Regulation of growth hormone signaling by selective estrogen receptor modulators occurs through suppression of protein tyrosine phosphatases.

Activation of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) pathway by GH is terminated by the suppressors of cytokine signaling (SOCSs) and protein tyrosine phosphatases, Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 and SHP-2. Based on our recent report that estrogen inhibits GH signaling by stimulating SOCS-2 expression, we investigated the effects of selective estrogen receptor modulators (SERMs) on GH signaling in human embryonic kidney (HEK293) and breast cancer (MDA-MB-231) cells expressing human GH receptor and estrogen receptor-alpha. 17beta-estradiol (E(2)) suppressed GH activation of a STAT5-responsive luciferase reporter and JAK2 phosphorylation in both cell models. 4-hydroxytamoxifen and raloxifene augmented these actions of GH in HEK293 cells but not breast cancer cells. SOCS-2 expression in both cell types was stimulated by E(2) but unaffected by SERMs. In HEK293 cells, SHP-1 was inhibited by raloxifene and 4-hydroxytamoxifen, whereas the latter additionally inhibited SHP-2. The phosphatases were unaffected by E(2). In breast cancer cells, phosphatase activity was not altered by SERMs or E(2). In summary, estrogen inhibited the JAK2/STAT5 signaling of GH and stimulated SOCS-2 expression in both HEK293 and breast cancer cells. By contrast, SERMs augmented GH signaling by reducing SHP activities in HEK293 cells and had no effect on both in breast cancer cells. We provide the first evidence for a novel mechanism regulating GH signaling, in which SERMs enhance GH activation of the JAK2/STAT5 pathway in a cell-type-dependent manner by attenuating protein tyrosine phosphatase activities.

Inactivation of p16INK4a, with retention of pRB and p53/p21cip1 function, in human MRC5 fibroblasts that overcome a telomere-independent crisis during immortalization.

Recent investigations, including our own, have shown that specific strains of fibroblasts expressing telomerase reverse transcriptase (hTERT) have an extended lifespan, but are not immortal. We previously demonstrated that hTERT-transduced MRC5 fetal lung fibroblasts (MRC5hTERTs) bypassed senescence but eventually succumbed to a second mortality barrier (crisis). In the present study, 67 MRC5hTERT clones were established by limiting dilution of a mass culture. Whereas 39/67 clones had an extended lifespan, all 39 extended lifespan clones underwent crisis. 11 of 39 clones escaped crisis and were immortalized. There was no apparent relationship between the fate of clones at crisis and the level of telomerase activity. Telomeres were hyperextended in the majority of the clones analyzed. There was no difference in telomere length of pre-crisis compared with post-crisis and immortal clones, indicating that hyperextended telomeres were conducive for immortalization and confirming that crisis was independent of telomere length. Immortalization of MRC5hTERT cells was associated with repression of the cyclin-dependent kinase inhibitor p16INK4a and up-regulation of pRB. However, the regulation of pRB phosphorylation and the response of the p53/p21cip1/waf1 pathway were normal in immortal cells subject to genotoxic stress. Overexpression of oncogenic ras failed to de-repress p16INK4a in immortal cells. Furthermore, expression of ras enforced senescent-like growth arrest in p16INK4a-positive, but not p16INK4a-negative MRC5hTERT cells. Immortal cells expressing ras formed small, infrequent colonies in soft agarose, but were non-tumorigenic. Overall, these results implicate the inactivation of p16INK4a as a critical event for overcoming telomere-independent crisis, immortalizing MRC5 fibroblasts and overcoming ras-induced premature senescence.

Estrogen up-regulates hepatic expression of suppressors of cytokine signaling-2 and -3 in vivo and in vitro.

Suppressors of cytokine signaling (SOCS) are important negative regulators of cytokine action. We recently reported that estrogen stimulates SOCS-2 expression and inhibits GH signaling in kidney cells. The effects of estrogen on SOCS expression in other tissues are unclear. The aim of this study was to investigate in vivo and in vitro whether estrogen affected SOCS expression in the liver, a major target organ of GH. The in vivo hepatic effects of estrogen on ovariectomized mice lacking estrogen receptor (ER)-alpha, ERbeta, or both and their wild-type littermates were examined by DNA microarray analysis. In vitro, the effects of estrogen on SOCS expression in human hepatoma cells were examined by reverse transcription quantitative PCR. Long-term (3 wk) estrogen treatment induced a 2- to 3-fold increase in hepatic expression of SOCS-2 and -3 in wild-type and ERbeta knockout mice but not in those lacking ERalpha or both ER subtypes. Short-term treatment (at 24 h) increased the mRNA level of SOCS-3 but not SOCS-2. In cultured hepatoma cells, estrogen increased SOCS-2 and -3 mRNA levels by 2-fold in a time- and dose-dependent manner (P < 0.05). Estrogen induced murine SOCS-3 promoter activity by 2-fold (P < 0.05) in constructs containing a region between nucleotides -1862 and -855. Moreover, estrogen and GH had additive effects on the SOCS-3 promoter activity. In summary, estrogen, via ERalpha, up-regulated hepatic expression of SOCS-2 and -3, probably through transcriptional activation. This indicates a novel mechanism of estrogen regulation of cytokine action.