic target to prevent, delay or reverse fibrotic diseases. HSP47 and fibrotic diseases Increased expression of HSP47 with excessive accumulation of collagens is consistently observed in various human and experimental fibrotic diseases [58,66,76–81]. Whether overexpression of HSP47 in fibrotic diseases is an epiphenomenon has not been obvious in earlier studies. Subsequent in vivo studies, however, showed that blocking the bioactivities of HSP47 did not only alter collagen production, but also reduced the progression of fibrotic lesions, directly implicating HSP47 in fibrogenesis [80,82]. Increased glomerular expression of HSP47 correlated with increased deposition of collagens in scleroproliferative glomeruli in anti-thymocyte serum-induced experimental model of glomerulosclerosis [80]. Such glomerulosclerosis in the in vivo experimental model of nephritis could be delayed by blocking bioactivities of HSP47 using anti-sense therapy [82]. These studies provide a pathological basis for the possible role of HSP47 in fibrogenesis. A similar induction of HSP47 expression, with excessive accumulation of collagens, is also noted in numerous other experimental models of renal fibrosis, such as diabetic nephropathy and hypertensive nephrosclerosis [65,66,83]. Increased glomerular expression of HSP47 is likely to be involved in overproduction of collagens that ultimately promote glomerular sclerosis. Furthermore, increased expression of HSP47 was also consistently detected in collagen-producing interstitial myofibroblasts and tubular epithelial cells in various experimental models of renal tubulointerstitial fibrosis, such as elderly F-344 rats and rats with radiation-induced tubulointerstitial nephritis [62,74]. One of the unique features of HSP47 is that its overexpression is always detected in all the studied fibrotic diseases, irrespective of tissue or organ involvement. Similar to the renal fibrotic diseases mentioned above, induction of HSP47 is consistently observed in other fibrotic diseases such as those affecting the lung, liver, heart, eye and skin [60,66,76,78,79]. For example, increased expression of HSP47 is mostly evident in and around the areas of excessive accumulation of interstitial collagen in bleomycininduced experimental pulmonary fibrosis [84] (Figure 3). The results of in vivo experimental studies have formed the basis to determine the role of HSP47 in human fibrotic diseases [60,66,76,78,79]. One of such studies was performed on human renal biopsy tissues obtained from IgA nephropathy and diabetic nephropathy [66]. In these samples, increased expression of HSP47 in glomeruli and the tubulointerstitium correlated with glomerular and tubulointerstitial accumulation of type IV collagen and type III collagen, respectively [66]. A similar correlation was also noted in human pulmonary fibrotic diseases [79]. Similarly, in patients with keloid [78] and cicatricial pemphigoid [59] increased dermal and ocular expression of HSP47 correlated with excessive accumulation of interstitial collagens in and around the areas of dermal and conjunctival www.sciencedirect.com

Figure 3. Expression of HSP47 in control rat lung (a) and bleomycin-induced pulmonary fibrotic lung (b), showing markedly increased expression of HSP47 (arrows) in the fibrotic lung. (c) Double staining of HSP47 (black) and type III collagen (red) showing increased expression of HSP47 mostly in the areas of increased accumulation of type III collagen (arrows).

fibrosis [60]. The profibrogenic role of HSP47 has also been proposed in the development of fibrotic lesions in human heart diseases [68]. It is therefore evident from numerous human and experimental studies that, irrespective of the primary disease, upregulation of HSP47 is a common phenomenon during collagenization of the affected organ. It is, hence, conceivable that monitoring expression of HSP47 might help to define those patients at risk of developing fibrotic complications, and in assessing


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the response to conventional and selective anti-fibrotic therapies. Reversibility of fibrosis The prospects of reversal of chronic progressive fibrotic diseases have generated huge research interest; such therapeutic maneuver has enormous clinical importance and profound impact on the prognosis of patients with such conditions. Can fibrosis be completely reversed? Some experts have reasonable doubt about complete resolution of fibrosis [85]. In most cases, it is not only the disorganized matrix accumulation but also its destructive effect on the surrounding structures that adversely affect the function of the involved organ. Any clinical benefits are more likely to be achieved if fibrosis can be delayed or reversed before the occurrence of severe cellular instability. Once cellular homeostasis is severely compromised, the regenerative ability of the affected cell and/or organ is impaired and reversal of the fibrotic process becomes unachievable. Therefore, the identification of the point of irreversibility of the fibrotic process is crucial. Experimental studies suggest that, once there is a significant amount of collagen cross-linked by tissue transglutaminase together with marked destruction of cellular components, there is little hope for reversal of the fibrotic process [86]. However, unless it reaches the terminal stage, it is generally believed that the progression of fibrosis might be delayed or even reversed to some extent by the use of anti-fibrotic therapy. Thus, development of potent anti-fibrotic agents is essential for effective treatment of fibrotic diseases. Recent studies with collagen-binding HSP47 have provided important in vivo evidence of a close association between overproduction of HSP47 and progression of fibrosis, and draw attention to this molecule as a potential anti-fibrotic target [77–82]. We further discuss the utility of anti-HSP47 therapy and the rationale for blocking the bioactivities of HSP47 to control the progression of fibrotic diseases in the subsequent sections. Targeting HSP47 in fibrotic diseases No effective anti-fibrotic therapy is available to date that can be used for patients with fibrotic diseases. Although several important fibrogenic molecules have been identified, some of these molecules are not suitable therapeutic targets because of their widespread vital systemic biological functions. Another drawback is that, despite their in vitro efficacy, the in vivo targeting of some of the circulating fibrogenic molecules does not always show similar effectiveness in the complex in vivo microenvironment. Because excessive and disorganized accumulation of collagen is the main pathological process of fibrotic diseases, strategies designed to prevent excessive synthesis and accumulation of collagens might provide the most desirable anti-fibrotic responses. HSP47 is involved in the molecular maturation of collagens, and selective blockade of its activities in fibrotic diseases might offer a novel therapeutic target. Using murine fibroblast cell line, in vitro studies have shown that antisense oligodeoxynucleotides against HSP47 could inhibit collagen production [39]. Similarly, a HSP47-specific ribozyme that could cleave its mRNA suppressed as www.sciencedirect.com

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much as 75% of production and secretion of collagen from human primary fibroblasts [87]. In agreement with the in vitro observations, in vivo studies have also demonstrated the beneficial effects of modulating the expression of HSP47 in various fibrotic diseases [82,88–90]. For example, suppression of HSP47 expression by the administration of antisense oligodeoxynucleotides against HSP47 delayed the progression of glomerular sclerotic process by reducing accumulation of collagens in the glomeruli [82]. Similar anti-fibrotic response using antisense HSP47 therapy is effective in reducing wound-associated scarring [88,89]. These studies, although preliminary, clearly suggest a profibrotic role of HSP47 in various fibrotic diseases and, more importantly, provide the in vivo evidence of how HSP47 might be a potential anti-fibrotic therapeutic target. Similarly, the chlorhexidine gluconateinduced peritoneal fibrosis in rats has been shown to be associated with increased expression of HSP47 and excessive accumulation of interstitial collagens [90]. Treating these rats with antisense phosphorothioate, HSP47 markedly reduced deposition of collagens and eventually reduced the extent of peritoneal fibrosis [90]. Age-associated kidney pathology is usually related to fibrotic changes due to upregulation of HSP47 with increased accumulation of collagens [91]. Such aging-related renal fibrosis could be delayed by reducing renal expression of HSP47 through chronic caloric restriction [92], reinforcing the idea that suppression of HSP47, either by genetic manipulation in experimentally induced fibrotic diseases or merely by caloric restriction in aged animals, might delay the progression of fibrotic diseases, a phenomenon that has great clinical importance. Furthermore, certain investigational drugs seem to exert their anti-fibrotic effects by suppressing the expression of HSP47. For example, in a clinical trial, pirfenidone stabilized pulmonary functions and improved prognosis of patients with idiopathic pulmonary fibrosis [93]. Recently, administration of pirfenidone was reported to reduce the accumulation of collagens in bleomycin-induced pulmonary fibrosis possibly by suppressing the expression of HSP47 [94]. Taken together, the above studies provide the experimental basis for targeting HSP47 with the aim of controlling the progression of chronic fibroproliferative diseases. Although these studies are promising, additional studies are needed to determine the adverse effects of long-term in vivo suppression of HSP47, because fibrotic diseases are usually chronic and progressive in nature. Another issue that need to be resolved is the type of therapy that will have best therapeutic effects. The therapeutic targeting of HSP47 is significantly enhanced by recently identified chemical compounds or small-molecule chemical inhibitors that effectively inhibit the bioactivity of HSP47 [75]. Thomson et al. [75] have recently identified inhibitors of HSP47 activity using a chemical library that contain 2080 compounds. The identified low-molecularweight compounds could interfere with the chaperone activity of HSP47, as demonstrated by inhibition of collagen fibrillogenesis [75]. These small-molecule chemical inhibitors of HSP47 might provide viable therapeutic option to control the progression of fibrotic diseases, an approach that is more likely to be clinically applicable than


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antisense therapy or other molecular manipulations such as ribozymes. Future directions Despite existence of preliminary data that suggest the possibility of targeting HSP47 to delay or minimize the progression of fibrotic lesions in experimental animals, the important question that remains to be answered is how to modulate the expression of HSP47. Because HSP47 is a physiologically important molecule, modulation of its expression in pathological conditions such as fibrosis requires strictly controlled manipulation. Given the fact that HSP47 is an intracellular protein, before designing any therapeutic strategy one needs to consider the best delivery system for neutralizing the pathological effects of HSP47. Even when these earlier technical hurdles of controlled manipulation and ideal delivery system are attained, another important issue that needs careful consideration is whether HSP47 can be used as an anti-fibrotic target without disrupting cellular homeostasis (Box 1). Once selective suppression of HSP47 is achieved, it is crucial to know how abnormally aggregated or incorrectly folded procollagen molecules will affect cellular functions. Although downregulation of harmful factors might benefit cell survival and functions, overload of misfolded proteins might put extra burden on cells and compromise their function. Any investigational drug that targets HSP47, therefore, needs a delicate balance between inducing minimal side effects and maximal efficacy. As discussed above, in Hsp47 / cells, there seems to be an abnormal accumulation of the immature form of procollagen molecules [46], although the cellular phenotype is expected to be different in complete ablation versus controlled suppression of HSP47. Is clinical application of HSP therapy feasible? Particularly promising seems the area of neurodegenerative disorders, where aberrant protein aggregation and neuron degeneration are the common pathological features. Studies have demonstrated that induction of the expression of HSPs, particularly of HSP70 by gene transfer, can reduce aberrant protein misfolding and inhibit the apoptotic deletion of cells to attenuate dopamine-neuron degeneration in Parkinson’s disease [95]. In contrast to the induction of HSPs for therapeutic purposes, a reverse strategy will be needed to minimize the over-activity of HSP47 in fibrotic diseases, which will be clinically more challenging, although advancement of gene therapy in the

Box 1. Outstanding questions  Does HSP47 physically interact with all types of collagens?  How and where does HSP47 bind to the native collagen precursors?  What are the factors that directly induce and regulate the synthesis of HSP47?  How can normal physiological and abnormal pathological responses of HSP47 be differentiated?  Can HSP47 be used as an anti-fibrotic target without disrupting cellular homeostasis?  Does genetic alteration of HSP47 result in distinct human diseases? www.sciencedirect.com

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form of RNA interference (RNAi) or carrier-mediated drug and/or protein delivery system might enable to achieve the ultimate goal of clinically targeting HSP47 to delay the progression of fibrotic diseases. As mentioned above, the therapeutic targeting of HSP47 is also enhanced by the recently identified chemical compounds or small-molecule chemical inhibitors that were shown to be able to suppress the functional activities of HSP47 [75]. Furthermore, recent understanding of the structural basis of the HSP47–collagen interaction will facilitate drug designing by taking the advantage of pharmacophore-based strategies [44,96,97] One of the reasons why treatment of fibrotic diseases is not always effective is that it is not always feasible to provide organ- or tissue-specific therapy when the targeting factor is a circulating protein. It will be, therefore, interesting to know whether HSP47 can be used to block organ-specific fibrosis selectively and, if so, to determine what will be the best pharmacological mean and delivery system to target HSP47 in fibrotic diseases. Concluding remarks In contrast to most, if not all, molecular chaperones that recognize several target proteins, HSP47 has a single substrate protein, collagen. Therefore, HSP47 provides a selective target to manipulate collagen production, a phenomenon that might have huge impact on controlling fibrotic diseases. Can in vivo experimental anti-fibrotic effects of targeting HSP47 be translated in the human diseases? We have discussed several issues that need further clarification before such clinical consideration. Any pharmacological treatment of fibrosis will require stage-specific modulation of those factors that are involved in a particular stage of the disease. Because HSP47 is involved in nearly all stages of the fibrotic process by facilitating increased production of collagen, selective inactivation of HSP47 might present a unique therapeutic target to either prevent or delay the progression of fibrotic diseases. Development of a novel therapeutic strategy is essential to treat fibrotic diseases effectively. The growing body of evidence presented suggests a strong rationale for blocking the bioactivities of collagen-binding HSP47 as one of the options to control the progression of fibrotic diseases. Here, we have highlighted two important aspects of HSP47: (i) is there a role for HSP47 in fibrosis? The existing studies clearly suggest a profibrogenic role of HSP47 in both human and experimental diseases. (ii) Can HSP47 be a therapeutic target to delay progression of fibrotic diseases? Preliminary results seem to suggest this, but need extensive controlled in vivo studies that will not only look for the favorable effects of HSP47 suppression, but also examine any possible side effects related to such suppression. Acknowledgements The authors thank to Dr. Ming Cheng, Dr. Arifa Nazneen, Dr. Diange Liu, Dr. Viet Thang Le and Dr. Yan Zha of the Department of Pathology of Nagasaki University, School of Biomedical Sciences (Nagasaki, Japan) for conducting original studies on various fibrotic models that have been cited in this article. Part of those original studies were supported by Grants-in-aid for scientific research to Dr. Razzaque (grant no. 09670192) from the Ministry of Education, Science and Culture, Japan. Authors are


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grateful to Dr. Bjorn R. Olsen of Harvard School of Dental Medicine for critically reading the manuscript and for helpful suggestions. The Institutional supports to Dr. Razzaque from Nagasaki University, School of Biomedical Sciences (Nagasaki, Japan) and Harvard School of Dental Medicine (Boston, USA) are gratefully acknowledged.

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