P2Y2 receptors regulate osteoblast mechanosensitivity during fluid flow.
Mechanical stimulation of osteoblasts activates many cellular mechanisms including the release of ATP. Binding of ATP to purinergic receptors is key to load-induced osteogenesis. Osteoblasts also respond to fluid shear stress (FSS) with increased actin stress fiber formation (ASFF) that we postulate is in response to activation of the P2Y2 receptor (P2Y2R). Furthermore, we predict that ASFF increases cell stiffness and reduces the sensitivity to further mechanical stimulation. We found that small interfering RNA (siRNA) suppression of P2Y2R attenuated ASFF in response to FSS and ATP treatment. In addition, RhoA GTPase was activated within 15 min after the onset of FSS or ATP treatment and mediated ASFF following P2Y2R activation via the Rho kinase (ROCK)1/LIM kinase 2/cofilin pathway. We also observed that ASFF in response to FSS or ATP treatment increased the cell stiffness and was prevented by knocking down P2Y2R. Finally, we confirmed that the enhanced cell stiffness and ASFF in response to RhoA GTPase activation during FSS drastically reduced the mechanosensitivity of the osteoblasts based on the intracellular Ca(2+) concentration ([Ca(2+)]i) response to consecutive bouts of FSS. These data suggest that osteoblasts can regulate their mechanosensitivity to continued load through P2Y2R activation of the RhoA GTPase signaling cascade, leading to ASFF and increased cell stiffness.
Biomechanics and Movement Science, University of Delaware, Newark, Delaware;
Joseph
J
Gardinier
Department of Biological Sciences, University of Delaware, Newark, Delaware; and.
Weidong
W
Yang
Department of Biological Sciences, University of Delaware, Newark, Delaware; and.
Gregory R
GR
Madden
Department of Biological Sciences, University of Delaware, Newark, Delaware; and.
Andris
A
Kronbergs
Department of Biological Sciences, University of Delaware, Newark, Delaware; and.
Vimal
V
Gangadharan
Bioimaging Center, Delaware Biotechnology Institute, Newark, Delaware.
Elizabeth
E
Adams
Department of Biological Sciences, University of Delaware, Newark, Delaware; and Bioimaging Center, Delaware Biotechnology Institute, Newark, Delaware.
Kirk
K
Czymmek
Biomechanics and Movement Science, University of Delaware, Newark, Delaware; Department of Biological Sciences, University of Delaware, Newark, Delaware; and Bioimaging Center, Delaware Biotechnology Institute, Newark, Delaware rlduncan@udel.edu.
Randall L
RL
Duncan
D000818:Animals; D002460:Cell Line; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D008560:Membrane Fluidity / Q000502:physiology*; D051379:Mice; D010006:Osteoblasts / Q000502:physiology*; D051381:Rats; D058473:Receptors, Purinergic P2Y2 / Q000502:physiology*; D013314:Stress, Mechanical
D058473:Receptors, Purinergic P2Y2
P2Y2 receptor; Rho GTPases; cytoskeleton; fluid flow; osteoblast mechanosensitivity