HSP47 Research Articles

Decreased Expression of Heat Shock Protein 47 Is Associated with T-2 Toxin and Low Selenium-Induced Matrix Degradation in Cartilages of Kashin-Beck Disease.

Recent evidence suggests a role of type II collagen in Kashin-Beck disease (KBD) degeneration. We aimed to assess the abnormal expression of heat shock protein 47 (HSP47) which is associated with a decrease in type II collagen and an increase in cartilage degradation in KBD. Hand phalange cartilages were collected from KBD and healthy children. Rats were administered with T-2 toxin under the selenium (Se)-deficient diet. ATDC5 cells were seeded on bone matrix gelatin to construct engineered cartilaginous tissue. C28/I2 and ATDC5 cells and engineered tissue were exposed to different concentrations of T-2 toxin with or without Se. Cartilage degeneration was determined through histological evaluation. The distribution and expression of type II collagen and HSP47 were investigated through immunohistochemistry, western blotting, and real-time PCR. KBD cartilages showed increased chondronecrosis and extracellular matrix degradation in deep zone with decreased type II collagen and HSP47 expression. The low-Se + T-2 toxin animal group showed a significantly lower type II collagen expression along with decreased HSP47 expression. Decreased type II collagen and HSP47 in C28/I2 and ATDC5 cells induced by T-2 toxin showed a dose-dependent manner. Hyaline-like cartilage with zonal layers was developed in engineered cartilaginous tissues, with decreased type II collagen and HSP47 expression found in T-2 toxin-treated group. Se-supplementation partially antagonized the inhibitory effects of T-2 toxin in chondrocytes and cartilages. HSP47 plays a role in the degenerative changes of KBD and associated with T-2 toxin-induced decreased type II collagen expression, further promoting matrix degradation.

Type I Collagen Suspension Induces Neocollagenesis and Myodifferentiation in Fibroblasts In Vitro.

The ability of a collagen-based matrix to support cell proliferation, migration, and infiltration has been reported; however, the direct effect of an aqueous collagen suspension on cell cultures has not been studied yet. In this work, the effects of a high-concentration aqueous suspension of a micronized type I equine collagen (EC-I) have been evaluated on a normal mouse fibroblast cell line. Immunofluorescence analysis showed the ability of EC-I to induce a significant increase of type I and III collagen levels, parallel with overexpression of crucial proteins in collagen biosynthesis, maturation, and secretion, prolyl 4-hydroxylase (P4H) and heat shock protein 47 (HSP47), as demonstrated by western blot experiments. The treatment led, also, to an increase of α-smooth muscle actin (α-SMA) expression, evaluated through western blot analysis, and cytoskeletal reorganization, as assessed by phalloidin staining. Moreover, scanning electron microscopy analysis highlighted the appearance of plasma membrane extensions and blebbing of extracellular vesicles. Altogether, these results strongly suggest that an aqueous collagen type I suspension is able to induce fibroblast myodifferentiation. Moreover, our findings also support in vitro models as a useful tool to evaluate the effects of a collagen suspension and understand the molecular signaling pathways possibly involved in the effects observed following collagen treatment in vivo.

Therapeutic effect of 1,25(OH)2-VitaminD3 on fibrosis and angiogenesis of peritoneum induced by chlorhexidine

The biological activity of vitamin D, which mediated by the vitamin D receptor, is widespread throughout the body. The present study aimed to define whether 1,25-dihydroxy vitamin D3 (1,25-(OH)2D3) can protect against the progression of peritoneum fibrosis (PF) through its impact on the expression of connective tissue growth factor (CTGF) and heat shock protein 47 (HSP47) in vivo and in vitro. The male Sprague-Dawley (SD) rats of PF were induced by daily intraperitoneally injection of chlorhexidine gluconate (CG) for 4 wks. PF Rats were also treated with calcitriol (i.p. 6 ng/100g*d) from initiation of the CG. In calcitriol rats, the ultrafiltration and the ratio of dialysate urea nitrogen to blood urea nitrogen were improved (P < 0.05), pathological changes and peritoneal thickness were milder than that of the PF group. Calcitriol ameliorated high glucose-induced HSP47 expression in peritoneal mesothelial cells via CTGF down-regulation both at the mRNA level and protein level. Furthermore, calcitriol prevented angiogenic mediators of fibrosis by reduced the expression of CD34 and vascular endothelial growth factor (VEGF). The present study demonstrated that 1,25-(OH)2D3 intervention had a partially protective effect on peritoneum fibrosis, which might inhibit CTGF/HSP47 and CD34/VEGF in the peritoneum tissues.

P0734THE DELETION OF AKT1 PROMOTES THE RENAL FIBROSIS IN THE MURINE MODEL OF UNILATERAL URETERAL OBSTRUCTION

Abstract Background and Aims Previous studies have found the increased Akt activity in experimental renal fibrosis. We investigated the role of Akt1, one of the three Akt isoforms, in renal fibrosis using the murine model of unilateral ureteral obstruction (UUO). Method In vivo, we subjected the wild type and Akt1−/− mice to UUO. In vitro, gene silencing of Akt1 was achieved using the short hairpin RNA delivered by the lentiviral vector in immortalized human proximal tubular cells (HK2 cells) and rat kidney fibroblasts (NRK-49F cells). Results In immunohistochemical stain, the expression of Akt1 was significantly higher in obstructed kidneys of wild type mice compared with control sham kidneys and increased gradually as UUO progressed. The fibronectin, type I collagen, and heat shock protein 47 (HSP47) were markedly more expressed in obstructed kidneys of Akt1−/− mice than in those of the wild type mice. Transforming growth factor β1 (TGFβ1) was highly induced within 1 day of UUO in obstructed kidneys of Akt1−/− mice and the expression of TGFβ1 was significantly higher in the Akt1−/− mice than in the wild type mice as UUO progressed. Western blot showed that silencing of Akt1 increased the expression of TGFβ1, which was enhanced by angiotensin II stimulation in HK2 cells, but not in NRK-49F cells. Immunohistochemical stain demonstrated that the expression of cleaved caspase-3 in renal tubules was significantly higher in the Akt1−/− mice than in the wild type mice. Western blot showed that silencing of Akt1 increased the expression of cleaved caspase-3 in HK2 cells, but not in NRK-49F cells. Conclusion Our findings suggest that the deletion of Akt1 might contribute to tubulointerstitial fibrosis and tubular apoptosis via TGFβ1 induction. We also showed that TGFβ1 upregulation by genetic deletion of Akt1 is associated with activation of STAT3 independently of the TGFβ1/Smad signaling pathway.

Brilliant blue G, a P2X7 receptor antagonist, attenuates early phase of renal inflammation, interstitial fibrosis and is associated with renal cell proliferation in ureteral obstruction in rats

BackgroundPrevious study showed that purinergic P2X7 receptors (P2X7R) reach the highest expression in the first week after unilateral ureteral obstruction (UUO) in mice, and are involved in the process of inflammation, apoptosis and fibrosis of renal tissue. We, herein, document the role of purinergic P2X7 receptors activation on the third day of UUO, as assessed by means of BBG as its selective inhibitor.MethodsWe investigated the effects of brilliant blue G (BBG), a P2X7R antagonist, in the third day of kidney tissue response to UUO in rats. For this purpose, male Wistar rats submitted to UUO or sham operated, received BBG or vehicle (V), comprising four groups: UUO-BBG, UUO-V, sham-BBG and sham-V. The kidneys were harvested on day 3 UUO and prepared for histology, immunohistochemistry (P2X7R, PCNA, CD-68, α-sma, TGF-β1, Heat-shock protein-47, TUNEL assay), quantitative real-time PCR (IL-1β, procollagens type I, III, and IV) for mRNA quantification.ResultsThe group UUO-V presented an enhancement in tubular cell P2X7-R expression, increase influx of macrophages and myofibroblasts, HSP-47 and TGF- β1 expression. Also, upregulation of procollagen types I, III, and IV, and IL-1β mRNAs were seen. On the other hand, group UUO-BBG showed lower expression of procollagens and IL-1β mRNAs, as well as less immunoreactivity of HSP-47, TGF-β, macrophages, myofibroblasts, and tubular apoptosis. This group also presented increased epithelial cell proliferation.ConclusionBBG, a known highly selective inhibitor of P2X7R, attenuated renal inflammation, collagen synthesis, renal cell apoptosis, and enhanced renal cell proliferation in the early phase of rat model of UUO.

Liver-Targeted siRNA Lipid Nanoparticles Treat Hepatic Cirrhosis by Dual Antifibrotic and Anti-inflammatory Activities

Previous studies on the treatment of hepatic cirrhosis have been focusing on how to inhibit liver fibrosis, while ignoring liver inflammation, a key and underlying factor that promotes cirrhosis. High mobility group box-1 (HMGB1) protein, a pro-inflammatory factor and fibroblast chemokine, can promote the proliferation of hepatic stellate cells (HSCs) and the development of hepatic inflammation and fibrosis, playing a key role in cirrhosis formation. In this study, we prepared pPB peptide (C*SRNLIDC*)-modified and HMGB1-siRNA-loaded stable nucleic acid lipid nanoparticles (HMGB1-siRNA@SNALP-pPB) to effectively treat hepatic cirrhosis by their dual antifibrotic and anti-inflammatory activities. The pPB peptide-modified and heat shock protein 47 (HSP47)-siRNA-loaded stable nucleic acid lipid nanoparticles (HSP47-siRNA@SNALP-pPB), which have only an antifibrotic effect without an anti-inflammatory effect, was used as control. The results demonstrated that HMGB1-siRNA@SNALP-pPB were actively targeted to HSCs by the mediation of pPB peptide, effectively silenced the HMGB1 gene, inhibited the activation and proliferation of HSCs, reduced the release of HMGB1 protein, inhibited collagen deposition and fibrosis formation in the liver, and significantly prolonged the survival time of cirrhotic mice models. HMGB1-siRNA@SNALP-pPB showed a stronger therapeutic effect on liver cirrhosis than HSP47-siRNA@SNALP-pPB. This study provides an actively targeted siRNA delivery system for cirrhosis treatment based on the dual antifibrotic and anti-inflammatory effects. In addition, this study clarified the role of inflammatory problems in cirrhosis treatment in addition to liver fibrosis, providing a useful idea and scientific basis for the development of cirrhosis treatment strategies in the future.

HSP47 promotes metastasis of breast cancer by interacting with myosin IIA via the unfolded protein response transducer IRE1α.

Breast cancer (BC) is an aggressive cancer that is a leading cause of cancer-associated death in women worldwide. Although increased expression of heat shock protein 47 (HSP47), a collagen-specific chaperone, is associated with the high malignancy of BC, its role in BC remains largely unclear. Here we show that a small population of high-invasive BC cells expresses HSP47 and that HSP47-positive high-invasive BC cells have a high metastatic potential that is completely abolished by disruption of HSP47. HSP47 interacts with non-muscle myosin IIA (NMIIA) via the unfolded protein response transducer IRE1α, resulting in enhancement of the metastatic potential of high-invasive BC cells by augmenting the contractile force of actin filaments. Ablation of NMIIA abrogates the metastatic potential of HSP47-positive high-invasive BC cells. We further show that forced expression of NMIIA confers a high metastatic potential on low-invasive BC cells in which HSP47 but not NMIIA is expressed. Overall, our study indicates that HSP47 acts as a stimulator for metastasis of BC cells and suggest that HSP47 may be a candidate for a therapeutic target against BC.

Connexin 43 upregulation in burns promotes burn conversion through spread of apoptotic death signals.

Burn wounds continue to worsen after initial injury in a process known as burn conversion, which lasts about 3-5 days. It causes burn wounds to enlarge and deepen, leading to greater morbidity. Apoptosis is one of the factors contributing to the conversion of the zone of stasis into the zone of coagulation. Suppression of apoptosis has been associated with reducing burn conversion. Connexin 43 (Cx43) gap junctions facilitate the spread of apoptotic signals from dying cells to healthy neighbouring cells in injured tissues through the bystander effect. The study is to understand the role of Cx43 in burn conversion. In our study, 15 burn tissue samples were arranged into three groups as early (beginning of burn conversion), intermediate (extensive burn conversion) and late (established burn conversion) burns. We found a striking increase in the amount of Cx43 protein expressed in the dermal fibroblasts (identified with heat shock protein 47 (HSP47) staining) in the zone of stasis in early and intermediate burns. These dermal fibroblasts also express high levels of cleaved-Caspase 3 indicating on-going apoptosis. Our findings suggest that elevation of Cx43 may play an active role in burn conversion spreading apoptosis in the early and intermediate burn wound.

Nintedanib inhibits keloid fibroblast functions by blocking the phosphorylation of multiple kinases and enhancing receptor internalization.

Keloid is a benign skin tumor characterized by its cell hyperproliferative activity, invasion into normal skin, uncontrolled growth, overproduction and deposition of extracellular matrices and high recurrence rate after various therapies. Nintedanib is a receptor tyrosine kinase inhibitor targeting VEGF, PDGF, FGF, and TGF-β receptors with proved efficacy in anti-angiogenesis and in treating various types of cancers. In this study, we investigated the effects of nintedanib on keloid fibroblasts in both in vitro and ex vivo models. Keloid fibroblasts were prepared from 54 keloid scar samples in active stages collected from 49 patients. We found that nintedanib (1−4 μM) dose-dependently suppressed cell proliferation, induced G0/G1 cell cycle arrest, and inhibited migration and invasion of keloid fibroblasts. The drug also significantly inhibited the gene and protein expression of collagen I (COL-1) and III (COL-3), fibronectin (FN), and connective growth factor (CTGF), as well as the gene expression of other pathological factors, such as alpha smooth muscle actin (α-SMA), plasminogen activator inhibitor-1 (PAI-1), FK506-binding protein 10 (FKBP10), and heat shock protein 47 (HSP47) in keloid fibroblasts. Furthermore, nintedanib treatment significantly suppressed the phosphorylation of p38, JNK, ERK, STAT3, and Smad, enhanced endocytosis of various growth factor receptors. Using an ex vivo tissue explant model, we showed that nintedanib significantly suppressed cell proliferation, migration, and collagen production. The drug also significantly disrupted microvessel structure ex vivo. In summary, our results demonstrate that nintedanib is likely to become a potential targeted drug for keloid systemic therapy.

Expression of Collagen (Types I, III, and V), HSP47, MMP-2, and TIMP-1 in Retrobulbar Adipose Tissue of Patients with Thyroid-Associated Orbitopathy.

Objective This study aimed to investigate the expression of collagen (types I, III, and V), heat shock protein 47 (HSP47), matrix metalloproteinase-2 (MMP-2), and tissue inhibitors of metalloproteinase-1 (TIMP-1) in the retrobulbar adipose tissues of patients with thyroid-associated orbitopathy (TAO). Materials and Methods The retrobulbar adipose tissues were collected from 4 TAO patients undergoing orbital decompression and 4 ocular enucleation patients with atrophic eyeball caused by ocular trauma between May 2019 and September 2019. Masson staining was performed to analyze the differences in collagen expression and degree of histologic fibrosis in each sample. The protein expressions of collagen (types I, III, and V), HSP47, MMP-2, and TIMP-1 were determined by western blotting. The data of western blotting were analyzed using SPSS version 17.0, with independent t-tests. Results The results of Masson staining showed that the expression of collagen fibers in the TAO group was significantly higher than that in the control group, and the fibers were diffuse and irregular in distribution. The expression level of collagen (types I, III, and V), HSP47, MMP-2, and TIMP-1 in the TAO group were significantly higher than that in the control group (P < 0.05). Conclusion The proliferation and fibrosis of retrobulbar adipose tissue in TAO patients might be related to the increased expression of collagen (types I, III, and V) and HSP47 and decreased degradation of extracellular matrix.

The effect and mechanism of heat shock protein 47 on streptozotocin-induced diabetic cardiomyopathy

Objective: To investigate the effect and specific mechanism of heat shock protein 47 (HSP47) on streptozotocin (STZ)-induced diabetic cardiomyopathy (DCM). Methods: A mouse model of type 1 diabetic cardiomyopathy was established by intraperitoneal injection of STZ. After 8 weeks of successful modeling, HSP47 was overexpressed by tail vein injection, and the heart of the mouse was harvested after 6 weeks. Hematoxylin-eosin (HE) staining and Sirius red (PSR) staining were used to detect the cross-sectional area of myocardial cells and myocardial fibrosis, respectively. Immunofluorescence staining with α-smooth muscle actin (α-SMA) and collagen Ⅰ was used to detect the degree of fibrosis activation. The expression level of fibrosis-related proteins was determined by Western blot. Results: The expression level of HSP47 in the myocardium of the diabetic group up-regulated (2.014±0.264 vs 1.004±0.064, P<0.001). The area of myocardial cells in the diabetic group was increased compared with the control group [(235.3±20.7) μm(2) vs (172.8±13.6) μm(2), P<0.001] and the cross-sectional area of myocardial cells in the HSP47 overexpression-diabetes group was further increased [(302.2±41.0) μm(2) vs (235.3±20.7) μm(2), P=0.009], while the mRNA levels of mouse cardiac hypertrophic markers atrial natriuretic peptide (ANP), type B brain natriuretic peptide (BNP), myosin heavy chain β (β-MHC) further upregulated (all P<0.001). Compared with the control group, the myocardial fibrosis content in the diabetic group increased [(7.333±1.127)% vs (4.837±0.775)%, P=0.002] and the left ventricular fibrosis content of the HSP47 overexpressing diabetic group further increased [(9.175±1.008)% vs (7.333±1.127)%, P=0.025] and the mRNA levels of fibrosis index collagenⅠ, collagen Ⅲ, connective tissue growth factor (CTGF) and transforming growth factor β (TGFβ) further up-regulated (all P<0.001). Immunofluorescence results showed that compared with the control group, the expression of collagenⅠ up-regulated in the endothelial stroma of the diabetic group and the content of collagenⅠ in the HSP47 over-expressing diabetic group was higher (P<0.001). Western blot results indicated that the phosphorylation level of Smad3 and the protein levels of α-SMA and TGFβ in HSP47 overexpressing diabetic group increased, compared with those of diabetic group (all P<0.001). Conclusion: HSP47 ameliorates STZ-induced diabetic myocardial fibrosis by activating the TGFβ/Smad3 signaling pathway.

Effects of radiofrequency and ultrasound on the turnover rate of skin aging components (skin extracellular matrix and epidermis) via HSP47-induced stimulation

Skin aging cannot be escaped, being due to both intrinsic and extrinsic stimuli. They lead to a reduced extracellular collagen matrix in the dermis, along with a higher degradation by metalloproteases (MMPs) activity, as well as a lower differentiation and function of epidermis keratinocytes, characterized by wrinkling and loss of skin elasticity. One of the recent technology to overcome this skin aging process is the use of radiofrequency (RF) and ultrasound (US) technologies which use thermal stimulation to induce neocollagenesis in the skin. But no explanations exist on the involved pathways. Our hypothesis is that RF-US generated heat increases the collagen formation via the heat shock protein 47 (HSP47) induction, a heat sensitive protein related to the collagen expression. To confirm this hypothesis, normal human skin substitutes were subjected to RF-US treatment and results were monitored after 24 and 44 h. RNA sequencing showed a significant induction for the genes related to the epidermis differentiation processes. Almost all keratin genes were thus found upregulated from 2 to 15 times, while collagen type XVII and collagen type IV were increased 12 and 5 times respectively. In parallel, most of MMP genes were observed downregulated. RF-US treatment significantly increased levels of HSP47 proteins, while collagen XVII proteins showed a tendency to be increased and glycosaminoglycans were found 1.4 times significantly enhanced. Finally, histology assessment showed a higher expression of cytokeratins 10 and 14 which can testify a possible reactivation of the skin proliferative state as a rejuvenation strategy.

Heat shock protein 47 promotes tumor survival and therapy resistance by modulating AKT signaling via PHLPP1 in colorectal cancer.

Objective: Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone that facilitates collagen maturation. Its role in cancer remains largely unknown. In this study, we investigated the roles of HSP47 in colorectal cancer (CRC) and therapy resistance.Methods: Expression of HSP47 in CRC tissues was examined (1) in paired human CRC/adjacent normal tissues, using real time quantitative reverse transcription polymerase chain reaction (qRT-PCR), The Cancer Genome Atlas (TCGA) database, and 22 independent microarray databases (curated CRC). In vitro studies on several CRC cell lines (HCT116, RKO and CCL228) with modulated HSP47 expression were conducted to assess cell viability and apoptosis (TUNEL assay and caspase-3/-7) during exposure to chemotherapy. AKT signaling and co-immunoprecipitation studies were performed to examine HSP47 and PHLPP1 interaction. In vivo studies using tumor xenografts were conducted to assess the effects of HSP47 modulation on tumor growth and therapy response.Results: HSP47 was upregulated in CRC and was associated with poor prognosis in individuals with CRC. In vitro, HSP47 overexpression supported the survival of CRC cells, whereas its knockdown sensitized cells to 5-fluorouracil (5-FU). HSP47 promoted survival by inhibiting apoptosis, enhancing AKT phosphorylation, and decreasing expression of the AKT-specific phosphatase PHLPP1 when cells were exposed to chemotherapy. These effects were partly results of the interaction between HSP47 and PHLPP1, which decreased PHLPP1 stability and led to more persistent AKT activity. In vivo, HSP47 supported tumor growth despite 5-FU treatment.Conclusions: HSP47 supports the growth of CRC tumors and suppresses the efficacy of chemotherapy via modulation of AKT signaling.

TGFβ1 induces bone formation from BMP9-activated Bone Mesenchymal Stem Cells, with possible involvement of non-canonical pathways.

Reconstruction of bone defects is one of the most substantial and difficult clinical challenges in orthopedics. Transforming growth factor beta 1 (TGFβ1) might play an important role in stimulating osteogenic differentiation of bone morphogenetic protein 9 (BMP9)-induced C3H10T1/2 mesenchymal stem cells. In our current study, we examined the potential synergy between TGFβ1 and BMP9 in promoting the osteogenesis of C3H10T1/2 cells, and whether such effects could contribute to bone formation in vivo. Our experiment data indicated that TGFβ1 could increase the expression of osteogenic markers and the formation of mineralized calcium nodules in, while suppressing the proliferation of, BMP9-induced C3H10T1/2 cells. Furthermore, mice intramuscularly injected with BMP9/TGFβ1-transduced C3H10T1/2 cells into the gastrocnemius muscle on their tibiae developed ectopic bone masses with more mature osteoid structures, compared to those grafted with cells expressing BMP9/RFP. Subsequent mechanistic studies found that TGFβ1-induced enhancement of osteogenesis in BMP9-overexpressing C3H10T1/2 cells was accompanied by augmented expression of heat shock protein 47 (HSP47), a collagen-specific molecular chaperone essential for collagen biosynthesis, and can be attenuated by pirfenidone, a known anti-fibrotic inhibitor. Interestingly, protein microarray analysis suggested that TGFβ1/BMP9-dependent osteogenesis of C3H10T1/2 cells seemed to involve several non-canonical signaling pathways such as Janus kinase-signal transducer and activator of transcription, phosphoinositide-3-kinase-protein kinase B, and mitogen-activated protein kinase. These results provided further evidence that TGFβ1 could promote bone formation from BMP9-induced C3H10T1/2 cells and shed important light on the underlying molecular mechanisms.

Lowering the culture temperature corrects collagen abnormalities caused by HSP47 gene knockout

Heat shock protein 47 (HSP47) is an endoplasmic reticulum (ER)-resident molecular chaperone that specifically recognizes triple helical portions of procollagens. The chaperone function of HSP47 is indispensable in mammals, and hsp47-null mice show an embryonic lethal phenotype accompanied by severe abnormalities in collagen-based tissue structures. Two leading hypotheses are currently accepted for the molecular function of HSP47 as a procollagen-specific chaperone. One is facilitation of procollagen folding by stabilizing thermally unstable triple helical folding intermediates, and the other is inhibition of procollagen aggregation or lateral association in the ER. The aim of this study was to elucidate the functional essence of this unique chaperone using fibroblasts established from hsp47−/− mouse embryos. When the cells were cultured at 37 °C, various defects in procollagen biosynthesis were observed, such as accumulation in the ER, over-modifications including prolyl hydroxylation, lysyl hydroxylation, and further glycosylation, and unusual secretion of type I collagen homotrimer. All defects were corrected by culturing the cells at a lower temperature of 33 °C. These results indicated that lowering the culture temperature compensated for the loss of HSP47. This study elucidated that HSP47 stabilizes the elongating triple helix of procollagens, which is otherwise unstable at the body temperature of mammals.

Heat shock protein 47 (HSP47) binds to discoidin domain–containing receptor 2 (DDR2) and regulates its protein stability

Cell-collagen interactions are crucial for cell migration and invasion during cancer development and progression. Heat shock protein 47 (HSP47) is an endoplasmic reticulum-resident molecular chaperone that facilitates collagen maturation and deposition. It has been previously shown that HSP47 expression in cancer cells is crucial for cancer invasiveness. However, exogenous collagen cannot rescue cell invasion in HSP47-silenced cancer cells, suggesting that other HSP47 targets contribute to cancer cell invasion. Here, we show that HSP47 expression is required for the stability and cell-surface expression of discoidin domain-containing receptor 2 (DDR2) in breast cancer tissues. HSP47 silencing reduced DDR2 protein stability, accompanied by suppressed cell migration and invasion. Co-immunoprecipitation results revealed that HSP47 binds to the DDR2 ectodomain. Using a photoconvertible technique and total internal reflection fluorescence microscopy, we further demonstrate that HSP47 expression significantly sustains the membrane localization of the DDR2 protein. These results suggest that binding of HSP47 to DDR2 increases DDR2 stability and regulates its membrane dynamics and thereby enhances cancer cell migration and invasion. Given that DDR2 has a crucial role in the epithelial-to-mesenchymal transition and cancer progression, targeting the HSP47-DDR2 interaction might be a potential strategy for inhibiting DDR2-dependent cancer progression.

Roles of transforming growth factor - β1 and heat shock protein 47 in progression of Schistosoma japonicum-induced hepatic fibrosis

To investigate the dynamic expression of transforming growth factor-β1 (TGF-β1) and heat shock protein 47 (HSP47) and explore their roles in the progression of hepatic fibrosis induced by Schistosoma japonicum infection. Fifty female mice of the ICR strain were randomly divided into the infection group and the normal control group, of 25 mice in each group. Each mouse in the infection group was infected with 20 ± 1 cercariae of S. japonicum via the abdominal skin, while uninfected animals served as normal control. Five mice were sacrificed 4, 6, 8, 10 and 12 weeks post-infection and liver tissues were sampled. Serum HSP47 and TGF-β1 was determined using enzyme-linked immunosorbent assay (ELISA), and the pathological changes of liver specimens were observed with hematoxylin & eosin (HE) staining. In addition, the synthesis of alpha 1 chain of type I collagen (COL1A1) was measured using Masson staining, and the mRNA expression of TGF-β1, HSP47 and COL1A1 was determined using real-time fluorescent quantitative PCR (qPCR) assay. During the period of S. japonicum-induced hepatic fibrosis, the serum HSP47 and TGF-β1 levels and the mRNA expression of TGF - β1, HSP47 and COL1A1 gradually increased with the progression of hepatic fibrosis. The serum levels of HSP47 and TGF-β1 were (179.26 ± 29.87) pg/mL and (22.37 ± 5.21) ng/mL 6 weeks post-infection, respectively, which were significantly greater than those [(150.29 ± 34.91) pg/mL and (18.54 ± 7.78) ng/mL, respectively] in the normal control group (both P values < 0.05). In addition, the mRNA expression of HSP47, COL1A1 and TGF-β1 was (0.86 ± 0.04), (1.17 ± 0.06) and (0.64 ± 0.13) in mouse liver specimens, which was significantly higher than that (0.23 ± 0.03, 0.20 ± 0.02 and 0.38 ± 0.02) in the normal control group (all P values < 0.01). The expression of TGF-β1 and HSP47 during the period of S. japonicum-induced hepatic fibrosis is consistent with the progression of the hepatic fibrosis, and exhibits the same tendency with type I collagen expression. HSP47 is a novel promising diagnosis marker and therapeutic target for S. japonicum-induced hepatic fibrosis.

S100A4: a novel partner for heat shock protein 47 in antler stem cells and insight into the calcium ion-induced conformational changes

S100A4 is a multiple-function protein highly expressed in tumor or stem cells. We found S100A4 was a novel protein partner for heat shock protein 47 (HSP47) in deer antlerogenic periosteum cells (AP cells), indicating that S100A4 could bind with HSP47. S100A4 had both calcium-dependent and calcium-independent patterns (labeled as SCd and SCi, respectively) to execute different biological activities. Homology models of HSP47, SCd and SCi were constructed. HSP47:collagen model, HSP47:collagen I-V, HSP47:SCd and HSP47:SCi complexes were built using ZDOCK software. Together with free SCd and SCi, 200 ns molecular dynamic (MD) simulations were performed to analyze binding free energies and SCi/SCd conformational changes. The energetic results showed that SCi had the strongest affinity to HSP47, and followed by collagens. SCd had little interaction with HSP47. Decomposition energy results showed that collagen model interacted with HSP47 mainly though neutral amino acids. When SCi bound with HSP47, the majority of mediated amino acids were charged. These results indicated that SCi could compete with collagen on the binding site of HSP47. Root mean square fluctuation (RMSF) values and cross-correlation matrices of principal component analysis (PCA) were calculated to evaluate the SCi/SCd structural variation during MD simulation. Both HSP47 and Ca2+ could stabilize the conformation of SCi/SCd. The loops interacting with Ca2+s and linking the two EF-hand motifs were impacted particularly. The relative moving directions of α-helices in EF-hands were distinct by the binding effect of HSP47 and Ca2+. We found that SCi may regulate the differentiation of AP cells by disturbing the interaction between HSP47 and collagen. Communicated by Ramaswamy H. Sarma

Ocular instillation of vitamin A–coupled liposomes containing HSP47 siRNA ameliorates dry eye syndrome in chronic GVHD

AbstractChronic graft-versus-host disease (GVHD) profoundly affects the quality of life of long-term survivors of allogeneic hematopoietic stem cell transplantation (SCT). The eyes are frequently involved, and dry eye syndrome is the most common manifestation of ocular chronic GVHD. We explored the role of heat shock protein 47 (HSP47) in ocular GVHD and developed a novel antifibrotic topical therapy using vitamin A–coupled liposomes containing HSP47 small interfering RNA (siRNA) against HSP47 (VA-lip HSP47). In a mouse model of chronic GVHD, infiltration of HSP47+ fibroblasts and massive fibrosis surrounding the lacrimal ducts were observed after allogeneic SCT, leading to impaired tear secretion. After ocular instillation, VA-lip HSP47 was distributed to the lacrimal glands, knocked down HSP47 expression in fibroblasts, reduced collagen deposition, and restored tear secretion after allogeneic SCT. Ocular instillation of VA-lip HSP47 also ameliorated established lacrimal gland fibrosis and dry eye syndrome. VA-lip HSP47 eye drops are a promising prophylactic and therapeutic option against dry eye syndrome in chronic GVHD.

Unilateral Ureteral Obstruction as a Model to Investigate Fibrosis-Attenuating Treatments.

Renal fibrosis is the common pathway for most forms of progressive renal disease. The Unilateral Ureteral Obstruction (UUO) model is used to cause renal fibrosis, where the primary feature of UUO is tubular injury as a result of obstructed urine flow. Furthermore, experimental UUO in rodents is believed to mimic human chronic obstructive nephropathy in an accelerated manner. Renal fibrosis is the common pathway for most forms of progressive renal disease. Removing the obstruction may not be sufficient to reverse fibrosis, so an accompanying treatment may be of benefit. In this review, we have done a revision on treatments shown to ameliorate fibrosis in the context of the UUO experimental model. The treatments inhibit the production of fibrotic and inflammatory proteins such as Transforming Growth Factor β1 (TGF-β1), Tumor Necrosis Factor α (TNF-α), collagen and fibronectin, Heat Shock Protein 47 (HSP47), suppress the proliferation of fibroblasts, prevent epithelial-to-mesenchymal transition, reduce oxidative stress, inhibit the action of the Nuclear Factor κB (NF-κB), reduce the phosphorylation of mothers against decapentaplegic homolog (SMAD) family members 2 and 3 (Smad2/3) or Mitogen-Activated Protein Kinases (MAPKs), inhibit the activation of the renin-angiotensin system. Summaries of the UUO experimental methods and alterations observed in the UUO experiments are included.