L-type Calcium Channel Cav1.2 Regulates BMP9-induced Osteogenic Differentiation Of Mesenchymal Stem Cells

Objective:To explore the effect of Cav1.2,a subtype of L-calcium channel,on the osteogenic differentiation of Mesenchymal Stem Cells（MSCs）induced by Bone Morphogenetic Protein 9（BMP9）,and to explore its specific mechanismMethods:MSCs were treated with BMP9,qRT-PCR was used to detect the gene expression levels of voltage-gated calcium channel subtypes,and then flow cytometry and laser scanning confocal microscopy were used to observe the intracellular Ca²⁺concentration in MSCs after BMP9 stimulation.MSCs were then treated with BMP9 in the presence of Cav1.2 activator Bay K8644 or inhibitor Nisoldipine,respectively.Cytochemical staining was used to detect the activity of alkaline phosphatase（ALP）,a marker of early osteogenesis.Alizarin red S staining was used to detect calcium deposition.qRT-PCR and western blot were used to detect the expressions of Osteopontin（OPN）and Osteocalcin（OCN）,markers of late osteogenesis,and Runx2,Id1,Id2,and Id3downstream osteogenic target molecules of BMP9.The effects of Cav1.2on BMP9-induced phosphorylation of Smad1/5/9,β-catenin,GSK3βand total MAPKs and phosphorylation levels were detected by western blot.Then crystal purple staining and MTT assay were used to detect cell proliferation.Cell cycle was measured by flow cytometry.MSCs were treated wth small interfence RNA targeting Cav1.2（si Cav1.2）.Then the effect of si Cav1.2 on BMP9-induced ALP activity and calcium deposition osteogenic differentiation of MSCs was analyzed by cytochemical staining and alizarin red S,respectively.Results:BMP9 could promote the expression of L-type calcium channels,especially Cav1.2,and meanwhile promote the intracellular accumulation of Ca²⁺.BMP9-induced ALP activity and calcium deposition was enhanced by Cav1.2 agonist Bay K8644,while inhibited by Cav1.2inhibitor Nisoldipine.BMP9-induced expression of Id1,Id2,Id3 and Runx2was further increased by Bay K8644,whereas decreased by Nisoldipine.Bay K8644 could promote BMP9-induced phosphorylation of Smad1/5/9GSK3β,p38,JNK but inhibit BMP9-induced phosphorylation of ERK1/2,on the contrary,Nisoldipine could suppress BMP9-induced phosphorylation of Smad1/5/9 GSK3β,p38,JNK while enhance BMP9-induce phosphorylation of ERK1/2.In addition,Bay K8644 and Nisoldipine had no effect on cell activity and cell cycle at the early stage of culture.si Cav1.2 could significantly inhibit BMP9-induced ALP activity and calcium deposition of MSCs.Conclusions:L-type calcium channel Cav1.2 may regulate bone differentiation of BMP9-induced MSCs possibly by affecting the activity of Smad1/5/9,Wnt/β-catenin and MAPKs signaling pathways.