TRPV4-mediated mechanotransduction in marrow stromal cells
Objective Marrow stromal cells(MSCs)are key cells in the process of fracture heal-ing,and mechanical stimulation can regulate the process of osteogenic differentiation.This study explores the localization of TRPV4 and F-actin as well as the calcium influx during osteogenic differentiation of MSCs.TRPV4 is therefore proposed as a potential clinical therapeutic target.Methods In vitro,the rat MSCs were divided into a control group and a stretching mechanical group.Osteogenesis induction culture was conducted in vitro for 7 days,14 days,and 21 days.Alizarin red staining and polymerase chain reac-tion(PCR)were performed to detect the activity of osteogenic genes[bone morphogenetic protein-2(BMP-2)and Runx2],and Western blotting was used to detect the expression of alkaline phosphatase(ALP)and TRPV4 proteins.A calcium ion fluorescence probe(Fura-4)was used to detect the intracellu-lar calcium ion influx in the control group and the stretching mechanical group,and the intracellular locali-zation was studied by immunofluorescence staining of TRPV4 and F-actin protein.After preliminarily deter-mining the impact of TRPV4,MSCs were treated with TRPV4 agonist(GSK101)and divided into a control group and GSK101 group.Changes in osteogenic genes(BMP-2 and Runx2)were detected.The paired t-test was done for comparison between two groups.Results After osteogenic induction culture of rat MSCs,tensile mechanical stimulation significantly promoted osteogenic differentiation of MSCs,with en-hanced Alizarin Red staining.Mechanical stimulation significantly increased the transcriptional activity of BMP-2 gene and Runx2 gene at 7th day after surgery(t=5.252,2.851,P＜0.05),with changes of(3.97±0.82)times and(3.47±0.42)times,respectively.The expression of ALP protein increased(t=4.069,P＜0.05),with a fold change of 2.42±0.86.At this time,the expression level of TRPV4 protein remained unchanged(t=1.115,P＞0.05),with a change multiple of(1.21±0.78)times.The results of immunofluorescence staining confirmed that the expression and localization of TRPV4 and mechanical re-sponsive F-actin protein were enhanced after mechanical stimulation.Fura-4 probe results showed that me-chanical stimulation promoted calcium ion influx in MSCs(t=7.048,P＜0.01),with a change multiple of 4.29±0.72.After treating MSCs with TRPV4 agonist GSK101,the transcriptional activity of osteogenic genes(BMP-2 and ALP)was significantly enhanced(t=3.762,3.977,P＜0.05),with changes of(3.68±1.05)times and(4.33±0.63)times,respectively.Conclusion Stretching stimulation can acti-vate the TRPV4 protein in MSCs,co localize with F-actin,mediate calcium ion influx,and thereby en-hance osteogenic differentiation activity.
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