| The relationship of signal molecules in signaling pathway related to lactation and proliferation is not clear in detail. In recent years, researchers take efforts to find important node molecules associated with lactation, in order to further improve milk protein and milk fat synthesis. Suppressor of cytokine signaling3(SOCS3) is a cytokine-induced negative feedback-loop regulator of cytokine signaling. More and more evidence has proved it to be an inhibitor of signal transducers and activators of transcription5(STAT5). But it is not fully known whether this is a common occurrence in bovine mammary epithelial cells. In the mammary gland, prolactin receptor (PRLR) signaling through PRLR-JAK2-STAT5a is crucial for lactation, so it would be interesting to examine whether SOCS3is involved in PRLR-JAK2-STAT5a pathway. On the other hands, SOCS3seems to regulate SREBP-1c to participate in milk fat synthesis. In the present study, we assessed the effects of SOCS3on JAK2/STAT5a signaling and explored the consequences of this selective local blockade of JAK2/STAT5a on downstream signaling, so as to explore the the mechanism of SOCS3on lactation and proliferation in dairy cow mammary epithelial cells (DCMECs).Here, DCMECs were used to analyze the function of SOCS3and the interaction between SOCS3and STAT5. Eukaryotic expression vector pGCMV-IRES-EGFP-SOCS3and SOCS3siRNA were constructed, DCMECs were transfected with pGCMV-IRES-EGFP-SOCS3eukaryotic expression vector or SOCS3siRNA by transient transfection into DCMECs using Lipofectamine TM2000, respectively. Cell viability and proliferation were determined with CASY-TT after transfection with SOCS3gene; By the experiments of overexpression and inhibition of SOCS3, we examined the JAK2/STAT5a pathway activity, the expression and synthesis of β-casein, the mRNA levels and protein expressions of key genes on milk protein synthesis (mTOR) and key genes on lipid metabolism (SREBP-lc, FAS, ACC, SCD), which were verified using qRT-PCR and Western blotting, respectively; we used immunofluorescence to observe the expression and subcellular localization of STAT5a, and to further elucidate the interaction between SOCS3and STAT5a, we also inhibited STAT5a activation, the mRNA levels and protein expressions of SOCS3and β-casein were detected by qRT-PCR and WB, respectively. Furthermore, DCMECs treated with L-Met, the expressions of SOCS3and related genes were detected by qRT-PCR and western blotting, respectively.Data showed that SOCS3could decrease cell proliferation and viability by CASY-TT detection. Overexpression and inhibition of SOCS3brought a remarkable milk protein synthesis change through the regulation of JAK2/STAT5a pathway activity, and SOCS3expression also decrease SREBP-1c expression and fatty acid synthesis. We found that SOCS3protein was distributed both in the cytoplasm and nucleus of DCMECs by fluorescent immunostaining, overexpressed SOCS3protein primarily localized in the nucleus, where STAT5a was significantly inhibited; The level of p-STAT5a increased in the nucleus after inhibition of SOCS3, which was consistent with the result of WB. Inhibited STAT5a activation accompanied with reduced SOCS3expression which indicated that SOCS3gene might be target of activated transcription factor STAT5a, and DCMECs treated with L-Met resulted in reduced SOCS3expression.Together, our findings indicate that SOCS3acts as an inhibitor of JAK2/STAT5a pathway, and its expression in DCMECs was regulated by STAT5a, thus we inferred that SOCS3brought milk protein synthesis change through the negative regulation of milk protein gene. SOCS3disturbs fatty acid synthesis by decreasing SREBP-1c expression and its downstream regulatory targets, FAS, SCD and ACC in DCMECs, which validates its involvement in both milk protein synthesis and fat synthesis. In aggregate, these results reveal that low SOCS3expression is required for milk synthesis and proliferation of DCMECs in vitro. |
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