Hydraulic Pressure During Fluid Flow Regulates Purinergic Signaling and Cytoskeleton Organization of Osteoblasts

Abstract

During physiological activities, osteoblasts experience a variety of mechanical forces that stimulate anabolic responses at the cellular level necessary for the formation of new bone. Previous studies have primarily investigated the osteoblastic response to individual forms of mechanical stimuli. However in this study, we evaluated the response of osteoblasts to two simultaneous, but independently controlled stimuli; fluid flow-induced shear stress (FSS) and static or cyclic hydrostatic pressure (SHP or CHP, respectively). MC3T3-E1 osteoblasts-like cells were subjected to 12dyn/cm2 FSS along with SHP or CHP of varying magnitudes to determine if pressure enhances the anabolic response of osteoblasts during FSS. For both SHP and CHP, the magnitude of hydraulic pressure that induced the greatest release of ATP during FSS was 15 mmHg. Increasing the hydraulic pressure to 50 mmHg or 100 mmHg during FSS attenuated the ATP release compared to 15 mmHg during FSS. Decreasing the magnitude of pressure during FSS to atmospheric pressure reduced ATP release to that of basal ATP release from static cells and inhibited actin reorganization into stress fibers that normally occurred during FSS with 15 mmHg of pressure. In contrast, translocation of nuclear factor kappa B (NFκB) to the nucleus was independent of the magnitude of hydraulic pressure and was found to be mediated through the activation of phospholipase-C (PLC), but not src kinase. In conclusion, hydraulic pressure during FSS was found to regulate purinergic signaling and actin cytoskeleton reorganization in the osteoblasts in a biphasic manner, while FSS alone appeared to stimulate NFκB translocation. Understanding the effects of hydraulic pressure on the anabolic responses of osteoblasts during FSS may provide much needed insights into the physiologic effects of coupled mechanical stimuli on osteogenesis.

Physiological cyclic hydrostatic pressure induces osteogenic lineage commitment of human bone marrow stem cells: a systematic study

“…Moreover, this ATP release was magnitude depedent over time at each loading event. To date, only osteoblasts have been shown to elicit an ATP response to CHP, whereas, stem cells were found to secrete ATP in a fluid flow magnitude dependent manner [ 44 – 46 ]. Purinergic signalling plays a crucial role in bone anabolism, therefore ATP synthesis in response to mechanical stimulation may highlight initiation of mechanotransduction events, irrespective of type of stimulus [ 47 – 49 ].…”

P2Y2 receptor modulates shear stress-induced cell alignment and actin stress fibers in human umbilical vein endothelial cells

“…HUVECs express P2X as well as P2Y receptors, and are shown to have high ATP signaling activity on their cell surface [ 37 – 40 ]. Previously, we reported that shear stress upregulated the P2Y 2 receptor in HUVECs [ 12 , 41 ] and recent studies from others have suggested that these receptors mediate mechanotransduction [ 14 , 15 , 26 ]. Our primary findings in the present study are that P2Y 2 receptors modulate endothelial cell alignment, ASF formation, and cell migration in HUVECs and we thereby propose a novel role for P2Y 2 receptors in shear stress-induced endothelial cell remodeling.…”

“…It is well documented that endothelial cells release ATP and UTP, in response to various stimuli including hypoxia, vascular injury, and mechanical stimulation, and that these extracellular nucleotides act as paracrine or autocrine mediators via activation of purinergic P2 receptors [ 9 , 10 ]. Purinergic receptors are classified into P2X ligand-gated ion channel receptors and P2Y G protein-coupled receptors [ 11 ] and among these receptors, the P2X4 receptor is implicated in flow-induced calcium flux, vasodilation, and atheroprotective gene expression [ 12 , 13 ] while the P2Y 2 receptor is associated with mechanosensitivity in osteoblasts [ 14 , 15 ]. Both ATP and/or UTP are shown to influence endothelial cell cytoskeletal changes such as actin filament formation and cell motility, associated with activation of integrins and growth factor receptors [ 16 – 18 ].…”

A two-phase response of endothelial cells to hydrostatic pressure

“…Furthermore, it remains elusive how cells can sense hydrostatic pressure in the first place, as the molecular nature of a potential pressure sensor is still unknown. There is some evidence for Ca 2+ , purinergic signaling, RhoA, Rac1 and NF-KB being involved in the adaption to changes in ambient (the background; ∼760 mmHg, 101 kPa) pressure (Gardinier et al, 2014;Mandal et al, 2010;Sappington et al, 2009;Zhao et al, 2015). However, these messenger ions/signaling molecules are likely downstream targets of the actual pressure sensor.…”