| Transforming growth factor-β(TGFβ) plays fundamental roles in cell proliferation, differentiation and apoptosis, and TGFβsignal is transduced through induction of Smads nuclear translocation. Insulin is a main growth factor that regulates cellular carbohydrate metabolism, and antagonizes TGFβsignaling. In the present study, by using a fluorescence cell imaging instrument IN Cell Analyzer 1000, we investigated the cross-talk between insulin and TGFβsignaling. We demonstrated that insulin could inhibit the TGFβ1-induced Smad2 activation in a PI3K/AKT dependent manner. Next, we showed that overexpression of protein tyrosine phosphatase 1B (PTP1B), a negative regulator of insulin signaling, effectively abolished insulin inhibition of Smad2 activation. Moreover, compound-2, a reported PTP1B inhibitor, could restore insulin inhibition effects through neutralizing PTP1B activity.However, when sodium orthovanadate (SOV), a non-specific PTP inhibitor was tested for its ability to regulate insulin/TGFβcross-talk, we surprisingly found that SOV could increase Smad2 action induced by TGFβ1. We presumed that the SOV effects on TGFβsignaling was not PTP1B dependent but through other mechanisms. Further studies indicated that SOV could potentiate Smad2 activation through a sensitization mechanism, which was PI3K/AKT independent and caveolae involved. Based on the result that SOV treatment resulted in an increased sensitivity for cells in response to TGFβ1 stimulation, we hypothesized a possible mechanism through which SOV affects TGFβsignal. Since SOV was reported to induce nitric oxide (NO) production in endothelial cells and Smad2 was recognized as a transcription factor of endothelial nitric oxide synthase (eNOS), our study may provide a new explanation for SOV mediated NO synthesis in endothelium.Since PTP1B knock-out mice have been shown to be healthy and obesity-resistant, PTP1B inhibitors are therefore thought to be promising therapeutic agents for the treatment of obesity and type 2 diabetes. In this study, we discovered a new PTP1B inhibitor (GYW-YF-002) from marine natural products (provided by Prof. Y. W. Guo) with an IC50 value of 42μM. Further study indicated that this compound was a non-competitive PTP1B inhibitor with a Ki value of 35μM. Cellular effects of the compound were evaluated using the high-content drug screening system IN Cell Analyzer 1000. Our results showed that the inhibitor could enhance AKT activation and GLUT4 membrane translocation in response to insulin in PTP1B overexpressed CHO cell. Moreover, the effects of GYW-YF-002 on TGFβsignaling also confirmed our previous findings that PTP1B may regulate Smad2 activation through insulin pathway.Estrogen receptor (ER) belongs to steroid hormone receptor superfamily, and two main ER isoforms (ERαand ERβ) have been found in mammals. Upon ligand binding, ER undergoes conformational changes and translocates to nuclei where ER functions as a transcription factor to facilitate downstream gene transcription. The coactivator recruitment is essential for ER to effectively initiate gene transcription. In this study, 3 estrogens and 2 SERMs were examined for their capacities to modulate ERα/coactivator interactions. Three coactivators SRC-1, CBP and RIP140 were used in the yeast two-hybrid system, and all estrogens were shown to increase ERα/coactivator binding. However, Raloxifene was shown to be an ER agonist in yeast and an antagonist by SPR assay. Only Tamoxifen exhibited full antagonist activity both in yeast and in SPR experiment. To our knowledge, this might be the first evidence that indicates Raloxifene may induce coactivator recruitment to ERα. Our study therefore suggests that Raloxifene possibly use different mechanisms for coactivator recruitment in vivo and in vitro.
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