OR31-3 The RhoA Gtpase Activating Protein ARHGAP18 Regulates Mesenchymal Stem Cell Lineage Commitment

Program: Abstracts - Orals, Poster Preview Presentations, and Posters
Session: OR31-Novel Signaling Mechanisms and Bone Cell Biology
Basic
Monday, June 23, 2014: 11:30 AM-1:00 PM
Presentation Start Time: 12:00 PM
W475 (McCormick Place West Building)

Outstanding Abstract Award
Sherwin S. Yen, MD MPH, William R. Thompson, DPT PhD, Gunes Uzer, PhD, Zhihui Xie, MD, Buer Sen, MD, Maya Styner, MD, Keith Burridge, PhD and Janet E Rubin, MD
University of North Carolina Chapel Hill
Exercise loading of the skeleton promotes bone formation through biasing marrow MSC toward osteoblasts and away from adipocytes. We have shown that application of mechanical strain suppresses adipogenesis in marrow derived MSC in part by enhancing cytoskeletal structure and stiffness through activation of RhoA. The proximal steps leading to mechanical RhoA activation involve a Fyn-FAK-mTORC2-Akt cascade initiated in the focal adhesion mechanosome. Exchange of GDP for GTP is the crucial last step governing RhoA activity and is regulated by Guanine nucleotide Exchange Factors (RhoA activators) and GTP-ase Activating Proteins (RhoA deactivators). We asked if strain activates RhoA by GEF stimulation and/or by GAP inhibition. We selected ARHGAP18 as a RhoA specific GAP candidate because it has been shown in previous reports to downregulate RhoA and decrease actin stress fiber formation in fibroblasts. ARHGAP18 deficient marrow derived MSC were generated through stable shRNA knockdown. ARHGAP18 deficient MSCs demonstrated increased basal RhoA activation measured by RhoA bead binding assay. This suggested that ARHGAP18 might function as a tonic inhibitor of RhoA in MSC. Imaging showed the actin cytoskeleton to be increased in the ARHGAP18 deficient MSC compared to controls. Application of 200 cycles x 2% strain using the Flexcell system induced a further increase in RhoA activation compared to controls and, strain-induced cytoskeletal reorganization (increased actin bundling and connectivity) was more robust in the ARHGAP18 KO cells. As the actin cytoskeleton not only responds to dynamic mechanical input, but also to the static substrate stiffness, we reasoned that a loss of ARHGAP18 might influence MSC differentiation due to enhanced cytoskeletal structure. After 4 d in adipogenic medium, ARHGAP18 deficient MSC resisted adipogenesis as compared to control MSC, assessed by Oil-Red-O lipid stain and measures of fat markers by immunoblot analysis. Furthermore, when cultured in 7 d of osteogenic media, ARHGAP18 deficient cells displayed accelerated osteogenesis with increased alkaline phosphatase staining and osteogenic gene expression. These data demonstrate that ARHGAP18 is a primary negative regulator of RhoA activity in MSCs, and alternate GAPs do not compensate for its absence. The stiffer cytoskeleton results in an anti-adipogenic, pro-osteogenic lineage bias. Our data suggest that MSC lineage is exquisitely sensitive to factors that alter cytoskeletal dynamics. By identifying the key regulators of RhoA activity during mechanical strain we aim to further elucidate how a dynamic physical environment can control MSC fate.

Nothing to Disclose: SSY, WRT, GU, ZX, BS, MS, KB, JER

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