title: Mechanical force-activated phospholipase D is mediated by Galpha12/13-Rho and calmodulin-dependent kinase in renal epithelial cells.
abstract: The renal glomerulus, the site of plasma ultrafiltration, is exposed to mechanical force in vivo arising from capillary blood pressure and fluid flow. Studies of cultured podocytes demonstrate that they respond to stretch by altering the structure of the actin cytoskeleton, but the mechanisms by which physical force triggers this architectural change and the signaling pathways that lead to generation of second messengers are not defined. In the present study, we found that in renal epithelial cells [podocytes and Madin-Darby canine kidney (MDCK) cells], application of mechanical force to the cell surface through fibronectin-coated ferric beads and exposure of the cells to magnetic force lead to Rho translocation and actin cytoskeleton reorganization. This application of force recruited Rho and filamentous actin (F-actin) to bead loci and subsequently stimulated phospholipase D (PLD), a downstream effector of Rho. Using MDCK cells that stably express regulators of G protein-signaling (RGS) proteins [RGS4 attenuates Galpha(i) and Galpha(q), and the p115RhoGEF-RGS domain (p115-RGS) attenuates Galpha(12/13)] to define the signaling pathway, we found that mechanical force induced Galpha(12/13)-Rho activation and increased F-actin to stimulate PLD activity. The activation can be partially prevented by the C(3) exoenzyme. Pretreatment of the cells with chemical inhibitors of several kinases showed that calmodulin-dependent kinase is also involved in stretch-induced PLD activation by a separate pathway. Taken together, our data demonstrate that in cultured podocytes and MDCK cells, mechanical force leads to actin cytoskeleton reorganization and PLD activation. The signaling pathways for PLD activation involve Galpha(12/13)/Rho/F-actin and calmodulin-dependent kinase.
authors.affiliation: Dept. of Medicine, Case Western Reserve Univ., Louis Stokes Veterans Affairs Medical Ctr., 10701 East Blvd., 151W, Cleveland, OH 44106, USA.
authors.forename: Jenny
authors.initials: J
authors.lastname: Ziembicki
authors.forename: Rajnish
authors.initials: R
authors.lastname: Tandon
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Schelling
authors.forename: John R
authors.initials: JR
authors.lastname: Sedor
authors.forename: R Tyler
authors.initials: RT
authors.lastname: Miller
authors.forename: Chunfa
authors.initials: C
authors.lastname: Huang
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000502:physiology*; D002460:Cell Line; D002474:Cell-Free System; D003599:Cytoskeleton / Q000502:physiology; D004285:Dogs; D004847:Epithelial Cells / Q000201:enzymology / Q000502:physiology*; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000502:physiology*; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D008465:Mechanoreceptors / Q000502:physiology; D009119:Muscle Contraction / Q000502:physiology; D010739:Phospholipase D / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology
chemical_list: D000199:Actins; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010739:Phospholipase D; D044365:GTP-Binding Protein alpha Subunits, G12-G13