The collagen-specific molecular chaperone HSP47: is there a role in fibrosis?

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.     

HSP47 as a collagen-specific molecular chaperone: function and expression in normal mouse development

A large family of molecular chaperones can be divided into two major groups: general chaperone and substrate-specific chaperone. HSP47 is a collagen-specific molecular chaperone residing in the endoplasmic reticulum (ER). Recent studies revealed that HSP47 is essential molecular chaperone for mouse development and is essential for collagen molecular maturation in the ER. In the absence of HSP47, collagen microfibril formation and basement membrane formation are impaired in mouse embryos because the failure in the molecular maturation of types I and IV collagens, respectively. The tissue-specific expression of HSP47 is always correlated with that of various types of collagens and closely related with the collagen-related diseases including fibrosis in various organs. The importance of HSP47 in the therapeutic strategy for fibrotic diseases as well as for a marker of collagen-related autoimmune diseases will also be discussed.     

Increased expression of collagen-binding heat shock protein 47 in murine bleomycin-induced pneumopathy

The 47-kDa heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone that has been shown to play a major role during the processing and/or secretion of procollagen. Expression of HSP47 has been reported to increase in parallel with expression of collagens during the progression of various fibrosis models. The aim of the present study was to investigate the association between HSP47 expression and collagen accumulation in bleomycin (BLM)-induced murine fibrosis. We investigated the expression of HSP47 protein and mRNA using immunohistochemical analysis and semi-quantitative RT-PCR in murine BLM-induced pulmonary fibrosis. Immunohistochemical analysis showed that higher expression of HSP47 protein was present in BLM-induced pulmonary fibrosis compared with controls. HSP47 was localized predominantly in alpha-smooth muscle actin-positive myofibroblasts, F4/80 negative, surfactant protein-A-positive type II pneumocytes, and F4/80-positive macrophages. RT-PCR also demonstrated an increase of HSP47 mRNA expression in BLM-treated lungs. Moreover, the relative amounts of HSP47 mRNA correlated significantly with the lung hydroxyproline content as an indicator of pulmonary fibrosis in BLM-treated lungs (r = 0.406, P <0.05). Our results suggest that these cells may play a role in the fibrotic process of BLM-treated lungs through upregulation of HSP47.     

Pirfenidone inhibits the expression of HSP47 in TGF-beta1-stimulated human lung fibroblasts

Pirfenidone (5-methyl-1-phenyl-2-(1H)-pyridone) is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and patients with idiopathic pulmonary fibrosis (IPF). Heat shock protein (HSP) 47, a collagen-specific molecular chaperone, is involved in the processing and/or secretion of procollagen and plays an important role in the pathogenesis of IPF. The present study evaluated the in vitro effects of pirfenidone on expression of HSP47 and collagen type I in cultured normal human lung fibroblasts (NHLF). Expression levels of HSP47 and collagen type I in NHLF stimulated by transforming growth factor (TGF)-beta1 were evaluated genetically, immunologically and immunocytochemically. Treatment with TGF-beta1 stimulated both mRNA and protein expressions of both HSP47 and collagen type I in NHLF, and pirfenidone significantly inhibited this TGF-beta1-enhanced expression in a dose-dependent manner. We concluded that the anti-fibrotic effect of pirfenidone may be mediated not only through direct inhibition of collagen type I expression but also at least partly through inhibition of HSP47 expression in lung fibroblasts, with a resultant reduction of collagen synthesis in lung fibrosis.     

HSP47 regulates ECM accumulation in renal proximal tubular cells induced by TGF-β1 through ERK1/2 and JNK MAPK pathways

Heat shock protein (HSP)47 is a collagen-specific molecular chaperone that is essential for the biosynthesis of collagen molecules. It is likely that increased levels of HSP47 contribute to the assembly of procollagen and thereby cause an excessive accumulation of collagens in disease processes associated with fibrosis. Although HSP47 promotes renal fibrosis, the underlying mechanism and associated signaling events have not been clearly delineated. We examined the role of HSP47 in renal fibrosis using a rat unilateral ureteral obstruction model and transforming growth factor (TGF)-β(1)-treated human proximal tubular epithelial (HK-2) cells. An upregulation of HSP47 in both in vivo and in vitro models was observed, which correlated with the increased synthesis of extracellular matrix (ECM) proteins and expression of tissue-type plasminogen activator inhibitor (PAI)-1. Blockade of HSP47 by short interfering RNA suppressed the expression of ECM proteins and PAI-1. In addition, TGF-β(1)-induced HSP47 expression in HK-2 cells was attenuated by ERK1/2 and JNK MAPK inhibitors. These data suggest that ERK1/2 and JNK signaling events are involved in modulating the expression of HSP47, the chaperoning effect of which on TGF-β(1) would ultimately contribute to renal fibrosis by enhancing the synthesis and deposition of ECM proteins.     

Role of heat shock protein 47 in intestinal fibrosis of experimental colitis

Background and aims

Intestinal fibrosis is a clinically important issue of inflammatory bowel disease (IBD). It is unclear whether or not heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, plays a critical role in intestinal fibrosis. The aim of this study is to investigate the role of HSP47 in intestinal fibrosis of murine colitis.

Methods

HSP47 expression and localization were evaluated in interleukin-10 knockout (IL-10KO) and wild-type (WT, C57BL/6) mice by immunohistochemistry. Expression of HSP47 and transforming growth factor-β1 (TGF-β1) in colonic tissue was measured. In vitro studies were conducted in NIH/3T3 cells and primary culture of myofibroblasts separated from colonic tissue of IL-10KO (PMF KO) and WT mice (PMF WT) with stimulation of several cytokines. We evaluated the inhibitory effect of administration of small interfering RNA (siRNA) targeting HSP47 on intestinal fibrosis in IL-10KO mice in vivo.

Results

Immunohistochemistry revealed HSP47 positive cells were observed in the mesenchymal and submucosal area of both WT and IL-10 KO mice. Gene expressions of HSP47 and TGF-β1 were significantly higher in IL-10KO mice than in WT mice and correlated with the severity of inflammation. In vitro experiments with NIH3T3 cells, TGF-β1 only induced HSP47 gene expression. There was a significant difference of HSP47 gene expression between PMF KO and PMF WT. Administration of siRNA targeting HSP47 remarkably reduced collagen deposition in colonic tissue of IL-10KO mice.

Conclusions

Our results indicate that HSP47 plays an essential role in intestinal fibrosis of IL-10KO mice, and may be a potential target for intestinal fibrosis associated with IBD.     

Antisense oligonucleotide against collagen-specific molecular chaperone 47-kDa heat shock protein suppresses scar formation in rat wounds

The 47-kDa heat shock protein (HSP47) is a molecular chaperone specifically targeting the processing and quality control of collagen molecules. This study was performed to investigate whether antisense therapy preventing HSP47 expression might affect the scar formation occurring during wound healing of skin. In wound healing of neonatal rat skin, the number of HSP47-positive cells and the amount of HSP47 protein consistently increased up to 7 days after surgical wounding. The increase in HSP47-positive cell number and protein content was efficiently suppressed by daily injections of HSP47-antisense deoxynucleotide (30 nmol) for 7 days. This treatment also suppressed the accumulation of collagen type I in the wound. Moreover, the disorder of collagenous fibers was relieved in the healed portion of the wounds subjected to the antisense treatment. Taken together, the authors propose that HSP47 is an important determinant in scar formation and that the antisense treatment against HSP47 gene may have a therapeutic potential to suppress the scar formation of skin.     

Intracellular interaction of collagen-specific stress protein HSP47 with newly synthesized procollagen

Heat shock protein 47 (HSP47), a collagen-specific stress protein, has been postulated to be a collagen-specific molecular chaperone localized in the ER. We previously demonstrated that HSP47 transiently associated with newly synthesized procollagen in the ER (Nakai, A., M. Satoh, K. Hirayoshi, and K. Nagata. 1992. J. Cell Biol. 117:903-914). In the present work, we examined the location where HSP47 binds to and dissociates from newly synthesized procollagen within the cells, and whether HSP47 associates with nascent single procollagen polypeptide chains and/or with mature triple-helix procollagen. This was accomplished by biochemical coprecipitation with anti-HSP47 and anticollagen antibodies, combined with pulse-label and chase experiments in the presence or absence of various inhibitors for protein secretion, as well as by confocal laser microscopic observation of the cells double stained with both antibodies. We further examined whether the RDEL (Arg-Asp-Glu-Leu) sequence at the COOH terminus of HSP47 can act as an ER-retention signal, as the KDEL sequence does. When the secretion of procollagen was inhibited by the presence of alpha, alpha'-dipyridyl, an iron chelator that inhibits procollagen triple-helix formation, or by the presence of brefeldin A. which inhibits protein transport between the ER and the Golgi apparatus, procollagen was found to be bound to HSP47 during the chase period in the intermediate compartment. In contrast, the dissociation of procollagen chains from HSP47 was not inhibited when procollagen secretion was inhibited by monensin or bafilomycin A1, both of which are known to be inhibitors of post-cis-Golgi transport. These findings suggest that HSP47 and procollagen dissociated between the post-ER and the cis-Golgi compartments. HSP47 was shown to bind to nascent, single- polypeptide chains of newly synthesized procollagen, as well as to the mature triple-helix form of procollagen. HSP47 with the RDEL sequence deleted was secreted out of the cells, which suggests that the RDEL sequence actually acts as an ER-retention signal, as the KDEL sequence does. This secreted HSP47 did not acquire endoglycosidase H resistance. The biological significance of the interaction between HSP47 and procollagen in the central secretory pathway, as well as possible mechanisms for this pathway, will be discussed.     

The change of HSP47, collagen specific molecular chaperone, expression in rat skeletal muscle may regulate collagen production with gravitational conditions.

It is well known that unloading of skeletal muscle with spaceflight leads skeletal muscle atrophy. However, it remains unclear how the extracellular matrix within the muscle and the connective tissues such as tendon and ligament respond to reduced mechanical load including microgravity, although they have been thought to play important roles in both the transmission of force and the signal transduction between cells and tissues. Type-I collagen and type-IV collagen, both of the major components of extracellular matrix and connective tissues. We focused on change of these collagen synthesis with mechanical load. To obtain an insight into the effects of gravitational changing on the protein metabolism of collagen in skeletal muscle during mechanical unloading, reloading after unloading, we investigated changes in the amount of Heat shock protein 47 (HSP47), has been postulated to be a collagen-specific molecular chaperone localized in the ER (Nagata et al, 1992). Western blot analysis revealed that HSP47 in rat soleus muscle decreases at 5 days after hindlimb suspension (HS). On the other hand, HSP47 in rat soleus muscle increases at 5 days after hypergravity (HG) induced by the centrifugation. RT-PCR analysis showed HSP47 mRNA decreased with HS earlier, as compared with collagen type-I and type-IV mRNA. From these results, the amount of HSP47 changing by gravitational condition may effect on signal transfers in the primary stage of adaptation and the change of HSP47 expression in skeletal muscle may regulate collagen production with gravitational conditions.     

The possible role of colligin/HSP47, a collagen-binding protein, in the pathogenesis of human and experimental fibrotic diseases.

Colligin or heat shock protein 47 (HSP47) is a stress protein that resides in the endoplasmic reticulum and is thought to participate in intracellular processing, folding, assembly and secretion of procollagens. Irrespective of the tissue site and organ, induction of colligin/HSP47 expression is always noted during the process of fibrosis, particularly in and around the fibrotic lesions in both humans and experimental models. Its expression is highly tissue- and cell-specific, and restricted to mostly phenotypically altered collagen-producing cells. These observations suggest that upregulation of this collagen-specific chaperone-colligin/HSP47 may play an important role in the subsequent fibrotic process, possibly by regulating increased synthesis/assembly of collagens.     

Involvement of collagen-binding heat shock protein 47 in scleroderma-associated fibrosis

Uncontrolled fibrosis of skin and internal organs is the main characteristic of scleroderma, and collagen is a major extracellular matrix protein that deposits in the fibrotic organs. As the chaperone of collagen, heat shock protein 47 (HSP47) is closely related with the development of fibrosis. To explore the potential function of HSP47 in the pathogenesis of scleroderma, the clinical, in vivo and in vitro studies were performed. In clinical, the increased mRNA level of HSP47 was observed in the skin fibroblasts and PBMC from scleroderma patients, and the enhanced protein level of HSP47 was also detected in the skin biopsy and plasma of the above patients. Unexpectedly, the enhanced levels of HSP47 were positively correlated with the presence of anti-centromere antibody in scleroderma patients. Moreover, a high expression of HSP47 was found in the skin lesion of BLM-induced scleroderma mouse model. Further in vitro studies demonstrated that HSP47 knockdown could block the intracellular and extracellular collagen over-productions induced by exogenous TGF-β. Therefore, the results in this study provide direct evidence that HSP47 is involved in the pathogenesis of scleroderma. The high expression of HSP47 can be detected in the circulatory system of scleroderma patients, indicating that HSP47 may become a pathological marker to assess the progression of scleroderma, and also explain the systemic fibrosis of scleroderma. Meanwhile, collagen over-expression is blocked by HSP47 knockdown, suggesting the possibility that HSP47 can be a potential therapeutic target for scleroderma.     

HSP47 contributes to angiogenesis by induction of CCL2 in bladder cancer

Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone and is involved in tumor progression by promoting angiogenesis. However, the regulatory network of HSP47 in angiogenesis remains elusive. In this study, we report a novel mechanism of HSP47-induced angiogenesis in bladder cancer (BC). We find that HSP47 is abnormally overexpressed in BC and is correlated with poor prognosis. HSP47 down-regulation suppresses angiogenesis in BC cells. Mechanistically, activation of the ERK pathway and induction of C-C Motif Chemokine Ligand 2 (CCL2) are responsible for HSP47-induced angiogenesis. The correlation between HSP47 with CCL2 and angiogenesis is further confirmed in BC clinical samples. Taken together, our findings suggest that HSP47 contributes to BC angiogenesis by induction of CCL2 and provide a potential anti-angiogenesis target for BC therapy.     

Prevalence of HSP47 antigen and autoantibodies to HSP47 in the sera of patients with mixed connective tissue disease

The 47-kDa heat shock protein (HSP47) is an endoplasmic reticulum molecular chaperone that assists in the maturation of collagen molecules and whose expression is known to be upregulated in lesions of fibrotic diseases. We examined the levels of HSP47 protein and autoantibodies to HSP47 in the sera of patients with rheumatic autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, Sj?gren's syndrome, and mixed connective tissue disease (MCTD) by enzyme-linked immunosorbent assay and immunoblot analysis. Patients with idiopathic pulmonary fibrosis (IPF) were assessed as an example of non-autoimmune fibrotic disease. HSP47 antigen and autoantibody levels are significantly elevated in the sera of the rheumatic autoimmune disease patients, but not in the sera of the IPF patients. The sera of the MCTD patients showed particularly high levels of HSP47 antigen relative to healthy controls (1.99+/-0.22 vs 0.41+/-0.07 ng/ml). Autoantibodies to HSP47 were also in high levels in the sera of MCTD patients. These results suggest that simultaneous occurrence of systemic inflammation and upregulation of HSP47 caused leakage of HSP47 from fibrotic lesions into the peripheral blood, and the leaked antigen induced high titer of autoantibodies to HSP47. The high levels of HSP47 antigen and autoantibody may be useful blood markers of MCTD.     

Advanced glycation end products increase collagen-specific chaperone protein in mouse diabetic nephropathy

Advanced glycation end products (AGEs) appear to contribute to the diabetic complications. This study reports the inhibitory effect of OPB-9195 (OPB), an inhibitor of AGEs formation, and the role of a collagen-specific molecular chaperone, a 47-kDa heat shock protein (HSP47) in diabetic nephropathy. Transgenic mice carrying nitric-oxide synthase cDNA fused with insulin promoter (iNOSTg) leads to diabetes mellitus. The iNOSTg mice at 6 months of age represented diffuse glomerulosclerosis, and the expression of HSP47 was markedly increased in the mesangial area in parallel with increased expression of types I and IV collagens. OPB treatment ameliorated glomerulosclerosis in the iNOSTg mice associated with the decreased expression of HSP47 and types I and IV collagens. The expression of transforming growth factor-beta (TGF-beta) was increased in glomeruli of iNOSTg mice and decreased after treatment with OPB. To confirm these mechanisms, cultured mesangial cells were stimulated with AGEs. AGEs significantly increased the expression of HSP47, type IV collagen, and TGF-beta mRNA. Neutralizing antibody for TGF-beta inhibited the overexpression of both HSP47 and type IV collagen in vitro. In conclusion, AGEs increase the expression of HSP47 in association with collagens, both in vivo and in vitro. The processes may be mediated by TGF-beta.     

Serum heat shock protein 47 levels are elevated in acute exacerbation of idiopathic pulmonary fibrosis

Little is known about the pathophysiology of acute exacerbation (AE) of idiopathic pulmonary fibrosis (IPF). Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, is essential for biosynthesis and secretion of collagen molecules. Previous studies in experimental animal fibrosis models have shown that downregulation of HSP47 expression reduces collagen production and diminishes fibrosis progression. In this study, serum HSP47 levels were evaluated to elucidate pathogenic differences involving HSP47 between AE-IPF and stable (S)-IPF. Subjects comprised 20 AE-IPF and 33 S-IPF patients. Serum levels of HSP47, Krebs von den Lungen-6 (KL-6), surfactant protein (SP)-A, SP-D, and lactate dehydrogenase (LDH) were measured. Immunohistochemical analysis of lung HSP47 expression was determined in biopsy and autopsy tissues diagnosed as diffuse alveolar damage (DAD) and usual interstitial pneumonia (UIP). Serum levels of HSP47 were significantly higher in AE-IPF than in S-IPF patients, whereas serum levels of KL-6, SP-A, and SP-D did not differ significantly. Receiver operating characteristic curves revealed that HSP47 was superior for discriminating AE-IPF and S-IPF. The cutoff for HSP47 resulting in the highest diagnostic accuracy was 559.4 pg/mL; sensitivity, specificity, and diagnostic accuracy were 100.0 %, 93.9 %, and 96.2 %, respectively. Immunohistochemical analysis revealed that pulmonary HSP47 expression was greater in DAD than UIP tissues. Serum HSP47 was significantly higher in AE-IPF than in S-IPF patients, suggesting that underlying fibrogenic mechanisms involving HSP47 differ in the two conditions.     

HSP47 binds cooperatively to triple helical type I collagen but has little effect on the thermal stability or rate of refolding

HSP47, a collagen-specific molecular chaperone, interacts with unfolded and folded procollagens. Binding of chicken HSP47 to native bovine type I collagen was studied by fluorescence quenching and cooperative binding with a collagen concentration at half saturation (K(half)) of 1.4 x 10(-7) m, and a Hill coefficient of 4.3 was observed. Similar results are observed for the binding of mouse HSP47 recombinantly expressed in Escherichia coli. Chicken HSP47 binds equally well to native type II and type III procollagen without the carboxyl-terminal propeptide (pN type III collagen), but binding to triple helical collagen-like peptides is much weaker. Weak binding occurred to both hydroxylated and nonhydroxylated collagen-like peptides, and a significant chain length dependence was observed. Binding of HSP47 to native type I collagen had no effect on the thermal stability of the triple helix. Refolding of type I collagen in the presence of HSP47 showed minor changes, but these are probably not biologically significant. Binding of HSP47 to bovine pN type III collagen has only minor effects on the thermal stability of the triple helix and does not influence the refolding kinetics of the triple helix.     

Expression of HSP47 in Usual Interstitial Pneumonia and Nonspecific Interstitial Pneumonia

Background

Heat shock protein (HSP) 47, a collagen-specific molecular chaperone, is involved in the processing and/or secretion of procollagens, and its expression is increased in various fibrotic diseases. The aim of this study was to determine whether quantitative immunohistochemical evaluation of the expression levels of HSP47, type I procollagen and α-smooth muscle actin (SMA) allows the differentiation of idiopathic usual interstitial pneumonia (UIP) from UIP associated with collagen vascular disease (CVD) and idiopathic nonspecific interstitial pneumonia (NSIP).

Methods

We reviewed surgical lung biopsy specimens of 19 patients with idiopathic UIP, 7 with CVD-associated UIP and 16 with idiopathic NSIP and assigned a score for the expression of HSP47, type I procollagen and α-SMA in type II pneumocytes and/or lung fibroblasts (score 0 = no; 1 = weak; 2 = moderate; 3 = strong staining).

Results

The expression level of HSP47 in type II pneumocytes of idiopathic UIP was significantly higher than in CVD-associated UIP and idiopathic NSIP. The expression of HSP47 in fibroblasts was significantly higher in idiopathic UIP and idiopathic NSIP than in CVD-associated UIP. The expression of type I procollagen in type II pneumocytes was significantly higher in idiopathic UIP than in idiopathic NSIP. The expression of type I procollagen in fibroblasts was not different in the three groups, while the expression of α-SMA in fibroblasts was significantly higher in idiopathic UIP than in idiopathic NSIP.

Conclusion

Our results suggest the existence of different fibrotic pathways among these groups involved in the expression of HSP47 and type I procollagen.     

Enhancement of secretion of human procollagen I in mouse HSP47-expressing insect cells

We previously demonstrated that insect cells were able to synthesize recombinant human procollagen I as triple-helical heterotrimers when transfected with cDNAs of both proalpha1(I) and proalpha2(I) chains. However, most of the heterotrimers were retained within the cells, unlike in the case of mammalian cells [Tomita, M., Kitajima, T., and Yoshizato, K. (1997) J. Biochem. 1061-1069]. In an attempt to improve the secretion of the heterotrimers, we introduced the putative collagen-specific chaperone HSP47 into this insect expression model. Mouse HSP47 produced by the insect cells bound intracellularly to both human proalpha1(I) and proalpha2(I) chains and enhanced the secretion of procollagen I heterotrimers. HSP47 was also coexpressed with either proalpha1(I) chains or proalpha2(I) chains, which showed that it enhanced the secretion of the former but not the latter. This selective effect of HSP47 was similarly observed in the cells treated with inhibitors of procollagen triple helix formation, indicating that HSP47 can also accelerate the secretion of non-helical procollagens. HSP47 did not change the intracellular solubility of proalpha1(I) and proalpha2(I) chains in 1% NP-40, eliminating the possibility that it prevents proalpha chains from aggregating into insoluble forms within the insect cells. We concluded that HSP47 can play a role in the secretion of alpha1(I)-procollagen chains in the insect cell model. The present study also demonstrated the dissimilarity in the mechanism of folding and secretion of the expressed procollagen I between the insect and mammalian cells.     

Collagens,collagen-binding heat shock protein 47 and transforming growth factor-beta 1 are induced in cicatricial pemphigoid: possible role(s) in dermal fibrosis

Cicatricial pemphigoid (CP) is an autoimmune mucocutaneous blistering disease associated with scarring. Heat shock protein 47 (HSP47) is thought to play an important role in fibrogenesis, but its role in skin lesions of cicatricial pemphigoid is not yet known. In the present study, we examined the role of HSP47 in dermal fibrosis in cutaneous lesions of a CP patient. Skin biopsies from a patient with CP, and from normal subjects were studied for the expression of HSP47, and interstitial collagens (type I and type III collagens) by immunohistochemistry. Dermal fibroblasts isolated from skin of normal individuals and from fibrotic skin of a CP patient were used to study the expression of HSP47, transforming growth factor beta 1 (TGF-beta 1), type I and type III collagens. Compared to the control skin sections, an increased expression of HSP47 was associated with an increased deposition of interstitial collagens in the fibrotic skin section of the CP patient. Similarly, in contrast to control dermal fibroblasts, the fibroblasts isolated and cultured from fibrotic skin of the CP patient, and grown in vitro, exhibited increased expression of HSP47, type I and type III collagens. Furthermore, compared to the normal control fibroblasts, an increased expression of TGF-beta 1 was detected in the dermal fibroblasts isolated from fibrotic skin of the CP patient. When dermal fibroblasts were treated with various concentrations of TGF-beta 1 (6.25, 12.5, 25, 50 and 100 ng/ml for 24 h), it induced the expression of both type I collagen and HSP47, as determined by quantitative real-time PCR. In conclusion, the expression of TGF-beta 1, HSP47, type I collagen and type III collagen was up-regulated in the fibrotic skin of CP patient, and a complex interaction of these molecules may initiate and propagate the fibrotic cascade in the skin of CP patients.