Phospholipase C-gamma modulates epithelial tight junction permeability through hyperphosphorylation of tight junction proteins.
Phospholipase C-gamma (PLC-gamma) is stimulated by epidermal growth factor via activation of the epidermal growth factor receptors. The PLC inhibitor, 3-nitrocoumarin (3-NC), selectively inhibited PLC-gamma in Madin-Darby canine kidney cells without affecting the activity of PLC-beta. In contrast, inhibitors of PLC-beta, hexadecylphosphocholine and, had no effect on the activity of PLC-gamma. Inhibition of PLC-gamma by 3-NC was associated with an increase in tight junction permeability across Madin-Darby canine kidney cell monolayers, as evidenced by 3-NC-induced decrease in transepithelial electrical resistance and increase in mannitol flux over a concentration range that was inhibitory to PLC-gamma. An analog of 3-NC, 7-hydroxy-3-NC (7-OH-3-NC), which was inactive as an inhibitor of PLC-gamma, also had no effect on tight junction permeability. Treatment with 3-NC caused punctate disruption in the cortical actin filaments. The PLC-gamma inhibitor, 3-NC, but not the inactive analog, 7-OH-3-NC, caused hyperphosphorylation of the tight junction proteins, occludin, ZO-1, and ZO-2. The serine/threonine kinase inhibitor, staurosporine (50-200 nm), significantly attenuated 3-NC-induced hyperphosphorylation of ZO-2. This corresponded with attenuation by staurosporine of 3-NC-induced increase in tight junction permeability, suggesting a relationship between ZO-2 phosphorylation and tight junction permeability.
Ward
Peter D
PD
Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7360, USA.
Klein
Ryan R
RR
Troutman
Matthew D
MD
Desai
Shaleish
S
Thakker
Dhiren R
DR
D000199:Actins / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000502:physiology*; D003374:Coumarins / Q000494:pharmacology; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism*; D009574:Nitro Compounds / Q000494:pharmacology; D051966:Phospholipase C gamma; D010766:Phosphorylation; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D019108:Tight Junctions / Q000378:metabolism / Q000502:physiology*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000502:physiology*
C446262:3-nitrocoumarin; D000199:Actins; D003374:Coumarins; D004791:Enzyme Inhibitors; D004963:Estrenes; D007527:Isoenzymes; D008565:Membrane Proteins; D009574:Nitro Compounds; D011760:Pyrrolidinones; D010767:Phosphorylcholine; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C039128:miltefosine; D010738:Type C Phospholipases; D051966:Phospholipase C gamma