Sodium-calcium exchanger 1 regulates epithelial cell migration via calcium-dependent extracellular signal-regulated kinase signaling.

Na(+)/Ca(2+) exchanger-1 (NCX1) is a major calcium extrusion mechanism in renal epithelial cells enabling the efflux of one Ca(2+) ion and the influx of three Na(+) ions. The gradient for this exchange activity is provided by Na,K-ATPase, a hetero-oligomer consisting of a catalytic α-subunit and a regulatory ß-subunit (Na,K-ß) that also functions as a motility and tumor suppressor. We showed earlier that mice with heart-specific ablation (KO) of Na,K-ß had a specific reduction in NCX1 protein and were ouabain-insensitive. Here, we demonstrate that Na,K-ß associates with NCX1 and regulates its localization to the cell surface. Madin-Darby canine kidney cells with Na,K-ß knockdown have reduced NCX1 protein and function accompanied by 2.1-fold increase in free intracellular calcium and a corresponding increase in the rate of cell migration. Increased intracellular calcium up-regulated ERK1/2 via calmodulin-dependent activation of PI3K. Both myosin light chain kinase and Rho-associated kinase acted as mediators of ERK1/2-dependent migration. Restoring NCX1 expression in ß-KD cells reduced migration rate and ERK1/2 activation, suggesting that NCX1 functions downstream of Na,K-ß in regulating cell migration. In parallel, inhibition of NCX1 by KB-R7943 in Madin-Darby canine kidney cells, LLC-PK1, and human primary renal epithelial cells (HREpiC) increased ERK1/2 activation and cell migration. This increased migration was associated with high myosin light chain phosphorylation by PI3K/ERK-dependent mechanism in HREpiC cells. These data confirm the role of NCX1 activity in regulating renal epithelial cell migration.

Caveolin-1 regulates P2X7 receptor signaling in osteoblasts.

The synthesis of new bone in response to a novel applied mechanical load requires a complex series of cellular signaling events in osteoblasts and osteocytes. The activation of the purinergic receptor P2X(7)R is central to this mechanotransduction signaling cascade. Recently, P2X(7)R have been found to be associated with caveolae, a subset of lipid microdomains found in several cell types. Deletion of caveolin-1 (CAV1), the primary protein constituent of caveolae in osteoblasts, results in increased bone mass, leading us to hypothesize that the P2X(7)R is scaffolded to caveolae in osteoblasts. Thus, upon activation of the P2X(7)R, we postulate that caveolae are endocytosed, thereby modulating the downstream signal. Sucrose gradient fractionation of MC3T3-E1 preosteoblasts showed that CAV1 was translocated to the denser cytosolic fractions upon stimulation with ATP. Both ATP and the more specific P2X(7)R agonist 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP) induced endocytosis of CAV1, which was inhibited when MC3T3-E1 cells were pretreated with the specific P2X7R antagonist A-839977. The P2X7R cofractionated with CAV1, but, using superresolution structured illumination microscopy, we found only a subpopulation of P2X(7)R in these lipid microdomains on the membrane of MC3T3-E1 cells. Suppression of CAV1 enhanced the intracellular Ca(2+) response to BzATP, suggesting that caveolae regulate P2X(7)R signaling. This proposed mechanism is supported by increased mineralization in CAV1 knockdown MC3T3-E1 cells treated with BzATP. These data suggest that caveolae regulate P2X(7)R signaling upon activation by undergoing endocytosis and potentially carrying with it other signaling proteins, hence controlling the spatiotemporal signaling of P2X(7)R in osteoblasts.

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.

Insulin-like growth factor-1 regulates the mechanosensitivity of chondrocytes by modulating TRPV4.

Both mechanical and biochemical stimulation are required for maintaining the integrity of articular cartilage. However, chondrocytes respond differently to mechanical stimuli in osteoarthritic cartilage when biochemical signaling pathways, such as Insulin-like Growth Factor-1 (IGF-1), are altered. The Transient Receptor Potential Vanilloid 4 (TRPV4) channel is central to chondrocyte mechanotransduction and regulation of cartilage homeostasis. Here, we propose that changes in IGF-1 can modulate TRPV4 channel activity. We demonstrate that physiologic levels of IGF-1 suppress hypotonic-induced TRPV4 currents and intracellular calcium flux by increasing apparent cell stiffness that correlates with actin stress fiber formation. Disruption of F-actin following IGF-1 treatment results in the return of the intracellular calcium response to hypotonic swelling. Using point mutations of the TRPV4 channel at the microtubule-associated protein 7 (MAP-7) site shows that regulation of TRPV4 by actin is mediated via the interaction of actin with the MAP-7 domain of TRPV4. We further highlight that ATP release, a down-stream response to mechanical stimulation in chondrocytes, is mediated by TRPV4 during hypotonic challenge. This response is significantly abrogated with IGF-1 treatment. As chondrocyte mechanosensitivity is greatly altered during osteoarthritis progression, IGF-1 presents as a promising candidate for prevention and treatment of articular cartilage damage.

Multi-scale 3D Cryo-Correlative Microscopy for Vitrified Cells.

Three-dimensional (3D) visualization of vitrified cells can uncover structures of subcellular complexes without chemical fixation or staining. Here, we present a pipeline integrating three imaging modalities to visualize the same specimen at cryogenic temperature at different scales: cryo-fluorescence confocal microscopy, volume cryo-focused ion beam scanning electron microscopy, and transmission cryo-electron tomography. Our proof-of-concept benchmark revealed the 3D distribution of organelles and subcellular structures in whole heat-shocked yeast cells, including the ultrastructure of protein inclusions that recruit fluorescently-labeled chaperone Hsp104. Since our workflow efficiently integrates imaging at three different scales and can be applied to other types of cells, it could be used for large-scale phenotypic studies of frozen-hydrated specimens in a variety of healthy and diseased conditions with and without treatments.

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

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.