Osteopontin mediates hypoxia-induced proliferation of cultured mesangial cells: role of PKC and p38 MAPK.
BACKGROUND
We previously reported that hypoxia induces the proliferation of cultured mesangial cells mediated by the stimulation of intracellular calcium and the activation of protein kinase C (PKC). In the present study, we examined the roles of mesangial cell specific growth factors (platelet-derived growth factor and endothelin-1) and osteopontin (OPN) in hypoxia-induced proliferation of mesangial cells. In addition, we determined the effect of hypoxia on p38 mitogen-activated protein (MAP) kinase activity and the roles of both PKC and p38 MAP kinase in hypoxia-induced alterations in OPN and mesangial cell growth.


METHODS
Quiescent cultures of mesangial cells were exposed to hypoxia (3% O2) or normoxia (18% O2) in a serum-free medium, and [3H]-thymidine incorporation, OPN protein and mRNA expression, and p38 MAP kinase activity were assessed.


RESULTS
Hypoxic-conditioned medium mimicked the effect of hypoxia on thymidine incorporation, suggesting the release of diffusable growth promoting factor(s) by hypoxia. Neither anti-endothelin-1 nor anti-platelet-derived growth factor-neutralizing antibodies had an effect on increased thymidine incorporation induced by hypoxia. However, blocking the effects of OPN either with anti-OPN antibody or its beta3 integrin receptor antibody completely prevented the hypoxia-induced increase in thymidine incorporation. Hypoxia also stimulated OPN protein and mRNA levels. Hypoxia caused an acute activation of p38 MAP kinase, which was inhibited by both verapamil and an inhibitor of PKC (calph C). PKC inhibitor and an inhibitor of p38 MAP kinase (SB203580) reduced the hypoxia-induced stimulation of both OPN and cell growth.


CONCLUSIONS
These studies provide, to our knowledge, the first evidence demonstrating the role of OPN in hypoxia-induced proliferation of mesangial cells. In addition, hypoxia causes an activation of p38 MAP kinase in a calcium- and PKC-dependent manner, and the activation of PKC and p38 MAP kinase appears to be involved in the stimulation of both OPN and mesangial cell proliferation induced by hypoxia.
Division of Nephrology and Hypertension, Northwestern University Medical School, Chicago, IL 60611, USA.
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D000818:Animals; D001798:Blood Proteins / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D015687:Cell Hypoxia / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D019332:Endothelin-1 / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D005920:Glomerular Mesangium / Q000166:cytology* / Q000201:enzymology*; D005923:Glomerulosclerosis, Focal Segmental / Q000378:metabolism; D007511:Ischemia / Q000378:metabolism; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D053495:Osteopontin; D010100:Oxygen / Q000494:pharmacology; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D012795:Sialoglycoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases
D001798:Blood Proteins; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D019332:Endothelin-1; D010982:Platelet-Derived Growth Factor; D012333:RNA, Messenger; D012795:Sialoglycoproteins; C506897:Spp1 protein, rat; D053495:Osteopontin; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D010100:Oxygen