NO inhibits stretch-induced MAPK activity by cytoskeletal disruption.
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 strain-induced ERK activity by cytoskeletal destabilization.
Ingram
A J
AJ
Department of Medicine, McMaster University, Hamilton, Ontario L8N 1Y2, Canada. ingrama@fhs.mcmaster.ca
James
L
L
Cai
L
L
Thai
K
K
Ly
H
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Scholey
J W
JW
D000199:Actins / Q000378:metabolism; D000818:Animals; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D047630:Depsipeptides; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009569:Nitric Oxide / Q000502:physiology*; D010456:Peptides, Cyclic / Q000494:pharmacology; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D018808:Transcription Factor AP-1 / Q000378:metabolism
D000199:Actins; D047630:Depsipeptides; D010456:Peptides, Cyclic; D018808:Transcription Factor AP-1; C057531:jasplakinolide; C016276:8-bromocyclic GMP; D009569:Nitric Oxide; D020928:Mitogen-Activated Protein Kinases; D006152:Cyclic GMP