Expression of transforming growth factor-β1 and β2 in rat glomeruli. The transforming growth factors-β are potent modulators of cell growth and extracellular matrix metabolism in most types of ...cultured cells. The distribution and functions of TGF-β in vivo are less well known. We utilized several different techniques including northern blots, a CC1-64 cell growth inhibition assay, and sandwich enzyme-linked immunosorbent assays (SELISA) to examine the expression of TGF-β1 and TGF-β2 in rat glomeruli. High levels of TGF-β1 mRNA and protein were found in glomeruli (56 ± 22 ng TGF-β1/g tissue). These levels were several-fold higher than those present in whole kidney (10 ± 5 ng/g). TGF-β2 mRNA was present in glomeruli but was not detected in whole kidney. TGF-β2 concentrations by SELISA were 19 ± 8 ng TGF-β2/g in glomeruli and less than 5 ng/g in whole kidney. Since TGF-β has such marked effects on cell growth, we also examined whether alterations in TGF-β expression were associated with the renal hypertrophy which follows unilateral nephrectomy. Expression of TGF-β1 mRNA decreased in glomeruli following nephrectomy. However, this was not associated with a significant fall in glomerular TGF-βl protein concentration. Whole kidney levels of TGF-β1 and its mRNA were unchanged following nephrectomy. Similar results were obtained for TGF-β2. Our data document the presence of high concentrations of TGF-β1 and β2 and their corresponding mRNAs in normal rat glomeruli. These results suggest that TGF-β may play important regulatory roles in the normal glomerulus.
Recent experimental work has identified a novel intracellular binding site for the synthetic progestin, Gestodene, that appears to be uniquely expressed in human breast cancer cells. Gestodene is ...shown here to inhibit the growth of human breast cancer cells in a dose-dependent fashion, but has no effect on endocrine-responsive human endometrial cancer cells. Gestodene induced a 90-fold increase in the secretion of transforming growth factor-beta (TGF-beta) by T47D human breast cancer cells. Other synthetic progestins had no effect, indicating that this induction is mediated by the novel Gestodene binding site and not by the conventional progesterone receptor. Furthermore, in four breast cancer cell lines, the extent of induction of TGF-beta correlated with intracellular levels of Gestodene binding site. No induction of TGF-beta was observed with the endometrial cancer line, HECl-B, which lacks the Gestodene binding site, but which expresses high levels of progesterone receptor. The inhibition of growth of T47D cells by Gestodene is partly reversible by a polyclonal antiserum to TGF-beta. These data indicate that the growth-inhibitory action of Gestodene may be mediated in part by an autocrine induction of TGF-beta.
Transforming growth factor beta 1 (TGF-beta 1) has been shown to play a prominent role in controlling proteoglycan synthesis and breakdown as measured following addition to organ cultures of calf ...articular cartilage (Morales, T. I., and Roberts, A. B., J. Biol. Chem., 263, 12,828-12,831, 1988). In this study, we compare two closely related TGF-beta isoforms, TGF-beta 1 and TGF-beta 2, both by assessing the effects of exogenous peptide as well as by analyzing the biosynthesis and total amount of these two isoforms in cartilage explants. Added exogenously, TGF-beta 1 and TGF-beta 2 induce a comparable increase in proteoglycan synthesis over basal controls with saturation at approximately 5 ng/ml. Synthesis of TGF-beta by basal calf cartilage cultures is demonstrated by (i) immunolocalization of intracellular TGF-beta, (ii) Northern blot analysis of steady-state mRNA levels, and (iii) immunoprecipitation of metabolically labeled TGF-beta from tissue extracts and conditioned culture medium. The net amount of extractable TGF-beta 1 and TGF-beta 2 in the basal cartilage cultures was assessed by a functional assay involving inhibition of proliferation of CCL-64 mink lung epithelial cells and by sandwich enzyme-linked immunosorbent assay. The predominant isoform was TGF-beta 1 (60-85%) and the total TGF-beta 1 + TGF-beta 2 was in excess of the amount required for maximal activation of proteoglycan synthesis. The level of both isoforms was maintained relatively constant between Days 2 and 7 of culture despite a sharp (approximately two to fourfold) drop in proteoglycan synthesis. This suggests that cartilage contains a large pool of TGF-beta which is not readily accessible to the chondrocyte. We propose that much of the polypeptide is sequestered in the matrix awaiting release upon demand.
The mechanism by which transforming growth factor-beta1 (TGF-beta1) induces apoptosis of prostate epithelial cells was studied in the NRP-154 rat prostate epithelial cell line. TGF-beta 1 ...down-regulates expression of Bcl-xL and poly(ADP-ribosyl)polymerase (PARP), promotes cytochrome c release, up-regulates expression of latent caspase-3, and activates caspases 3 and 9. We tested the role of Bcl-xL in this cascade by stably overexpressing Bcl-xL to prevent loss by TGF-beta 1. Clones overexpressing Bcl-xL are resistant to TGF-beta 1 with respect to induction of apoptosis, cytochrome c release, activation of caspases 9 and 3, and cleavage of PARP; yet they remain sensitive to TGF-beta 1 by cell cycle arrest, induction of both fibronectin and latent caspase-3 expression, and loss of PARP expression. We show that Bcl-xL associates with Apaf-1 in NRP-154 cells; but this association does not inhibit the activation of caspases 9 and 3 by cytochrome c. Together, our data suggest that TGF-beta1 induces apoptosis through loss of Bcl-xL, leading to cytochrome c release and the subsequent activation of caspases 9 and 3. Moreover, our data demonstrate that the antiapoptotic effect of Bcl-xL occurs by inhibition of mitochondrial cytochrome c release and not through antagonizing Apaf-1-dependent processing of caspases 9 and 3.
Survivin is a prosurvival protein overexpressed in many cancers through mechanisms that remain poorly explored, and is implicated in control of tumor progression and resistance to cancer ...chemotherapeutics. Here, we report a critical role for survivin in the induction of apoptosis by transforming growth factor-beta (TGF-beta). We show that TGF-beta rapidly downregulates survivin expression in prostate epithelial cells, through a unique mechanism of transcriptional suppression involving Smads 2 and 3, Rb/E2F4, and the cell-cycle repressor elements CDE and CHR. This TGF-beta response is triggered through a Smad2/3-dependent hypophosphorylation of Rb and the subsequent association of the Rb/E2F4 repressive complex to CDE/CHR elements in the proximal region of the survivin promoter. Viral-mediated gene delivery experiments, involving overexpressing or silencing survivin, reveal critical roles of survivin in apoptosis induced by TGF-beta alone or in cooperation with cancer therapeutic agents. We propose a novel TGF-beta/Rb/survivin axis with a putative role in the functional switch of TGF-beta from tumor suppressor to tumor promoter. PUBLICATION ABSTRACT
Survivin is a prosurvival protein overexpressed in many cancers through mechanisms that remain poorly explored, and is implicated in control of tumor progression and resistance to cancer ...chemotherapeutics. Here, we report a critical role for survivin in the induction of apoptosis by transforming growth factor-b (TGF-b). We show that TGF-b rapidly downregulates survivin expression in prostate epithelial cells, through a unique mechanism of transcriptional suppression involving Smads 2 and 3, Rb/E2F4, and the cell-cycle repressor elements CDE and CHR. This TGF-b response is triggered through a Smad2/3-dependent hypophosphorylation of Rb and the subsequent association of the Rb/E2F4 repressive complex to CDE/CHR elements in the proximal region of the survivin promoter. Viral-mediated gene delivery experiments, involving overexpressing or silencing survivin, reveal critical roles of survivin in apoptosis induced by TGF-b alone or in cooperation with cancer therapeutic agents. We propose a novel TGF-b/Rb/survivin axis with a putative role in the functional switch of TGF-b from tumor suppressor to tumor promoter.Oncogene (2008) 27, 5326-5338; doi:10.1038/onc.2008.165; published online 26 May 2008
The standard therapy for advanced prostate cancer is androgen ablation. Despite transitory responses, hormonally treated patients ultimately relapse with androgen-independent disease that is ...resistant to further hormonal manipulation and cytotoxic chemotherapy. To develop an additional approach to the treatment of advanced prostate cancer, we have been studying the signal transductions controlling the growth of human androgen-independent prostate carcinoma cell lines. We report here that elevation of intracellular cAMP markedly inhibits the growth of the hormone-refractory cell line PC-3. To examine the mechanism of cAMP action in PC-3 cells, we tested the effect of the cAMP analog dibutyryl cAMP (Bt2-cAMP) on the regulation of the potent negative growth factor transforming growth factor beta (TGF-beta). Bt2-cAMP selectively induced the secretion of TGF-beta 2 and not TGF-beta 1 by PC-3 cells. This TGF-beta 2 was shown to be bioactive by using the CCL-64 mink lung cell assay. TGF-beta 1 was not activated despite being present at 3-fold higher concentrations than TGF-beta 2. Northern analysis showed that Bt2-cAMP induced an increase in the five characteristic TGF-beta 2 transcripts and had no effect on the level of TGF-beta 1 or TGF-beta 3 transcripts. TGF-beta 2 induction was only weakly enhanced by cycloheximide and was completely inhibited by actinomycin D. These data show that Bt2-cAMP induces the expression of active TGF-beta 2 by PC-3 prostate carcinoma cells, suggesting a new approach to the treatment of prostate cancer and a new molecular mechanism of cAMP action.
Transforming growth factor- beta 1 (TGF- beta 1), which is induced in the prostate following castration, has been speculated to mediate apoptosis of epithelial cells during prostatic involution. ...Here, we report the first evidence of a direct effect of TGF- beta on induction of apoptosis in prostatic epithelial cells in vitro, using NRP-152 nontumorigenic and NRP-154 tumorigenic rat prostatic epithelial cell lines. TGF- beta 1 induces apoptosis of both cell lines within 24 h, as shown by a decrease in cell viability, in situ DNA nick-end labeling, and internucleosomal DNA fragmentation. Moreover, the ability of TGF- beta to induce apoptosis of NRP-152 is strictly dependent on culture conditions, because dexamethasone enhances while insulin and insulin-like growth factor-I specifically block apoptosis induced by TGF- beta . We suggest that TGF- beta s are direct physiological regulators of apoptosis of prostatic epithelial cells.
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