Regulation Of Milk Protein Synthesis In Bovine Mammary Epithelial Cells By Integrin β1

At least two types of signals are required to induce lactogenic differentiation in mammary epithelial cells,which are related to soluble hormones and growth factors,and to specialized extracellular matrix(ECM)—basement membrane.Various endocrine,paracrine and autocrine hormones or growth factors can bind to their receptors to activate adjacent signaling molecules to initiate cascades,to induce cell proliferation,apoptosis and milk protein synthesis ultimately.Laminin is a kind of component of the basement membrane,which combined with prolactin play a viral role for lactogenic differentiation in mouse mammary epithelial cells.In the absence of Lamin,the JAK2-STAT5 pathway can only be transiently activated by prolactin,milk protein synthesis cannot be initiated.At the presence of laminin-rich matrix as substrate,signal molecules can be recruited to cell basal side where the platform forms,which would assist prolactin receptor to activate the JAK2-STAT5 pathway.On the cell surface,integrins as adhesion receptors for ECM exist as heterodimers.They are involved in cell-ECM interactions and are linked to the cytoskeleton to help transport intracellular signaling molecules.A few integrin receptors for laminin consist of β1 subunit and certain α subunit.The mouse mammary gland deletion of β1 integrin gene is characterized of failed acinar morphological development because that STAT5 can not be translocated into the nucleus,inhibiting lactogenic differentiation.But,there are a few differences between rodents and ruminants.The effects of laminin and β1 integrin on lactogenic differentiation in bovine mammary epithelial cells(BMECs)haven’t been confirmed yet.In this study,we used mammary gland epithelial cells and mammary gland tissue from lacting Chinese Holstein cows.With laminin as a substrate,we mimic lactation physiological microenvironment in Transwell chamber,The cell top-bottom polarity and secretory differentiation were induced by differentiation medium containing HIP complex(prolactin + insulin + hydrocortisone).All experimental work was carried out,such as laser confocal fluorescence microscopy,flow cytometry,mass spectrometry,q RT-PCR and Western blotting,which were used to detect the expression of integrin subunits β1 and α6.The function-block antibody was added to regulate integrin β1 activity.Data shown below:(1)Monolayer mammary epithelial cells obtained by primary cell culture have two cells molecule markers,which are luminal epithelial cells marker CK18 and basal epithelial cells marker integrin β1;(2)In adherent BMECs,integrin subunits β1 and α6 are mainly located on basal side membrane,whether on uncoated plates or coated(BSA and laminin)plates;(3)In Co-IP assay,total protein sample was immunoprecipitated by integrin β1 monoclone antibody(rat Ig G1 kappa).Integrin subunits β1 and α6 were found in both of Input sample and IP sample,but the counterpart in Ig G control group couldn’t be detected.It’s obviously that β1 subunit binded to α6 subunit and formed the heterodimer— α6β1,which is one of integrin receptors for laminin.(4)Compared with DMEM/F12 control medium,HIP complex could induce lactogenic differentiation in BMECs on laminin substrate: the higher protein abundance of integrin subunits β1 and α6,β-casein,PRLR and STAT5 and the higher p-STAT5 level;a similar profile for their m RNA abundance.(5)Compared with BSA control substrate,laminin had a significant lactogenic effect on BMECs in HIP medium: up-regulation of β-casein expression induced by HIP occurred at both m RNA and protein levels;integrin subunits β1 and α6 protein levels were significantly up-regulated,but there were no significant change at their m RNA levels.(6)Compared with Ig G isotype control group,integrin subunits β1 and α6 expression had no significant change,but integrin subunit β1 was inactivated by AIIB2,which inhibited lactogenic differentiation in BMECs on laminin substrate at the presence of HIP complex: the protein abundance of β-casein,STAT5 and p-STAT5 were significantly reduced;however,there was no significantly change in PRLR protein expression;the m RNA abundance of PRLR,β-casein and STAT5 A decreased significantly,but STAT5 B had no obviously change.In conclusion,the β-casein expression in BMECs can be induced by HIP complex.Laminin substrate can up-regulate the expression of integrin β1 while enhance the expression of β-casein induced.When inactivation integrin subunit β1,the protein expression and phosphorylation activity of STAT5 was inhibited in accordance with down-regulated β-casein expression.Therefore,This study demonstrates that integrin subunit β1 mediates lactogenic effect of laminin.This study regulates milk protein synthesis and provides new research ideas and experimental data about the regulation of lactation in cows.