Transforming growth factor-beta1 stimulates collagen matrix remodeling through increased adhesive and contractive potential by human renal fibroblasts

Renal tubulointerstitial fibrosis is the common final pathway leading to end-stage renal failure. Tubulointerstitial fibrosis is characterized by fibroblast proliferation and excessive matrix accumulation. Transforming growth factor-beta1 (TGF-beta1) has been implicated in the development of renal fibrosis accompanied by alpha-smooth muscle actin (alpha-SMA) expression in renal fibroblasts. To investigate the molecular and cellular mechanisms involved in tubulointerstitial fibrosis, we examined the effect of TGF-beta1 on collagen type I (collagen) gel contraction, an in vitro model of scar collagen remodeling. TGF-beta1 enhanced collagen gel contraction by human renal fibroblasts in a dose- and time-dependent manner. Function-blocking anti-alpha1 or anti-alpha2 integrin subunit antibodies significantly suppressed TGF-beta1-stimulated collagen gel contraction. Scanning electron microscopy showed that TGF-beta1 enhanced the formation of the collagen fibrils by cell attachment to collagen via alpha1beta1 and alpha2beta1 integrins. Flow cytometry and cell adhesion analyses revealed that the stimulation of renal fibroblasts with TGF-beta1 enhanced cell adhesion to collagen via the increased expression of alpha1 and alpha2 integrin subunits within collagen gels. Fibroblast migration to collagen was not up-regulated by TGF-beta1. Furthermore, TGF-beta1 increased the expression of a putative contractile protein, alpha-SMA, by human renal fibroblasts in collagen gels. These results suggest that TGF-beta1 stimulates fibroblast-collagen matrix remodeling by increasing both integrin-mediated cell attachment to collagen and alpha-SMA expression, thereby contributing to pathological tubulointerstitial collagen matrix reorganization in renal fibrosis.     

Transforming growth factor-beta stimulates collagen VII expression by cutaneous cells in vitro

Collagen VII, the major component of cutaneous anchoring fibrils is expressed at a low level by normal human keratinocytes and fibroblasts in vitro. In cocultures of these two cell types, signals from fibroblasts enhance expression of collagen VII by keratinocytes and vice versa. In this study, the effects of a possible mediator of such a stimulation, transforming growth factor-beta (TGF-beta), were investigated. Its effect on the expression and deposition of the highly insoluble collagen VII was assessed in a semiquantitative manner by a newly developed enzyme-linked immunoassay which is based on immunoblotting. In keratinocyte monocultures, 0.5-20 ng/ml of TGF-beta 2 induced a dose-dependent stimulation of collagen VII expression as measured per microgram of DNA. The maximal enhancement was about sevenfold compared to controls. The effect of TGF-beta 2 was observed already after 12 h, with a steady increase at least up to 3 d. As previous studies have implicated, untreated cocultures of keratinocytes and fibroblasts exhibited a higher basic level of collagen VII expression, which could be further stimulated about twofold by TGF-beta 2. Fibroblasts alone synthesized very minor quantities of collagen VII and could be only weakly stimulated by TGF-beta 2. This growth factor seems a specific enhancer of collagen VII since the expression of laminin, collagen IV, as well as total protein was increased to a much lesser extent. Our data suggest that TGF-beta may be an important mediator of epithelial-mesenchymal interactions and may regulate the synthesis of the anchoring fibrils at the skin basement membrane zone.     

Hypoxia modulates the effects of transforming growth factor-beta isoforms on matrix-formation by primary human lung fibroblasts

Chronic hypoxia is implicated in lung fibrosis, which is characterized by enhanced deposition of extracellular matrix (ECM) molecules. Transforming growth factor-beta (TGF-beta) plays a key role in fibroblast homeostasis and is involved in disease states characterized by excessive fibrosis, such as pulmonary fibrosis. In this study, we investigated if hypoxia modulates the effects of TGF-beta on the expression of gelatinases: matrix metalloproteinase (MMP)-2 and MMP-9, interstitial collagenases: MMP-1 and MMP-13, tissue inhibitors of MMP (TIMP), collagen type I and interleukin-6 (IL-6). Primary human lung fibroblasts, established from tissue biopsies, were cultivated under normoxia or hypoxia in the presence of TGF-beta1, TGF-beta2 or TGF-beta3. Gelatinases were assessed by gelatin zymography and collagenases, TIMP, collagen type I and IL-6 by ELISA. Under normoxia fibroblasts secreted MMP-2, collagenases, TIMP, collagen type I and IL-6. TGF-betas significantly decreased MMP-1 and increased TIMP-1, IL-6 and collagen type I. Hypoxia significantly enhanced MMP-2, and collagenases. Compared to normoxia, the combination of TGF-beta and hypoxia reduced MMP-1, and further amplified the level of TIMP, IL-6, and collagen type I. Thus, in human lung fibroblasts hypoxia significantly increases the TGF-betas-induced secretion of collagen type I and may be associated to the accumulation of ECM observed in lung fibrosis.     

Human type VII collagen: genetic linkage of the gene (COL7A1) on chromosome 3 to dominant dystrophic epidermolysis bullosa.

Epidermolysis bullosa (EB) is a heterogeneous group of heritable blistering disorders affecting the skin and the mucous membranes. Previous ultrastructural studies on the dystrophic (scarring) forms of EB have demonstrated abnormalities in the anchoring fibrils, morphologically distinct structures below the basal lamina at the dermal/epidermal basement membrane zone. Type VII collagen is the major collagenous component of the anchoring fibrils, and it is therefore a candidate gene for mutations in some families with dystrophic forms of EB. In this study, we performed genetic linkage analyses in a large kindred with dominant dystrophic EB. A 1.9-kb type VII collagen cDNA clone was used to identify a PvuII RFLP to follow the inheritance of the gene. This RFLP cosegregated with the EB phenotype in this family, strongly supporting genetic linkage (Z = 5.37; theta = .0). In addition, we assigned the type VII collagen gene (COL7A1) to chromosome 3 by hybridization to a panel of human x rodent somatic cell hybrids. These data demonstrate very close genetic linkage between the clinical phenotype in this family and the polymorphism in the type VII collagen gene mapped to chromosome 3. The absence of recombination between EB and the type VII collagen gene locus, as well as the observed abnormalities in the anchoring fibrils, strongly suggest that this collagen gene is the mutant locus in this kindred.     

Transforming growth factor (TGF)-beta-activated kinase 1 mimics and mediates TGF-beta-induced stimulation of type II collagen synthesis in chondrocytes independent of Col2a1 transcription and Smad3 signaling

Transforming growth factor (TGF)-beta, bone morphogenetic protein (BMP), and interleukin-1beta activate TGF-beta-activated kinase 1 (TAK1), which lies upstream of the p38 MAPK, JNK, and NF-kappaB pathways. Our knowledge remains incomplete of TAK1 target genes, requirement for cooperative signaling, and capacity for shared or segregated ligand-dependent responses. We show that adenoviral overexpression of TAK1a in articular chondrocytes stimulated type II collagen protein synthesis 3-6-fold and mimicked the response to TGF-beta1 and BMP2. Both factors activated endogenous TAK1 and its activating protein, TAB1, and the collagen response was inhibited by dominant-negative TAK1a. Isoform-specific antibodies to TGF-beta blocked the response to endogenous and exogenous TGF-beta but not the response to TAK1a. Expression of Smad3 did not stimulate type II collagen synthesis or enhance that caused by TGF-beta1 or TAK1a, in contrast to its effects on its endogenous targets, CTGF and plasminogen-activated inhibitor-1. TAK1a, overexpressed alone and immunoprecipitated, phosphorylated MKK6 and stimulated the plasminogen-activated inhibitor-1 promoter following transient transfection; both effects were enhanced by TAB1 coexpression, but type II collagen synthesis was not. Stimulation by TAK1a or TGF-beta did not require increased Col2a1 mRNA, and TAK1 actually reduced Col2a1 mRNA in parallel with the cartilage markers, SRY-type HMG box 9 (Sox9) and aggrecan. Thus, TAK1 increased target gene expression (Col2a1) by translational or posttranslational mechanisms as a Smad3-independent response shared by TGF-beta1 and BMP2.     

Smad3 mediates the TGF-beta-induced contraction of type I collagen gels by mouse embryo fibroblasts

TGF-beta signals through TGF-beta receptors and Smad proteins. TGF-beta also augments fibroblast-mediated collagen gel contraction, an in vitro model of connective tissue remodeling. To investigate the importance of Smad2 or Smad3 in this augmentation process, embryo-derived fibroblasts from mice lacking expression of Smad2 or Smad3 genes were cast into native type I collagen gels. Fibroblast-populated gels were then released into 0.2% FCS-DMEM alone or with recombinant human TGF-beta1, beta2, beta3, or recombinant rat PDGF-BB. Gel contraction was determined using an image analyzer. All three isoforms of TGF-beta significantly augmented contraction of collagen gels mediated by fibroblasts with genotypes of Smad2 knockout (S2KO), Smad2 wildtype (S2WT), and Smad3 wildtype (S3WT), but not Smad3 knockout (S3KO) mice. PDGF-BB augmented collagen gel contraction by all fibroblast types. These results suggest that expression of Smad3 but not Smad2 may be critical in TGF-beta augmentation of fibroblast-mediated collagen gel contraction. Thus, the Smad3 gene could be a target for blocking contraction of fibrotic tissue induced by TGF-beta.     

Development and characterization of a recombinant truncated type VII collagen "minigene". Implication for gene therapy of dystrophic epidermolysis bullosa

Dystrophic epidermolysis bullosa (DEB) is an inherited mechano-bullous disorder of skin caused by mutations in the type VII collagen gene. The lack of therapy for DEB provides an impetus to develop gene therapy strategies. However, the full-length 9-kilobase type VII collagen cDNA exceeds the cloning capacity of current viral delivery vectors. In this study, we produced a recombinant type VII minicollagen containing the intact noncollagenous domains, NC1 and NC2, and part of the central collagenous domain using stably transfected human 293 cell clones and purified large quantities of the recombinant minicollagen VII from culture media. Minicollagen VII was secreted as correctly-folded, disulfide-bonded, helical trimers resistant to protease degradation. Purified minicollagen VII bound to fibronectin, laminin-5, type I collagen, and type IV collagen. Furthermore, retroviral-mediated transduction of the minigene construct into DEB keratinocytes (in which type VII collagen was absent) resulted in persistent synthesis and secretion of a 230-kDa recombinant minicollagen VII. In comparison with parent DEB keratinocytes, the gene-corrected DEB keratinocytes demonstrated enhanced cell-substratum adhesion, increased proliferative potential, and reduced cell motility, features that reversed the DEB phenotype toward normal. We conclude that the use of the minicollagen VII may provide a strategy to correct the cellular manifestations of gene defects in DEB.     

Mice lacking serum amyloid P component do not necessarily develop severe autoimmune disease

Interleukin-10 (IL-10) is a cytokine with many regulatory functions. In particular, IL-10 exerts neutralizing effect on other cytokines, and therefore IL-10 is thought to have important therapeutic implications. Recent reports suggest that IL-10 regulates not only immunocytes but also collagen and collagenase gene expression in fibroblasts. In this study, we investigated the effect of IL-10 on gene expression of extracellular matrix (ECM) proteins, such as type I collagen, fibronectin, and decorin, in human skin fibroblasts. Results of Northern blot analysis showed that both collagen I and fibronectin mRNAs were downregulated, while decorin gene expression was enhanced by IL-10 (10 ng/ml) time-dependently (6-24 h). alpha1(I) collagen and fibronectin mRNAs were decreased to one-third and one-fourth, respectively, by 50 ng/ml IL-10, whereas decorin mRNA was increased up to 2.7-fold by 50 ng/ml IL-10. Response to IL-10 by scleroderma fibroblasts was similar to that in normal dermal fibroblasts, with decreased expression levels of collagen and fibronectin and induced decorin mRNA levels. Transforming growth factor-beta (TGF-beta) is a crucial fibrogenic cytokine which upregulates the mRNA expression of collagen and fibronectin, whereas it downregulates decorin mRNA expression in fibroblasts. Monocyte chemoattractant protein-1 (MCP-1) has recently been shown to upregulate the type I collagen mRNA expression in cultured fibroblasts. We therefore examined whether IL-10 alters gene expression of ECM elicited by TGF-beta and MCP-1. Our results demonstrated that IL-10 downregulated the TGF-beta-elicited increase of mRNA expression of type I collagen and fibronectin, while partially recovering TGF-beta-elicited decrease of decorin expression in normal skin fibroblasts. By contrast, IL-10 did not alter the MCP-1-elicited upregulation of mRNA expression of either alpha1(I) collagen and decorin. Our data indicate that IL-10 differentially regulates TGF-beta and MCP-1 in the modulation of ECM proteins and therefore suggest that IL-10 plays a role in the regulation of tissue remodeling.     

Demonstration of the interaction of transforming growth factor beta 2 and type X collagen using a modified tandem affinity purification tag

Like other members of the transforming growth factor beta (TGF-beta) family of growth factors, the biological activity of TGF-beta2 is believed to be regulated by the formation and dissociation of multiprotein complexes. To isolate the molecular complex formed by TGF-beta2 secreted by hypertrophic chondrocytes we have used expression of TGF-beta2 fused with the humanized, tandem affinity purification (hTAP) tag and mass spectrometry for the identification of interacting proteins. The hTAP synthetic gene was assembled by systematically replacing the rare codons of the original TAP tag with codons most preferred in highly expressed human genes to circumvent the poor translation efficiency of the original TAP tag in animal cells. TGF-beta2 was shown to interact with Type X collagen and this interaction confirmed using V5 tagged TGF-beta2. Functional interaction was suggested by the inhibition of TGF-beta2 activity by type X collagen in culture and the influence of a mutation in type X collagen on the distribution of TGF-beta2 in growth cartilage.     

Antagonistic effects of TNF-alpha on TGF-beta signaling through down-regulation of TGF-beta receptor type II in human dermal fibroblasts

Transforming growth factor-beta stimulates the production of the extracellular matrix, whereas TNF-alpha has antifibrotic activity. Understanding the molecular mechanism underlying the antagonistic activities of TNF-alpha against TGF-beta is critical in the context of tissue repair and maintenance of tissue homeostasis. In the present study, we demonstrated a novel mechanism by which TNF-alpha blocks TGF-beta-induced gene and signaling pathways in human dermal fibroblasts. We showed that TNF-alpha prevents TGF-beta-induced gene trans activation, such as alpha2(I) collagen or tissue inhibitor of metalloproteinases 1, and TGF-beta signaling pathways, such as Smad3, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinases, without inducing levels of inhibitory Smad7 in human dermal fibroblasts. TNF-alpha down-regulates the expression of type II TGF-beta receptor (TbetaRII) proteins, but not type I TGF-beta receptor (TbetaRI), in human dermal fibroblasts. However, neither TbetaRII mRNA nor TbetaRII promoter activity was decreased by TNF-alpha. TNF-alpha-mediated decrease of TbetaRII protein expression was not inhibited by the treatment of fibroblasts with either a selective inhibitor of I-kappaB-alpha phosphorylation, BAY 11-7082, or a mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor, PD98059. Calpain inhibitor I (ALLN), a protease inhibitor, inhibits TNF-alpha-mediated down-regulation of TbetaRII. We found that TNF-alpha triggered down-regulation of TbetaRII, leading to desensitization of human dermal fibroblasts toward TGF-beta. Furthermore, these events seemed to cause a dramatic down-regulation of alpha2(I) collagen and tissue inhibitor of metalloproteinases 1 in systemic sclerosis fibroblasts. These results indicated that TNF-alpha impaired the response of the cells to TGF-beta by regulating the turnover of TbetaRII.     

Proliferation and collagen production of human patellar tendon fibroblasts in response to cyclic uniaxial stretching in serum-free conditions

We studied the effect of cyclic mechanical stretching on the proliferation and collagen mRNA expression and protein production of human patellar tendon fibroblasts under serum-free conditions. The role of transforming growth factor-beta1 (TGF-beta1) in collagen production by cyclically stretched tendon fibroblasts was also investigated. The tendon fibroblasts were grown in microgrooved silicone dishes, where the cells were highly elongated and aligned with the microgrooves. Cyclic uniaxial stretching with constant frequency and duration (0.5 Hz, 4 h) but varying magnitude of stretch (no stretch, 4%, and 8%) was applied to the silicone dishes. Following the period of stretching, the cells were rested for 20 h in stretching-conditioned medium to allow for cell proliferation. In separate experiments, the cells were stretched for 4h and then rested for another 4 h. Samples of the medium, total cellular RNA and protein were used for analysis of collagen and TGF-beta1 gene expression and production. It was found that there was a slight increase in fibroblast proliferation at 4% and 8% stretch, compared to that of non-stretched fibroblasts, where at 8% stretch the increase was significant. It was also found that the gene expression and protein production of collagen type I and TGF-beta1 increased in a stretching-magnitude-dependent manner. And, levels of collagen type III were not changed, despite gene expression levels of the protein being slightly increased. Furthermore, the exogenous addition of anti-TGF-beta1 antibody eliminated the increase in collagen type I production under cyclic uniaxial stretching conditions. The results suggest that mechanical stretching can modulate proliferation of human tendon fibroblasts in the absence of serum and increase the cellular production of collagen type I, which is at least in part mediated by TGF-beta1.     

P-glycoprotein expression in extracellular matrix formation of chondrogenic differentiation of human adult stem cells

Mesenchymal stem cell (MSC) has been known as a good source of progenitor for multiple connective tissue including cartilage, muscle, adipocyte, and bone. P-glycoproteins (P-gps) also known as ABCB1 that exports diverse substrates are the product of the multidrug resistance-1 (MDR-1) gene. P-gp expression has been reported in chondrosarcoma and hypertrophic chondrocyte in the human growth plate. This study was designed to investigate the expression of P-gp during chondrogenic differentiation of adult human stem cells. Bone marrow samples were obtained from nine human donors after informed consent. The isolated mononuclear cells (MNCs) were incubated as one pellet/tube and 0.5ml chondrogenic medium in the presence of 10ng/ml of TGF-beta 1 and TGF-beta 3 for 28 days. The expression of surface P-gps was analyzed by flow cytometry and quantitative RT-PCR was performed for the detection of mRNA expression of MDR-1 and type II collagen gene. Total collagen and glycosaminoglycan (GAG) contents of the pellets were measured. Surface P-gp expression of the MSCs was decreased during chondrogenic differentiation. MDR-1 gene was decreased 10-fold after the 2-week incubation whereas type II collagen gene was increased 491-fold after the 4-week incubation in chondrogenic medium. The total amount of collagen and GAG were increased during pellet culture. This study has demonstrated a decrease in expression of P-gp and down regulation of MDR-1 gene consistently by flow cytometry and quantitative RT-PCR, but an increased expression of type II collagen on MSC during chondrogenesis.     

Gene expression of type I and type III collagen by mechanical stretch in anterior cruciate ligament cells

Mechanical stretch affects the healing and remodeling process of the anterior cruciate ligament (ACL) after surgery in important ways. In this study, the effects of mechanical stress on gene expression of type I and III collagen by cultured human ACL cells and roles of transforming growth factor (TGF)-beta1 in the regulation of mechanical strain-induced gene expression were investigated. Uniaxial cyclic stretch was applied on ACL cells at 10 cycles/min with 10% length stretch for 24 h. mRNA expression of the type I and type III collagen was increased by the cyclic stretch. TGF-beta1 protein in the cell culture supernatant was also increased by the stretch. In the presence of anti-TGF-beta1 antibody, stretch-induced increase in type I and type III mRNA expression was markedly ablated. The results suggest that the stretch-induced mRNA expression of the type I and type III collagen is mediated via an autocrine mechanism of TGF-beta1 released from ligament cells.