title: Mechanical strain downregulates C/EBPβ in MSC and decreases endoplasmic reticulum stress.
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.
authors.affiliation: Department of Medicine, University of North Carolina, Chapel Hill, North Carolina, USA. mstyner@med.unc.edu
authors.forename: Maya
authors.initials: M
authors.lastname: Styner
authors.forename: Mark B
authors.initials: MB
authors.lastname: Meyer
authors.forename: Kornelia
authors.initials: K
authors.lastname: Galior
authors.forename: Natasha
authors.initials: N
authors.lastname: Case
authors.forename: Zhihui
authors.initials: Z
authors.lastname: Xie
authors.forename: Buer
authors.initials: B
authors.lastname: Sen
authors.forename: William R
authors.initials: WR
authors.lastname: Thompson
authors.forename: John Wesley
authors.initials: JW
authors.lastname: Pike
authors.forename: Janet
authors.initials: J
authors.lastname: Rubin
mesh_terms: D050156:Adipogenesis; D000818:Animals; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000235:genetics*; D015536:Down-Regulation / Q000187:drug effects; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects; D059630:Mesenchymal Stem Cells / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D013314:Stress, Mechanical; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: D022782:CCAAT-Enhancer-Binding Protein-beta; D014415:Tunicamycin