Hedgehog Signaling Pathway Regulates The Proliferation And Differentiation Of Bovine Muscle Satellite Cells

Hedgehog (Hh) signaling pathway is crucial for animal growth and development. Almostthe normal functions of all tissues are under the control of Hh signaling pathway. Studies thatfocus on many kinds of mesoderm-derived cell suggested that, the proliferaton anddifferentiation of different cell types were regulated by Hh signaling. The muscle satellitecells (MuSCs) which are located beneath the basement membrane of muscle fibers are as thereserve for the renewing and repairing of muscle fiber, which decided the performance of beefcattle production. We studied in vivo expression level of bovine tissues and in vitro expressionlevel of bovine MuSC for several genes associatied with the signaling transduction, cellproliferation and differentiation. Cyclopamine and purmpahamine, inhibitor and agonist of Hhsignaling pathway saperately, were used to study the effect of Hh signaling pathway on theproliferation and differentiation of bovin MuSC.We investigated expression level of several genes in different tissues of Qinchuan cattleand cells populations derived from muscle tissue at the stage of proliferation anddifferentiation. Besides that, flow cytometry assay was used to analyze cell cycle under thetreatment of cyclopamine and purmorphamine. Medium containing only2%horse serumwere used to induce the myogenesis and adipogenesis was induced by the medium containinginsulin or overexpression of C/EBPα or ADIG and then differentiation of bovine MuSC wasanalyzed.The expression profile showed that expression level of Ptch1, Smo, Gli1and Gli2werelowest in muscle compared with other12tissues detected in our experiments, and all of thesegenes are key components of Hh signaling pathway. In consistent, cell populations fromprimary culture, expression level of the above genes are lower in cells adhered later than cellsadhered earlier. Cells adhered in later time were proved to contain more myoblasts. Therefore,usually genes reltive to Hh signaling pathway were less expressed in muscle of adult cattleand in the myoblasts.One population of MuSC containing abundance of myoblast was isolated. Most of thecells were CD34+and myogenic markers positive. During the proliferation of bovine MuSC, cyclopamint suppressed cell growth and elevated the rate of cells in G1phase. The emergenceof the expression peak of cyclin E2was later in cyclopamine treated group, compared tocontrol and purmorphamine groups. The inhibition ability of cyclopamine to MuSCproliferation was dose dependent, which was revealed by the growth curve.The formation of myotubes was depressed when cyclopamine was supplemented duringthe myognic differentiation. In contrast, purmophamine enhanced this process, the numberand extended period of myotubes are all increased after purmophamine treatment. After theinitiation of myogenic differentiation, expression level of components of Hh signalingpathway were up-regulated. Expression level of myogenic transcriptive factors MyoD, MyoGand Pax7were higher in purmorphamine treated group, and lower in cyclopamine treatedgroup. Therefore, it was concluded that gene expression of components of Hh signalingpathway was activated after the inition of myogenic differentiation. As the Hh signaling wasblocked, myogenesis was suppressed. On the contrary, activation of Hh signaling promotedthe process of myogenesis.During adipogenic differentiation of MuSC, the adipogenic level in cyclopamine treatedgroup was similar to control group, but the number and size of lipid droplet are smaller in thepurmorphamine treated group. Expression levels of the components of Hh signaling pathwaydeclined in the adipogenesis process regardless of the induce assays. The expression levels ofPPARγ and C/EBPα were not different significantly between the groups in whichcyclopamine or purmorphamine or solvent alone was supplemented during the adipogincdifferentiation of MuSC. Although the further inhibition of Hh did not affect the formation oflipid droplet in cells, adipogenesis was depressed by increase the activity of Hh signaling.In conclusion, activity of Hh signaling is positive to proliferation and myogenicdifferentiation of bovine MuSC, and there was a negative relationship between Hh signalingand adipogenesis.