Laminin Regulates Lactation Differentiation Of Bovine Mammary Epithelial Cells Via Integrin Beta1 Signaling Pathway

The development and functional differentiation of the mammary gland is regulated by the interaction of the mammary epithelium cells with the extracellular matrix(ECM)environment.Cells receive cues from the ECM through a family of adhesion receptors called integrins.I ntegrins assist cells in sensing their appropriate developmental context in response to both hormones and growth factors.The BM protein laminin supports PRL-dependent activation of the JAK2–STAT5 signaling pathway and the transcription of PRL-and STAT5-regulated milk protein genes(e.g.,CSN2).Various β1 integrin heterodimers formed by β1 subunit are cell surface receptors of laminin.Integrin β1 is actively required for PRL signaling both in culture and in vivo.However,it is not clear how laminin and integrin β1 regulate PRL-STAT5 an important signal pathway of lactation differentiation and tissue-specific gene expression in the bovine mammary gland.In this study,primary bovine mammary epithelial cells were used as experimental materials.Cell culture was performed using transwell technology with Matrigel(positive control),a major component of the mammary basement membrane,laminin(LN),and non-basement membrane component BSA(negative control),as the substrates for culture.Complexes(hydrocortisone,insulin and prolactin)activate JAK2-STAT5 signaling pathway and induce milk protein synthesis in bovine mammary epithelial cells to simulate the microenvironment of the growth and differentiation of bovine mammary epithelial cells in vivo to obtain an experimental model of functional regulation of bovine mammary epithelial cells,Analyze the signal pathways of integrin-mediated ECM in this process and its relationship with prolactin(receptor)signals,and determine the key signal molecu les associated with the two pathways.The expression and localization of integrin β1 and PRLR in mammary gland tissue and cultured mammary epithelial cells were observed by laser confocal microscopy.The growth characteristics and polarity of cells on diff erent substrates were observed by inverted microscope and transmission electron microscope.Integrin β1 and PRLR were detected by Co-IP in cells.The regulation of laminin and integrin β1 activity on lactation differentiation of bovine mammary epithelial cells were detected q RT-PCR and Western blotting.The results are as follows:In bovine mammary gland,integrin β1 was mainly located in the basement membrane of acinar cells,while PRLR surrounds the whole acinar cell membrane.In both BSA and LN,integrin β1 showed the characteristics of basolateral localization,but it was more clustered in the basolateral membrane of cells in contact with LN.Inverted microscope showed that the mammary epithelial cells on LN showed differentiation characteristics as dome structures,interconnecting structures and formation of acini-like structures.The ultrastructural characterization results of cells growing on L N showed typical polar characteristics of differentiation,that is,the nucleus sinks,the nucleus-to-cytoplasm ratio decreases,and the intracrine vesicles show a directional secretion trend.Integrin β1 and PRLR were detected by Co-IP with integrin β1 antibody.The results showed that integrin β1 and PRLR were detected in the same protein complex in IP group,suggesting that there might be interaction between the two signaling pathways.Moreover,in the presence of LN,the expression level of integrin β1 and PRLR was significantly increased.Under the induction of HIP differentiation medium,LN can promote the lactation differentiation of bovine mammary epithelial cells.At the protein level,the expression of integrin β1,ILK,Rac1,PRLR,p-STAT5 and β-casein was significantly up-regulated.At the m RNA level,the expression of integrin β1,PRLR,STAT5 and CSN2 was significantly up-regulated.Under the induction of HIP differentiation medium,AIIB2,an integrin β1 function blocking antibody,was added to inhibit the effect of LN on lactation differentiation of bovine mammary epithelial cells.The protein expression levels of ILK,p-STAT5 and β-casein were significantly down-regulated,while that of Rac1 was significantly up-regulated.In conclusion,PRLR and integrin β1 co-localize in the basal membrane of cells,which has the spatial conditions for cell-ECM interaction and signal crosstalk.And integrin β1 and PRLR exist in the same protein complex.Under the induction of HIP differentiation medium,LN can enhance the expression of integrin β1 and activate the integrin β1-ILK signaling pathway,and then regulate the lactation differentiation of mammary epithelial cells and improve the synthesis and secretion of β-casein by up regulating the activity level of PRL(PRLR)-STAT5 signaling pathway.