| Milk,originally a nutrient for mammals to feed their offspring,has become an indispensable and important food in modern human life.Milk is known as "the nearest perfect food",which contains a large number of high-quality proteins,lipids,essential vitamins and minerals.Mammary epithelial cells are the most basic structure and function unit of lactation.Through the study of lactation mechanism of mammary epithelial cells,it will provide theoretical basis and technical approach for establishing efficient lactation regulation technology.These basic theories and techniques are of great significance to the development of dairy and animal husbandry.In addition,in the field of biology,it is also an important scientific issue to explore the molecular mechanism regulating milk protein synthesis.Prolactin is a critical exogenous hormone which is responsible for regulating the synthesis of milk proteins and promoting cell proliferation and differentiation,and it has been proved to control the proliferation of mammary epithelial cells and the synthesis of milk proteins through regulating Tudor-SN(tudor staphylococcal nuclease).Although various evidence have shown that Tudor-SN is involved in the regulation of cell proliferation and milk protein synthesis as a multifunctional protein,the detailed molecular regulatory mechanism remains to be elucidated.In the present study,it was proved that JNK(c-Jun N-terminal kinase)was phosphorylated in bovine mammary epithelial cells(BMECs)upon prolactin stimulation,and the phosphorylated JNK further phosphorylated Tudor-SN in cytoplasm,and the phosphorylated Tudor-SN translocated into the nucleus to interact with STAT5(signal transducer and activator of transcription)to upregulate target gene expression to regulate milk protein synthesis and cell proliferation.This study reveals the molecular mechanism of Tudor-SN regulating the proliferation of BMECs and milk protein synthesis through phosphorylation activation under the action of prolactin.In this study,we found that prolactin activates Thr103 phosphorylation of Tudor-SN and phosphorylated Tudor-SN enters the nucleus of BMECs.Western blot experiments showed that prolactin significantly increased the expression of phosphorylated Tudor-SN.Then,it was demonstrated that prolactin further stimulated the translocation of phosphorylated Tudor-SN into the nucleus by immunofluorescence assays and western blot analysis.In addition,it was proved that Tudor-SN could not be translocated into the nucleus after Thr103 in Tudor-SN was mutated to Ala.Taken together,these results suggested that prolactin stimulated the phosphorylation of Tudor-SN and its translocation into the nucleus of BMECs.To explore the upstream activation mechanism of Tudor-SN,Co-IP(co-immunoprecipitation)assay was used to prove that Tudor-SN was phosphorylated by JNK.Then the phosphorylation of Tudor-SN upon the stimulation of prolactin was determined after JNK was inhibited or activated by adding its inhibitor SP600125 or activator anisomycin into the culture medium of BMECs.Western blot and immunofluorescence results of Tudor-SN protein in the cytoplasm and nucleus showed that Tudor-SN was not phosphorylated after JNK was inhibited by SP600125,and the phosphorylation of Tudor-SN was dramatically enhanced after JNK was activated by anisomycin.Therefore,all the above results indicated that JNK was the upstream signaling for the activation of Tudor-SN in response to prolactin stimulation.To figure out whether JNK mediated cell proliferation and milk protein synthesis induced by Tudor-SN upon prolactin stimulation,cell growth and milk protein levels were determined in the presence of prolactin or Tudor-SN overexpression after the inhibitor of JNK was added by Western blot and flow cytometry analysis.The results showed that JNK inhibitor arrested much more cells in G0/G1 phase and inhibited milk protein levels under the stimulation of prolactin or Tudor-SN overexpression.Thus,these results indicated that the regulation of prolactin on cell proliferation and milk protein in BMECs depends on JNK activation.To explore the biological role of phosphorylated Tudor-SN in the nucleus of BMECs,Co-IP assay was used to determine whether Tudor-SN interacts with STAT5.Western blot and immunofluorescence results indicated that Tudor-SN and STAT5 were both activated by prolactin.Cell proliferation and milk protein synthesis were detected upon prolactin stimulation or Tudor-SN overepression together with STAT5 knockdown by si RNA transfection.The results demonstrated that the promotion of cell proliferation and milk protein synthesis by prolactin and Tudor-SN were inhibited significantly by STAT5 inhibition,and the transition from G1 phase to S phase was also blocked.Therefore,these results indicated that phosphorylated Tudor-SN interacted with STAT5 to regulate cell growth and milk protein synthesis.Hence,we discovered a new molecular regulatory mechanism of prolactin on cell proliferation and milk protein synthesis mediated by Tudor-SN.In brief,JNK is first activated by prolactin,and Tudor-SN is activated and phosphorylated by JNK;then phosphorylated Tudor-SN is further translocated into the nucleus to interact with the transcription activator STAT5,thereby regulates proliferation of BMECs and milk protein synthesis. |
