Mechanical Sensing Element PDLIM5 Promotes Osteogensis Of Human Fibroblasts By Affecting The Activity Of Microfilaments

BackgroundBone defects caused by fractures and diseases are currently existing problems.In this process,fibroblasts can synthesize and secrete collagen fibers and matrix components that promote bone healing.And under specific induction,human skin fibroblasts(HSFs)can differentiate into osteoblasts.HSFs have the characteristics of easy to fetch material,wide source,and suitable for in vitro proliferation,they are regarded as one of the important seed sources for bone tissue engineering applications.As a bridge connecting the extracellular environment,the cytoskeleton participates in the perception,conduction and response of many signal pathways.Among them,microfilament is one of the cytoskeletons,and its integrity is the basis for many signaling molecules to function.Studies have shown that the expression of microfilament increases in the process of osteogenesis,and the shape of microfilament is filamentous and orderly,which plays an important role in osteogenesis.PDLIM5 is a cytoplasmic protein about 63kDa in molecular size,which is composed of N-terminal PDZ domain and C-terminal LIM domain.PDLIM5 plays an important role in the formation of specific protein macromolecular complexes,which has attracted attention as an cytoskeleton adaptor protein of microfilaments.It binds to cytoskeleton and membrane proteins through the PDZ domain,and interacts with various signal molecules through the LIM domain,which also plays a key role in the differentiation of various cell types and other cell functions.Therefore,this subject intends to explore the relationship between the cytoskeleton-related protein PDLIM5 and microfilaments,and to explore the potential mechanism of osteogenic differentiation in human fibroblasts.This will lay a theoretical foundation for the research of stem cells and the treatment of bone tissue engineering-related diseases such as bone defects,as well as for researchers to design suitable bone filling materials for patients.PurposeTo explore the relationship between PDLIM5 and the microfilament skeleton in human skin fibroblast osteogenesis,to confirm that PDLIM5 participates in mediating the osteogenic differentiation of fibroblasts by affecting the expression and changes of the microfilament skeleton.MethodsThe osteogenic medium was used to induce osteogenic differentiation of human fibroblasts,and the expression and localization of PDLIM5 in osteogenic differentiation were verified by WesternBlot,qRT-PCR and immunofluorescence.And then PDLIM5 was knocked down by lentiviral transfection to verify the effect of PDLIM5 on microfilament and osteogenic differentiation.Finally,the molecular mechanism of PDLIM5-F-actin mediated osteogenic differentiation was discussed by using microfilament stabilizers.Results1.On the 4 d,7 d,14 d,21d of osteogenic differentiation of HSFs,Western blot and qRT-PCR quantitative analysis were performed on PDLIM5.With the increase of osteogenic differentiation time,the expression of PDLIM5 protein and mRNA levels were up-regulated,and the difference was statistically significant(P<0.05).Immunofluorescence and immunoprecipitation found that PDLIM5 was localized to the cytoskeleton microfilaments,and PDLIM5 was detected in ?-actininl immunoprecipitation.2.Lentivirus transfected human skin firoblast successfully down-regulated PDLIM5.CCK8,wound healing,and transwell cell migration test of the transfected HSFs showed that after knocking down PDLIM5,compared with the control group,the cell proliferation and migration ability of the shPDIM5 group were significantly decreased,and the difference was statistically significant(P<0.001).3.The transfected HSFs were treated with osteogenic medium.ALP staining,qRT-PCR and WesternBlot all showed that after knocking down PDLIM5,the osteogenic differentiation ability of HSFs decreased significantly,and the expression of cytoskeletal microfilaments and related proteins and mRNA levels also decreased.The difference was statistically significant(P<0.05).Immunofluorescence showed that after knocking down PDLIM5,it was found that the cytoskeleton microfilament morphology changed from the original thin and long fiber shape to short,wide and thick,but there was no change in the uninduced group.4.After knocking down PDLIM5,adding microfilament stabilizer(Jasplakinolide,JAS)to the osteogenic medium,it is found that JAS can reverse the inhibitory effect of knocking down PDLIM5 on HSF osteogenesis.ALP staining and WestemBlot showed that,compared with the control group(shPDLIM5),the osteogenic ability was significantly enhanced in(shPDLIM5+JAS)group;The expression of osteogenesis marker proteins(RUNX2,ALP)and other related proteins(?-actin,a-actininl,PDLIM5)were significantly up-regulated.ConclusionThis study confirmed that knocking down PDLIM5 significantly inhibited the proliferation and movement ability of fibroblasts.The osteogenic medium was used to induce the osteogenic differentiation of fibroblasts,verifying that PDLIM5 can mediate the osteogenic differentiation of fibroblasts by affecting the expression the arrangement of microfilaments.