The Abnormal Regulation of Integrin α2in Osteogenic Differentiation of hBMSCs from Senile Osteoporosis
|School||Fourth Military Medical University|
|Keywords||Osteoporosis Human Bone Marrow Stromal Cells Integrin ERK Pathway|
Osteoporosis is defined by low bone mass and deteriorating bone structure, whichlead to increased bone fragility and susceptibility to fracture. An imbalance betweenosteoblast-mediated bone formation and osteoclast-mediated bone resorption in themarrow microenvironment results in the pathogenesis of osteoporosis. Multipleendogenous and exogenous factors have been shown to be involved in regulating boneremodeling. Although osteoporosis risks may be prevented by lifestyle changes,catastrophic effects on disability and mortality, still severely impact the life quality of theaging population, especially in patients65years of age and older. However, the keyregulating factor(s) and the underlying mechanism of male senile osteoporosis are notclearly defined or fully understood. Therefore, we have focused on the molecularmechanism underlying male senile osteoporosis to seek more effective therapeutic targets,irrespective of estrogen and glucocorticoid influences. Previous study showed that the defective bone formation during the process of boneremodeling seemed to be the principal pathophysiological mechanism responsible forage-related bone loss. Bone marrow mesenchymal cells (BMSCs) in marrow pool are themajor source of osteogenitor cells, contributing to bone remodeling of adults. Therefore,understanding the factors regulating osteogenic differentiation of BMSCs andstrenghthening BMSCs’s osteogenic differentiation are essential for further exploringbetter strategy of osteoporosis therapy.Adhesion and differentiation of cells are initiated and mediated by the activation of αand β trans-membrane Integrins, which are the principle mediators of the moleculardialogue between a cell and its ECM environment. Previous studies showed that αV, α5β1,αvβ3, andβ3/β5Integrins were involved interaction between osteoblasts and extracellularmatrix (ECM) affected osteoblast function and bone remodeling. Osteoblastmineralization was reduced significantly during osteogenesis following perturbation withα5or β1Integrin subunit antibody by approximately20%and45%, respectively—αVβ3Integrin by nearly65%, and α2β1Integrin by nearly95%. Moreover, Integrin α2β1, α5β1and αVβ3were also found to activate intracellular signaling cascades, and subsequentlyup－regulate the expressions of alkaline phosphatase (ALP) and osteocalcin (OCN) duringosteogenic differentiation in hBMSCs. However, whether there is any defect of Integrinsin the pathophysiology of senile osteoporosis and what is the regulatory role of individualIntegrin in hBMSCs are still unclear. Thus, our study focused on Integrin in senileosteoporotic hBMSCs.【Aims】(1) Separate, cultivation, and detection of osteogenesis ability of hBMSCs fromsenile osteoporosis patient.(2) The expression of α2, α5, αv, β1, and β3Integrin subunitsby real time-PCR of hBMSCs from senile osteoporosis patient.(3) To construct thelentiviral vector of Integrin α2gene and restore Integrin α2expression in osteoporotichBMSCs.(4) To determine the value of Integrin α2upon the regulation of osteogenicdifferentiation and gain further insight into the potential mechanism. 【Methods】(1) Stratified centrifuge technology was used to separate MSCs from donated bonemarrow. The cells consistently differentiated into bone was determined by differentiationassay. The cell surface markers were detected by FACS and the clonogenic potential wasperformed by CFU-F assay.(2) PCR analysis were used to examined the expression of α2,α5, αv, β1, and β3Integrin subunits.(3) DNA ligation and DNA sequence analysis wereused to construct plasmid containing α2Integrin gene. To investigate the optimumcondition for restore Integrin α2expression in osteoporotic hBMSCs.(4) RT-PCR, MTT,alkaline phosphatase staining and alizarin red staining techniques, immunofluorescencestaining were used to determine the stem cell propoties of transduced MSCs. Westernblotting assays were used to investigated the phosphorylation status of ERK, JNK, p38,and Akt for pinpoint a key signaling pathways.【Results】1Separate, cultivation, and detection of osteogenesis ability of hBMSCs fromsenile osteoporosis patienthBMSCs were isolated from NDs and OPs with an average age of69.375years.hBMSCs from NDs and OPs shared a similar fibroblast-like spindle shape. We alsoobserved that osteoporotic hBMSCs were less confluent than control cells. FACS analysesshowed that osteoporotic hBMSCs were positive for CD44and CD105and negative forCD34and CD45, similar to control donor cells.Furthermore, we evaluated and comparedthe proliferative and osteogenic differentiation capabilities of hBMSCs isolated from NDsand OPs. Based on MTT analysis and cell counts, the cell growth rate of osteoporoticBMSCs was significantly slower than that of hBMSCs from normal donors, and thisprominent difference persisted for8days of cell culture (p<0.001). The colony formationassay showed similar results (p<0.01). We evaluated and compared the osteogenicdifferentiation ability of hBMSCs from OP and ND groups by ALP activity detection andalizarin red S staining. ALP staining and activity assay showed that hBMSCs from NDsdisplayed a significantly higher ALP level compared to those from OPs. In contrast to the increased ALP activity in normal hBMSCs at day14, the ALP activity of osteoporotichBMSCs was somewhat decreased (p<0.01). Similarly, alizarin red S staining showed adefect in the mineralization ability of osteoporotic hBMSCs.Taken together, thesefindings indicate that, under similar culture conditions, the proliferation capacity ofhBMSCs isolated from OPs is retarded and that these cells gradually lose the ability todifferentiate into the osteogenic lineage during osteoporosis.2The expression of Integrin subunits in hBMSCs from senile osteoporosispatientTo explore the possible contribution of Integrins to the inhibited osteogenesis ofosteoporotic hBMSCs, we performed qPCR to detect the expression levels of α2, α5, αv,β1, and β3Integrin subunits. On day3of osteogenic differentiation, there was weak orabsent expression of the α2and β1chains in senile osteoporotic hBMSCs, Furthermore,we examined the expression level of Integrin α2and β1and osteogenic markers such asRunt-related transcription factor2(Runx2) and Osterix (Osx) in hBMSCs derived from2groups cultured in osteogenic differentiation medium. The expression level of Integrin α2was significantly reduced in osteoporotic hBMSCs compared with the normal groupduring osteogenic differentiation, which was consistent with the change in the trend of theosteogenesis-related genes (Runx2and Osx), suggesting that the down-regulation ofIntegrin α2might be involved in the functional paralysis of osteoporotic hBMSCs. Thus,Integrin α2might play an important role in the retardative osteogenesis of osteoporotichBMSCs though mediating the regulation of Runx2and Osx.3To construct the lentiviral vector of Integrin α2gene and restore Integrin α2expression of senile osteoporotic hBMSCsWe investigate the optimum condition for restore Integrin α2expression inosteoporotic hBMSCs and used lentiviral vectors to restore Integrin α2expression inosteoporotic hBMSCs. Stable over-expressing Integrin α2clones (LV-α2) were identifiedby Western blotting. 4Forced Integrin α2expression promotes cell growth and osteogenicdifferentiation and the ERK pathway is required for Integrin α2-mediatedosteoblastic differentiation of senile osteoporotic hBMSCsWe also found that the high level of Integrin α2in Lv-α2hBMSCs was accompaniedby increases in Runx2and Osx expression. Furthermore, we examined the effect ofIntegrin α2overexpression on cell proliferation. The MTT assay showed that forcedIntegrin α2expression promotes cell growth (p<0.01). We also investigated the effect ofIntegrin α2on the osteogenic differentiation of hBMSCs. Compared to Lv-ctr cells, theALP activity and alizarin red S staining of Lv-α2-infected cells was shown to be increased.These results indicate that Integrin α2promotes the proliferation and osteogenicdifferentiation of osteoporotic hBMSCs.To gain further insight into the potential mechanism underlying the osteogenicdifferentiation mediated by Integrin α2, we examined the phosphorylation status ofdownstream Integrin targets, including ERK, JNK, P38, and Akt, by Western blotting.Upon the stimulation of osteogenic differentiation, compared with the Lv-ctr, thephosphorylation of ERK1/2of Lv-α2-infected hBMSCs was rapidly activated. We foundthat Integrin α2-mediated the elevated RUNX2、 osteogenic differentiation andmineralization were significantly inhibited by PD98059during osteogenesis.【Conclusions】Our study demonstrates that hBMSCs derived from senile osteoporosis have aweakened capacity for cell proliferation and osteogenic differentiation due to thedown-regulation of Integrin α2. Moreover, the overexpression of Integrin α2can reversethe reduced proliferation and osteoblast differentiation capacity of hBMSCs. Furthermore,a mechanistic analysis suggests that Integrin α2activates ERK pathways byphosphorylation, thus promoting the expression of related downstream targets of Runx2and causing osteoblast proliferation and differentiation. Taken together, our findings suggestthat Integrin α2/ERK/Runx2pathways play an important role in senile osteoporosis and mayprovide a novel molecular target for the prevention and treatment of this disease.