Mechanical inhibition of RANKL expression is regulated by H-Ras-GTPase.
Mechanical input is known to regulate bone remodeling, yet the molecular events involved in mechanical signal transduction are poorly understood. We here investigate proximal events leading to the ERK1/2 activation that is required for mechanical repression of RANKL (receptor activator of NF-kappaB ligand) expression, the factor that controls local recruitment of osteoclasts. Using primary murine bone stromal cells we show that dynamic mechanical strain via substrate deformation activates Ras-GTPase, in particular the H-Ras isoform. Pharmacological inhibition of H-Ras function prevents strain activation of H-Ras as well as the downstream mechanical repression of RANKL. Furthermore, small interfering RNA silencing of H-Ras, but not K-Ras, abrogates mechanical strain repression of RANKL. H-Ras-specific inhibition of mechanorepression of RANKL was also demonstrated in a murine pre-osteoblast cell line (CIMC-4). The requirement of cholesterol for H-Ras activation was probed; cholesterol depletion of rafts using methyl-betacyclodextrin prevented mechanical H-Ras activation. That the mechanical repression of RANKL requires activation of H-Ras, a specific isoform of Ras-GTP that is known to reside in the lipid raft microdomain, suggests that spatial arrangements are critical for generation of specific downstream events in response to mechanical signals. By partitioning signals this way, cells may be able to generate different downstream responses through seemingly similar signaling cascades.
Rubin
Janet
J
Department of Medicine, Emory University School of Medicine and Veterans Affairs Medical Center, Atlanta, Georgia 30033, USA. janet.rubin@emory.edu
Murphy
Tamara C
TC
Rahnert
Jill
J
Song
Hannah
H
Nanes
Mark S
MS
Greenfield
Edward M
EM
Jo
Hanjoong
H
Fan
Xian
X
D000818:Animals; D001696:Biomechanical Phenomena; D001854:Bone Marrow Cells / Q000166:cytology; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics*; D002460:Cell Line; D002478:Cells, Cultured; D004789:Enzyme Activation; D051231:Farnesyltranstransferase / Q000037:antagonists & inhibitors; D005786:Gene Expression Regulation / Q000502:physiology*; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000235:genetics*; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000502:physiology; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D017154:Stromal Cells / Q000166:cytology / Q000502:physiology; D020703:ras GTPase-Activating Proteins / Q000378:metabolism*
D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; D020703:ras GTPase-Activating Proteins; D051231:Farnesyltranstransferase; C495971:Hras protein, mouse; D016283:Proto-Oncogene Proteins p21(ras)