Androgen influences transforming growth factor-beta1 gene expression in human adrenocortical cells

Sex steroid hormones have been shown to affect adrenocortical function and trophism, yet little is known about androgen action in human adrenocortical gland. In this study we examined the effects of androgens on transforming growth factor-beta1 (TGF/beta1) production by the human adrenocortical cell line, NCI-H295, which we recently demonstrated to express androgen receptor and whose growth is significantly reduced by dihydrotestosterone (DHT) treatment. TGFbeta1 is an important regulator of human adrenal development, with marked effects on steroid-producing cell function, and the production of distinct TGFbeta subtypes has been suggested to be regulated by steroid hormones in several tissues. To address potential TGFbeta1 induction by DHT, quantitative PCR and enzyme-linked immunoadsorbent assay were performed in NCI-H295 cells treated with DHT (from 10(-12)-10(-9) mol/L). DHT led to a significant dose-dependent increase in TGFbeta1 messenger ribonucleic acid expression and in biologically active TGFbeta1 protein levels in the conditioned media of NCI-H295 cells, demonstrating that androgen can induce TGFbeta1 expression and production. TGFbeta1 (10(-7)-10(-6) mol/L) was capable of significantly reducing cell proliferation (P < 0.05) after 24 h of treatment, as assessed by measuring [3H]thymidine incorporation in NCI-H295 cells. The addition of TGFbeta1-neutralizing antibody to cell cultures treated with different DHT concentrations (10(-9) and 10(-10) mol/L) blocked the inhibitory effect of TGF/beta1 on adrenocortical cell proliferation. These findings suggest that TGFbeta1 exerts an inhibitory action on adrenocortical cell proliferation. Therefore, it might be reasonable to suppose that DHT could also influence human adrenocortical cell growth by involving TGFbeta1.     

Androgen regulation of prostate morphoregulatory gene expression: Fgf10-dependent and -independent pathways

Androgens are essential and sufficient for prostate gland morphogenesis; however, the downstream gene targets that mediate this action are unclear. To identify androgen-regulated genes involved in prostate development, we used short-term organ culture and examined the effect of testosterone on the expression of several critical prostate morphoregulatory genes. Rat ventral prostates (VP) and lateral prostates (LP) were collected at birth, and contralateral lobes were cultured for 18 h in the presence or absence of 10 nM testosterone with or without OH-flutamide to block residual androgens. Gene expression was quantitated using real-time RT-PCR. Although expression of Fgf10, Nkx3.1, and Ptc was increased in both prostate lobes, other genes were regulated by testosterone in a lobe-specific manner. This included up-regulation of epithelial genes FgfR2iiib, Shh, Hoxb13, and Bmp7 in the VP specifically and down-regulation of mesenchymal genes Wnt5a (VP) and Bmp4 (LP). Thus, in addition to stimulation of homeobox genes and paracrine-acting growth factors, androgens may positively regulate prostatic development through suppression of growth inhibitory genes. Because previous studies revealed a similar gene regulation pattern in response to exogenous Fgf10, experiments were performed to identify androgen-regulated genes mediated through Fgf10 signaling. Short-term VP and LP cultures with FgfR antagonist PD173074 and Mek inhibitor U0126 identified epithelial Shh and Hoxb13 up-regulation by androgens to be Fgf10-dependent. We propose that androgen regulation of prostate development is mediated through positive and negative regulation of multiple morphoregulatory genes acting in combination through complex gene networks. Lobe-specific responses may provide a developmental basis for prostate gland heterogeneity.     

Promoter polymorphism of transforming growth factor-β1 gene and ulcerative colitis

AIM： To elucidate the possible difference in two promoter polymorphisms of the transforming growth factor-β1 （TGF-β1） gene （-800G 〉 A, -509C 〉 T） between ulcerative colitis （UC） patients and normal subjects.
METHODS： A total of 155 patients with established ulcerative colitis and 139 normal subjects were selected as controls. Two single nucleotide polymorphisms within the promoter region of TGF-β1 gene （-509C 〉 T and -800G 〉 A） were genotyped using PCR-RFLP. RESULTS： There was a statistically significant difference in genotype and allele frequency distributions between UC patients and controls for the -800G 〉 A polymorphism of the TGF-β1 gene （P 〈 0.05）. The frequency of the TGF-β1 gene polymorphism at position -800 showed that the AA genotype and the allele A frequencies significantly differed between the patients and healthy controls （P 〈 0.05）. At position -509, there was no statically significant difference in genotype and allele frequency between the patients and control subjects.
CONCLUSION： The results of our study indicate that there is a significant difference in both allele and genotype frequency at position -800G 〉 A of TGF-β1 gene promoter between Iranian patients with UC and normal subjects.     

Differential effects of transforming growth factor-beta1 on cellular proliferation in the developing prostate

TGFbeta1 plays an important role in the growth of the prostate and has been reported to stimulate or inhibit the proliferation of prostatic epithelia. We show here that Tgfbeta1, Tgfbeta2, and Tgfbeta3 mRNA expression correlated with developmental growth of the prostate. Recombinant TGFbeta1 inhibited the growth of the prostate when added to cultures of ventral prostate (VP) organs grown in vitro. Interestingly, TGFbeta1 had contrasting effects on cellular proliferation; it stimulated proliferation at the periphery of the organs (distal to urethra), but inhibited proliferation in the center of the organs (proximal to urethra). We speculate that differential effects on proliferation may be determined by the level of cellular differentiation, because cells at the periphery are undifferentiated whereas those in the center are more highly differentiated. TGFbeta1 also stimulated branching morphogenesis at growing ductal tips at the perimeter of the VP. To investigate potential mechanisms of TGFbeta1 action, we examined the three-dimensional distribution of smooth muscle in prostatic organs after treatment with TGFbeta1. TGFbeta1 showed a significant effect on the distribution of smooth muscle within VPs, which may mediate part of its effect on proliferation. Finally, we addressed how testosterone and TGFbeta1 might affect gene expression in our developmental system. Testosterone repressed the expression of Tgfbeta2 mRNA in the prostate, whereas TGFbeta1 showed a modest repression of fibroblast growth factor-10 mRNA. It appeared that the effects of these factors were more pronounced in a model of prostatic mesenchyme devoid of epithelia than in prostatic organs (containing epithelia).     

Regulation of transforming growth factor-beta1 expression by granulocyte macrophage-colony-stimulating factor in leiomyoma and myometrial smooth muscle cells

Human myometrium and leiomyomas express granulocyte macrophage-colony-stimulating factor (GM-CSF), transforming growth factor-beta (TGFbeta), and their receptors. Overexpression of TGFbeta and, to a limited extent, GM-CSF has been associated with tissue fibrosis throughout the body, including leiomyomas. The objective of the present study was to determine the action of GM-CSF on leiomyoma and myometrial smooth muscle cells (LSMC and MSMC) and examine whether the action of GM-CSF is mediated through the induction of TGFbeta1 expression. Using competitive quantitative RT-PCR and enzyme-linked immunosorbent assay, we found that LSMC express significantly higher GM-CSF messenger ribonucleic acid (mRNA; 0.6 +/- 0.1 x 10(3) copies of mRNA/microg total RNA) and protein (0.75 +/- 0.2 ng/mL) than MSMC (0.5 +/- 0.1 x 10(2) copies of mRNA and 0.45 +/- 0.07 ng/mL protein; P < 0.05). In addition, LSMC expressed significantly higher TGFbeta1 mRNA (1.6 +/- 0.3 x 10(4) copies of mRNA/microg total RNA) than MSMC (2.4 +/- 0.4 x 10(3) copies) and synthesized and secreted more TGFbeta1 protein (1.7 +/- 0.2 vs. 0.5 +/- 0.02 ng/mL); whereas MSMC contained more cell-associated TGFbeta1 (56.2 +/- 1.2 ng/mL) than LSMC (35.2 +/- 1.2 ng/mL; P < 0.05). We found that GM-CSF (0.01-100 ng/mL) has limited mitogenic activity for LSMC but not for MSMC determined by the rate of [3H]thymidine incorporation and cell proliferation assay. However, GM-CSF at 1 ng/mL increased its own production, the expression of TGFbeta1 mRNA, the cell-associated TGFbeta1 protein content in both cell types, and TGFbeta1 released into the culture-conditioned medium of LSMC (P < 0.05). TGFbeta1 also increased its own mRNA and protein expression, but had no effect on cell-associated TGFbeta1 in both cell types (P < 0.05). Cotreatment of LSMC and MSMC with GM-CSF and TGFbeta1 induced changes similar to those produced by GM-CSF in both cells. In conclusion, our data suggest that GM-CSF is not a mitogen for MSMC and LSMC, but it regulates its own expression and the expression of TGFbeta1 by these cells, a regulatory interaction that may account for the GM-CSF-induced tissue fibrosis that occurs in leiomyomas.     

Regulation of transforming growth factor beta 1 gene expression by the product of the retinoblastoma-susceptibility gene.

Transforming growth factor beta (TGF-beta) isoforms inhibit the growth of many cell types and block progression of the cell cycle by inhibiting events in late G1 phase. The retinoblastoma gene product, RB, also has properties of a cell-cycle regulatory factor. It remains underphosphorylated in the presence of TGF-beta and has been shown to repress the activity of the c-fos promoter, resulting in inhibition of transit through the cell cycle. These observations led us to examine effects of human RB on the expression of the human TGF-beta 1 gene. Using chimeric TGF-beta 1 promoter-chloramphenicol acetyltransferase gene constructs, we show that RB induces TGF-beta 1 gene expression in CCL-64 mink lung epithelial cells and A-549 human lung adenocarcinoma cells but represses its expression in NIH 3T3 and AKR-2B mouse cells. Several sequences homologous to the c-fos RB control element were identified in the TGF-beta 1 promoter. These results demonstrate that human RB can regulate TGF-beta 1 gene expression negatively or positively depending on the cell type.     

Quercetin inhibits the growth of leukemic progenitors and induces the expression of transforming growth factor-beta 1 in these cells

We previously showed that quercetin (3,3',4',5,7 pentahydroxyflavone) inhibits in a dose-dependent manner the growth of acute leukemias and is able to enhance the antiproliferative activity of cytosine arabinoside. We show here that quercetin inhibits the clonogenic activity of 20 of 22 acute leukemias (AL; 4 M1-AML, 3 M2-AML, 2 M3-AML, 3 M4-AML, 3 M5-AML, and 7 ALL). In the present report, we show that the induction of transforming growth factor-beta 1 (TGF-beta 1) in leukemic blasts is one of the growth-inhibitory mechanisms of quercetin in these cells. This observation was supported by the following data. (1) Quercetin-sensitive leukemic blasts, when treated with quercetin, secrete large amounts of TGF-beta 1 in the medium and show positivity for TGF-beta 1-immunoreactive material in the cytoplasm. (2) At a concentration of 8 mumol/L, antisense TGF-beta 1 oligonucleotides prevent the growth-inhibitory action of quercetin. (3) Anti-TGF-beta 1 neutralizing monoclonal antibodies can prevent almost completely the growth-inhibitory activity of quercetin. The analysis of quercetin- resistant cases confirmed as well the central role of TGF-beta 1 in the growth-inhibitory activity of quercetin. In conclusion, quercetin can act as a cytostatic agent for leukemic cells by modulating the production of TGF-beta 1.     

Autoimmune manifestations in the transforming growth factor-beta 1 knockout mouse

Targeted disruption of the transforming growth factor-beta 1 (TGF-beta 1) gene in mice results in the development of a massive multifocal inflammatory disease in many tissues. Because no detectable pathogen was identified, we examined whether autoimmune mechanisms played a role in initiating or maintaining the inflammatory disease. The serum of TGF- beta 1 knockout mice contained elevated titers of antibodies to nuclear antigens (ssDNA, dsDNA, Sm, and RNP) as well as reactivity against the 16/6 idiotype (16/6 Id). In addition, Ig deposits were detected in renal glomeruli of TGF- beta 1 knockout mice. Transplantation of TGF- beta 1 knockout hematopoietic cells into normal irradiated recipients resulted in a similar profile of autoantibody production as well as in the induction of inflammatory lesions. Our results describe autoimmune activity that ensues when the TGF-beta 1 cytokine is absent.     

心房颤动患者心房组织碱性成纤维细胞生长因子及转化生长因子-β_1基因表达与心房纤维化的关系

目的探讨心房颤动(简称房颤)患者心房纤维化的分子机制。方法75例风湿性心脏瓣膜病(简称风心病)接受换瓣手术者按术前心脏节律分为窦性心律组(n=34),阵发性房颤组(n=11),持续性房颤组(n=30);分别采用半定量逆转录聚合酶链反应和免疫组化技术测定Ⅰ型胶原(ColⅠ)、碱性成纤维细胞生长因子(bFGF)和转化生长因子-β1(TGFβ-1)的mRNA和蛋白含量。结果阵发性房颤组、持续性房颤组心房组织胶原容积分数明显高于窦性心律组;持续性房颤组增加更明显(P均<0.05)。心房组织胶原容积分数与左房内径、房颤持续时间呈正相关。与窦性心律组比较,ColⅠ、bFGF和TGFβ-1的mRNA和蛋白表达水平在阵发性房颤组、持续性房颤组均显著上调,持续性房颤组相对于阵发性房颤组亦明显增高(P均<0.05)。同时ColⅠ、bFGF的mRNA、蛋白表达均与房颤持续时间及左房内径呈正相关。结论心房组织中bFGF和TGFβ-1的基因表达上调可能是成纤维细胞增殖导致胶原增加和心房纤维化的分子机制之一。     

Regulation of human lung fibroblast C1q-receptors by transforming growth factor-beta and tumor necrosis factor-alpha

Transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) are two polypeptide mediators which are believed to play a role in the evolution of idiopathic pulmonary fibrosis (IPF). We have evaluated the effect of these two substances on the expression of receptors for collagen (cC1q-R) and globular (gC1q-R) domains of C1q and on type I collagen in human lung fibroblasts. Two fibroblast subpopulations differing in C1q receptor expression were obtained by culturing human lung explants in medium containing fresh human serum and heated plasma-derived serum and separating them based on C1q binding [Narayanan, Lurton and Raghu: Am J Resp Cell Mol Biol. 1998; 17:84]. The cells, referred to as HH and NL cells, respectively, were exposed to TGF-beta and TNF-alpha in serum-free conditions. The levels of mRNA were assessed by in situ hybridization and Northern analysis, and protein levels compared after SDS-polyacrylamide gel electrophoresis and Western blotting. NL cells exposed to TGF-beta and TNF-alpha contained 1.4 and 1.6 times as much cC1q-R mRNA, respectively, whereas in HH cells cC1q-R mRNA increased 2.0- and 2.4-fold. The gC1q-R mRNA levels increased to a lesser extent in both cells. These increases were not reflected in protein levels of CC1q-R and gC1q-R, which were similar to or less than controls. Both TGF-beta and TNF-alpha also increased procollagen [I] mRNA levels in both cells. Overall, TNF-alpha caused a greater increase and the degree of response by HH fibroblasts to both TGF-beta and TNF-alpha was higher than NL cells. These results indicated that TGF-beta and TNF-alpha upregulate the mRNA levels for cC1q-R and collagen and that they do not affect gC1q-R mRNA levels significantly. They also indicated different subsets of human lung fibroblasts respond differently to inflammatory mediators.     

风湿性心脏病心房颤动患者心房组织胶原及转化生长因子β基因表达的研究

目的　观察风湿性心脏病 (风心病 )心房颤动 (房颤 )患者心房组织中Ⅰ型和Ⅲ型胶原和转化生长因子 β(TGF β1、TGF β2 )基因表达的改变。方法　将 4 9例风心病二尖瓣病变接受换瓣手术者于术中获取的右心耳 (约 10 0mg)分为三组 ,其中窦性心律组 2 0例 ,阵发性房颤组 8例 ,持续性房颤组2 1例 (≥ 6个月 ) ,以 β肌动蛋白为内参照基因 ,通过半定量逆转录 聚合酶链反应 (RT PCR)技术 ,测定各组心房组织中Ⅰ型和Ⅲ型胶原和TGF β1、TGF β2 的mRNA的含量。结果　与风心病窦性心律组相比 ,Ⅰ型胶原、Ⅲ型胶原、TGF β1mRNA表达在阵发性和持续性房颤组均显著增加 ;与阵发性房颤组相比 ,持续性房颤组中这三种基因的表达继续明显增加 ;三组间TGF β2 mRNA表达差异无显著性。结论　风心病患者心房组织中Ⅰ型胶原、Ⅲ型胶原和TGF β1mRNA表达上调可能是心房纤维化发生的分子机制之一 ,为风心病患者房颤的发生和维持提供了结构基础