Wiesław H. Trzeciak

Steroidogenic factor 1 gene transcription is inhibited by transforming growth factor β

The orphan nuclear receptor, steroidogenic factor 1 (SF-1), plays a major role in adrenal and gonadal development, as well as in sexual differentiation. It has been demonstrated that the expression of a number of genes regulated by SF-1 is inhibited by the transforming growth factor β (TGF-β). To date, however, the influence of TGF-β on the expression of SF-1 gene has not been reported. A Northern blot analysis with the use of a radiolabeled cDNA probe, and immunodetection with antibodies directed against SF-1, demonstrated that the Sf-1 transcript and the SF-1 protein levels were lowered by TGF-β in Y-1 adrenocortical cells, both in untreated and adenylyl cyclase activator, forskolintreated cells. An examination of the Sf-1 transcript stability in the presence of actinomycin D revealed no influence of TGF-β on the rate of Sf-1 mRNA decay. Inhibition of Sf-1 expression by TGF-β was abolished by cycloheximide, suggesting that the growth factor inhibitory effect requires ongoing protein synthesis. We conclude that in Y-1 cells TGF-β inhibits the expression of SF-1 gene at a transcriptional level, and we postulate that the inhibitory effect of TGF-β on steroid hormone synthesis in the adrenal cortex could be due to an attenuated transcription of Sf-1.

Expression of three negative regulators of CYP17 gene transcription in adrenocortical cells.

The expression of negative regulators of CYP17 gene expression: DAX-1, COUP-TF and N-CoR was investigated in bovine adrenocortical cells in primary culture. The cells were incubated for 6 hours in a defined medium, containing ACTH (10 nM) or forskolin (25 μM). Total RNA was isolated, the concentration of CYP17 gene transcript was determined by hybridization analysis, and polyA RNA was reverse-transcribed to obtain cDNA. Fragments of CYP17, DAX-1, COUP-TF, N-CoR and β-actin cDNA were amplified by PCR and the amplified cDNAs were analyzed by agarose gel electrophoresis followed by densitometry. It was found that DAX-1 and COUP-TF mRNA levels were unaffected by treatment with ACTH or forskolin. On the contrary, both compounds enhanced the level of mRNA encoding N-CoR. Under the same conditions, ACTH or forskolin substantially stimulated CYP17 mRNA accumulation. Our results suggest that in the adrenocortical cells in culture, the expression of DAX1 and COUP-TF is constitutive, whereas N-CoR expression might be inducible.

Binding of low mobility group protein from rat liver chromatin with histones studied by chemical cross-linking

The protein of molecular weight about 160 kD (designated LMG160) was isolated from purified low mobility group chromatin proteins. Polyclonal antibody directed against the LMG160 protein in mouse was raised. The specificity of the antibody was determined with the use of ELISA. Using chemical cross-linking procedure followed by immunoprecipitation with the antiLMG160 antibody complex formation with chromatin proteins was demonstrated. Among the proteins that form complexes with LMG160, histones H3, H2A, and H4 were identified (Western blotting technique).

Regulation of 11β-hydroxysteroid dehydrogenase type II in rat adrenocortical cells

The regulation of 11beta-hydroxysteroid dehydrogenase type II (11beta-HSD2) gene expression was studied in primary cultures of rat adrenocortical cells. The protein kinase A (PKA) pathway agonists forskolin, dibutyryl cAMP and ACTH caused a 5-10 fold increase in 11beta-HSD2 mRNA as determined by semiquantitative PCR. The effect of forskolin could be partially inhibited by the addition of the phorbol ester TPA, an activator of the protein kinase C (PKC) pathway. The increase in mRNA encoding 11beta-HSD2 was accompanied by increased synthesis of 11beta-HSD2 as measured by immunoprecipitation of labeled protein. It is concluded that both the PKA and PKC pathways are involved in the regulation of rat adrenal 11beta-HSD2 gene expression.

TGF-beta inhibits CYP17 transcription in H295R cells acting via activin receptor-like kinase 5.

Objective. Transforming growth factor β (TGF-β) is a potent inhibitor of 17α-hydroxylase/17,20 lyase activity and CYP17 gene expression. We investigated the mechanism how CYP17 is inhibited by TGF-β in adrenocortical cells. Methods. H295R cells were culture and incubated with TGF-β, transcription inhibitor (DRB), activin receptor-like kinase 5 ALK5 (TβRII) inhibitor (SB431542), mitogen activated kinases inhibitors (PD98059 and SB203580), subsequently using reverse transcription and quantitative PCR (RT-qPCR) we determined CYP17 expression. Results. TGF-β significantly decreased the level of cytochrome P450c17 mRNA and this inhibitory effect of TGF-β on CYP17 expression required activin receptor-like kinase 5 (ALK5) and on-going transcription. Mitogen activated kinases MEK1 and p38 MAPK are not involved it the inhibitory effect of TGF-β on CYP17 expression. Conclusion. We concluded that the TGF-β-dependent decrease of 17α-hydroxylase/17,20 lyase activity in the H295R cells is caused by inhibition of CYP17 transcription and is mediated by the ALK5 receptor.

Binding of low mobility group proteins with DNA examined in homologous and heterologous systems by Gel retardation assay.

The interaction of low mobility group proteins (LMG), isolated from chromatin of pancreatic carcinoma cells (CAPAN‐2), with fragments of 5′‐flanking region of the antigen 17‐1A gene was studied by gel retardation assay. The LMG proteins, which formed complexes with DNA were extracted from the gels and identified by polyacrylamide gel electrophoresis under denaturing conditions. The proteins of Mw about 100, 60, 55 and 48 kDa, which formed specific complexes with fragments of 5′‐flanking region of the antigen 17‐1A gene, were identified.

STUDIES ON 17-1A ANTIGEN GENE REGULATION IN NONEXPRESSING A549 AND A431 CELLS, AS COMPARED TO EXPRESSING PANCREATIC CARCINOMA (CAPAN 2) CELLS, REVEAL A COMPLEX MECHANISM OF REPRESSION OF THIS GENE

Elements controlling high expression of the 17‐1A antigen gene in pancreatic carcinoma cells (Capan 2) reside within the two regions: proximal (−193 to +3) and distal (−877 to −518). We demonstrate here that some factors present in nuclear extracts from nonexpressing cells bind specifically to the control elements, important for gene expression. Our results suggest that nonexpressing cells may either lack at least one of the factors necessary for activation or may contain their modified forms. A major difference between expressing and nonexpressing cells was found in the region containing core enhancer sequence. Moreover, nonexpressing cells display a complex pattern of DNA‐protein interactions in this region, suggesting that these cells contain factors acting negatively mainly on the enhancer sequence. Our results however, indicate that the mechanism of repression is much more complicated than expected.