NO Inhibits Stretch-induced MAPK Activity by Cytoskeletal Disruption

Abstract

Mesangial cells (MC) grown on extracellular matrix protein-coated plates and exposed to cyclic strain/relaxation proliferate and produce extracellular matrix protein, providing an in vitro model of signaling in stretched MC. Intracellular transduction of mechanical strain involves mitogen-activated protein kinases, and we have shown that p42/44 mitogen-activated protein kinase (extracellular signal-regulated kinase (ERK)) is activated by cyclic strain in MC. In vivo studies show that increased production of nitric oxide (NO) in the remnant kidney limits glomerular injury without reducing glomerular capillary pressure, and we have observed that NO attenuates stretch-induced ERK activity in MC via generation of cyclic guanosine monophosphate (cGMP). Accordingly, we sought to determine whether NO affects strain-induced ERK activity after strain and how this is mediated. Strain-induced ERK activity was dependent on time and magnitude of stretch and was maximal after 10 min at ؊27 kilopascals. Actin cytoskeleton disruption with cytochalasin D abrogated this. The non-metabolizable cGMP analogue 8-bromo cyclic GMP (8-Br-cGMP) dose-dependently attenuated strain-induced ERK activity. Cytoskeletal stabilization with jasplakinolide prevented this inhibitory effect of 8-Br-cGMP. Cyclic strain increased nuclear translocation of phospho-ERK by immunofluorescent microscopy, again attenuated by 8-Br-cGMP. Jasplakinolide prevented the inhibitory effect of 8-Br-cGMP on activated ERK nuclear translocation after strain. Strain increased ERK-dependent AP-1 nuclear protein binding, which was attenuated by cytochalasin D and 8-Br-cGMP. These data indicate that cGMP can inhibit cyclic strain-induced ERK activity, nuclear translocation, and AP-1 nuclear protein binding. Cytoskeletal disruption leads to the same effect, whereas cytoskeleton stabilization reverses the effect of 8-Br-cGMP. Thus, NO inhibits straininduced ERK activity by cytoskeletal destabilization.Glomerular mesangial cells (MC) 1 are positioned as architectural supports for capillary loops and are therefore exposed to pulsatile stretch/relaxation (1). Whereas little resident glomerular cell proliferation or sclerosis is demonstrable in normal animals (2), MC proliferation and matrix production, eventually resulting in sclerosis, can be induced by maneuvers that increase intraglomerular pressure by 10 mmHg (3-5). Moreover, in these models, preventing the intraglomerular pressure rise attenuates sclerosis (5-7). We and others have shown reduction of sclerosis and MC proliferation in remnant glomeruli by oral L-arginine supplementation to increase NO production (8, 9). L-Arginine increases nitric oxide production by the remnant kidney and reduces glomerular endothelin-1 expression but does not lower glomerular capillary pressure (8).The effects of mechanical forces on MC in vitro can be modelled by culturing cells in wells with deformable bottoms and then applying a vacuum to the wells to generate alternating cycles of strain and relaxation. Initial experiments using this m...

Hypertonic Preconditioning Inhibits Macrophage Responsiveness to Endotoxin

“…Stress fiber polymerization after endotoxin stimulation is necessary for optimal cellular adhesion, migration, intracellular signaling, and gene transcription and translation (42). Recent work has demonstrated that inhibition of endotoxin-induced stress fiber polymerization by cytochalasin D and other inhibitors results in an inhibition of MAPK signaling and attenuation of TNF-␣ production in mononuclear cells (43)(44)(45). These effects are similar to those demonstrated by extracellular hypertonic conditioning.…”

Integration of signal pathways for stretch-dependent growth and differentiation in vascular smooth muscle

“…Aikawa and co-workers (1) showed a small but significant activation of ERK after transfection of cardiac myocytes with constitutively active mutants of Rho. Furthermore, stabilization of actin filaments with jasplakinolide prevented dephosphorylation of ERK in response to actin depolymerization by nitric oxide (20). Our observations suggest that the state of actin polymerization, regardless of applied stretch, is able to regulate the activity of the MAPK cascade.…”

Akt Mediates Mechanical Strain-Induced Collagen Production by Mesangial Cells

“…The events leading to PI3K/Akt activation by mechanical strain, however, remain largely unexplored. We first investigated the role of the actin cytoskeleton in this regard given its importance in the stretch-induced activation of MAPK pathways, as we and others have published (9,32,48,49). Disruption of the actin cytoskeleton with cytochalasin D, however, did not inhibit straininduced Akt activation.…”

“…We first hypothesized that the cytoskeleton might be an important mediator because we have previously shown that actin reorganization and integrity was critical to activation of MAPK by stretch in MC (9,32). We used cytochalasin D (200 ng/ml, 1 h) to disrupt the actin cytoskeleton, having previously shown this to be effective in MC by rhodamine phalloidin imaging of F-actin (9).…”