Cell-type-specific localization of transforming growth factor-beta 2 and transforming growth factor-beta 1 in the hamster ovary: differential regulation by follicle-stimulating hormone and luteinizing hormone.

Cell-type-specific localization and gonadotropin regulation of transforming growth factor-beta 1 (TGF-beta 1) and transforming growth factor-beta 2 (TGF-beta 2) in the hamster ovary were evaluated immunohistochemically under three conditions: (1) during the estrous cycle (Day 1 = estrus; Day 4 = proestrus); (2) after the blockade of periovulatory gonadotropin surges by phenobarbital, and (3) after FSH and/or LH treatment of long-term hypophysectomized hamsters. Ovarian TGF-beta 1 activity was primarily localized in theca and interstitial cells. The activity increased moderately but significantly after the preovulatory LH surge and reached a peak at 0900 h, Day 2 h; oocytes showed considerable activity. TGF-beta 1 immunoreactivity subsequently fell to low levels in theca-interstitial cells through 0900 h, Day 4. Significant TGF-beta 2 immunoreactivity appeared after the surge, mainly in the granulosa cells of both preantral and antral follicles; a few interstitial cells surrounding preantral follicles showed discrete staining. TGF-beta 2 immunoreactivity in granulosa cells and in interstitial cells next to preantral follicles reached a peak at 0900 h, Day 1, and persisted up to 0900 h, Day 2; oocytes showed no staining. Phenobarbital treatment blocked the appearance of TGF-beta 1 and TGF-beta 2 immunoreactivities at 1600 h, Day 4; however, a rebound in immunoreactivities was observed with the onset of the surge after a 1-day delay. Replacement of LH to long-term hypophysectomized hamsters resulted in a marked increase in TGF-beta 1 immunoreactivity in the interstitial cells, but FSH, although it induced follicular development, did not influence ovarian TGF-beta 1 activity. Treatment with FSH, however, induced a massive increase in TGF-beta 2 immunoreactivity in the granulosa cells of newly developed antral and preantral follicles but not in the interstitial cells; LH, on the other hand, had no significant effect on TGF-beta 2 activity. Treatment with FSH and LH combined resulted in a dramatic increase in TGF-beta 2 immunoreactivity in granulosa and interstitial cells and in TGF-beta 1 in theca and interstitial cells comparable to their peak activity in intact animals. Western analyses substantiated the presence of TGF-beta 1 and TGF-beta 2 in the hamster ovary and the specificity of immunolocalization. These studies, therefore, provide critical evidence that TGF-beta 1 and TGF-beta 2 in the hamster ovary are expressed in specific cell types and that their expression is differentially regulated by LH and FSH, respectively.     

Negative regulation of hepatocyte growth factor gene expression in human lung fibroblasts and leukemic cells by transforming growth factor-beta 1 and glucocorticoids.

Hepatocyte growth factor (HGF), a mesenchymal-derived factor which regulates growth, motility, and morphogenesis of epithelial and endothelial cells, functions as a hepatotrophic and renotrophic factor for regeneration of the liver and kidney. We have now obtained evidence that transforming growth factor-beta 1 (TGF-beta 1) and glucocorticoids are negative regulators for HGF gene expression. When TGF-beta 1 or dexamethasone was added to cultures of MRC-5 human embryonic lung fibroblasts and HL-60 human promyelocytic leukemic cells, the amount of HGF secreted into the culture medium was inhibited to 30-40% of that of control cultures by 10 ng/ml TGF-beta 1 and to 40-50% by 10(-6) M dexamethasone. The inhibitory effect of TGF-beta 1 and dexamethasone on HGF synthesis in MRC-5 cells was additive, thereby suggesting that TGF-beta 1 and dexamethasone exert effects through distinct mechanisms. Hydrocortisone also inhibited HGF synthesis with the same potency as dexamethasone; however, testosterone, estriol, and beta-estradiol had no effect. The rate of HGF synthesis in MRC-5 cells, as measured by pulse labeling with [35S]methionine and subsequent immunoprecipitation, was suppressed to 30-40% of the control with 10 ng/ml TGF-beta 1, and to 30-45% by 10(-6) M dexamethasone. HGF mRNA levels in MRC-5 cells and HL-60 cells were dose-dependently suppressed by TGF-beta 1 and dexamethasone; 10 ng/ml TGF-beta 1 suppressed HGF mRNA levels to 32% and 35% of control culture, respectively, in MRC-5 cells and HL-60 cells, and 10(-6) M dexamethasone suppressed to 43% and 38%, respectively. Thus, TGF-beta 1 and glucocorticoids seem to inhibit HGF synthesis by suppressing the expression of the HGF gene. We propose that a negative regulation of HGF gene expression by TGF-beta 1 or glucocorticoids may be involved in physiological or pathological processes during tissue regeneration.     

Endothelial cell regulation by transforming growth factor-beta.

Pronounced changes including growth inhibition, increased matrix deposition and suppression of cell-associated proteolytic activity, take place in endothelial cells (EC) upon the application of TGF-beta. Interrelationships between these effects have shed some light on the mechanism of action of TGF-beta and on its role in regulating EC function vis-a-vis angiogenesis. For instance, preliminary evidence has indicated that increased levels of certain matrix components may be partly responsible for the antiproliferative action of TGF-beta. In addition, TGF-beta and bFGF have opposing effects on cellular proteolytic balance which may contribute to the antagonistic effect that TGF-beta has on bFGF-induced EC growth and possibly to the anti-angiogenic effect exerted by TGF-beta under certain circumstances. Of particular interest in this regard is the fact that physical contact between EC and vascular mural cells in EC:mural cell cocultures has been found to generate active TGF-beta, thus further implicating TGF-beta in the maintenance of the quiescent, differentiated aggregation of EC as found in vascular structures in vivo. While more information is needed to define what, if any role TGF-beta plays in endothelial differentiation, it is to be noted that many of the cellular and biochemical processes affected by TGF-beta are linked to differentiation. It is therefore possible that the growth inhibition of EC by TGF-beta primes them for differentiation and/or is critical for the maintenance of a differentiated state.     

Transforming growth factor-beta1, transforming growth factor-beta2, and transforming growth factor-beta3 enhance ovarian cancer metastatic potential by inducing a Smad3-dependent epithelial-to-mesenchymal transition

Transforming growth factor-beta (TGF-beta) is thought to play a role in the pathobiological progression of ovarian cancer because this peptide hormone is overexpressed in cancer tissue, plasma, and peritoneal fluid. In the current study, we investigated the role of the TGF-beta/Smad3 pathway in ovarian cancer metastasis by regulation of an epithelial-to-mesenchymal transition. When cancer cells were cultured on plastic, TGF-beta1, TGF-beta2, and TGF-beta3 induced pro-matrix metalloproteinase (MMP) secretion, loss of cell-cell junctions, down-regulation of E-cadherin, up-regulation of N-cadherin, and acquisition of a fibroblastoid phenotype, consistent with an epithelial-to-mesenchymal transition. Furthermore, Smad3 small interfering RNA transfection inhibited TGF-beta-mediated changes to a fibroblastic morphology, but not MMP secretion. When cancer cells were cultured on a three-dimensional collagen matrix, TGF-beta1, TGF-beta2, and TGF-beta3 stimulated both pro-MMP and active MMP secretion and invasion. Smad3 small interfering RNA transfection of cells cultured on a collagen matrix abrogated TGF-beta-stimulated invasion and MMP secretion. Analysis of Smad3 nuclear expression in microarrays of serous benign tumors, borderline tumors, and cystadenocarcinoma revealed that Smad3 expression could be used to distinguish benign and borderline tumors from carcinoma (P = 0.006). Higher Smad3 expression also correlated with poor survival (P = 0.031). Furthermore, a direct relationship exists between Smad3 nuclear expression and expression of the mesenchymal marker N-cadherin in cancer patients (P = 0.0057). Collectively, these results implicate an important role for the TGF-beta/Smad3 pathway in mediating ovarian oncogenesis by enhancing metastatic potential.     

Identification of a gene family regulated by transforming growth factor-beta

We have identified two related genes whose mRNAs are increased after treatment with transforming growth factor-beta (TGF-beta 1). Mouse AKR-2B cells were treated with TGF-beta 1 in the presence of cyclohexamide and a cDNA library was subjected to differential screening. Several TGF-beta-induced genes (beta IG) were isolated and two of these, beta IG-M1 and beta IG-M2, were characterized. beta IG-M1 and beta IG-M2 RNAs were significantly increased after TGF-beta 1 treatment and both were superinduced in the presence of cyclohexamide. cDNA sequence analysis of beta IG-M1 showed that it encoded a 379-amino-acid protein which was 81% homologous to CEF-10, a v-src and TPA-inducible gene, and identical to cyr61, a gene induced by serum in growth-arrested BALB-3T3 cells. cDNA sequence analysis of beta IG-M2 showed that it encoded a 348-amino-acid protein that was 50% homologous to beta IG-M1. Thirty-eight cysteine residues are conserved between beta IG-M1 and beta IG-M2, which are clustered at the amino and carboxy ends: The middle regions of the two proteins are cysteine free and display the highest degree of nonhomology. Both proteins contain an amino-terminal cysteine-rich motif common to insulin-like growth factor binding proteins and a carboxy-terminal domain with strong homology to a motif found near the carboxy-terminal of the malarial circumsporozoite protein which may be involved in cell adhesion. The regulation of mRNA encoding these proteins by TGF-beta 1 suggests that they may be involved in mediating some of the pleiotropic effects of this multipotent modulator of cell growth and differentiation.