A study of bovine adrenocortical cell shape on adrenocorticotropic hormone (ACTH) challenge showed that the cells round up and develop arborized processes. This effect was found to be (1) specific for ACTH because angiotensin II and basic fibroblast growth factor have no effect; (2) mediated by a cAMP-dependent pathway because forskolin reproduces the effect of the hormone; (3) inhibited by sodium orthovanadate, a phosphotyrosine phosphatase inhibitor, but unchanged by okadaic acid, a serine/threonine phosphatase inhibitor; and (4) correlated with a complete loss of focal adhesions. Biochemical studies of the focal-adhesion-associated proteins showed that pp125fak, vinculin (110 kDa) and paxillin (70 kDa) were detected in the Triton X-100-insoluble fraction from adrenocortical cells. During cell adhesion on fibronectin as substratum, two major phosphotyrosine-containing proteins of molecular masses 125 and 68 kDa were immunodetected in the same fraction. A dramatic decrease in the extent of tyrosine phosphorylation of these proteins was observed within 60 min after treatment with ACTH. No change in pp125fak tyrosine phosphorylation nor in Src activity was detected. In contrast, paxillin was found to be tyrosine-dephosphorylated in a time-dependent manner in ACTH-treated cells. Sodium orthovanadate completely prevented the effect of ACTH. These observations suggest a possible role for phosphotyrosine phosphatases in hormone-dependent cellular regulatory processes.

Transforming growth factor-␤s (TGF-␤s) constitute a family of dimeric proteins that affect growth and differentiation of many cell types. TGF-␤ 1 has also been proposed to be an autocrine regulator of adrenocortical steroidogenesis, acting mainly by decreasing the expression of cytochrome P450c17. Here, we demonstrate that TGF-␤ 1 has a second target in bovine adrenocortical cells, namely the steroidogenic acute regulatory protein (StAR). Indeed, supplying cells with steroid precursors revealed that TGF-␤ 1 inhibited two steps in the steroid synthesis pathway, one prior to pregnenolone production and another corresponding to P450c17. More specifically, TGF-␤ 1 inhibited pregnenolone production but neither the conversion of 25-hydroxycholesterol to pregnenolone nor P450scc activity. Thus, TGF-␤ 1 must decrease the cholesterol supply to P450scc. We therefore examined the effect of TGF-␤ 1 on the expression of StAR, a mitochondrial protein implicated in intramitochondrial cholesterol transport. TGF-␤ 1 decreased the steady state level of StAR mRNA in a time-and concentrationdependent manner. This inhibition occurs at the level of StAR transcription and depends on RNA and protein synthesis. It is likely that the TGF-␤ 1 -induced decrease of StAR expression that we report here may be expanded to other steroidogenic cells in which a decrease of cholesterol accessibility to P450scc by TGF-␤ 1 has been hypothesized.

Matrix attachment regions (MARs) are thought to participate in the organization and segregation of independent chromosomal loop domains. Although there are several reports on the action of MARs in the context of heterologous genes, information is more limited on the role of MARs associated with plant genes. Transgenic studies suggest that the upstream, intron and downstream regions of the developmentally regulated heat shock cognate 80 gene (HSC80) of tomato participate in chromatin organization. In this study, we tested the in vitro affinity of the HSC80 gene to chromosomal scaffolds prepared from shoot apices of tomato. We found that a 1.5 kb upstream region and a 1.4 kb downstream region, but not the intron region, are MARs. These MARs interact with tomato and pea scaffolds and bind regardless of the expression status of HSC80 in the tissue from which the nuclei were isolated. Comparison to two known yeast MARs, ARS1 and CENIII, showed that the HSC80 5'MAR binds more avidly to tomato scaffolds than ARS1, while no binding of CENIII was observed. Competition binding between the two HSC80 MARs indicated that the 5'MAR can outcompete the 3'MAR and not vice versa. Last, we observed that the interaction of the 3'MAR with the scaffold could result in an electrophoretic mobility shift resistant to SDS, protease, and phenol treatment. In conclusion, MARs whose binding properties can be clearly differentiated are closely flanking the HSC80 gene. The discovery of MARs in regions which have a distinct function in HSC80 transgenes but not in transient expression assays, is consistent with a chromosomal scaffold role in HSC80 gene regulation.