Abstract
Class IA phosphoinositide 3-kinase (PI3K) is involved in regulating many cellular functions including cell growth, proliferation, cell survival, and differentiation. The p85 regulatory subunit is a critical component of the PI3K signaling pathway. Mesenchymal stem cells (MSC) are multipotent cells that can be differentiated into osteoblasts (OBs), adipocytes, and chondrocytes under defined culture conditions. To determine whether p85α subunit of PI3K affects biological functions of MSCs, bone marrow-derived wild type (WT) and p85α-deficient (p85α(-/-)) cells were employed in this study. Increased cell growth, higher proliferation rate and reduced number of senescent cells were observed in MSCs lacking p85α compare with WT MSCs as evaluated by CFU-F assay, thymidine incorporation assay, and β-galactosidase staining, respectively. These functional changes are associated with the increased cell cycle, increased expression of cyclin D, cyclin E, and reduced expression of p16 and p19 in p85α(-/-) MSCs. In addition, a time-dependent reduction in alkaline phosphatase (ALP) activity and osteocalcin mRNA expression was observed in p85α(-/-) MSCs compared with WT MSCs, suggesting impaired osteoblast differentiation due to p85α deficiency in MSCs. The impaired p85α(-/-) osteoblast differentiation was associated with increased activation of Akt and MAPK. Importantly, bone morphogenic protein 2 (BMP2) was able to intensify the differentiation of osteoblasts derived from WT MSCs, whereas this process was significantly impaired as a result of p85α deficiency. Addition of LY294002, a PI3K inhibitor, did not alter the differentiation of osteoblasts in either genotype. However, application of PD98059, a Mek/MAPK inhibitor, significantly enhanced osteoblast differentiation in WT and p85α(-/-) MSCs. These results suggest that p85α plays an essential role in osteoblast differentiation from MSCs by repressing the activation of MAPK pathway.
Highlights
Class IA phosphatidylinositol-3-kinases (PI3Ks) are critical regulators of a broad range of cellular processes including cell proliferation, differentiation, survival, and migration [12, 13]
While wild type (WT) and p85␣Ϫ/Ϫ Mesenchymal stem cells (MSC) grew slowly during early passages; a steady increase in the number of MSCs was observed over 10 passages in both the genotypes
This is consistent with studies by our group and others [27, 30, 31] and after passage 3, p85␣Ϫ/Ϫ MSCs demonstrated faster cell growth compared with WT MSCs at each time point examined (Fig. 2A)
Summary
Animals and Materials—p85␣Ϫ/Ϫ mice in a C57BL/6J strain have been described previously [23]. p85␣Ϫ/Ϫ mice were genotyped by polymerase chain reaction (PCR) as previously described and shown in Fig. 1 [24]. Differentiation Assays and Histochemical Staining—To induce osteogenic differentiation, WT, and p85␣Ϫ/Ϫ MSCs were plated at 5 ϫ 104/ml in osteogenic differentiation medium (MesenCult proliferation kit supplemented with 10Ϫ8 mol/liter dexamethasone, 5 g/ml ascorbic acid 2-phosphate, and 10mmol/liter -glycerophosphate) in 12-well plate. For Von Kossa staining, cells were cultured for 2 weeks; for ALP activity assay, cells were maintained in osteogenic differentiation medium for 1, 3, 5, and 7 days. WT and p85␣Ϫ/Ϫ MSCs were maintained in chondrogenic differentiation medium (MesenCult proliferation kit supplemented with 10Ϫ8 mol/liter dexamethasone, 5 g/ml ascorbic acid 2-phosphate, 10 mmol/liter -glycerophosphate, and 10 ng/ml TGF3) for 2 weeks. WT and p85␣Ϫ/Ϫ MSCs were maintained in adipogenic differentiation medium (MesenCult proliferation kit supplemented with 10Ϫ8 mol/liter dexamethasone and 10 g/ml of insulin) for 2 weeks.
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