Bernard P. Schimmer

Adrenocortical cell lines

The human adrenal cortex is a complex endocrine organ that secretes mineralocorticoids, glucocorticoids and adrenal androgens. These steroids arise from morphologically and biochemically distinct zones of the adrenal gland. Studying secretion of these distinct steroid hormones can make use of cells isolated from the adrenal gland but this requires animal sacrifice and the need for continued isolation for long-term studies. In addition primary cultures of adrenal cells have a limited life-span in culture and the cultured cells are often contaminated by the presence of non-steroidogenic cells. For that reason in vitro cell culture models have several benefits for research on adrenocortical function. Herein we discuss the available adrenocortical cell lines and their uses as model systems for adrenal studies. Focus is placed on the human NCI-H295 and mouse Y-1 adrenal cell lines, which have been used extensively as adrenocortical model systems. These cell lines have proven to be of considerable value in studying the molecular and biochemical mechanisms controlling adrenal steroidogenesis. The current review will discuss the attributes and limitations of the currently available adrenocortical cell lines as models for adrenal studies.

Minireview: steroidogenic factor 1: its roles in differentiation, development, and disease.

The orphan nuclear receptor steroidogenic factor 1 (SF-1, also called Ad4BP, encoded by the NR5A1 gene) is an essential regulator of endocrine development and function. Initially identified as a tissue-specific transcriptional regulator of cytochrome P450 steroid hydroxylases, studies of both global and tissue-specific knockout mice have demonstrated that SF-1 is required for the development of the adrenal glands, gonads, and ventromedial hypothalamus and for the proper functioning of pituitary gonadotropes. Many genes are transcriptionally regulated by SF-1, and many proteins, in turn, interact with SF-1 and modulate its activity. Whereas mice with heterozygous mutations that disrupt SF-1 function have only subtle abnormalities, humans with heterozygous SF-1 mutations can present with XY sex reversal (i.e. testicular failure), ovarian failure, and occasionally adrenal insufficiency; dysregulation of SF-1 has been linked to diseases such as endometriosis and adrenocortical carcinoma. The current state of knowledge of this important transcription factor will be reviewed with a particular emphasis on the pioneering work on SF-1 by the late Keith Parker.

Transcriptional Regulation of the Genes Encoding the Cytochrome P-450 Steroid Hydroxylases

Steroid hormone biosynthesis requires the concerted action of a related group of cytochrome P-450 steroid hydroxylases. In recent years considerable effort has been directed toward defining the molecular basis for the cell-selective expression of these genes and their transcriptional regulation by trophic hormones. The orphan nuclear receptor SF-1, acting through a conserved element found in the proximal promoter regions of all steroid hydroxylase genes, seems to be a major, but not exclusive, determinant of cell-selective gene expression. In contrast, the coordinate responses of the steroid hydroxylases to trophic hormones apparently involves an interplay of multiple proteins that collectively lead to a synchronous induction of gene expression. In some instances these interactions apparently involve transcription factors that also contribute to the cell-selective expression of these genes.

Unmasking a growth-promoting effect of the adrenocorticotropic hormone in Y1 mouse adrenocortical tumor cells.

The adrenocorticotropic hormone (ACTH) inhibits the growth of Y1 mouse adrenocortical tumor cells as well as normal adrenocortical cells in culture but stimulates adrenocortical cell growth in vivo. In this study, we investigated this paradoxical effect of ACTH on cell proliferation in Y1 adrenal cells and have unmasked a growth-promoting effect of the hormone. Y1 cells were arrested in the G1 phase of the cell cycle by serum starvation and monitored for progression through S phase by measuring [3H]thymidine incorporation into DNA and by measuring the number of nuclei labeled with bromodeoxyuridine. Y1 cells were stimulated to progress through S phase and to divide after a brief pulse of ACTH (up to 2 h). This effect of ACTH appeared to be cAMP independent, since ACTH also induced cell cycle progression in Kin-8, a Y1 mutant with defective cAMP-dependent protein kinase activity. The growth-promoting effect of ACTH in Y1 was preceded by the rapid activation of p44 and p42 mitogen-activated protein kinases and by the accumulation of c-FOS protein. In contrast, continuous treatment with ACTH (14 h) inhibited cell cycle progression in Y1 cells by a cAMP-dependent pathway. The inhibitory effect of ACTH mapped to the midpoint of G1. Together, the results demonstrate a dual effect of ACTH on cell cycle progress, a cAMP-independent growth-promoting effect early in G1possibly mediated by mitogen-activated protein kinase and c-FOS, and a cAMP-dependent inhibitory effect at mid-G1. It is suggested that the growth-inhibitory effect of ACTH at mid-G1 represents an ACTH-regulated check point that limits cell cycle progression.

Genes essential for early events in gonadal development

Abstract. The acquisition of a sexually dimorphic phenotype is a critical event in mammalian development. The basic underlying principle of sexual development is that genetic sex — determined at fertilization by the presence or absence of the Y chromosome — directs the embryonic gonads to differentiate into either testes or ovaries. Thereafter, hormones produced by the testes direct the developmental program that leads to male sexual differentiation. In the absence of testicular hormones, the female pathway of sexual differentiation occurs. Recent studies have defined key roles in gonadal development for two transcription factors: Wilms’ tumor suppressor 1 (WT1) and steroidogenic factor 1 (SF-1). After presenting a brief overview of gonadal development and sexual differentiation, this paper reviews the studies that led to the isolation and characterization of WT1 and SF-1, and then discusses how interactions between these two genes may mediate their key roles in a common developmental pathway.

Effects of catecholamines and monovalent cations on adenylate cyclase activity in cultured glial tumor cells.

Abstract 1. 1. Adenylate cyclase activity was demonstrated in homogenates of rat glial tumor cells grown in culture. 2. 2. Catecholamines, in an order of potency related to their activity ad β-adrenergic agonists, and fluoride stimulated glial cell adenylate cyclase activity without altering the rate of adenosine 3′,5′-monophosphate breakdown. 3. 3. Lithium chloride (10 mM) enhanced epinephrine stimulated adenylate cyclase activity more than 2-fold while affecting fluoride stimulated levels of adenylate cyclase activity only slightly. 4. 4. Other monovalent cations also potentiated epinephrine stimulated adenylate cyclase activity in the order Li + > Na + > K + . 5. 5. These results strongly suggest that girl cells contribute to levels of adenylate cyclase activity measured in brain tissue. Lithium ions may exert their effects through the molecular processes involved in epinephrine stimulation of adenylate cyclase activity.

Transcriptional regulation of the adrenal steroidogenic enzymes

Corticosteroid biosynthesis requires the concerted action of a related group of cytochrome P450 steroid hydroxylases. The genes encoding these steroid hydroxylases exhibit two distinct levels of transcriptional regulation: selective expression in steroidogenic cells and induction in response to trophic hormones. With respect to cell-selective expression, recent studies have identified a nuclear receptor protein expressed only in steroidogenic cells that is postulated to regulate the expression of all cytochrome P450 steroid hydroxylases through common promoter elements. In contrast, the coordinate responses of these genes to trophic hormones are not readily explained by a unifying mechanism, and their hormone responsive expression probably involves multiple promoter elements.

The roles of the nuclear receptor steroidogenic factor 1 in endocrine differentiation and development.

The orphan nuclear receptor steroidogenic factor 1 (SF-1) has emerged as a critical determinant of adrenal and gonadal differentiation, development, and function. SF-1 was initially isolated as a positive regulator of the cytochrome P450 steroid hydroxylases in the adrenal glands and gonads; developmental analyses subsequently showed that SF-1 was also expressed in the diencephalon and anterior pituitary, suggesting additional roles in endocrine function. Analyses of knockout mice deficient in SF-1 revealed multiple abnormalities, including adrenal and gonadal agenesis, male to female sex reversal of the internal genitalia, impaired gonadotrope function, and absence of the ventromedial hypothalamic nucleus. Taken together, these results implicate SF-1 as a global regulator within the hypothalamic-pituitary-gonadal axis and the adrenal cortex.

The Roles of camp and camp-Dependent Protein Kinase in Forskolin's Actions on Y1 Adrenocortical Tumor Cells

The effects of forskolin on the regulation of steroidogenesis and growth were examined in the Y1 adrenocortical tumor cell line, and the roles of cAMP and cAMP-dependent protein kinase in these actions of forskolin were evaluated. Forskolin, like corticotropin, stimulated steroidogenesis 3-fold and inhibited growth by 90%. In mutants of the Y1 cell line harboring specific defects in cAMP-dependent protein kinase activity, the responses to forskolin were attenuated. The resistance of the protein kinase mutants to the diterpene was closely correlated with their resistance to corticotropin and with impaired responses of their protein kinases to cAMP. These results indicate that cAMP and cAMP-dependent protein kinase are obligatory components of forskolins actions on Y1 adrenal cells. Forskolin, at concentrations which were approximately 100-times greater than those required to stimulate steroidogenesis, caused cAMP to accumulate. Apparently, only a small fraction of the cAMP generated in response to forskolin was required to stimulate steroidogenesis or inhibit growth.

Inhibitory properties of the regulatory domains of human protein kinase Calpha and mouse protein kinase Cepsilon.

Two fusion proteins in which the regulatory domains of human protein kinase Cα (Rα; amino acids 1–270) or mouse protein kinase Cε (Rε; amino acids 1–385) were linked in frame with glutathione S-transferase (GST) were examined for their abilities to inhibit the catalytic activities of protein kinase Cα (PKCα) and other protein kinases in vitro. Both GST-Rα and GST-Rε but not GST itself potently inhibited the activities of lipid-activated rat brain PKCα. In contrast, the fusion proteins had little or no inhibitory effect on the activities of the Ser/Thr protein kinases cAMP-dependent protein kinase, cGMP-dependent protein kinase, casein kinase II, myosin light chain kinase, and mitogen activated protein kinase or on the src Tyr kinase. GST-Rα and GST-Rε, on a molar basis, were 100–200-fold more potent inhibitors of PKCα activity than was the pseudosubstrate peptide PKC19–36. In addition, a GST-Rα fusion protein in which the first 32 amino acids of Rα were deleted (including the pseudosubstrate sequence from amino acids 19–31) was an effective competitive inhibitor of PKCα activity. The three GST-R fusion proteins also inhibited protamine-activated PKCα and proteolytically activated PKCα (PKM), two lipid-independent forms of PKCα; however, the IC50values for inhibition were 1 order of magnitude greater than the IC50 values obtained in the presence of lipid. These results suggest that part of the inhibitory effect of the GST-R fusion proteins on lipid-activated PKCα may have resulted from sequestration of lipid activators. Nonetheless, as evidenced by their abilities to inhibit the lipid-independent forms of the enzyme, the GST-R fusion proteins also inhibited PKCα catalytic activity through direct interactions. These data indicate that the R domains of PKCα and PKCε are specific inhibitors of protein kinase Cα activity and suggest that regions of the R domain outside the pseudosubstrate sequence contribute to autoinhibition of the enzyme.