Gordon M. Ringold

TNFα-Mediated Inhibition and Reversal of Adipocyte Differentiation Is Accompanied by Suppressed Expression of PPARγ without Effects on Pref-1 Expression1

Tumor necrosis factor α (TNFα) is a polypeptide hormone with pleiotropic effects on cellular proliferation and differentiation. To investigate how TNFα inhibits and reverses adipocyte differentiation, we studied the expression of two factors involved in the adipocyte differentiation process. Peroxisome proliferator-activated receptor γ (PPARγ) is a positive regulator of adipogenesis, whereas preadipocyte factor 1 (Pref-1) inhibits adipocyte differentiation. The expression patterns of both PPARγ and Pref-1 change during early stages of adipocyte differentiation. Decreased expression of Pref-1 and increased expression of PPARγ occur 1 day and 2 days, respectively, after 3T3-L1 cells reach confluence. During TNFα-mediated inhibition of adipocyte differentiation, PPARγ messenger RNA (mRNA) expression stays at low levels. In contrast, TNFα treatment has no effect on the normal decrease in Pref-1 gene expression that occurs during adipogenesis. We observed that certain cytokine and growth factors[ such as TNFα,...

Glucocorticoid regulation of mouse mammary tumor virus gene expression.

Glucocorticoid hormones act rapidly and specifically to stimulate the synthesis of mouse mammary tumor virus RNA in a variety of mouse mammary tumor cells and infected heterologous cells. The increase in viral RNA production appears to be mediated by receptor proteins and requires the presence of basal levels of viral RNA. Infection of heterologous cells with MMTV may alter host cell responses to glucocorticoids; in addition, production of unintegrated viral DNA in these cells has provided reagents required for studying the structure and function of the viral DNA itself. The advent of new techniques for genetic manipulation of eukaryotic cells and for isolation of large amounts of specific DNA sequences should now permit detailed analyses of steroid hormone action in this system.

Isolation of glucocorticoid-unresponsive rat hepatoma cells by fluorescence-activated cell sorting

We have used a mouse mammary tumor virus (MMTV)-infected rat hepatoma cell line as a model system for studying glucocorticoid action. These cells induce tyrosine aminotransferase and MMTV in response to the synthetic glucocorticoid, dexamethasone. The major viral antigen, a glycoprotein of 52,000 daltons (gp52), appears on the surface of infected cells in amounts which reflect the cytoplasmic content of viral RNA. Using an anti-gp52 antiserum and a fluorescence-activated cell sorter (FACS), we have selected variants which display low levels of pg52 in the presence of the hormone. Multiple cycles of enrichment for cells that fluoresce weakly in the presence of hormone have generated a population which fails to produce a detectable increase in cell surface gp52 in response to dexamethasone. This population of nonresponders and a number of independent clones derived from this population were analyzed for their ability to induce gp52 and TAT and for these presence of glucocorticoid receptors. All nonresponder clones exhibited little or no induction of either glucocorticoid-inducible marker. Two of the clones contained reduced levels of glucocrticoid receptor, while the remainder of the clones showed no detectable specific hormone binding. These results provide genetic evidence that a single class of glucocorticoid receptors is involved in the induction of both MMTV and TAT in HTC cells.

Hormonal Control of Adipocyte Differentiation and Adipocyte Gene Expression

Publisher Summary This chapter presents a summarization of a large number of studies aimed at elucidating the signaling system by which determined cells decide to activate a terminal differentiation program. Using adipogenic cells in culture, it can be documented that the hormonal milieu plays a major role in dictating the decision to trigger differentiation. The effects of indomethacin suggest that the products of arachidonic acid may play a key role in the regulation of cell differentiation. Inhibition of protein kinase C activity is important for triggering differentiation as its activation by phorbol esters or certain growth factors inhibit expression of the differentiated phenotype. The phorbol ester and the growth factor repressible clone 5 gene play an important role in defining the biochemical state associated with the potential for differentiation; in particular, suppression of clone 5 gene expression appears to correlate with entry of cells into a nonproliferative state within the G1 portion of the cell cycle in which cells are incapable of differentiating.

Glucocorticoid control of developmentally regulated adipose genes

We have analyzed the hormonal basis for the acceleration of differentiation by dexamethasone in the stable adipogenic cell line TA1. These cells, which were derived from 5-azacytidine-treated 10T1/2 mouse embryo fibroblasts, undergo differentiation in culture after reaching confluence. Using cDNA clones corresponding to mRNAs that are induced during adipogenesis, we find that dexamethasone elicits the precocious accumulation of differentiation-specific gene products. This effect appears to be mediated by the glucocorticoid receptor, yet unlike standard steroid inductions, most of the RNAs reach the same maximal levels in the absence of dexamethasone. Glucocorticoids thus may increase the expression of a regulatory factor required for activating the entire set of differentiation-dependent genes. We also describe a gene whose transcription is not only activated during adipogenesis, but is also specifically inducible by dexamethasone in the mature adipocyte. Moreover, the glucocorticoid responsiveness of this gene in differentiated cells appears to be dependent on its prior developmental activation.

Glucocorticoid Regulation of Gene Expression: Mouse Mammary Tumor Virus as a Model System

Publisher Summary Glucocorticoids—as well as other classes of steroid hormones—appear to function via the two-step model. It is generally accepted that steroids interact with a soluble receptor protein inducing a structural alteration that increases the receptors affinity for DNA or chromatin. This so-called activated form of the steroid–receptor complex accumulates within the nucleus of the cell leading to increased—and perhaps in some cases, decreased—transcription of specific genes. This chapter describes the glucocorticoid regulation of gene expression. The classes of new messenger RNAs (mRNAs) produced in response to a given steroid are in large part cell or tissue specific and their utilization in production of new proteins leads to the characteristic hormonal response of the target cell. The primary role of the steroid is to act as an allosteric effector that unmasks a DNA-binding site on the receptor protein. The chapter briefly reviews some of the studies on the glucocorticoid regulation of gene expression and presents a detailed account of the use of mouse mammary tumor virus (MMTV) in the mechanisms by which glucocorticoids regulate gene expression.

Hormonal control of adipogenesis

The concept that hormonal substances can alter the expression of entire developmental programs is in itself not particularly new. The ability to define conditions under which a specific hormone can precociously activate the differentiation of a well-defined population of cells and under which another hormone can both block and reverse such a developmental progression, however, provides a major step forward toward unraveling the biochemical events that define the transition from a committed precursor to a fully differentiated cell. Further analysis of the molecular events initiated by glucocorticoids and TNF should provide insights into the control of adipogenesis and may generate a foundation for understanding the mechanisms by which other cells enter a particular differentiative lineage. In a more applied sense, such knowledge may also provide a rational approach to controlling metabolic disease syndromes related to adipogenesis gone awry such as obesity-associated diabetes and cachexia.