Abstract
Exercise prevents marrow mesenchymal stem cell (MSC) adipogenesis, reversing trends that accompany aging and osteoporosis. Mechanical input, the in-vitro analogue to exercise, limits PPARγ expression and adipogenesis in MSC. We considered whether C/EBPβ might be mechanoresponsive as it is upstream to PPARγ, 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/EBPβ mRNA (0.6-fold ±0.07, p<0.05) and protein (0.4-fold ±0.1, p<0.01) in MSC. SiRNA silencing of β-catenin prevented mechanical repression of C/EBPβ. C/EBPβ overexpression did not override strain’s inhibition of adipogenesis, which suggests that mechanical control of C/EBPβ is not the primary site at which adipogenesis is regulated. Mechanical inhibition of C/EBPβ, however, might be critical for further processes that regulate MSC health. Indeed, overexpression of C/EBPβ 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/EBPβ and both CHOP and BiP genes. The strain regimen, in addition to decreasing C/EBPβ 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/EBPβ, increased BiP and decreased CHOP protein expression. Thus, C/EBPβ is a mechanically responsive transcription factor and its repression should counter increases in marrow fat as well as improve skeletal resistance to ER stress.
Highlights
Exercise increases skeletal strength through mechanical effects that improve bone mineral content and architecture [1,2,3]
C/EBPb is a Mechanical Target in mesenchymal stem cells (MSC) We have previously shown that mechanical input inhibits the expression of PPARc protein in MSC, suggesting that there should be a previously unidentified mechanically sensitive target proximal to PPARc [6]
Application of mechanical strain to cells cultured in adipogenic medium significantly limited the rise in C/EBPb mRNA expression which occurred in early adipogenesis, compared to unloaded control cultures (p,0.05, see figure 1A)
Summary
Exercise increases skeletal strength through mechanical effects that improve bone mineral content and architecture [1,2,3]. The positive effect of exercise on the skeleton depends, at least partially, on the ability of mechanical input to regulate output of osteoblasts from progenitor mesenchymal stem cells (MSC). Decreased adipocytes and increased pre-osteoblasts have been demonstrated in the marrow of running rats [4] and climbing mice [5], indicating that MSCs are targeted by mechanical input. Several studies demonstrate that negative regulators of adipogenesis exert their effects via a critical transcription factor upstream of PPARc, C/EBPb [9,10,11,12,13]. As mechanical input leads to reduced expression of PPARc [6], we hypothesized that mechanical signals might regulate C/EBPb
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
