Temporal expression of autocrine growth factors corresponds to morphological features of mesangial proliferation in Habu snake venom-induced glomerulonephritis.

Habu snake venom induces an accelerated mesangial proliferative glomerulonephritis that follows a predictable course from early capillary aneurysms to micronodules comprised of confluent mesangial cells within 72 hours. We examined morphologically the course of mesangial cell proliferation and correlated it with the expression of messenger (m) RNA encoding two peptide growth factors, platelet-derived growth factor (PDGF) A and B chains and transforming growth factor-beta (TGF-beta). Rats were uninephrectomized and 24 hours later injected with Habu snake venom or saline. Kidney cortex and isolated glomeruli were obtained 24, 48, and 72 hours later for histological assessment, preparation and Northern analysis of mRNA, and immunohistochemical localization of PDGF using a polyclonal antibody that recognizes A and B chains. Maximal expression of PDGF B chain mRNA occurred at 24 hours and before the onset of mesangial cell proliferation; whereas maximal expression of PDGF A chain and TGF-beta mRNA occurred at 48 hours and during active mesangial cell proliferation. Expression of TGF-beta mRNA persisted at 72 hours at a time when PDGF A chain declined and PDGF B chain was not expressed compared to uninephrectomy and saline controls and at a time when mesangial cells within lesions reached confluence and proliferation subsided. PDGF protein localized in glomerular lesions associated with platelets at 24 and 48 hours and within mesangial cells at 48 and 72 hours. These results agree with the known roles of PDGF and TGF-beta as positive and negative modulators, respectively, of mesangial cell growth in vitro and suggest that a relative balance of the expression of these factors may operate in glomerular disease in vivo.     

Mannose-6-phosphate/IGF-II receptors mediate the effects of IGF-1-induced latent transforming growth factor beta 1 on expression of type I collagen and collagenase in dermal fibroblasts

We have previously shown that insulin-like growth factor-1 (IGF-1) induces the expression of latent transforming growth factor beta 1 (LTGF-beta 1) through activation of c-fos and c-jun oncogenes. In this study we investigated whether IGF-1 induced latent TGF-beta 1 has autocrine effects on dermal fibroblasts and described a possible mechanism. Human dermal fibroblasts were treated with either vehicle, IGF-1 alone, or IGF-1 with either anti-TGF-beta 1 neutralizing antibody or mannose-6-phosphate (M6P) and levels of mRNAs for TGF-beta 1, collagenase and the pro alpha 1(I) chain of type I collagen were then evaluated by Northern analysis. Conditioned medium was also collected from treated and untreated cells and assayed for TGF-beta 1 protein by enzyme-linked immunosorbent assay. The results of the Northern analysis revealed a differential effect on the expression of the pro alpha 1(I) chain of type I collagen and collagenase in dermal fibroblasts: mRNA for the former being significantly increased in response to IGF-1 treatment while that for collagenase was markedly suppressed. These effects of IGF-1 were blocked to a significant extent by TGF-beta 1 neutralizing antibody at a concentration of 0.5-2.0 micrograms/ml. As the TGF-beta 1 induced by IGF-1 is inactive in the traditionally used mink lung epithelial cell growth inhibition assay, we explored the possible role of IGF-II/M6P receptors in facilitating these autocrine effects. The results showed that the greater than two-fold increase (201.9 +/- 38 vs 81.8 +/- 13, p < 0.05) in mRNA for the pro alpha 1(I) chain of type I collagen induced by IGF-1 was at least 60% inhibited by M6P in a time-dependent fashion. A direct correlation between the expression of TGF-beta 1 and the pro alpha 1(I) chain of type I collagen was found in response to either IGF-1 alone or IGF-1 with M6P. Treatment of cell cultures with TGF-beta 1 neutralizing antibody mimicked the effect of M6P. In contrast to the effects on expression of type I collagen, the level of collagenase mRNA was markedly reduced by IGF-1 alone and was restored by the administration of M6P. The levels of TGF-beta 1 in conditioned medium from treated and untreated cells showed a similar pattern to that of the mRNA detected by Northern analysis. These findings suggest that IGF-1 induces latent TGF-beta 1 and that the matrix-modulating autocrine effects of LTGF-beta 1 on dermal fibroblasts are facilitated by M6P/IGF-II receptors on these cells.     

Transforming growth factor-beta inhibits lipopolysaccharide-stimulated expression of inflammatory cytokines in mouse macrophages through downregulation of activation protein 1 and CD14 receptor expression

The septic shock that occurs in gram-negative infections is caused by a cascade of inflammatory cytokines. Several studies showed that transforming growth factor-beta1 (TGF-beta1) inhibits this septic shock through suppression of expression of the lipopolysaccharide (LPS)-induced inflammatory cytokines. In this study, we investigated whether TGF-beta1 inhibition of LPS-induced expression of inflammatory cytokines in the septic shock results from downregulation of LPS-stimulated expression of CD14, an LPS receptor. TGF-beta1 markedly inhibited LPS stimulation of CD14 mRNA and protein levels in mouse macrophages. LPS-stimulated expression of CD14 was dramatically inhibited by addition of antisense, but not sense, c-fos and c-jun oligonucleotides. Since TGF-beta1 pretreatment inhibited LPS-stimulated expression of c-fos and c-jun genes and also the binding of nuclear proteins to the consensus sequence of the binding site for activation protein 1 (AP-1), a heterodimer of c-Fos and c-Jun, in the cells, TGF-beta1 inhibition of CD14 expression may be a consequence of downregulation of AP-1. LPS-stimulated expression of interleukin-1beta and tumor necrosis factor alpha genes in the cells was inhibited by addition of CD14 antisense oligonucleotide. Also, TGF-beta1 inhibited the LPS-stimulated production of both inflammatory cytokines by the macrophages. In addition, TGF-beta1 inhibited expression of the two cytokines in several organs of mice receiving LPS. Thus, our results suggest that TGF-beta1 inhibition of LPS-stimulated inflammatory responses resulted from downregulation of CD14 and also may be a possible mechanism of TGF-beta1 inhibition of LPS-induced septic shock.     

Connective tissue growth factor and its correlation to other growth factors in experimental granulation tissue

Connective tissue growth factor (CTGF) is upregulated in a variety of fibrotic disorders, probably secondary to the activation and production of transforming growth factor-beta (TGF-beta). We have studied the expression of CTGF in a rat wound-healing model using Northern blot, in situ hybridization, and immunohistochemistry. The expression of CTGF mRNA in Northern blot and immunohistochemistry were correlated to the expression of TGF-beta1 and platelet-derived growth factor (PDGF). Northern hybridization showed the maximum expression of CTGF mRNA on day 14, whereas TGF-beta1 expression was maximal on days 7 and 14 and the time-related changes were smaller than for CTGF. PDGF A and PDGF B mRNA expressions were at maximum on day 14 and on day 21, respectively. In situ hybridization showed that fibroblast-like cells expressed CTGF most intensively, expression declining rapidly after day 14. CTGF mRNA and protein were found in blood vessel cells during the first week. In immunohistochemistry, all growth factors were expressed by fibroblast-like cells, macrophage-like cells, and blood vessels but CTGF-positive cells were fewer and were more restricted on days 5 and 7. These results demonstrate that CTGF expression together with TGF-beta and PDGF are up-regulated in wound healing, and CTGF expression in blood vessels suggests that CTGF is involved in angiogenesis.     

Differential modulation of basement membrane gene expression in human fibrosarcoma HT-1080 cells by transforming growth factor-beta 1. Enhanced type IV collagen and fibronectin gene expression correlates with altered culture phenotype of the cells

The effects of transforming growth factor-beta 1 (TGF-beta 1) on the regulation of basement membrane gene expression were studied in human fibrosarcoma HT-1080 cell cultures. Treatment of cells with TGF-beta 1 resulted in a time- and dose-dependent enhancement of type IV collagen and fibronectin gene expression, as detected both at the mRNA level by Northern hybridizations and at the protein level by semi-quantitative indirect immunofluorescence analyses. These changes were accompanied by profoundly altered morphology of the cell cultures. In contrast, laminin B1 and B2 chain, and nidogen mRNA levels remained unaltered by TGF-beta 1 in the same cultures, indicating uncoordinate modulation of the expression of basement membrane components by TGF-beta 1. Cycloheximide experiments provided evidence that the TGF-beta 1-elicited upregulation of the expression of the fibronectin gene is, but that of type IV collagen is not, entirely dependent on protein synthesis, suggesting two different mechanisms for enhancement of gene expression. Incubation of HT-1080 cells with TGF-beta 1 also resulted in a slightly enhanced expression of beta 1 and alpha 5 integrin mRNAs, as well as beta 1 integrin epitopes. Furthermore, incubation of the cells with anti-beta 1 integrin antibodies partially counteracted the TGF-beta 1-induced morphologic alterations. These studies provide evidence for the role of beta 1 integrins in TGF-beta 1 elicited alterations in HT-1080 cell culture morphology, possibly mediated by the expression of ligand proteins for these integrins, such as type IV collagen and fibronectin.     

Effect of CMW 1 bone cement on transforming growth factor-beta 1 expression by endothelial cells.

The present study examined the effects of in vitro challenge with an acrylic bone cement CMW 1 on the expression of transforming growth factor-beta 1 (TGF-beta 1) in human umbilical vein endothelial cells (HUVEC). The extracts in cell culture medium of the cements were tested, after 1 h and 7-day curing. Some cultures were also stimulated with interleukin-1 beta (IL-1 beta) or all-trans retinoic acid (ATRA). The expression of mRNA was evaluated by RT-PCR with specific primers. The release of TGF-beta 1 into the conditioned medium was evaluated by enzyme immunoassay. TGF-beta 1 mRNA was constitutively expressed by endothelial cells in the culture medium after 24 h. The incubation with the extracts of CMW 1, cured both for 1 h and 7 days, induced changes neither in mRNA expression, nor in the release of TGF-beta 1 into the conditioned medium, compared to the unstimulated cells. Even stimulation with ATRA, alone or added to the extracts at both curing times, affected neither mRNA expression nor TGF-beta 1 release, compared to the cells incubated with the cement alone or with the unstimulated cultures. The mRNA expression and the release were not changed by the stimulation with IL-1beta alone or added to the extract cured for 1 h. A significant decrease compared to the unstimulated cells was observed after the addition of IL-1 beta to the extract cured for 7 days. It was concluded that CMW 1 extract did not significantly modify TGF-beta 1 expression after 1-h curing, or after 7-day curing. Incubation with CMW 1 added with ATRA did not produce any changes in TGF-beta 1 synthesis. Incubation with cement extract after 7-day curing added with IL-beta 1 produced a significant reduction in TGF-beta 1 release.     

Transforming growth factor-beta 1 in experimental autoimmune neuritis. Cellular localization and time course.

Experimental autoimmune neuritis (EAN) is a monophasic inflammatory disorder of the peripheral nervous system that resolves spontaneously by molecular mechanisms as yet unknown. We have investigated whether the immunosuppressive cytokine transforming growth factor-beta 1 (TGF-beta 1) might be endogenously expressed in the peripheral nervous system of Lewis rats with actively induced and adoptive transfer EAN. TGF-beta 1 mRNA was upregulated to high levels in sensory and motor roots, spinal ganglia, and sciatic nerve as revealed by quantitative Northern blot analysis and in situ hybridization histochemistry, with peak levels just preceding the first signs of clinical recovery. TGF-beta 1 mRNA was localized to scattered round cells and dense cellular infiltrates, but only rarely to Schwann cell profiles. Double labeling studies revealed macrophages and subpopulations of T cells as the major cellular source of TGF-beta 1 mRNA. TGF-beta 1 protein was visualized immunocytochemically and localized to infiltrating mononuclear cells with peak expression around the same time as mRNA, in addition to some constitutive expression in axons and Schwann cells. Our studies suggest that the spontaneous recovery observed in Lewis rat EAN might be mediated by the endogenous elaboration of TGF-beta 1 within the peripheral nerve, and that macrophages might control their own cytotoxicity by expressing TGF-beta 1.     

ALK-5 mediates endogenous and TGF-beta1-induced expression of connective tissue growth factor in embryonic lung

Transforming growth factor-beta1 (TGF-beta1) has been implicated as a major negative regulator of lung branching morphogenesis. Since connective tissue growth factor (CTGF) is a downstream mediator of TGF-beta1 effects on mesenchymal cells, we hypothesized that TGF-beta1 induces CTGF expression in mouse embryonic lung explants and that CTGF mediates TGF-beta1 inhibition of branching morphogenesis. We show that addition of TGF-beta1 to the serum-free medium of embryonic day (E)12.5 lung explant cultures inhibited branching morphogenesis and induced CTGF mRNA expression in time- and dose-dependent manners. In contrast to basal endogenous CTGF protein, which was exclusively localized in the distal airway epithelium, TGF-beta1-induced CTGF protein was localized in both the epithelium and the mesenchyme. Addition of exogenous CTGF to culture medium directly inhibited branching morphogenesis. To identify the signal transduction pathway through which TGF-beta1 induces CTGF, we used SB431542, a specific inhibitor for TGF-beta type I receptor (TbetaRI)/ALK-5 to block TGF-beta1-induced Smad2/3 phosphorylation. Consequently, SB431542 stimulated normal branching morphogenesis and blocked TGF-beta1 inhibition of branching. Furthermore, SB-431542 blocked both endogenous and TGF-beta1-induced expression of CTGF mRNA and protein. These results demonstrate for the first time that TGF-beta1 induces CTGF expression in mouse embryonic lung explants, that CTGF inhibits branching morphogenesis, and that both endogenous and TGF-beta1-induced CTGF expression are mediated by the TbetaRI/ALK-5-dependent Smad2 signaling pathway.     

Regulation of mesangial cell growth by polypeptide mitogens. Inhibitory role of transforming growth factor beta.

Proliferation of mesangial cells is a common histologic abnormality in glomerular diseases. In vivo studies suggest a role for platelets and monocytes-macrophages in mediating glomerular hypercellularity. The authors recently reported that several peptide growth factors stimulate DNA synthesis and growth of human mesangial cells. This article reports that transforming growth factor beta (TGF-beta), a peptide released by inflammatory cells and platelets, inhibits DNA synthesis and growth of human mesangial cells. The stimulatory and inhibitory effects of these mitogens on DNA synthesis and growth was confirmed by autoradiography and cell counting. The inhibitory effect of TGF-beta is not mediated at the receptor level because TGF-beta did not inhibit the binding of epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) to mesangial cells. Because peptide growth factors that stimulate DNA synthesis in mesangial cells induce expression of PDGF mRNAs, the effect of TGF-beta on PDGF mRNAs expression induced by peptide growth factors was studied. TGF-beta did not lower the increased levels of PDGF mRNAs caused by EGF or PDGF. These data show that TGF-beta is a potent inhibitor of DNA synthesis and growth of mesangial cells. The mechanism of the inhibitory effect of TGF-beta remains to be determined.     

胶质瘤细胞c—fos和c—myc基因表达与血小板源生长因子B链的？…

目的 探讨胶质瘤细胞ｃ－ｆｏｓ和ｃ－ｍｙｃ基因表达和血小板源生长因子Ｂ链的纯合二聚体自分泌环活性的改变及其相互关系。方法 用原位杂交和免疫组化方法观察了６７例人胶质瘤组织。结果 ６７例中,ｃ－ｆｏｓ ｍＲＮＡ,ｃ－ｆｏｓ蛋白,ｃ－ｍｙｃ ｍＲＮＡ及ｃ－ｍｙｃ蛋白阳性率分别为;１００％,１００％,８５．１％,８３．６％。     

Advanced glycation end-products induce connective tissue growth factor-mediated renal fibrosis predominantly through transforming growth factor beta-independent pathway

Advanced glycation end-products (AGEs) play a critical role in diabetic nephropathy by stimulating extracellular matrix (ECM) synthesis. Connective tissue growth factor (CTGF) is a potent inducer of ECM synthesis and increases in the diabetic kidneys. To determine the critical role of CTGF in AGE-induced ECM accumulation leading to diabetic nephropathy, rats were given AGEs by intravenous injection for 6 weeks. AGE treatment induced a significant renal ECM accumulation, as shown by increases in periodic acid-Schiff-positive materials, fibronectin, and type IV collagen (Col IV) accumulation in glomeruli, and a mild renal dysfunction, as shown by increases in urinary volume and protein content. AGE treatment also caused significant increases in renal CTGF and transforming growth factor (TGF)-beta 1 mRNA and protein expression. Direct exposure of rat mesangial cells to AGEs in vitro significantly induced increases in fibronectin and Col IV production, which could be completely prevented by pretreatment with anti-CTGF antibody. AGE treatment also significantly increased both TGF-beta 1 and CTGF mRNA expression; however, inhibition of TGF-beta 1 mRNA expression by shRNA or neutralization of TGF-beta 1 protein by anti-TGF-beta 1 antibody did not significantly prevent AGE-increased expression of CTGF mRNA and protein. These results suggest that AGE-induced CTGF expression, predominantly through a TGF-beta 1-independent pathway, plays a critical role in renal ECM accumulation leading to diabetic nephropathy.