Henry B. Sadowski

Stat3 Plays an Important Role in Oncogenic Ros- and Insulin-like Growth Factor I Receptor-induced Anchorage-independent Growth

The role of signal transducers and activators of transcription (STATs) in receptor protein-tyrosine kinase (PTK)-induced cell growth and transformation was investigated using an inducible epidermal growth factor receptor-Ros chimeric receptor called ER2 and a constitutively activated insulin-like growth factor I receptor called NM1, both of which are able to induce anchorage-independent growth of NIH 3T3 cells. ER2 and NM1 receptor PTKs are able to cause Stat3 activation. Co-expressing the dominant negative Stat3 mutant with ER2 or NM1 in transiently or stable transfected cells resulted in a dramatic inhibition of colonies induced by these receptor PTKs and a moderate inhibition of their mitogenicity in monolayer. Therefore, Stat3 is not only important for initiation of transformation, as demonstrated by inhibition of the epidermal growth factor-inducible colony formation of the ER2 cells by the mutant, but it is also required for the maintenance of transformation, as evidenced by reversion of the NM1 transformed cells. The DNA binding and transcriptional activities of the endogenous Stat3 were greatly inhibited in the ER2 and NM1 cells co-expressing the Stat3 mutants. We conclude that activated function of Stat3 is required for the establishment and maintenance of Ros and insulin-like growth factor I receptor PTK-induced cell transformation.

Insulin Induction of SOCS-2 and SOCS-3 mRNA Expression in C2C12 Skeletal Muscle Cells Is Mediated by Stat5*

Previously, by a yeast 2-hybrid screen, we identified signal transducer and activator of transcription 5b (Stat5b) as a substrate of the insulin receptor (IR). We demonstrated that refeeding of fasted mice leads to rapid activation of Stat5 proteins in liver, skeletal muscle, and fat, suggesting that Stat5b is a physiological target of insulin. Here, we show that injection of glucose or insulin into fasted mice leads to robust activation of both Stat5a and Stat5b in skeletal muscle. In C2C12 myotubes, we find that insulin stimulates tyrosine phosphorylation of Stat5a and Stat5b by 3–5-fold. This degree of Stat5 activation in vitro is significantly lower than what we observe in vivo and inversely correlates with IRS-1/2 levels. We can recapitulate robust insulin activation of Stat5 in C2C12 cells by stable overexpression of the human IR (hIR). To identify insulin-activated genes that are Stat5 targets, we also overexpressed an IR mutant (LA-hIR) that signals normally for mitogen-activated protein kinase- and phosphatidylinositol 3-kinase-dependent pathways but is deficient in Stat5 signaling in response to insulin. We demonstrate that insulin induces the expression of SOCS-2 mRNA in the wild type hIR but not in the LA-hIR-overexpressing cells. The induction of SOCS-3 by insulin is reduced but not lost in the LA-hIR cells. Therefore, our results suggest that insulin induction of SOCS-2, and in part SOCS-3 mRNA expression, is mediated by Stat5 and can be independent of mitogen-activated protein kinase and phosphatidylinositol 3-kinase-signaling pathways.

Stat5 phosphorylation status and DNA-binding activity in the bovine and murine mammary glands

The transcription factors Stat5a and Stat5b are mediators of prolactin signalling in mammary epithelial cells, and are thought to play a role in lactogenesis. In cultured cells, activation of Stat5 activity through phosphorylation results in Stat5 binding to the promoters of at least some of the milk protein genes, thereby stimulating their transcription. However, the mammary biology of Stat5 differs between species, and the role of Stat5 in the bovine mammary gland is not fully understood. We have generated an antibody that specifically recognises the phosphorylated forms of Stat5a and Stat5b and used it to compare the levels of phosphorylated Stat5 with Stat5 DNA-binding activity in bovine and murine mammary tissue. Both Stat5 DNA-binding activity and phosphorylation status in the bovine mammary gland were at near-maximal levels at late pregnancy (27-35 days prior to calving), when at least three of the major milk proteins are not highly expressed. In addition, these studies revealed significant animal-to-animal variation in the level of Stat5 activity in both species. The results are consistent with a role in terminal differentiation of mammary epithelial cells. They also suggest that the stimulation of high-level expression of milk protein genes in the bovine mammary gland is not through activation of the prolactin receptor-Jak2-Stat5 pathway.

Glucocorticoid and phorbol ester effects in 3T3-L1 fibroblasts suggest multiple and previously undescribed mechanisms of glucocorticoid receptor-AP-1 interaction

A number of mechanisms have been proposed to account for glucocorticoid hormone and mitogen effects observed on the transcription of engineered exogenous target genes. However, the effect of the co-addition of these agents on the full range of endogenous responsive genes in a given cell type has not been addressed. We detected 19 metabolically labeled proteins or in vitro translation products whose synthesis was altered in 3T3-L1 fibroblasts in response to glucocorticoid hormone using two-dimensional electrophoresis on large-format gels. Co-addition of the mitogenic phorbol ester, tetradecanoylphorbolacetate (TPA), with glucocorticoid hormone resulted in independent (one species), synergistic (six species), and antagonistic (five species) effects on the glucocorticoid regulation of individual gene products, while seven other glucocorticoid regulated species were not affected. These and additional observations suggest direct and gene-specific effects of glucocorticoid receptor and AP-1 on the transcription of responsive genes, and for some of these genes the pattern of regulation is not accounted for by mechanisms described to date. In addition, the pattern of regulation of five species is consistent with a role in mediating the opposing physiological effects of glucocorticoid hormone and growth factors in these cells.