Interactions in the transcriptional regulation exerted by Stat5 and by members of the steroid hormone receptor family

Abstract

The pathways which connect extracellular signals with the regulation of the activity of transcription factors are being investigated in molecular detail. Extensive progress has been made in the description of the mode of action of steroid hormones and of cytokines. Steroid hormones associate intracellularly with latent receptor molecules, cause the dissociation of masking proteins, the dimerization of receptors, and their binding to specific hormone response elements in the promoters of target genes. Cytokines also activate latent transcription factors (Stats—signal transducers and activators of transcription), but act through an enzymatic mechanism. Tyrosine kinases associated with the transmembrane cytokine receptors phosphorylate Stat molecules. The phosphorylated monomers dimerize and assume specific DNA binding ability. Both classes of transcription factors bind to different response elements and regulate different target genes and both signals, cytokines and steroid hormones, can affect growth differentiation and homeostasis of different cell types. Here, we describe that Stat5, a molecule activated by several essential cytokines, functionally interacts with members of the steroid receptor family. We find that glucocorticoid receptor, mineralocorticoid receptor and progesterone receptor synergize with Stat5 in the induction of the transcription from the β-casein gene promoter. The estrogen receptor diminishes Stat5 mediated induction and the androgen receptor has no effect. Conversely, Stat5 negatively interferes with glucocorticoid receptor, mineralocorticoid receptor and progesterone receptor induced transcription from the MMTV LTR and the estrogen receptor induced transcription from an ERE-containing promoter.

Introduction

Biological regulation is generally exerted through combinatorial events. Interaction of regulatory pathways and individual transcription factors results in cell type specific gene transcription. We have investigated the co-operation and interference between nuclear hormone receptors and Stat5.

Nuclear hormone receptors belong to a family of transcription factors dependent in their activity on hormonal ligands. The nuclear receptor family includes receptors for steroid hormones (estrogens, progesterons, glucocorticoids and mineralocorticoids) [1], [2], receptors for thyroid hormone, retinoids, vitamin D3 and receptors whose ligands are still unknown (orphan receptors) [3]. Steroid hormones regulate essential biological phenotypes such as growth, differentiation and homeostasis. They function through the regulation of target genes in receptor expressing cell types. The steroid hormone receptors are characterized by a modular domain structure, with a carboxyl terminal ligand binding domain, a central DNA binding domain and an amino terminal transcriptional regulatory function [4], [5], [6]. It is thought that ligand association induces the dissociation of the receptor molecule from heat shock proteins, conformational changes which allow dimerization of the receptors and binding to DNA as well as the recruitment of coactivators. These are prerequisites for the enhancement of transcription of target genes. Steroid receptors bind as homodimers to hormone response elements which are composed of two hexanucleotide recognition sequences organized as inverted palindromes. Three nucleotides serve as spacers between the half sides.

Although different in their physiological functions, several steroid hormone receptors share a common response element. The DNA binding site, initially found for the glucocorticoid receptor, is a 15 bp imperfect palindromic DNA sequence present in the promoter region of target genes. Four copies of the GRE have been found in the glucocorticoid inducible mouse mammary tumor virus LTR. The same DNA sequences can also confer transcriptional induction through the progesterone, androgen and mineralocorticoid receptors. The nucleotide sequences defining a progesterone response element, androgen response element or mineralocorticoid response element are identical to the glucocorticoid response element. A different situation exists for the estrogen receptor and the estrogen response element comprises a 13 bp perfect palindromic DNA sequence which is clearly distinct from the glucocorticoid response element [7]. Although additional components, such as coactivators and basal transcription factors are required for transcriptional induction, provision of the steroid receptors and a reporter indicator gene allows the study of steroid hormone action in transfected COS cells.

Stat proteins (signal transducers and activators of transcription) mediate the response to cytokines and growth factors and are comparable but distinct in their mode of action to steroid hormone receptors. Cytokines do not enter the cells but activate cell surface receptors and associated tyrosine kinases which in turn cause the tyrosine phosphorylation of Stat proteins [8], [9]. Phosphorylated Stat proteins dimerize, translocate to the nucleus and assume the ability to bind to specific DNA sequences in target gene promoters. Stat proteins bind as dimers to DNA target sites, comprising a 9 bp consensus sequence. This sequence is fundamentally different from the one to which nuclear hormone receptors bind [10], [11], [12]. Distinct functional domains have been identified within Stat molecules. An amino terminal domain mediates cooperativity between Stat molecules, a central domain confers the DNA binding specificity of the dimer and a SH-2 domain mediates dimerization through phosphotyrosine recognition. The carboxyl terminus comprises a transactivation domain [13].

The study of differentiation specific gene expression in mammary epithelial cells has provided first insights into the molecular mechanisms governing the co-operation between steroid hormone receptors and a Stat mediated pathway [14], [15], [16], [17], [18], [19], [20]. Prolactin and glucocorticoid hormones co-operate in the regulation of milk protein gene transcription and a novel role for the glucocorticoid receptor has been defined. The glucocorticoid receptor acts as a transcriptional coactivator for Stat5 and enhances Stat5 dependent transcription of the β-casein gene promoter. This action of the glucocorticoid receptor is dependent on the complex formation with Stat5 and recruitment to the β-casein gene promoter, but independent of a GRE. The complex formation between Stat5 and the glucocorticoid receptor diverts the glucocorticoid receptor from GRE containing promoters and diminishes the glucocorticoid response of for example, MMTV LTR transcription [21], [22].

We have extended our investigations and analysed functional interactions between Stat5 and the mineralocorticoid receptor, progesterone receptor, androgen receptor and estrogen receptor [23]. We find that the mineralocorticoid receptor and the progesterone receptor synergize with Stat5 in the induction of the β-casein gene promoter, although to a smaller extent than the glucocorticoid receptor. Activation of the estrogen receptor diminishes Stat5 dependent induction, whereas the androgen receptor has no effect. The inducibility of glucocorticoid, mineralocorticoid and progesterone responsive genes is diminished by the simultaneous activation of Stat5. The same is true for an estrogen receptor dependent indicator gene.