Function Of Glycoprotein M6B In Smooth Muscle Differentiation And Underlying Mechanism

BackgroundSmooth muscle cells are widely distributed in digestive,respiratory,circulatory,urinary and reproductive systems.Smooth muscle cells are a sort of highly differentiated cells that maintain tension through contractions to cause movement and hold deformation of organs.They also produce continuous contraction and tonic contraction,allowing the organ to withstand the load and maintain its original shape.The role of smooth muscle in the cardiovascular system is involved in the formation of blood vessels,the maintenance of blood pressure and other physiological processes as well as the pathogenesis of hypertension and atherosclerosis.In recent years,the prevalence of cardiovascular diseases increased year by year and cardiovascular diseases have become the first cause of death.According to the WTO’s statistics on the cause of death in the global population in 2008 and its future expectations,the number of deaths from cardiovascular diseases is 17 million,which account for nearly one-third of globle death.Among the cardiovascular deaths,the number of deaths due to hypertension is 9.4 million,accounting for about 45%.The mortality rate of stroke in China in 2008 was >130 per 100,000 people,which accounted for 51% of the total deaths.Hypertension and stroke were inextricably linked with smooth muscle function,differentiation,and proliferation.Therefore,it is of great significance to study the mechanism of smooth muscle differentiation for the controling the occurrence and development of cardiovascular disease.Highly differentiated smooth muscle cells express a variety of contractile and contractile-associated proteins,such as smooth muscle α-actin(α-SMA),smooth muscle myosin heavy chain(MYH11),and calponin.Various physical and chemical factors such as growth factors,inflammatory regulators,and shearing forces may regulate the differentiation of smooth muscle cells.Among them,tissue growth factor beta in TGF-beta superfamily induces smooth muscle(SMC)marker genes in many cell types including C3H10T1/2(10T1/2)cells,neural crest Mone-1 cells,mesenchyme cells,embryonic stem cells and other pluripotent stem cells.TGF-β is a multifunctional cytokine that regulates a variety of cellular activities including proliferation and differentiation.The TGF-β1 signal is received by type 1 and type 2 receptors of TGF-β1,before phosphorylating Smad2 and Smad3 proteins.When Smad2 and Smad3 proteins are activated by TGF-beta receptors,they bind to Smad4 proteins in the cytoplasm to form complexes and translocate to the nucleus,regulate the expression of the target gene with other transcription factors or individually.The GPM6 B gene encodes a transmembrane protein which belongs to proteolipid protein(PLP)family.GPM6 B was originally discovered as a structural protein in the nervous system of vertebrate embryos.Recent studies have shown that GPM6 B binds directly to the serotonin transporter(SERT)and decreases the expression of serotonin transporters on the cell membrane,leading to abnormalities in mood and motor function.The GPM6 B gene can be detected not only in the cerebellum,but also in the white matter of the brain and the dorsal root ganglion of the embryo,and it is expressed in the heart,smooth muscle,liver,skeletal muscle,kidney and spleen.The GPM6 B gene not only plays an important role in the central nervous system but also is active in peripheral tissues.In B lymphcytes,overexpression of GPM6 B induces overexpression of actin and microtubule systems,which makes GPM6 B a cancer-associated gene of lymphoma.Studies in some tumors show that GPM6 B may also be one of the markers of certain tumor angiogenesis.GPM6 B also affects osteoblast alkaline phosphatase(ALP)activity and extracellular matrix mineralization,and is also closely related to actin cytoskeleton and exocytosis of stromal vesicle.In summary,the current research on GPM6 B mainly focuses on 1.signal transduction of neurons;2.tumor cytoskeleton;3.angiogenesis;4.osteoblast differentiation and osteogenesis.There is no study on GPM6 B about cardiovascular system been reported in the literature.According to another report,the GPM6 B gene binds directly to the serotonin transporter(SERT)in the nervous system and regulates neuronal function.However,SERT also binds to TβRI and regulates the function of TβRI and regulates the phosphorylation of Smad2 and Smad3 by TβRI after cardiomyocyte injury.This process is also a classical pathway and regulatory site in the process of smooth muscle differentiation.Therefore,we speculated that GPM6 B may be a new regulatory protein of TGF-β-Smad2/3 pathway and thus regulate smooth muscle cell differentiation.Aims1 To investigate the expression of GPM6 B in smooth muscle cells and tissues.2 To clarify the role of GPM6 B in the differentiation of smooth muscle cells.3 To elucidate the molecular mechanism of GPM6 B regulating smooth muscle cell differentiation.Methods1 The differentiation of C3H10T1/2 cells into smooth muscle cells was induced by TGF-β1,and invesgate the expression of smooth muscle myosin heavy chain(SM-SMA),smooth muscle α-actin Calponin,as well as the expression of the GPM6 B gene.2 C3H10T1/2 cells were infected with lentivirus containing a shRNA knockdown the expression of GPM6 B to create a steady transfected cell lines sh-GPM6B1-5,and sh-Scramble containing a scramble plasmid shRNA fragment was used as a blank control.Differentiated sh-GPM6 B cells and sh-Scramble cells were prepared to observe the effect of GPM6 B knockdown on the differentiation of C3H10T1/2 cells into smooth muscle cells.3 After incubation with TGF-β,we observed the phosphorylation and nuclear translocation of TGF-β-Smad2/3 pathway after GPM6 B knockdown.4 Co-incubation with TGF-β and SRI-011381,a specific agonist of tissue growth factor receptor(TβRI),of sh-GPM6 B and sh-Scramble cells to observe whether the repressive effects on differentiation of smooth muscle cells and TGF-β-Smad2/3 activation can be restored by TβRI-specific agonists SRI-011381.5 GPM6 B and p-Smad2/3 were labeled by immunofluorescence in the aorta and small intestine sections of mouse embryonic thoracic and adult mice on day 12.5 to observe the expression of GPM6 B and Smad2/3 phosphorylation level in smooth muscle cells during development and in adult tissues.6 Expression of TβRI and TβRII in C3H10T1/2 cells after inducing smooth muscle differentiation,and the TGF-β-Smad2/3 pathway-related protein that directly bound to GPM6 B in C3H10T1/2 cells after 3 days of differentiation was detected by co-immunoprecipitation.Results1 The expression level of GPM6 B gradually increased during the differentiation of smooth muscle cells,and GPM6 B was highly expressed during the differentiation of mouse embryonic smooth muscle cells,but was lower in adult smooth muscle cells.2 Knockdown of GPM6 B expression induced smooth muscle cell-specific protein SM-MHC,α-SMA,and CALPONIN expression in smooth muscle cell differentiation.Therefore,the knockdown of GPM6 B expression will inhibit the differentiation of smooth muscle cells.3 Knockdown the expression of GPM6 B also inhibits the phosphorylation and nuclear translocation of Smad2/3 in the TGF-β-Smad2/3 pathway.Therefore,the knockdown of GPM6 B expression also inhibits the activation of the TGF-β-Smad2/3 pathway.4 Co-treatment of cells with SRI-011381,a specific agonist of TGF-β1 and TGF-β-Smad2/3 pathway,restored the inhibitory effects of GPM6 B on the differentiation of smooth muscle cells and the activation of TGF-β-Smad2/3 pathway.It is suggested that GPM6 B functions to regulate smooth muscle cell differentiation by regulating TGF-β-Smad2/3 pathway.5 The knockdown of GPM6 B expression does not affect TGF-beta receptor 1 and TGF-beta receptor 2 expression.Immunoprecipitation assays revealed that GPM6 B is directly associated with TGF-beta receptor 1 in differentiated C3H10T1/2 cells.Binds without direct binding to TGF-beta receptor 2 or Smad2/3.This indicates that GPM6 B exerts a regulatory effect on the TGF-β-Smad2/3 pathway through its direct interaction with TGF-β receptor 1.ConclusionsOur study has revealed that GPM6 B play a crucial role in SMC differention.We also demonstrated that GPM6 B promotes SMC differentiation through activation of TGF-β-Smad2/3 signaling by directly interacts with TβRI.These findings provide new insights into the biological function of GPM6 B,SMC differentiation and propagation of TGF-β signaling.Therefore,GPM6 B could be considered as a potential target influencing SMC differentiation and cardiovascular regenerative medicine.