The Effects Of Silencing Connective Tissue Growth Factor On The Expression Of Matrix Metalloproteinase-1 And -2 In Human Osteoblastic MG63 Cells

Part one The Effects of Silencing Connective Tissue GrowthFactor on the Expression of Matrix Metalloproteinase-1and-2 in Human Osteoblastic MG63 CellsObjective To investigate the inhibitory effect of small interferingRNA(siRNA) on the expression of connective tissue growth factor(CTGF), and to observe the effects of down-regulated CTGF on theexpression of type 1 collagen,alkaline phosphatase,matrixmetalloproteinase-1 (MMP-1), -2 (MMP-2) mRNA, metalloproteinase-1and -2 protein in human osteoblastic MG63 cells.Methods Three pairs of 21-nucleotide CTGF siRNAs directed to thehuman CTGF mRNA 440,875 and 910 targets were differentlytransfected into human osteoblastic MG63 cells with DMRIE-C reagentpackage, Untreated and transfected scramble siRNA served as the blankcontrol and nonspecific siRNA control, respecetively. Total RNA andprotein of the cells after 48h incubation were extracted. The expression ofCTGF in mRNA and protein level was assessed by northern blot andwestern blot,and the expression of type 1 collagen and alkalinephosphatase mRNA in human osteoblastic MG63 cells evaluated by semiquantitive RT-PCR, The expression of MMP-1 and -2 in mRNA andprotein level were assessed by semiquantitive RT-PCR and western blot.Cell viability was tested by MTT assay.Results Compared with the blank control group,the expression of CTGFmRNA and protein was markedly down-regulated by siRNA directed to440 and 910 target of human CTGF mRNA,siRNA directed to 875 targetand scramble siRNA showed no effect on the expression of CTGF.Theexpression of type 1 collagen and alkaline phosphatase mRNA wasreduced in human osteoblastic MG63 cells transfected with siRNAdirected to 440 and 910 target, accompanied by significantly decreasedMG63 cell viability.Using western immunoblot analyses, we found thatthe expression of CTGF and MMP-1 protein was markedlydown-regulated by siRNA directed to 440 and 910 target of human CTGFmRNA. Down-regulated CTGF had no impact on the level of activeMMP-2.Conclusion CTGF siRNA can effectively reduced the expression ofCTGF mRNA and protein, Inhibition of CTGF expression decreases theexpression of type 1 collagen, alkaline phosphatase and MMP-1 mRNAas well as MMP-1 protein level in human osteoblastic MG63 cells. Thesurvival of human osteoblastic-like MG63 cell was markedly reduced. Thus, CTGF may play an essential role in sustaining bone metabolichomeostasis.Part Two The Effects of Recombinant Human ConnectiveTissue Growth Factor on the Proliferation, Differentiation,and Apoptosis in Human OsteoblastsObjective To investigate the effects of recombinant human connectivetissue growth factor (rCTGF) on the cell proliferation, differentiation, andapoptosis in human osteoblasts(HOB).Methods Human osteoblasts were treated with rCTGF,cell proliferationwas measured by (~3H)-thymidine (~3H-TdR) incorporation method.Theactivity of alkaline phosphatase (ALP) was determined by usingα-nitrophenyl phosphate as a substrate.The effect of rCTGF on theexpression of type 1 collagen was determined by western blotting.Osteocalcin in conditioned media was measured by radioimmune assay.Cell apoptosis was assayed by flow cytometry.Results rCTGF promoted osteoblastic proliferation, and increased theALP activity, osteocalcin and type 1 collagen secretion in adose-depentent manner, rCTGF markedly inhibited human osteoblasticapoptosis induced by serum deprivation.Conclusion rCTGF can promote osteoblastic proliferation, differentiation,and protect osteoblast against apoptosis.These results indicated thatCTGF may play an essential role in bone metabolism through increasingosteoblast number and bone matrix synthesis.Part Three The Effects of Recombinant Human ConnectiveTissue Growth Factor on the Expression of Osteoprotegerin(OPG) and Receptor Activator of NF-κB Ligand (RANKL)Objective To investigate the effects of recombinant human connectivetissue growth factor on the expression of OPG and RANKL. Methods Total protein in human osteoblasts treated with rCTGF wasextracted and the expression of OPG and RANKL was assessed bywestern blotting.The expression of focal adhesion kinase (FAK),mitogen-activated protein kinase (MAPK) and AKT was also assessed bywestern blotting.Results rCTGF inhibited RANKL protein expression in a dose- andtime-depentent manner in human osteoblasts, rCTGF showed no effect onthe expression of OPG. rCTGF induced activation of p38 MAPK anddephosphorylation of FAK in human osteoblasts, p38 MAPK inhibitorSB23058 can abrogated the inhibitory effect of rCTGF on RANKL inhuman osteoblasts.Conclusion rCTGF can inhibit the expression of RANKL in humanosteoblasts via activation of p38 MAPK and dephosphorylation of FAK. Part Four The Effects of Recombinant Human ConnectiveTissue Growth Factor on the Expression of membranetype-1 matrix metalloproteinase and matrixmetalloproteinase-2 in Human OsteoblastsObjective To investigate the effects of recombinant human connectivetissue growth factor on the expression of membrane type-1 matrixmetalloproteinase (MT1-MMP) and matrix metalloproteinase-2 (MMP-2)in human osteoblastsMethods Total protein in human osteoblast treated with rCTGF wasextracted.The expression of membrane type-1 matrix metalloproteinaseand matrix metalloproteinase-2 was assessed by western blotting.Theexpression of mitogen-activated protein kinase (MAPK) and AKT wasalso assessed by western blotting.Results rCTGF increased the expression of membrane type-1 matrixmetalloproteinase and matrix metalloproteinase-2 protein in a dose- andtime-depentent manner in human osteoblasts, rCTGF induced activationof p38 MAPK in human osteoblasts, p38 MAPK inhibitor SB23058 canabrogated the effect of rCTGF on expression of membrane type-1matrix metalloproteinase and matrix metalloproteinase-2 in human osteoblasts.Conclusion rCTGF can increase the expression of membrane type-1matrix metalloproteinase and matrix metalloproteinase-2 in humanosteoblasts via activation of p38 MAPK.CTGF may play an essential rolein bone remodeling through regulating the expressions of MMPs.