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.

In-silico analysis of heat shock protein 47 for identifying the novel therapeutic agents in the management of oral submucous fibrosis.

Background: Heat shock proteins-47 (HSP47) is a collagen specific molecular chaperone, involved in the processing and/or secretion of procollagen. It seems to be regularly upregulated in various fibrotic or collagen disorders. Hence, this protein can be a potential target for the treatment of various fibrotic diseases including oral submucous fibrosis (OSF), which is a collagen metabolic disorder of oral cavity and whose etiopathogeneic mechanism and therapeutic protocols are still not well documented. Aim: The aim of this study is to identify the novel therapeutic agents using in-silico methods for the management of OSF. Objectives: The objectives of this study are to identify the binding sites of HSP47 on the collagen molecule and to identify the lead compound with anti-HSP47 activity from the library of natural compounds, using in-silico methodology. Materials and Methods: The web-based and tool based in-silico analysis of the HSP47 and collagen molecules are used in this study. The crystal structure of collagen and HSP47 were retrieved from Protein Data Bank website. The binding site identification and the docking studies are done using Molegro Virtual Docker offline tool. Results: Out of the 104 Natural compounds, six ligands are found to possess best binding affinity to the binding amino acid residues. Silymarin binds with the 4AU2A receptor and the energy value are found to be −178.193 with four Hbonds. The other best five natural compounds are hesperidin, ginkgolides, withanolides, resveratrol, and gingerol. Our findings provide the basis for the in-vitro validation of the above specified compounds, which can possibly act as "lead" molecules in designing the drugs for OSF. Conclusion: HSP47 can be a potential candidate to target, in order to control the production of abundance collagen in OSF. Hence, the binding sites of HSP47 with collagen are identified and some natural compounds with a potential to bind with these binding receptors are also recognized. These natural compounds might act as anti-HSP47 lead molecules in designing novel therapeutic agents for OSF, which are so far unavailable.

Single-chain human anti-EGFR antibody/truncated protamine fusion protein carrying Hsp47 siRNA can induce apoptosis of human hepatic stellate cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To construct a single-chain human anti-EGFR antibody (scFv) and truncated protamine (tP) fusion protein, ScFv/tP, carrying small interfering (si)RNA directed against the heat shock protein Hsp47, a collagen-binding glycoprotein, in order to evaluate the role Hsp47 in apoptosis of hepatic stellate cells.</p><p><b>METHODS</b>A single chain of the human variable fragment was obtained by phage display and fused with the tP gene and with or without (negative control) the Hsp47 siRNA sequences. Following expression and purification of the scFv/tP fusion protein and the scFv/tPHsp47 siRNA fusion protein, internalization capabilities were tested in isolated human hepatic stellate cells and the QSG-7701 human hepatocyte cells with visualization by immunofluorescent staining. The DNA binding ability of the fusion proteins were verified by gel shift assay.Following ScFv/tP-Hsp47 siRNA fusion protein transfection into the human hepatic stellate cells, the levels of Hsp47 mRNA and protein expression were tested by RT-PCR and Western blotting; in addition, effects of siRNA-mediated silencing of Hsp47 on cell proliferation and apoptosis were analyzed by the cell counting kit (CCK)-8, flow cytometry and Western blot detection of the apoptosis marker poly (ADP-ribose) polymerase (PARP).</p><p><b>RESULTS</b>Indirect immunofluorescence revealed that the ScFv/tP fusion proteins were internalized into human hepatic stellate cells but not into the QSG-7701 cells.The ScFv/tP-Hsp47 siRNA fusion protein caused reduced expression of Hsp47 mRNA and protein expression in the human hepatic stellate cells, as well as increased the cells' apoptosis remarkably.</p><p><b>CONCLUSION</b>The ScFv/tP fusion protein can be used as a transfection reagent to deliver Hsp47 siRNA into hepatic stellate cells and to mediate apoptosis via blockade of Hsp47 expression.</p>

Expression of E-cadherin, Heat Shock Protein 47, Transforming Growth Factor beta1 and C4d in Chronic Allograft Nephropathy / 대한이식학회지

BACKGROUND: Chronic allograft nephropathy (CAN), which causes graft failure, is related to tubular atrophy and interstitial fibrosis. E-cadherin is a well-known epithelial marker and heat shock protein (HSP)-47 is a collagen-specific molecular chaperone that regulates collagen synthesis. Transforming growth factor (TGF)-beta1, a profibrotic cytokine, downregulates E-cadherin and induces expression of mesenchymal markers in an in vitro model. C4d expression is considered a poor prognostic marker for graft survival. This study evaluated the relationship between the expression of E-cadherin, HSP47, TGF-beta1, and C4d with the prognosis for CAN. METHODS: Between March 1991 and August 2007, we performed renal allograft biopsies on 42 recipients with deteriorating renal function. CAN was diagnosed according to the chronic allograft damage index (Banff classification). Renal allograft biopsies were examined for the expression of E-cadherin, HSP47, TGF-beta1, or C4d by immunohistochemistry. The HSP47, TGF-beta1, and E-cadherin staining was scored semiquantitatively by analyzing ten different fields of cortical interstitium and tubules. Biopsies with endothelial C4d staining in peri-tubular capillaries (> or =25%) were designated as C4d-positive. RESULTS: Of 42 recipients, 17 (40.5%) were in the graft survival group (GS) and 25 (59.5%) were in the graft failure group (GF). E-cadherin expression in tubular cells of the GS was much higher than that of the GF (94.1% vs 52%, P=0.04). HSP47 expression in tubular cells and interstitium in the GF was much higher than that in the GS (84% vs 35.3%, P=0.001). TGF-beta1 expression in tubular cells and interstitium in the GF was much higher than that in the GS (72% vs 23.5%, P=0.02). CONCLUSIONS: E-cadherin, HSP47, and TGF-beta1 expression was strongly correlated with the CAN prognosis.

The effect of recombinant HSP47siRNA on the pathological scar in nude mice: in vivo study / 中华整形外科杂志

<p><b>OBJECTIVE</b>To study the significance of HSP47 gene in the development of pathological scar.</p><p><b>METHODS</b>The nude mice were used to reconstruct animal model of pathological scar. 16 days later, the mixture of recombinant HSP47siRNA and liposome was injected into the pathological scar in experimental group. In the control group, 0.25 ml PBS was injected intraperitoneally. 7 days after injection, the specimens were collected for detection of mRNA of HSP47, the collagen and for immunohistochemical study.</p><p><b>RESULTS</b>In the control and experimental group, the collagen content was (91.71 +/- 1.24)% and (82.12 +/- 4.79)%, respectively; the expression of HSP47mRNA was 1 042 862.01 +/- 604 194.36 and 306 123.68 +/- 105 857.08, respectively; the expression of collagen I mRNA was 10 228 614.70 +/- 2 532 879.04 and 6 011 841.97 +/- 2 886 897.17, respectively;the scar volume was (255.60 +/- 21.34) mm3 and (132.99 +/- 24.06) mm3, respectively. All the above results showed significant difference between the two groups (P < 0.05).</p><p><b>CONCLUSIONS</b>The collagen production can be reduced through suppression of the expression of HSP47 gene. It indicates that HSP47 gene enhance the development of keloid and could be used as the target of treatment.</p>

Effect of heat shock protein 47 on the expression of collagen I induced by TGF-beta(1) in hepatic stellate cell-T6 cells / 中南大学学报(医学版)

OBJECTIVE@#To determine the effect of heat shock protein 47 (HSP47) on the expression of collagen I induced by transforming growth factor beta(1) (TGF-beta(1)) in hepatic stellate cell-T6 (HSC-T6) cells.@*METHODS@#We used 1 ng/mL and 10 ng/mL recombinant human TGF-beta(1) to stimulate the cultured HSC-T6 cells. Heat shock response (HSR) and antisense oligonucleotides of HSP47 were used to induce and block the expression of HSP47, respectively. The expressions of HSP47 and collagen I were detected by Western blot and the cell viability was observed by MTT assay.@*RESULTS@#Both HSP47 and collagen I were expressed in normal HSC-T6 cells. Collagen I and HSP47 expression could be induced by both 1 ng/mL and 10 ng/mL TGF-beta(1) and collagen I was expressed the most after the treatment with 10 ng/mL TGF-beta(1). Although HSR could not affect the synthesis of collagen I as it induced the HSP47 expression, HSR could promote the expression of collagen I induced by TGF-beta(1). With no effect on the cell viability, antisense oligonucleotides could significantly inhibit HSR-mediated HSP47 expression and TGF-beta(1)-induced collagen I synthesis.@*CONCLUSION@#Over-expression of HSP47 enhances TGF-beta(1)-induced expression of collagen I in HSC-T6 cells, and HSP47 may play important roles in the process of hepatic fibrosis.

Heat Shock Protein Induction By An Infrared Warm Compression Device

PURPOSE: To investigate if the use of the infrared warm compression device often used in clinical settings induced heat shock proteins. METHODS: Subjects were heat-treated with an infrared warm compression device for 20 minutes. We examined the temperature of the upper eyelid and cornea before and after heat treatment and images were obtained by Digital Infrared Thermal Imaging System. After 6 hours of heat treatment, conjunctival epithelial cells were obtained by gently pressing nitrocellulose paper on the conjunctival surface for 3 to 5 seconds Immunocytochemical staining analysis was performed on the obtained samples. Tear samples were obtained prior to heat treatment and Western blot was performed to observe the expression patterns of heat shock proteins 27, 47, 70, and 90. RESULTS: By Western blot and immunocytochemical analysis, heat shock proteins 70 and 27 were significantly increased in the heat-treated samples. However, no difference was observed for heat shock proteins 47 and 90 before and after heat treatment, according to the immunocytochemical analysis. On Western blot, heat shock protein 47 was slightly increased by heat treatment but heat shock protein 90 did not show a significant difference after heat treatment. CONCLUSIONS: It was observed that the infrared warm compression device significantly increased the induction of heat shock proteins 27 and 70, and that 47 was also slightly induced. This result suggests that the device developed herein could be used as a new therapeutic modality for the reduction of inflammatory cell injury through the induction of heat shock proteins.