Jennivine Tsao

Contributions of Steroidogenic Factor 1 to the Transcription Landscape of Y1 Mouse Adrenocortical Tumor Cells

The contribution of steroidogenic factor 1 (SF-1) to the gene expression profile of Y1 mouse adrenocortical cells was evaluated using short hairpin RNAs to knockdown SF-1. The reduced level of SF-1 RNA was associated with global changes that affected the accumulation of more than 2000 transcripts. Among the down-regulated transcripts were several with functions in steroidogenesis that were affected to different degrees--i.e., Mc2r>Scarb1>Star≥Hsd3b1>Cyp11b1. For Star and Cyp11b1, the different levels of expression correlated with the amount of residual SF-1 bound to the proximal promoter regions. The knockdown of SF-1 did not affect the accumulation of Cyp11a1 transcripts even though the amount of SF-1 bound to the proximal promoter of the gene was reduced to background levels. Our results indicate that transcripts with functions in steroidogenesis vary in their dependence on SF-1 for constitutive expression. On a more global scale, SF-1 knockdown affects the accumulation of a large number of transcripts, most of which are not recognizably involved in steroid hormone biosynthesis.

SF1 polymorphisms in the mouse and steroidogenic potential.

ACTH-resistance in four mutant derivatives of a mouse adrenocortical tumor cell line results from a defect that reduces the activity of steroidogenic factor-1 (SF1) thereby preventing expression of the ACTH receptor and other SF1-dependent genes. The SF1 genes from these mutants contain a sequence difference that changes an Ala to Ser at codon 172. Steroidogenic factor-1S172 represents a polymorphism rather than a spontaneous mutation since the two forms of SF1, SF1A172, and SF1S172, can be traced to the hybrid mouse strain (C57L/J × A/HeJ) from which the original adrenal tumor was derived. The SF1S172 allele is amplified in three of the four mutant clones together with the neighboring genes germ cell nuclear factor and LIM homeobox2. The two forms of SF1 had only modest differences in transcriptional activity in reporter gene assays, suggesting that the SF1 polymorphism per se is not directly responsible for the loss of mc2r expression. Rather, ACTH resistance in this family of adrenocortical tumor cell mutants may be due to a closely linked gene on the SF1S172 allele. Mouse strains with reportedly high steroidogenic capacity (C57Bl/6J, C57Bl/10J) also have the SF1A172 allele while mouse strains with low steroidogenic capacity (C3H/HeJ, DBA/2J) have the SF1S172 allele. These latter observations suggest that the two SF1 alleles also may be markers of steroidogenic potential among mouse strains.

Forskolin-resistant Y1 adrenal cell mutants are deficient in adenylyl cyclase type 4

Four mutant clones independently derived from the Y1 mouse adrenocortical tumor cell line have adenylyl cyclase (AC) activities that are resistant to forskolin, a direct activator of AC. In this study the AC isoform composition of the forskolin-resistant mutants was examined in order to explore the underlying basis for the resistance to forskolin. As determined by Western blot and RT-PCR analysis, the four forskolin-resistant mutants all were deficient in AC-4; the levels of other AC isoforms (AC-1, AC-3 and AC-5/6) were comparable to the levels in parent Y1 cells. Transfection of one of the mutant clones with an AC-4 expression vector increased forskolin-stimulated cAMP signaling, and restored forskolin-induced changes in cell morphology and growth. Taken together, these observations indicate that AC-4 deficiency is a hallmark of the forskolin-resistant phenotype of these mutants and suggest that AC-4 is an important target of forskolin action in the Y1 adrenal cell line.

Expression of adenylyl cyclase-4 (AC-4) in Y1 and forskolin-resistant adrenal cells.

Forskolin-resistant mutants of a mouse adrenocortical cell line present a complex phenotype in which adenylyl cyclase (AC) is resistant to activation by forskolin and by ACTH. ACTH-resistance results from a defect affecting transcription of the ACTH receptor and can be overcome by transfecting mutant cells with expression vectors encoding G beta/gamma. Forskolin-resistance results from an AC-4 deficiency. We now demonstrate that the AC-4 deficiency in forskolin-resistant mutants results from a transcription defect affecting the promoter activity of the AC-4 gene. Furthermore, the underlying defect leading to AC-4 deficiency and forskolin-resistance can be overcome by transfection of mutant clones with expression vectors encoding G beta/gamma. These data support our hypothesis that AC-4 is a preferred target of forskolin action in Y1 cells, demonstrate novel roles for G beta/gamma in gene expression and indicate that a common underlying defect, suppressible by G beta/gamma, accounts for both the resistance to ACTH and to forskolin.

A Polymorphic Form of Steroidogenic Factor 1 Associated with ACTH Receptor Deficiency in Mouse Adrenal Cell Mutants

Abstract: We have described a family of adrenocortical tumor cell mutants (including clones OS3, Y6, and 10r9) that are resistant to ACTH because they fail to express the gene encoding the ACTH receptor (MC2R). The MC2R deficiency results from a mutation that impairs the activity of the nuclear receptor steroidogenic factor 1 (SF1) at the MC2R promoter. In this report, we show that ACTH resistance in the mutant clones is associated with a Sf1 gene that has Ser at codon 172 instead of Ala. In two of the three mutant clones, this Sf1 allele is amplified together with flanking DNA from chromosome 2 that includes the genes encoding germ cell nuclear factor and the beta‐type proteosome subunit Psmb7. SF1A172 and SF1S172 exhibit little or no difference in transcriptional activity in SF1‐dependent reporter gene assays, suggesting that SF1S172 per se is not directly responsible for the loss of MC2R expression. Instead, the Sf1S172 allele appears to be a marker of ACTH resistance in this family of adrenocortical tumor cell mutants, possibly reflecting the activity of a neighboring gene.

ACTH-receptor deficient mutants of the Y1 mouse adrenocortical tumor cell line

Two mutant clones (Y6 and OS3) derived from the ACTH-responsive Y1 mouse adrenocortical tumor cell line fail to respond to ACTH with increased adenylyl cyclase activity and, as a consequence, are resistant to the steroidogenic effects of the hormone. As determined from Northern blot and RNase protection assays, ACTH resistance in these mutants results from the failure to accumulate ACTH receptor transcripts. The ACTH receptor gene appears to be present in these mutants as determined by Southern blot hybridization analysis and can be activated following the growth of the mutant cells as tumors in mice, suggesting that the ACTH receptor gene is modified in a reversible manner. When mutant cells are transformed with a gene encoding the mouse beta 2-adrenergic receptor they respond to beta-adrenergic agonists with increased adenylyl cyclase activity in a manner that is indistinguishable from a similarly transformed parent Y1 cell line. These results suggest that the adenylyl cyclase system in the mutants is otherwise intact and that the failure to express ACTH receptor transcripts limits the responsiveness of these clones to the hormone.

Regulation of adenylate cyclase activity in glial—adrenal hybrid cells

SOMATIC cell hybrids, formed by fusion of hormone-responsive and insensitive cells, have been used to evaluate the structure and genetic regulation of the hormone-sensitive adenylate cyclase system1–6. In the cell hybrids studied so far, catecholamine-sensitive adenylate cyclase activity was lost when catecholamine-responsive and insensitive cells were fused. Prostaglandin E1 (PGE1)-sensitive adenylate cyclase activity, on the other hand, was retained in hybrids between PGE1-sensitive and insensitive cells. Where examined, the presence or the absence of hormone sensitivity in the hybrids closely correlated with hormone receptor activity as measured by ligand-binding assays1–3. These observations suggest that genetic mechanisms may control the expression of hormone responsiveness at the level of the hormone receptor. To examine these patterns of regulation further, we have formed cell hybrids between rat glial tumour cells with adenylate cyclase activity responsive to β-adrenoceptor agonists7 and mouse adrenocortical tumour cells with adrenocorticotrophic hormone (ACTH)-sensitive adenylate cyclase activity8. Besides having distinct functional hormone receptors, the adenylate cyclases of parental cells exhibit marked quantitative differences in response to fluoride ion7,8. We find that the glial–adrenal hybrids retain the adrenaline-sensitive adenylate cyclase activity characteristic of the glial parent, and lose ACTH sensitivity. The level of the fluoride response, however, is characteristic of the adrenal parent. These data indicate that hormone receptor and catalytic unit activities in the adenylate cyclase system are independently regulated.

Agonist-Induced Desensitization of Adenylyl Cyclase in Y1 Adrenocortical Tumor Cells

Y1 adrenocortical tumor cells (Y1DS) and Y1 mutants resistant to ACTH-induced desensitization of adenylyl cyclase (Y1DR) were transfected with a gene encoding the mouse beta 2-adrenergic receptor (beta 2-AR). Transfectants expressed beta 2-ARs that were able to stimulate adenylyl cyclase activity and steroid biosynthesis. These transfectants were used to explore the basis for the DR mutation in Y1 cells. We demonstrate that beta-adrenergic agonists desensitize the adenylyl cyclase system in transfected Y1DS cells whereas transfected Y1DR cells are resistant to desensitization by beta-adrenergic agonists. The fate of the beta 2-ARs during desensitization was evaluated by photoaffinity labelling with [125I]iodocyanopindolol diazerine. Desensitization of Y1DS transfectants was accompanied by a modest loss in receptor density that was insufficient to account for the complete loss of responsiveness to beta-adrenergic agonists. The extent of receptor loss induced by beta-adrenergic agonists in Y1DR transfectants exceeded that in the Y1DS transfectants indicating that the mutation which protects Y1DR cells from agonist-induced desensitization is prior to receptor down-regulation in the desensitization pathway. From these results we infer that ACTH and isoproterenol desensitize adenylyl cyclase by a common pathway and that receptor loss is not a major component of the desensitization process in these cells.