Mechanical Strain Downregulates C/EBPβ in MSC and Decreases Endoplasmic Reticulum Stress

Exercise prevents marrow mesenchymal stem cell (MSC) adipogenesis, reversing trends that accompany aging and osteoporosis. Mechanical input, the in-vitro analogue to exercise, limits PPAR gamma expression and adipogenesis in MSC. We considered whether C/EBP beta might be mechanoresponsive as it is upstream to PPAR gamma, and also is known to upregulate endoplasmic reticulum (ER) stress. MSC (C3H10T1/2 pluripotent cells as well as mouse marrow-derived MSC) were cultured in adipogenic media and a daily mechanical strain regimen was applied. We demonstrate herein that mechanical strain represses C/EBP beta mRNA (0.6-fold +/- 0.07, p<0.05) and protein (0.4-fold +/- 0.1, p<0.01) in MSC. SiRNA silencing of beta-catenin prevented mechanical repression of C/EBP beta. C/EBP beta overexpression did not override strain's inhibition of adipogenesis, which suggests that mechanical control of C/EBP beta is not the primary site at which adipogenesis is regulated. Mechanical inhibition of C/EBP beta, however, might be critical for further processes that regulate MSC health. Indeed, overexpression of C/EBP beta in MSC induced ER stress evidenced by a dose-dependent increase in the pro-apoptotic CHOP (protein 4-fold +/- 0.5, p<0.05) and a threshold reduction in the chaperone BiP (protein 0.6-fold +/- 0.1, p = 0.2; mRNA 0.3-fold +/- 0.1, p<0.01). ChIP-seq demonstrated a significant association between C/EBP beta and both CHOP and BiP genes. The strain regimen, in addition to decreasing C/EBP beta mRNA (0.5-fold +/- 0.09, p<0.05), expanded ER capacity as measured by an increase in BiP mRNA (2-fold +/- 0.2, p<0.05) and protein. Finally, ER stress induced by tunicamycin was ameliorated by mechanical strain as demonstrated by decreased C/EBP beta, increased BiP and decreased CHOP protein expression. Thus, C/EBP beta is a mechanically responsive transcription factor and its repression should counter increases in marrow fat as well as improve skeletal resistance to ER stress.