TGFβ induction of extracellular matrix associated proteins in normal and transformed human mammary epithelial cells in culture is independent of growth effects

We have previously characterized a human mammary epithelial cell (HMEC) culture system for the effects of TGFβ1 on cell growth. In the current report, the effects of TGFβ 1 on synthesis and secretion of proteins associated with the extracellular matrix and proteolysis were examined. In particular, we compared the TGFβ responses of normal finite lifespan HMEC, which are growth inhibited by TGFβ, to two immortally transformed cell lines derived from the normal HMEC. One of these lines maintains active growth in the presence of TGFβ and the other shows partial growth inhibition. In contrast to the differing effects of TGFβ on cell growth, we found that all these cell types showed strong induction of most of the mRNA and protein species examined, including fibronectin, collagen IV, laminin, type IV collagenase, urokinase type plasminogen activator (uPA), and plasminogen activator inhibitor 1 (PAI-1). The profile of TGFβ 1 binding proteins was the same in HMEC that were, and were not growth suppressed by TFGβ. Therefore, the effects of TGFβ on cell growth could be dissociated from its effects on specialized responses, indicating that within this one cell type there must be at least two independent pathways for TGFβ activity, one which leads to cessation of proliferation and one which induces a specific set of cellular responses. This cell system may be useful for examining the pathway of TGFβ induced growth inhibition using closely matched cells which vary in their growth-induced response but retain similar specialized responses to TGFβ. © 1993 Wiley-Liss, Inc.     

Relationship between actions of transforming growth factor (TGF)-β and cell surface expression of its receptors in clonal osteoblastic cells

Various osteoblastic cell lines were examined for the relationship between the presence of cell-surface transforming growth factor (TGF)-β receptors and the synthesis of matrix proteins with their responsiveness to TGF-β. Treatment with TGF-β1 inhibited proliferation and stimulated proteoglycan and fibronectin synthesis in MC3T3-E1 and MG 63 cells. The major proteoglycans synthesized by these cells were decorin and biglycan, and TGF-β1 markedly stimulated the synthesis of decorin in MC3T3-E1 and of biglycan in MG 63 cells. SaOS 2 and UMR 106 cells synthesized barely detectable amounts of decorin or biglycan, and TGF-β1 did not stimulate the synthesis of these proteoglycans. In SaOS 2 cells, however, TGF-β1 enhanced fibronectin synthesis. TGF-β1 did not show any of these effects in UMR 106 cells. Receptor cross-linking studies revealed that only MC3T3-E1 and MG 63 cells had both types I and II signal-transducing receptors for TGF-β in addition to betaglycan. SaOS 2 cells possessed type I but no type II receptor on the cell surface. In contrast, SaOS 2 as well as MC3T3-E1 and MG 63 cells expressed type II receptor mRNA by Northern blot analysis, and cell lysates contained type II receptor by Western blot analysis. Thus, it appears that type II receptor present in SaOS 2 cells is not able to bind TGF-β1 under these conditions. UMR 106 cells with no response to TGF-β1 had neither of the signal-transducing receptors by any of the analyses. These observations using clonal osteoblastic cell lines demonstrate that the ability of osteoblastic cells to synthesize bone matrix proteoglycans is associated with the responsiveness of these cells to TGF-β1, that the responsiveness of osteoblastic cells to TGF-β1 in cell proliferation and proteoglycan synthesis correlates with the presence of both types I and II receptors, and that the effect of TGF-β1 on fibronectin synthesis can develop with little binding of TGF-β1 to type II receptor if type I receptor is present. It is suggested that the combination of cell-surface receptors for TGF-β determines the responsiveness of osteoblastic cells to TGF-β and that changes in cell-surface TGF-β receptors may play a role in the regulation of matrix protein synthesis and bone formation in osteoblasts. © 1995 Wiley-Liss, Inc.     

Effect of vasopressin on type IV collagen production in human mesangial cells

Production of extracellular matrix proteins, such as type IV collagen and fibronectin, by mesangial cells contributes to progressive glomerulosclerosis. In this study, the ability of vasopressin (AVP), which causes mesangial cell proliferation and hypertrophy, to stimulate type IV collagen production by cultured human mesangial cells was examined using an enzyme-linked immunosorbent assay. AVP induced a concentration-dependent increase in the production of type IV collagen and this effect was potently and concentration-dependently inhibited by AVP V_1A receptor antagonists, including YM218. AVP also induced a concentration-dependent increase in transforming growth factor (TGF)-β secretion by human mesangial cells and this effect was inhibited by V_1A receptor antagonists. Furthermore, TGF-β also induced an increase in the production of type IV collagen; the AVP-enhanced production of type IV collagen was inhibited by an anti-TGF-β antibody. These findings indicate that AVP stimulates synthesis of type IV collagen by cultured human mesangial cells through the induction of TGF-β synthesis mediated by V_1A receptors; consequently, AVP contributes to glomerular remodeling and extracellular matrix accumulation observed in glomerular diseases.     

TGFβ-Stimulated MicroRNA-21 Utilizes PTEN to Orchestrate AKT/mTORC1 Signaling for Mesangial Cell Hypertrophy and Matrix Expansion

Transforming growth factor-β (TGFβ) promotes glomerular hypertrophy and matrix expansion, leading to glomerulosclerosis. MicroRNAs are well suited to promote fibrosis because they can repress gene expression, which negatively regulate the fibrotic process. Recent cellular and animal studies have revealed enhanced expression of microRNA, miR-21, in renal cells in response to TGFβ. Specific miR-21 targets downstream of TGFβ receptor activation that control cell hypertrophy and matrix protein expression have not been studied. Using 3'UTR-driven luciferase reporter, we identified the tumor suppressor protein PTEN as a target of TGFβ-stimulated miR-21 in glomerular mesangial cells. Expression of miR-21 Sponge, which quenches endogenous miR-21 levels, reversed TGFβ-induced suppression of PTEN. Additionally, miR-21 Sponge inhibited TGFβ-stimulated phosphorylation of Akt kinase, resulting in attenuation of phosphorylation of its substrate GSK3β. Tuberin and PRAS40, two other Akt substrates, and endogenous inhibitors of mTORC1, regulate mesangial cell hypertrophy. Neutralization of endogenous miR-21 abrogated TGFβ-stimulated phosphorylation of tuberin and PRAS40, leading to inhibition of phosphorylation of S6 kinase, mTOR and 4EBP-1. Moreover, downregulation of miR-21 significantly suppressed TGFβ-induced protein synthesis and hypertrophy, which were reversed by siRNA-targeted inhibition of PTEN expression. Similarly, expression of constitutively active Akt kinase reversed the miR-21 Sponge-mediated inhibition of TGFβ-induced protein synthesis and hypertrophy. Furthermore, expression of constitutively active mTORC1 prevented the miR-21 Sponge-induced suppression of mesangial cell protein synthesis and hypertrophy by TGFβ. Finally, we show that miR-21 Sponge inhibited TGFβ-stimulated fibronectin and collagen expression. Suppression of PTEN expression and expression of both constitutively active Akt kinase and mTORC1 independently reversed this miR-21-mediated inhibition of TGFβ-induced fibronectin and collagen expression. Our results uncover an essential role of TGFβ-induced expression of miR-21, which targets PTEN to initiate a non-canonical signaling circuit involving Akt/mTORC1 axis for mesangial cell hypertrophy and matrix protein synthesis.     

Characterization and regulation of the latent transforming growth factor-β complex secreted by vascular pericytes

Transforming growth factor-β (TGF-β) stimulates the accumulation of extracellular matrix in renal and hepatic disease. Kidney glomerular mesangial cells (GMC) and liver fat-storing cells (FSC) produce latent or inactive TGF-β. In this study, we characterized the latent TGF-β complexes secreted by these cells. Human FSC produce a single latent TGF-β complex, predominantly of the TGF-β1 isoform, whereas GMC secrete multiple complexes of latent TGF-β, containing β1 and β2 isoforms. At least four forms were identified in GMC using ion exchange chromatography, including a peak not previously described in other cell types which eluted at 0.12 M NaCl, and predominantly of the β2 isoform. Both cell types secrete the latent TGF-β1 binding protein of 190 kDa, as part of a high molecular weight TGF-β complex. Epidermal growth factor stimulates the secretion of latent TGF-β and latent TGF-β binding protein in both cell types. Secretion of the latent TGF-β in both cell types was found to be associated with secretion of decorin. This study shows that vascular pericytes from the kidney and the liver have distinctly different profiles of latent TGF-β complexes, with GMC secreting a unique form of latent TGF-β2. The regulatory effect of epidermal growth factor and platelet-derived growth factor has potential implication for the pathophysiology of liver regeneration and chronic liver and kidney diseases. © 1996 Wiley-Liss, Inc.     

Interferon‐γ promotes vascular remodeling in human microvascular endothelial cells by upregulating endothelin (ET)‐1 and transforming growth factor (TGF) β2

Systemic sclerosis (SSc) is a complex disease characterized by vascular alterations, activation of the immune system and tissue fibrosis. Previous studies have implicated activation of the interferon pathways in the pathogenesis of SSc. The goal of this study was to determine whether interferon type I and/or type II could play a pathogenic role in SSc vasculopathy. Human dermal microvascular endothelial cells (HDMVECs) and fibroblasts were obtained from foreskins of healthy newborns. The RT Profiler PCR Array System was utilized to screen for EndoMT genes. Treatment with IFN‐α or IFN‐γ downregulated Fli1 and VE‐cadherin. In contrast, IFN‐α and IFN‐γ exerted opposite effects on the expression of α‐SMA, CTGF, ET‐1, and TGFβ2, with IFN‐α downregulating and IFN‐γ upregulating this set of genes. Blockade of TGFβ signaling normalized IFN‐γ‐mediated changes in Fli1, VE‐cadherin, CTGF, and ET‐1 levels, whereas upregulation of α‐SMA and TGFβ2 was not affected. Bosentan treatment was more effective than TGFβ blockade in reversing the actions of IFN‐γ, including downregulation of α‐SMA and TGFβ2, suggesting that activation of the ET‐1 pathway plays a main role in the IFN‐γ responses in HDMECs. IFN‐γ induced expression of selected genes related to endothelial‐to‐mesenchymal transition (EndoMT), including Snail1, FN1, PAI1, TWIST1, STAT3, RGS2, and components of the WNT pathway. The effect of IFN‐γ on EndoMT was mediated via TGFβ2 and ET‐1 signaling pathways. This study demonstrates distinct effects of IFN‐α and IFN‐γ on the biology of vascular endothelial cells. IFN‐γ may contribute to abnormal vascular remodeling and fibrogenesis in SSc, partially via induction of EndoMT. J. Cell. Physiol. 228: 1774–1783, 2013. © 2013 Wiley Periodicals, Inc.     

Differential effects of various growth factors and cytokines on the syntheses of DNA,type I collagen,laminin, fibronectin,osteonectin/secreted protein,acidic and rich in cysteine (SPARC), and alkaline phosphatase by human pulp cells in culture

The purpose of this study is to differentiate roles of several growth factors and cytokines in proliferation and differentiation of pulp cells during development and repair. In human pulp cell cultures, laminin and type I collagen levels per cell remained almost constant during the whole culture period (22 days). On the other hand, secreted protein, acidic and rich in cysteine (SPARC/osteonectin) and alkaline phosphatase (ALPase) levels markedly increased after the cultures reached confluence. Laminin and type I collagen, as well as fibronectin, stimulated the spreading of pulp cells within 1 h. Adding transforming growth factor-β (TGF-β) decreased laminin and ALPase levels, whereas it increased SPARC and fibronectin levels 3- to 10-fold. Western and Northern blots showed that TGF-β enhanced SPARC synthesis at the protein and mRNA levels. Basic fibroblast growth factor (bFGF) decreased type I collagen, laminin, SPARC, and ALPase levels without changing the fibronectin level. Platelet-derived growth factor (PDGF) selectively decreased laminin, SPARC, and ALPase levels. Epidermal growth factor (EGF) also decreased SPARC and ALPase levels. Tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) decreased type I collagen and laminin levels, and abolished SPARC and ALPase syntheses. Of these peptides, bFGF and PDGF showed the greatest stimulation of [³H]thymidine incorporation into DNA. TGF-β, EGF, and TNF-α had less effect on DNA synthesis, whereas IL-1β inhibited DNA synthesis. These findings demonstrated that TGF-β, bFGF, EGF, PDGF, TNF-α, and IL-1β have characteristically different patterns of actions on DNA, laminin, type I collagen, fibronectin, ALPase, and SPARC syntheses by pulp cells. J. Cell. Physiol. 174:194–205, 1998. © 1998 Wiley-Liss, Inc.     

TGFβ‐induced PI 3 kinase‐dependent Mnk‐1 activation is necessary for Ser‐209 phosphorylation of eIF4E and mesangial cell hypertrophy

Transforming growth factorβ (TGFβ)‐induced canonical signal transduction is involved in glomerular mesangial cell hypertrophy; however, the role played by the noncanonical TGFβ signaling remains largely unexplored. TGFβ time‐dependently stimulated eIF4E phosphorylation at Ser‐209 concomitant with enhanced phosphorylation of Erk1/2 (extracellular signal regulated kinase1/2) and MEK (mitogen‐activated and extracellular signal‐regulated kinase kinase) in mesangial cells. Inhibition of Erk1/2 by MEK inhibitor or by expression of dominant negative Erk2 blocked eIF4E phosphorylation, resulting in attenuation of TGFβ‐induced protein synthesis and mesangial cell hypertrophy. Expression of constitutively active (CA) MEK was sufficient to induce protein synthesis and hypertrophy similar to those induced by TGFβ. Pharmacological or dominant negative inhibition of phosphatidylinositol (PI) 3 kinase decreased MEK/Erk1/2 phosphorylation leading to suppression of eIF4E phosphorylation. Inducible phosphorylation of eIF4E at Ser‐209 is mediated by Mnk‐1 (mitogen‐activated protein kinase signal‐integrating kinase‐1). Both PI 3 kinase and Erk1/2 promoted phosphorylation of Mnk‐1 in response to TGFβ. Dominant negative Mnk‐1 significantly inhibited TGFβ‐stimulated protein synthesis and hypertrophy. Interestingly, inhibition of mTORC1 activity, which blocks dissociation of eIF4E‐4EBP‐1 complex, decreased TGFβ‐stimulated phosphorylation of eIF4E without any effect on Mnk‐1 phosphorylation. Furthermore, mutant eIF4E S209D, which mimics phosphorylated eIF4E, promoted protein synthesis and hypertrophy similar to TGFβ. These results were confirmed using phosphorylation deficient mutant of eIF4E. Together our results highlight a significant role of dissociation of 4EBP‐1‐eIF4E complex for Mnk‐1‐mediated phosphorylation of eIF4E. Moreover, we conclude that TGFβ‐induced noncanonical signaling circuit involving PI 3 kinase‐dependent Mnk‐1‐mediated phosphorylation of eIF4E at Ser‐209 is required to facilitate mesangial cell hypertrophy. J. Cell. Physiol. 228: 1617–1626, 2013. © 2013 Wiley Periodicals, Inc.     

Expression of transforming growth factor alpha (TGF alpha) in differentiated rat mammary tumors: estrogen induction of TGF alpha production

Primary well-differentiated dimethylbenzene alpha-anthracene (DMBA)-or nitrosomethylurea (NMU)-induced rat mammary adenocarcinomas that are estrogen dependent possess biologically active and immunoreactive transforming growth factor alpha (TGF alpha), which can be detected in a sort agar growth-promoting assay and by a specific liquid-phase competitive RIA, respectively. In contrast, tissue extracts prepared from transplantable undifferentiated DMBA-I and NMU-II rat mammary carcinomas that are estrogen independent and metastatic exhibit low or undetectable levels of TGF alpha. In addition, the primary DMBA- and NMU-induced rat mammary adenocarcinomas express a specific 4.8-kilobase TGF alpha mRNA species, whereas little or no TGF alpha mRNA can be detected in the transplantable DMBA-I and NMU-II tumors. Primary tumors synthesize type IV basement membrane collagen, whereas the transplantable tumors elaborate very little type IV collagen. Either TGF alpha or estrogens can differentially enhance the synthesis of type IV collagen by 0.5- to 4-fold over total protein synthesis in primary cultures of normal mouse mammary epithelial cells or in primary NMU-induced tumor cells, respectively. Therefore, TGF alpha could function as an estrogen-inducible autocrine growth factor for well differentiated rat mammary tumor cells by its ability to selectively regulate type IV collagen synthesis. Estrogens can modulate TGF alpha production in vivo in primary DMBA-induced rat mammary tumors, because ovariectomy results in a rapid decline (within 6 h) of TGF alpha mRNA levels. This response to estrogens can also be observed in vitro. Primary DMBA- or NMU-induced rat mammary tumor cells cultured in the presence of 17 beta-estradiol (10(-8) M) for 4 days show an increase in the level of TGF alpha mRNA over cells not treated with estrogen. This increase in TGF alpha mRNA is paralleled by a 2- to 3-fold increase in the levels of immunoreactive TGF alpha that can be detected and in the conditioned medium from estrogen-treated cells. These results suggest that TGF alpha may be an adjunct marker for those mammary tumors that are well differentiated adenocarcinomas and estrogen dependent and that estrogen-independent tumors do not constitutively produce TGF alpha or express TGF alpha mRNA.     

Transforming growth factor β blocks cystogenesis by MDCK epithelium in vitro by enhancing the paracellular flux: Implication of collagen V

Transforming growth factor β (TGFβ) determines a nearly complete inhibition of cystogenesis by MDCK cells grown in collagen I-enriched matrices in vitro. In order to elucidate the mechanism implicated in this phenomenon, we performed a series of experiments aimed at discovering a relevant role of extracellular matrix. TGFβ (2 ng/ml) played a marked stimulatory effect on the expression of extracellular matrix by MDCK with a selective effect on collagen V (three to fourfold increase of protein and mRNA) and in parallel inhibited cystogenesis by 95%. Cotreatment with TGFβ and anti-collagen V antibodies restored a normal cystogenesis. In analogy, when MDCK cells were grown in three-dimensional matrices containing collagen I and minor (10%) amounts of collagen V, cystogenesis was once again inhibited by 95%. To characterize the molecular mechanism activated by TGFβ and collagen V, we looked at the electrophysiological characteristics of MDCK monolayers and found a drastic fall of transepithelial electrical resistance (TER) in both conditions. In parallel with the decrease in TER, TGFβ and collagen V also induced the leakage of two high molecular weight tracers, i.e., [³H]-inulin and 150 kD FITC-Dextran, suggesting a perturbation of the paracellular permeability. Finally, TGFβ at the relevant concentration did not stimulate apoptosis in our cellular model, as judged by propidium iodide staining and by in situ end labeling of DNA fragments. These observations suggest that TGFβ inhibits cystogenesis by MDCK cells in vitro by altering the collagenic composition of the three-dimensional milieu where MDCK cells grow and form cysts. The molecular mechanism responsible for inhibition of cystogenesis is the increase of paracellular flux which overcomes the active transport of solutes and water inside cysts. J. Cell. Physiol. 177:214–223, 1998. © 1998 Wiley-Liss, Inc.     

Platelet-derived growth factor and inflammatory cytokines have differential effects on the expression of integrins α1β1 and α5β1 by human dermal fibroblasts in vitro

Dermal fibroblasts are essential for the repair of cutaneous wounds. Fibroblasts presumably use cell surface receptors of the integrin family during migration into a wound from the adjacent uninjured tissue and for the subsequent matrix repairs. We have investigated the possible roles of platelet-derived growth factor and inflammatory cytokines in the regulation of integrin expression on wound fibroblasts using a porcine cutaneous wound model and cultured human cells. Tissue specimens collected from 4-day pig wounds were stained with antibodies specific for the α1 and α5 integrin subunits. Staining for α1 was markedly decreased on fibroblasts adjacent to the wound and in the granulation tissue, while staining for α5 was clearly enhanced in both locations. Normal adult human dermal fibroblasts in culture express the integrins α1β1, a collagen receptor, and α5β1, a fibronectin receptor. Quantitative flow cytometry was used to measure cell surface integrin expression after treatment with platelet-derived growth factor (PDGF)-AA, PDGF-AB, or PDGF-BB. Each isoform of PDGF produced a significant decrease in the level of α1 present on the cell surface and an increase in the level of α5. Furthermore, PDGF-BB produced a corresponding decrease in α1 mRNA and an increase in α5 mRNA. In contrast, treatment with three inflammatory cytokines, IL-1β, TNF-α, and IFN-γ, produced clear increases in the levels of α1 and α5 present on the cell surface. Our observations suggest that the differential effects of PDGF and inflammatory cytokines may be part of the mechanism regulating the expression of α1 and α5 integrins by dermal fibroblasts during wound repair. © 1996 Wiley-Liss, Inc.     

Induction of extracellular matrix gene expression in normal human keratinocytes by transforming growth factor beta is altered by cellular differentiation

Changes in epithelial substrate have been related to the cellular capacity for proliferation and to changes in cellular behavior. The effect of TGF beta 1 on the expression of the basement membrane genes, fibronectin, laminin B1, and collagen alpha 1 (IV), was examined. Northern analysis revealed that treatment of normal human epidermal keratinocytes with 100 pM TGF beta 1 increased the expression of each extracellular matrix (ECM) gene within 4 h of treatment. Maximal induction was reached within 24 h after treatment. The induction of ECM mRNA expression was dose dependent and was observed at doses as low as 1-3 pM TGF beta 1. Incremental doses of TGF beta 1 also increased cellular levels of fibronectin protein in undifferentiated keratinocytes and resulted in increased secretion of fibronectin. Squamous-differentiated cultures of keratinocytes expressed lower levels of the extracellular matrix RNAs than did undifferentiated cells. Treatment of these differentiated cells with TGF beta 1 induced the expression of fibronectin mRNA to levels seen in TGF beta-treated, undifferentiated keratinocytes but only marginally increased the expression of collagen alpha 1 (IV) and laminin B1 mRNA. The increased fibronectin mRNA expression in the differentiated keratinocytes was also reflected by increased accumulation of cellular and secreted fibronectin protein. The inclusion of cycloheximide in the protocol indicated that TGF beta induction of collagen alpha 1 (IV) mRNA was signaled by proteins already present in the cells but that TGF beta required the synthesis of a protein(s) to fully induce expression of fibronectin and laminin B1 mRNA. The differential regulation of these genes in differentiated cells may be important to TGF beta action in regulating reepithelialization.     

Inflammatory cytokines promote growth of intestinal smooth muscle cells by induced expression of PDGF‐Rβ

Thickening of the inflamed intestinal wall involves growth of smooth muscle cells (SMC), which contributes to stricture formation. Earlier, the growth factor platelet‐derived growth factor (PDGF)‐BB was identified as a key mitogen for SMC from the rat colon (CSMC), and CSMC growth in colitis was associated with both appearance of its receptor, PDGF‐Rβ and modulation of phenotype. Here, we examined the role of inflammatory cytokines in inducing and modulating the growth response to PDGF‐BB. CSMC were enzymatically isolated from Sprague–Dawley rats, and the effect of tumour necrosis factor (TNF)‐α, interleukin (IL)‐1β, transforming growth factor (TGF), IL‐17A and IL‐2 on CSMC growth and responsiveness to PDGF‐BB were assessed using proliferation assays, PCR and western blotting. Conditioned medium (CM) was obtained at 48 hrs of trinitrobenzene sulphonic acid‐induced colitis. Neither CM alone nor cytokines caused proliferation of early‐passage CSMC. However, CM from inflamed, but not control colon significantly promoted the effect of PDGF‐BB. IL‐1β, TNF‐α and IL‐17A, but not other cytokines, increased the effect of PDGF‐BB because of up‐regulation of mRNA and protein for PDGF‐Rβ without change in receptor phosphorylation. PDGF‐BB was identified in adult rat serum (RS) and RS‐induced CSMC proliferation was inhibited by imatinib, suggesting that blood‐derived PDGF‐BB is a local mitogen in vivo. In freshly isolated CSMC, CM from the inflamed colon as well as IL‐1β and TNF‐α induced the early expression of PDGF‐Rβ, while imatinib blocked subsequent RS‐induced cell proliferation. Thus, pro‐inflammatory cytokines both initiate and maintain a growth response in CSMC via PDGF‐Rβ and serum‐derived PDGF‐BB, and control of PDGF‐Rβ expression may be beneficial in chronic intestinal inflammation.     

High glucose stimulates synthesis of fibronectin via a novel protein kinase C,Rap1b,and B-Raf signaling pathway

The molecular mechanism(s) by which high glucose induces fibronectin expression via G-protein activation in the kidney are largely unknown. This investigation describes the effect of high glucose (HG) on a small GTP-binding protein, Rap1b, expression and activation, and the relevance of protein kinase C (PKC) and Raf pathways in fibronectin synthesis in cultured renal glomerular mesangial cells (MCs). In vivo experiments revealed a dose-dependent increase in Rap1b expression in glomeruli of diabetic rat kidneys. Similarly, in vitro exposure of MCs to HG led to an up-regulation of Rap1b with concomitant increase in fibronectin (FN) mRNA and protein expression. The up-regulation of Rap1b mRNA was mitigated by the PKC inhibitors, calphostin C, and bisindolymaleimide, while also reducing HG- induced FN expression in non-transfected MCs. Overexpression of Rap1b by transfection with pcDNA 3.1/Rap1b in MCs resulted in the stimulation of FN synthesis; however, the PKC inhibitors had no significant effect in reducing FN expression in Rap1b-transfected MCs. Transfection of Rap1b mutants S17N (Ser --> Asn) or T61R (Thr --> Arg) in MCs inhibited the HG-induced increased FN synthesis. B-Raf and Raf-1 expression was investigated to assess whether Rap1b effects are mediated via the Raf pathway. B-Raf, and not Raf-1, expression was increased in MCs transfected with Rap1b. HG also caused activation of Rap1b, which was largely unaffected by anti-platelet-derived growth factor (PDGF) antibodies. HG-induced activation of Rap1b was specific, since Rap2b activation and expression of Rap2a and Rap2b were unaffected by HG. These findings indicate that hyperglycemia and HG cause an activation and up-regulation of Rap1b in renal glomeruli and in cultured MCs, which then stimulates FN synthesis. This effect appears to be PKC-dependent and PDGF-independent, but involves B-Raf, suggesting a novel PKC-Rap1b-B-Raf pathway responsible for HG-induced increased mesangial matrix synthesis, a hallmark of diabetic nephropathy.     

Transforming growth factor β1 increases the phosphoenolpyruvate carboxykinase mRNA level in cultured rat hepatocytes

Transforming growth factor β1 (TGFβ1) elevated the phosphoenolpyruvate carboxykinase (PEPCK) mRNA abundance in primary cultures of rat hepatocytes. Although this increase was not as large as the rise in PEPCK gene expression induced by the cAMP-elevating agents glucagon or isoproterenol, the effect of TGFβ1 was several-fold and concentration-dependent, with ED₅₀ at about 2.5 pM, which is in the same concentration range as the previously found growth-inhibitory effect of TGFβ. The data show that the level of mRNA for PEPCK, an enzyme typically expressed in the liver, can be regulated in the same direction by TGFβ1 and cAMP.     

Regulation of fibrogenic/fibrolytic genes by platelet-derived growth factor C, a novel growth factor, in human dermal fibroblasts

Platelet-derived growth factor (PDGF) is a potent mitogenic and chemotactic cytokine, and PDGF-C is a novel growth factor belonging to the PDGF family. In this study, to determine whether this growth factor can contribute to fibrosis and tissue remodeling, we examined the effect of PDGF-CC on the expression of fibrogenic/fibrolytic genes such as type I collagen, fibronectin (FN), matrix metalloproteinases (MMPs), and their inhibitors (TIMPs) in normal human dermal fibroblasts in vitro. PDGF elevated the levels of MMP-1 or TIMP-1 protein as well as mRNA, whereas this cytokine had no influence on the expression of type I collagen, FN, or TIMP-2. PDGF-CC also increased the levels of MMP-1 catalytic activity in the cultured media and mRNA expression, which was paralleled that on the levels of promoter activation. Additionally, PDGF-CC induced the mitogenic and migratory activity of human dermal fibroblasts in a dose-dependent manner. On the other hand, we also determined the specificity of the inhibitory effect of monoclonal antibodies against PDGF-CC generated by immunizing balb/c mice with recombinant human PDGF-CC. This antibody could inhibit the regulatory effects of MMP-1 or TIMP-1 synthesis as well as the mitogenic effects on human dermal fibroblasts induced by PDGF-CC, whereas this antibody did not affect those induced by other PDGF forms such as PDGF-AA, -AB, or -BB. These results suggest that this cytokine plays a role in the tissue remodeling.     

A factor from damaged rat kidney stimulates collagen biosynthesis by mesangial cells

Rats were administered CCl4, a well-defined nephrotoxin, for 20 weeks to produce glomerular sclerosis. Tubular degeneration and necrosis with interstitial fibrosis was clearly evident by histological examination. Kidneys were homogenized in phosphate-buffered saline and a collagen synthesis-stimulating factor was isolated by Sephadex G-50 gel filtration. The 5 kDa component stimulated both type I and type IV procollagen synthesis by mesangial cells and type I procollagen synthesis by rat skin fibroblasts. In each cell type, 2-6-fold increases in procollagen protein production or cell proliferation was noted. The steady-state levels of mRNA encoding for procollagen alpha 1(I) and procollagen alpha 1(IV) chains in mesangial cells were determined by by hybridization to their corresponding cDNA clones. The type I procollagen mRNA was elevated 1.4-fold compared to a 1.6-fold increase in mRNA encoding for type IV procollagen. The similar properties and chemical characteristics of this fibrogenic factor with a factor from fibrotic liver suggests they are the same and that a common endogenous collagen synthesis stimulator may be present in fibrosing organs, thus providing a driving force for collagen over-production.     

Plasminogen-dependent activation of latent transforming growth factor beta (TGFβ) by growing cultures of osteoblast-like cells

Osteoblasts secrete transforming growth factor beta (TGFβ) as a biologically inert, latent complex that must be dissociated before the growth factor can exert its effects. We have examined the production and proteolytic activation of latent TGFβ (LTGFβ) by clonal UMR 106-01 rat osteosarcoma cells and neonatal mouse calvarial (MC) osteoblast-like cells in vitro. Synthetic bPTH-(1–34) increased the activity of tissue-type (tPA) and urokinase-type (uPA) plasminogen activators (PA) in cell lysates (CL) of UMR 106-01 cells. The concentration of active TGFβ in serum-free CM from cultures treated with bPTH-(1–34) and plasminogen was significantly greater than in CM from untreated controls and cultures treated with either bPTH-(1–34) or plasminogen alone. This effect occurred at concentrations of PTH-(1–34) that increased PA activity and was prevented by aprotinin, an inhibitor of plasmin activity. Treatment with bPTH-(1–34) had no effect on the concentration of TGFβ in acid-activated samples of CM. Functional consequences of proteolytically activated TGFβ was examined in primary cultures of neonatal MC osteoblast-like cells. Human platelet TGFβ1 caused a dose-dependent increase in the migration of these cells in an in vitro wound healing assay. Cell migration was also stimulated in cultures treated with bPTH-(1–34) and plasminogen together. This effect was blocked by an anti-TGFβ1 antibody. The results of these studies demonstrate that (1) LTGFβ secreted by osteoblasts in vitro is activated under conditions where the plasmin activity in the cultures is increased, and (2) the TGFβ generated by plasmin-mediated proteolysis is biologically active. We suggest that the local concentration of TGFβ in bone may be controlled by the osteoblast-associated plasminogen activator/plasmin system. Furthermore, since several calciotropic factors influence osteoblast PA activity, this system may have an important role in mediating their anabolic and/or catabolic effects. © 1993 Wiley-Liss, Inc.