Z. Xie, N. Case, C. O'Conor, J. Rubin, M. Zou, B. Sen, M. Horowitz, M. Styner

Exercise combats obesity while promoting the formation of bone. Since adipocytes and osteoblasts originate from a common precursor, the mesenchymal stem cell (MSC), effects of exercise on both fat and bone may occur at the stage of mesenchymal lineage selection. We have investigated the usefulness of MSCs cloned from adult murine marrow in the study of mechanical regulation of MSC lineage. We first confirmed that MSCs made from C57BL/6 adult males would undergo controlled differentiation. After 4 days in adipogenic medium, MSCs expressed PPARγ and adiponectin protein and stained positively for oil-red-O. Adipogenesis was accompanied by a reduction in β-catenin levels. MSCs grown in osteogenic medium for 5 days showed positive alkaline phosphatase staining and increased osterix expression, while bone nodules stained by alizarin red were present after 21 days. Hematopoietic stem cells were excluded from the MSC cultures, as assessed by FACs analysis. In adipogenic medium, application of strain (3600 cycles, 2%, daily for 3 d) reduced PPARγ and adiponectin mRNA by 30% and 50%, with reduction confirmed by Western. The decrease in β-catenin accompanying adipogenesis was prevented by the strain regimen. These results are consistent with our prior work showing that strain prevented adipogenesis and involved β-catenin in C3H10T1/2 cells. We next evaluated MSCs made from adult Caveolin-1 null mice and found that adipogenesis was retarded: after 3 days in adipogenic medium, PPARγ and adiponectin mRNA levels in Cav-1 null MSCs were only 5% and 1% compared to concurrently differentiating caveolin-1 replete MSCs. However, adipogenesis had progressed in the Cav-1 null MSCs by 7 days. Importantly, strain effectively inhibited adipogenesis in the Cav-1 transgenic MSCs. We next considered strain effects on MSCs grown in osteogenic medium for 3 days.  Application of strain for 6 hours increased COX2 and WISP1 mRNA by 212% and 199% in wild-type MSCs, consistent with strain activation of β-catenin we previously described in pre-osteoblasts. In contrast, strain failed to increase COX2 or WISP1 in Cav-1 null MSCs, and these MSCs did not form bone nodules, suggesting that osteogenic differentiation was perturbed in the absence of caveolin-1. In conclusion, we have shown that C57BL/6 MSCs, which effectively undergo adipogenesis and osteogenesis, respond to mechanical strain in a manner similar to our prior work in murine cell lines. That the caveolin-1 null MSCs respond differently suggests that MSCs from transgenic mice can be expected to provide further insights into the mechanisms by which mechanical strain influences mesenchymal lineage selection.     

Disclosures: None