Endoglin modulation of TGF-beta1-induced collagen synthesis is dependent on ERK1/2 MAPK activation.

BACKGROUND/AIMS: Transforming growth factor-beta1 (TGF-beta1) plays a pivotal role in the extracellular matrix accumulation observed in fibrotic diseases. Endoglin is an important component of the TGF-beta receptor complex highly expressed in tissues undergoing fibrotic processes. Endoglin expression regulates the effect of TGF-beta on extracellular matrix synthesis. The purpose of our study has been to understand the molecular mechanism by which endoglin exerts its effects on fibrosis and the possible role of MAP kinases in these effects. METHODS: We have assessed in mock and in endoglin-transfected L6E9 myoblasts the effect of TGF-beta1 on collagen mRNA by Northern blot and effect of TGF-beta1 on collagen content in the cultured medium by [(3)H]-Proline incorporation into collagen proteins. Total and activated MAPK and their role on collagen synthesis were assessed by Western blot. RESULTS: TGF-beta1 induced an increase on alpha(2) (I) collagen mRNA expression and collagen accumulation in mock-transfected myoblasts, whereas the response was much lower in endoglintransfected cells. TGF-beta1 activated the ERK1/2 and p38 MAPK pathways but not the JNK pathway in L6E9 myoblasts. TGF-beta1-induced alpha(2) (I) collagen mRNA expression and collagen accumulation were completely inhibited by SB203580, in either mock or endoglintransfected myoblasts. PD98059 increased TGF-beta1 induced-collagen synthesis and accumulation in endoglin-transfected myoblasts but not in mock cells. CONCLUSION: Our studies demonstrate that TGF-beta1- induced collagen synthesis is mediated by p38 MAPK activation in L6E9 myoblasts. Furthermore, endoglin expression reduces basal and TGF-beta1 induced collagen synthesis when ERK1/2 pathway is operating.     

Temporal changes in renal endoglin and TGF-β1 expression following ureteral obstruction in rats

Chronic renal disease is characterized by the accumulation of extracellular matrix proteins in the kidney and a loss of renal function. Tubulointerstitial fibrosis has been reported to play an important role in the progression of chronic renal diseases. Transforming growth factor-beta1 (TGF-beta1) is a profibrotic cytokine playing a major contribution to fibrotic kidney disease. Endoglin is a membrane glycoprotein of the TGF-beta1 receptor system. The aim of this work was to determine the time-course expression of renal type I and IV collagens, endoglin and TGF-beta1 in a rat model of induced tubulointerstitial fibrosis at 1, 3, 10 and 17 days after unilateral ureteral obstruction (UUO). In 17 days-ligated (L)-renal samples, a marked interstitial fibrosis was detected by Masson's trichromic and Sirius red staining, accompanied by an increase in type I collagen expression as shown by immunohistochemical analysis. Northern blot studies revealed a progressive increase in collagen alpha2(I), TGF-beta1 and endoglin mRNA expression in L kidneys when compared with the corresponding non-ligated (NL) kidneys from the animals subjected to left UUO. Seventeen days after UUO, significant increases in collagen alpha2(I), collagen alpha1(IV), TGF-beta1 and endoglin mRNA levels were detected in L kidneys vs NL kidneys. Significantly higher levels of the protein endoglin were found in L kidneys than in NL kidneys 10 and 17 days following obstruction. A marked increase expression for endoglin and TGF-beta1 was localized in renal interstitium by immunohistochemical studies 17 days after obstruction. In conclusion, this work reports the upregulation of endoglin coincident to that of its ligand TGF-beta1 in the kidneys of rats with progressive tubulointerstitial fibrosis induced by UUO.     

Skeletal muscle cells express the profibrotic cytokine connective tissue growth factor (CTGF/CCN2), which induces their dedifferentiation

Fibrotic disorders are typified by excessive connective tissue and extracellular matrix (ECM) deposition that precludes normal healing processes of different tissues. Connective tissue growth factor (CTGF) seems to be involved in the fibrotic response. Several muscular dystrophies are characterized by a progressive weakness and wasting of the musculature, and by extensive fibrosis. However, the exact role of CTGF in skeletal muscle is unknown. Here we show that myoblasts and myotubes are able to synthesize CTGF in response to transforming growth factor type-beta (TGF-beta) and lysophosphatidic acid (LPA). CTGF induced several ECM constituents such as fibronectin, collagen type I and alpha4, 5, 6, and beta1 integrin subunits in myoblasts and myotubes. CTGF had an important inhibitory effect on muscle differentiation evaluated by the decrease in the nuclear translocation of the early muscle regulatory factor myogenin and myosin. Remarkable, CTGF treatment of myoblasts induced their dedifferentiation, characterized by down regulating MyoD and desmin, two markers of committed myoblasts, together with a strong reorganization of cytoskeletal filaments. These results provide novel evidence for the underlying mechanisms and participation of skeletal muscle cells in the synthesis and role of CTGF inducing fibrosis, inhibiting myogenesis and dedifferentiating myoblasts.     

Connective tissue growth factor (CTGF/CCN2) is a downstream mediator for TGF-beta1-induced extracellular matrix production in osteoblasts

Connective tissue growth factor (CTGF/CCN2) is a cysteine-rich, extracellular matrix (ECM) protein that acts as an anabolic growth factor to regulate osteoblast differentiation and function. Recent studies have identified CTGF as a downstream effector of transforming growth factor-beta1 (TGF-beta1) for certain functions in specific cell types. In this study, we examined the role of CTGF as a downstream mediator of TGF-beta1-induced ECM production and cell growth in osteoblasts. Using primary cultures, we demonstrated that TGF-beta1 is a potent inducer of CTGF expression in osteoblasts, and that this induction occurred at all stages of osteoblast differentiation from the proliferative through mineralization stages. TGF-beta1 treatment of osteoblasts increased the expression and synthesis of the ECM components, collagen and fibronectin. When CTGF-specific siRNA was used to prevent TGF-beta1 induction of CTGF expression, it also inhibited collagen and fibronectin production, thereby demonstrating the requirement of CTGF for their up-regulation. To examine the effects of TGF-beta1 on osteoblast cell growth, cultures were treated with TGF-beta1 during the proliferative stage. Cell number was significantly reduced and the cells exhibited a decrease in G1 cyclin expression, consistent with TGF-beta1-induced cell-cycle arrest. Cultures transfected with CTGF siRNA prior to TGF-beta1 treatment showed an even greater reduction in cell number, suggesting that TGF-beta1-induced growth arrest is independent of CTGF in osteoblasts. Collectively, these data demonstrate for the first time that CTGF is an essential downstream mediator for TGF-beta1-induced ECM production in osteoblasts, but these two growth factors function independently regarding their opposing effects on osteoblast proliferation.     

Transforming growth factor-beta1 induces collagen synthesis and accumulation via p38 mitogen-activated protein kinase (MAPK) pathway in cultured L(6)E(9) myoblasts

Transforming growth factor-beta (TGF-beta) plays a pivotal role in the extracellular matrix accumulation observed in chronic progressive tissue fibrosis, but the intracellular signaling mechanism by which TGF-beta stimulates this process remains poorly understood. We examined whether mitogen-activated protein kinase (MAPK) routes were involved in TGF-beta1-induced collagen expression in L(6)E(9) myoblasts. TGF-beta1 induced p38 and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation whereas no effect on Jun N-terminal kinase phosphorylation was observed. Biochemical blockade of p38 but not of the ERK MAPK pathway abolished TGF-beta1-induced alpha(2)(I) collagen mRNA expression and accumulation. These data indicate that TGF-beta1-induced p38 activation is involved in TGF-beta1-stimulated collagen synthesis.     

Endoglin overexpression modulates cellular morphology, migration, and adhesion of mouse fibroblasts.

Endoglin is the gene mutated in hereditary hemorrhagic telangiectasia type 1 (HHT1), a dominantly inherited vascular disorder. Endoglin glycoprotein is a component of the transforming growth factor type beta (TGF-beta) receptor system which is highly expressed by endothelial cells, and at lower levels on fibroblasts and smooth muscle cells, suggesting the involvement of these lineages in the HHT1 vascular dysplasia. Overexpression of endoglin in mouse NCTC929 fibroblasts led to decreased migration in chemotactic and wound healing assays, as well as changes in the cellular morphology. When plated on uncoated surfaces, endoglin transfectants formed intercellular clusters, endoglin being not specifically localized to the cell-cell junctions, but homogenously distributed on the cellular surface. Although the expression of alpha5beta1 integrin and of an activation epitope of beta1 integrin were unchanged, a polyclonal antibody to alpha5beta1 integrin was able to inhibit cluster formation, suggesting the involvement of integrin ligand/s. In fact, coating with fibronectin, laminin, or an RGD-containing 80 kDa fragment of fibronectin were able to prevent the cellular clustering. Furthermore, synthesis of plasminogen activator inhibitor 1 (PAI-1), and to a weak extent that of fibronectin, were inhibited in endoglin transfectants. Thus, the presence of endoglin in mouse NCTC929 fibroblasts is associated with reduced production of certain extracellular matrix (ECM) components, which might explain their altered morphology, migration and intercellular cluster formation.     

Cell adhesion to collagen and decreased myogenic gene expression implicated in the control of myogenesis by transforming growth factor beta

Transforming growth factor beta 1 (TGF-beta 1) is an inhibitor of skeletal muscle myoblast differentiation. Myoblast differentiation is dependent on the expression of certain myogenic differentiation genes and is affected by cell interaction with the extracellular matrix. We have searched for events in the differentiation process of L6E9 rat myoblasts that may be involved in the inhibitory action of TGF-beta 1. Elevated expression of the myogenic differentiation gene, myogenin, which is thought to play a central role in the differentiation process, occurs 10 h after the shift of L6E9 myoblasts to differentiation medium. Elevation of myogenin mRNA is blocked by TGF-beta 1 added at the time of the shift. This effect is preceded by, and might be related to, a rapid up-regulation of junB mRNA observed in TGF-beta 1-treated L6E9 myoblasts. However, TGF-beta 1 can also block myogenic differentiation in cells transfected with the myogenin gene under the control of a constitutive SV40 viral promoter. The nature of a mechanism that could mediate the inhibitory action of TGF-beta 1 without blocking myogenin mRNA expression is suggested by the observations that (a) TGF-beta 1 upregulates type I collagen expression and deposition in L6E9 myoblasts, (b) a fibrillar type I collagen layer inhibits L6E9 myoblast differentiation, and (c) inhibition of L6E9 myoblast differentiation by a type I collagen layer occurs without a block in myogenin expression. Thus, the data suggest that inhibition of L6E9 myoblast differentiation by TGF-beta 1 may be accomplished by at least two mechanisms acting in concert. One mechanism leads to a block in the expression of myogenin whereas the other mechanism may involve TGF-beta 1-induced changes in cell adhesion that either block the action of myogenic differentiation gene products or prevent the function of other as yet unknown components of the myogenic differentiation pathway.     

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.     

Regulation of fibronectin and type I collagen mRNA levels by transforming growth factor-beta

Human platelet-derived transforming growth factor-beta (TGF-beta 1) increases the accumulation of the extracellular matrix proteins, fibronectin and type I collagen, in mesenchymal and epithelial cells. To determine the basis for this effect, we have examined the levels of mRNAs corresponding to fibronectin and alpha 2(I) procollagen in NRK-49 rat fibroblasts and L6E9 rat myoblasts treated with TGF-beta 1. TGF-beta 1 increased severalfold the levels of mRNAs for both proteins. The kinetics of this effect were similar for both mRNA species. The increase in fibronectin and alpha 2(I) procollagen mRNAs was detectable 2 h after addition of TGF-beta 1 to the cells and their maximal levels remained constant for several days. Actinomycin D, but not cycloheximide, inhibited the increase in fibronectin and alpha 2(I) procollagen mRNA levels induced by TGF-beta 1. The results indicate that TGF-beta 1 controls the composition and abundance of extracellular matrices at least in part by inducing a coordinate increase in the levels of fibronectin and type I collagen mRNAs.     

Endoglin differentially modulates antagonistic transforming growth factor-beta1 and BMP-7 signaling

Transforming growth factor-beta1 (TGF-beta1) and BMP-7 (bone morphogenetic protein-7; OP-1) play central, antagonistic roles in kidney fibrosis, a setting in which the expression of endoglin (CD105), an accessory TGF-beta type III receptor, is increased. So far, endoglin is known as a negative regulator of TGF-beta/ALK-5 signaling. Here we analyzed the effect of BMP-7 on TGF-beta1 signaling and the role of endoglin for both pathways in endoglin-deficient L(6)E(9) cells. In this myoblastic cell line, TGF-beta1 and BMPs are opposing cytokines, interfering with myogenic differentiation. Both induce specific target genes of which Id1 (for BMPs) and collagen I (for TGF-beta1) are two examples. TGF-beta1 activated two distinct type I receptors, ALK-5 and ALK-1, in these cells. Although the ALK-5/Smad3 signaling pathway mediated collagen I expression, ALK-1/Smad1/Smad5 signaling mediated a transient Id1 up-regulation. In contrast, BMP-7 exclusively activated Smad1/Smad5 resulting in a more prolonged Id1 expression. Although BMP-7 had no impact on collagen I abundance, it antagonized TGF-beta1-induced collagen I expression and (CAGA)(12)-MLP-Luc activity, effects that are mediated by the ALK-5/Smad3 pathway. Finally, we found that the transient overexpression of endoglin, previously shown to inhibit TGF-beta1-induced ALK-5/Smad3 signaling, enhanced the BMP-7/Smad1/Smad5 pathway.     

Endoglin modulates cellular responses to TGF-beta 1

Endoglin is a homodimeric membrane glycoprotein which can bind the beta 1 and beta 3 isoforms of transforming growth factor-beta (TGF-beta). We reported previously that endoglin is upregulated during monocyte differentiation. We have now observed that TGF-beta itself can stimulate the expression of endoglin in cultured human monocytes and in the U-937 monocytic line. To study the functional role of endoglin, stable transfectants of U-937 cells were generated which overexpress L- or S- endoglin isoforms, differing in their cytoplasmic domain. Inhibition of cellular proliferation and downregulation of c-myc mRNA which are normally induced by TGF-beta 1 in U-937 cells were totally abrogated in L-endoglin transfectants and much reduced in the S- endoglin transfectants. Inhibition of proliferation by TGF-beta 2 was not altered in the transfectants, in agreement with the isoform specificity of endoglin. Additional responses of U-937 cells to TGF- beta 1, including stimulation of fibronectin synthesis, cellular adhesion, platelet/endothelial cell adhesion molecule 1 (PECAM-1) phosphorylation, and homotypic aggregation were also inhibited in the endoglin transfectants. However, modulation of integrin and PECAM-1 levels and stimulation of mRNA levels for TGF-beta 1 and its receptors R-I, R-II, and betaglycan occurred normally in the endoglin transfectants. No changes in total ligand binding were observed in L- endoglin transfectants relative to mock, while a 1.5-fold increase was seen in S-endoglin transfectants. The degradation rate of the ligand was the same in all transfectants. Elucidating the mechanism by which endoglin modulates several cellular responses to TGF-beta 1 without interfering with ligand binding or degradation should increase our understanding of the complex pathways which mediate the effects of this factor.     

Endoglin promotes endothelial cell proliferation and TGF-beta/ALK1 signal transduction

Endoglin is a transmembrane accessory receptor for transforming growth factor-beta (TGF-beta) that is predominantly expressed on proliferating endothelial cells in culture and on angiogenic blood vessels in vivo. Endoglin, as well as other TGF-beta signalling components, is essential during angiogenesis. Mutations in endoglin and activin receptor-like kinase 1 (ALK1), an endothelial specific TGF-beta type I receptor, have been linked to the vascular disorder, hereditary haemorrhagic telangiectasia. However, the function of endoglin in TGF-beta/ALK signalling has remained unclear. Here we report that endoglin is required for efficient TGF-beta/ALK1 signalling, which indirectly inhibits TGF-beta/ALK5 signalling. Endothelial cells lacking endoglin do not grow because TGF-beta/ALK1 signalling is reduced and TGF-beta/ALK5 signalling is increased. Surviving cells adapt to this imbalance by downregulating ALK5 expression in order to proliferate. The ability of endoglin to promote ALK1 signalling also explains why ectopic endoglin expression in endothelial cells promotes proliferation and blocks TGF-beta-induced growth arrest by indirectly reducing TGF-beta/ALK5 signalling. Our results indicate a pivotal role for endoglin in the balance of ALK1 and ALK5 signalling to regulate endothelial cell proliferation.     

Adenovirus conducted connective tissue growth factor on extracellular matrix in trabecular meshwork and its role on aqueous humor outflow facility

Deposition of extracellular matrix (ECM) in trabecular meshwork, such as fibronectin, collagen IV, elastin. leads to increased resistance of trabecular meshwork in primary open angle glaucoma (POAG). Connective tissue growth factor (CTGF) is known to regulate the ECM deposits. In this study, we detect the effect of adenovirus conducted CTGF (Adv-CTGF) transfection on either the expression of ECM components or aqueous humor outflow facility. Adv-CTGF was used to transfect rat trabecular meshwork cells in vivo and in vitro. Aqueous humor outflow facility was test by microbeads perfusion. Protein expression of CTGF, fibronectin, and collagen IV was determined using Western blot. In the Adv-CTGF group, the outflow facility displayed a significant decrease from baseline. It appears as though the transfection with Adv-CTGF significantly affects the aqueous humor outflow pattern. A negative correlation between IOP and PEFL indicated that a decrease in the area of bead deposition corresponded to an overall decrease of outflow, leading to an elevated IOP. Adv-CTGF can enhance the expression of CTGF, fibronectin and collagen IV. CTGF is the novel target for treatment of POAG. It is necessary to further study to test inhibition of CTGF expression for treatment of POAG.     

ALK1 heterozygosity increases extracellular matrix protein expression,proliferation and migration in fibroblasts

Fibrosis is a pathological situation in which excessive amounts of extracellular matrix (ECM) are deposited in the tissue. Myofibroblasts play a crucial role in the development and progress of fibrosis as they actively synthesize ECM components such as collagen I, fibronectin and connective tissue growth factor (CTGF) and cause organ fibrosis. Transforming growth factor beta 1 (TGF-β1) plays a major role in tissue fibrosis. Activin receptor-like kinase 1 (ALK1) is a type I receptor of TGF-β1 with an important role in angiogenesis whose function in cellular biology and TGF-β signaling is well known in endothelial cells, but its role in fibroblast biology and its contribution to fibrosis is poorly studied. We have recently demonstrated that ALK1 regulates ECM protein expression in a mouse model of obstructive nephropathy. Our aim was to evaluate the role of ALK1 in several processes involved in fibrosis such as ECM protein expression, proliferation and migration in ALK1^+/+ and ALK1^+/− mouse embryonic fibroblasts (MEFs) after TGF-β1 stimulations and inhibitors. ALK1 heterozygous MEFs show increased expression of ECM proteins (collagen I, fibronectin and CTGF/CCN2), cell proliferation and migration due to an alteration of TGF-β/Smad signaling. ALK1 heterozygous disruption shows an increase of Smad2 and Smad3 phosphorylation that explains the increases in CTGF/CCN2, fibronectin and collagen I, proliferation and cell motility observed in these cells. Therefore, we suggest that ALK1 plays an important role in the regulation of ECM protein expression, proliferation and migration.     

Regulation of pro-inflammatory and pro-fibrotic factors by CCN2/CTGF in H9c2 cardiomyocytes

Connective tissue growth factor (CTGF), also known as CCN2, is implicated in fibrosis through both extracellular matrix (ECM) induction and inhibition of ECM degradation. The role of CTGF in inflammation in cardiomyocytes is unknown. In some mesenchymal cell systems, CTGF mediates effects through TGF-β or tyrosine kinase cell surface receptor, TrkA, signalling. In this study, cellular mechanisms by which CTGF regulates pathways involved in fibrosis and inflammation were explored. Murine H9c2 cardiomyocytes were treated with recombinant human (rh)CTGF and ECM formation gene expression: fibronectin, collagen type -I and -III and ECM degradation genes: TIMP-1, TIMP-2 and PAI-1 were found to be induced. CTGF treatment also increased pro-inflammatory cytokines TNF-α, IL-6, MCP-1 and IL-8. CTGF upregulated TGF-β1 mRNA and rapidly induced phosphorylation of TrkA. The CTGF-induced pro-fibrotic and pro-inflammatory effects were blocked by anti-TGF-β neutralizing antibody and Alk 5 inhibitor (SB431542). A specific blocker of TrkA activation, k252a, also abrogated CTGF-induced effects on fibrosis and gene expresison of MCP-1 and IL-8, but not TNF-α or IL-6. Collectively, this data implicates CTGF in effects on pro-fibrotic genes and pro-inflammatory genes via TGF-β pathway signalling and partly through TrkA.     

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.