Expression profiles of collagens,HSP47, TGF-beta1, MMPs and TIMPs in epidermolysis bullosa acquisita

Epidermolysis bullosa acquisita (EBA) is a chronic, uncommon, sub-epidermal blistering disease involving the skin and mucous membranes that heals with scar formation and milia. Collagens, matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are important components that play an essential role(s) in matrix remodeling during scar formation. However, the possible involvement of these components in EBA-induced scarring is not yet known. In the present study, we examined the expression profile of collagens, collagen-binding heat shock protein 47 (HSP47), MMPs and their inhibitory enzymes, TIMPs, in matrix remodeling during conjunctival scarring. The involvement of TGF-beta1, a fibrogenic factor, was also studied. Compared to the controls, an increased expression of type I collagen, type III collagen and HSP47 was detected in conjunctival biopsy sections of patient with EBA using immunohistochemistry. Similar increase in the expression of type I collagen, type III collagen and HSP47 was noted in conjunctival fibroblasts obtained from the patient with EBA. Up-regulation in the expression of MMP-1 and MMP-14 was also noted in conjunctival fibroblasts isolated from the patient with EBA, while no significant changes in the expression of MMP-3, MMP-8, MMP-9 and MMP-13 were seen. As for TIMPs, conjunctival fibroblasts isolated from the patient with EBA, grown in vitro, exhibited increased expression of TIMP-1, TIMP-2 and TIMP-3, when compared with fibroblasts grown from control conjunctival tissues, although the expression level varies with different molecules of the same family. Additionally, compared to the control conjunctival fibroblasts, an increased expression of TGF-beta1 was detected in fibroblasts isolated from the conjunctival tissues of patient with EBA.This study suggests that there is up-regulation in the production of collagens (type I and III), collagen-binding protein (HSP47), matrix degrading collagenases (MMP-1 and 14), and their inhibitory enzymes (TIMP-1, 2 and 3) during the process of conjunctival matrix remodeling in the patient with EBA. The presented data is preliminary and could serve as a basis for further studies to enhance our understanding about the molecular mechanisms of conjunctival scarring in patients with EBA.     

Upregulation of HSP47 and collagen type III in the dermal fibrotic disease, keloid

Keloid is a dermal fibrotic disease characterized by excessive accumulation of mainly type I collagen in extracellular matrix of the dermis. We have studied the expression levels of collagen types I and III, and its molecular chaperone HSP47 in keloid lesions and surrounding unaffected skin using Northern and Western blotting and immunohistochemical analyses. Collagen types I and III mRNA levels were found to be upregulated 20-fold in keloid tissues, contradicting previous reports of nearly normal type III collagen levels in this disease. HSP47 expression in keloid lesions was also highly upregulated; eightfold at mRNA level and more than 16-fold at the protein level. Strong upregulation of these three proteins in keloid was confirmed by immunohistochemical staining. These results suggest that accumulation of both type I and type III collagen is important for the development of keloid lesions, and that HSP47 plays a role in the rapid and extensive synthesis of collagen in keloid tissues.     

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.     

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.     

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.     

Nitric oxide in the pathogenesis of diffuse pulmonary fibrosis

By studying the responses of nitric oxide in pulmonary fibrosis, the role of inducible nitric oxide synthase in diffuse pulmonary fibrosis as caused by lipopolysaccharide (LPS) treatment was investigated. When compared to rats treated with LPS only, the rats pretreated with 1400W (an iNOS-specific inhibitor) were found to exhibit a reduced level in: (i) NOx (nitrate/nitrite) production, (ii) collagen type I protein expression, (iv) soluble collagen production, and (iv) the loss of body weight and carotid artery PO2. In the pulmonary fibroblast culture, exogenous NO from LPS-stimulated secretion by macrophages or from a NO donor, such as DETA NONOate, was observed to induce the expression of TIMP-1, HSP47, TGF-beta1, and collagen type I as well as the phosphorylation of SMAD-2. After inhalation of NO for 24 h, an up-regulation of collagen type I protein was also noted to occur in rat pulmonary tissue. The results suggest that the NO signal pathway enhanced the expression of TGF-beta1, TIMP-1, and HSP47 in pulmonary fibroblasts, which collectively demonstrate that the NO signal pathway could activate the SMAD-signal cascade, by initiating a rapid increase in TGF-beta1, thereby increasing the expression of TIMP-1 and HSP47 in pulmonary fibroblasts, and play an important role in pulmonary fibrosis.     

The renal expression of heat shock protein 47 and collagens in acute and chronic experimental diabetes in rats

Glomerulosclerosis and tubulointerstitial fibrosis are the main structural changes found in the later stages of diabetic nephropathy, which is clinically characterized by proteinuria, and progressive renal insufficiency. Heat shock protein (HSP) 47, a collagen-binding stress protein, has a specific role in the intracellular processing of procollagen molecules during collagen synthesis. It is implicated in the pathogenesis of various fibrotic diseases. However, the expression and significance of HSP47 in acute and chronic phases of diabetic nephropathy is not yet known. In this study, we studied the expression of HSP47 in the kidneys obtained from streptozotocin-induced diabetic rats, in both short- and long-term diabetes. To determine the renal expression of HSP47, and collagens (type III and IV) in acute (days 1, 3 and 14) and chronic (weeks 4, 12 and 24) diabetes, we have performed a time-course study using streptozotocin-induced diabetic rats. The expression pattern of alpha-smooth muscle actin (to identify mesangial cell damage), vimentin (to identify tubular epithelial cell damage), and desmin (to identify glomerular epithelial cell damage) was also determined in kidneys of these diabetic rats. Antibodies specific for HSP47, type III and type IV collagens, alpha-smooth muscle actin, vimentin, and desmin were used to assess the relative expression of their proteins in paraffin-embedded kidney sections by immunohistochemistry. Compared to control rat kidneys, no significant changes in the expression of HSP47 was found in the kidneys of acute diabetic rats. However a significant increase in the expression of HSP47 was noted in the kidneys of chronic diabetic rats; increased expression of HSP47 correlated with an increased renal deposition of types III and IV collagens. Similarly, compared to kidneys of control and acute diabetic rats, an increased expression of alpha-smooth muscle actin (in mesangial cells), vimentin (in tubular epithelial cells), and desmin (in glomerular epithelial cells) was detected in the kidneys of chronic diabetic rats; by dual immunostaining, these phenotypically-altered renal cells in kidneys of chronic diabetic rats were found to be HSP47-producing cells. Importantly, HSP47 up-regulation coincided with the initiation and progression of renal fibrosis, as determined by the expression and deposition of collagens. Our results strongly support a pathological role for HSP47 in the later stages (sclerotic phase) of streptozotocin-induced diabetic nephropathy, which is associated with glomerulosclerosis and tubulointerstitial fibrosis.     

Localization of the expression of types I, III, and IV collagen, TGF-beta 1 and c-fos genes in developing human calvarial bones

Total RNA extracted from developing calvarial bones of 15- to 18-week human fetuses was studied by Northern hybridization: in addition to high levels of type I collagen mRNAs, the presence of mRNAs for type III and type IV collagen, TGF-beta and c-fos was observed. In situ hybridization of sections containing calvarial bone, overlying connective tissues, and skin was employed to identify the cells containing these mRNAs. Considerable variation was observed in the distribution of pro alpha 1(I) collagen mRNA in osteoblasts: the amount of the mRNA in cells at or near the upper surface of calvarial bone was distinctly greater than that in cells at the lower surface, indicating the direction of bone growth. High levels of type I collagen mRNAs were also detected in fibroblasts of periosteum, dura mater, and skin. Type III collagen mRNA revealed a considerably different distribution: the highest levels were detected in upper dermis, lower levels were seen in fibroblasts of the periosteum and the fibrous mesenchyme between bone spiculas, and none was seen in osteoblasts. Type IV collagen mRNAs were only observed in the endothelial cells of blood capillaries. Immunohistochemical localization of type III and IV collagens agreed well with these observations. The distribution of TGF-beta mRNA resembled that of type I collagen mRNA. In addition, high levels of TGF-beta mRNA were observed in osteoclasts of the calvarial bone. These cells, responsible for bone resorption, were also found to contain high levels of c-fos mRNA. Production of TGF-beta by osteoclasts and its activation by the acidic environment could form a link between bone resorption and new matrix formation.     

Life-long Dietary Restriction Modulates the Expression of Collagens and Collagen-binding Heat Shock Protein 47 in Aged Fischer 344 Rat Kidney

It has been shown that the expression of HSP47 and collagens is substantially increased in the sclerotic/fibrotic process in various organs, including kidney. However, the factors regulating the increased expression of HSP47 are not yet clear. In this study, we examined the effect of dietary restriction for the expression of collagens and collagen-binding HSP47 in the kidneys of 6- and 24-month-old male Fischer 344 (F 344) rats fed ad libitum or 30% diet-restricted. No significant histological alteration was found in the kidneys of 6-month-old fed or diet-restricted rats. Kidneys obtained from 24-month-old freely fed rats showed glomerulosclerosis with marked tubulointerstitial damage including interstitial fibrosis, while in the kidneys of 24-month-old diet-restricted rats, renal damage was remarkably less than those noted in 24-month-old freely fed rat kidneys. Immunohistochemical analysis showed an increased accumulation of type I, type III and type IV collagens in areas of glomerulosclerosis and interstitial fibrosis in old rat kidneys. Dietary restriction significantly reduces renal accumulation of collagens in old age. Aging enhanced expression of HSP47 in 24-month-old freely fed rat kidneys whereas dietary restriction suppressed its expression in 24-month-old diet-restricted rat kidneys. Also, phenotypic alterations of mesangial cells and interstitial cells (immunopositive for -smooth muscle actin), glomerular epithelial cells (immunopositive for desmin) and tubular epithelial cells (immunopositive for vimentin) were seen in 24-month-old freely fed rat kidneys and found to express HSP47. Dietary restriction significantly diminished phenotypically altered renal cells in 24-month-old rat kidneys. Our results suggest that increased expression of HSP47 is associated with age-related renal damage and that diet-restricted alteration of its expression is associated with the modulation of age-associated renal sclerosis/fibrosis.     

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.     

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.     

Age-related nephropathy in the Fischer 344 rat is associated with overexpression of collagens and collagen-binding heat shock protein 47

To explore the possible role of heat shock protein (HSP) 47 in the age-related renal changes in Fischer 344 (F 344) rats, the expression of collagen-binding HSP47 with various proteins implicated in phenotypic modulation (α-smooth muscle actin, desmin, and vimentin) and fibrosis (type I, type III, and type IV collagens) was examined in young and old F 344 rat kidneys. Male F 344 rats often develop spontaneous nephropathy in old age. Kidneys obtained from 24-month-old F 344 rats showed glomerulosclerosis with marked tubulointerstitial damage including interstitial fibrosis, while no significant histological alteration was found in the kidneys of 6-month-old rats. Immunohistochemical analysis showed an increased accumulation of type I, type III, and type IV collagens in areas of glomerulosclerosis and interstitial fibrosis in old rat kidneys. In kidneys of young rats, collagen-binding HSP47 expression was weak in the glomeruli and occasionally seen in the interstitial cells. In contrast, strong immunostaining for HSP47 was noted in the glomeruli, tubular epithelial cells, and interstitial cells in kidneys of old rats. In addition, phenotypic alterations of mesangial cells and interstitial cells (immunopositive for α-smooth muscle actin), glomerular epithelial cells (immunopositive for desmin), and tubular epithelial cells (immunopositive for vimentin) were found in the kidneys of old F 344 rats. Double immunostaining showed that all these phenotypically altered renal cells express HSP47 and that increased expression of HSP47 was always associated with increased expression of collagens in the old rat kidneys. From the above observations, it is concluded that overexpression of HSP47 by phenotypically altered renal cells might play an important role in the excessive assembly of collagens and could thereby contribute to the glomerulosclerosis and interstitial fibrosis found in kidneys of aged F 344 rats.     

Immunohistochemical and biochemical studies on the collagenous proteins of human osteosarcomas

The distribution of type I, II, III, IV, V and VI collagens in 20 cases of osteosarcoma was demonstrated immunohistochemically using monospecific antibodies to different collagen types. In addition, biochemical analysis was made on collagenous proteins synthesized by tumor cells in short-term cultures obtained from seven representative cases and compared with dermal fibroblasts. In osteoblastic areas, most of the tumor osteoid consisted exclusively of type I collagen. Type V collagen was associated in some of them. Type III and type VI collagens were mainly localized in the perivascular fibrous stroma. Cultured tumor cells from osteoblastic osteosarcomas produced type I collagen exclusively and small amount of type V collagen constantly, while the synthetic activity of type III collagen was extremely low. In contrast, fibroblastic areas were characterized by the codistribution of type I, III, VI collagens and chondroblastic areas by type I, V, VI collagens as well as type II. Furthermore, type IV collagen was demonstrated in the stroma, other than the basement membrane region of blood vessels, in fibroblastic, intramedullary well-differentiated and telangiectatic osteosarcomas. In vitro, the production of variable amounts of type IV collagen, which was not detected in cultured dermal fibroblasts, was also recognized in the osteoblastic, fibroblastic, undifferentiated and intramedullary well-differentiated osteosarcomas examined. These findings suggest that the immunohistochemical approach using monospecific antibodies to different collagen types is useful not only in identifying some specific organoid components, such as tumor osteoid, but also in disclosing the biological properties of osteosarcoma cells with diverse differentiation.     

Cysteine-rich protein 61 (CCN1) mediates replicative senescence-associated aberrant collagen homeostasis in human skin fibroblasts

Dermal fibroblasts produce a collagen-rich extracellular matrix, which confers mechanical strength and resiliency to human skin. During aging, collagen production is reduced and collagen fragmentation is increased, which is initiated by matrix metalloproteinase-1 (MMP-1). This aberrant collagen homeostasis results in net collagen deficiency, which impairs the structural integrity and function of skin. Cysteine-rich protein 61 (CCN1), a member of the CCN family, negatively regulates collagen homeostasis, in primary human skin dermal fibroblasts. As replicative senescence is a form of cellular aging, we have utilized replicative senescent dermal fibroblasts to further investigate the connection between elevated CCN1 and aberrant collagen homeostasis. CCN1 mRNA and protein levels were significantly elevated in replicative senescent dermal fibroblasts. Replicative senescent dermal fibroblasts also expressed significantly reduced levels of type I procollagen and increased levels of MMP-1. Knockdown of elevated CCN1 in senescent dermal fibroblasts partially normalized both type I procollagen and MMP-1 expression. These data further support a key role of CCN1 in regulation of collagen homeostasis. Elevated expression of CCN1 substantially increased collagen lattice contraction and fragmentation caused by replicative senescent dermal fibroblasts. Atomic force microscopy (AFM) further revealed collagen fibril fragmentation and disorganization were largely prevented by knockdown of CCN1 in replicative senescent dermal fibroblasts, suggesting CCN1 mediates MMP-1-induced alterations of collagen fibrils by replicative senescent dermal fibroblasts. Given the ability of CCN1 to regulate both production and degradation of type I collagen, it is likely that elevated-CCN1 functions as an important mediator of collagen loss, which is observed in aged human skin.     

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.