Alphav integrin regulates TNF-alpha-induced nitric oxide synthesis in rat mesangial cells--possible role of osteopontin.
BACKGROUND
Tumour necrosis factor-alpha (TNF-alpha) induces nitric oxide (NO) synthesis in rat mesangial cells (MCs). We previously demonstrated that osteopontin (OP), a matrix protein that mainly interacts with the alphav integrin family, increased time-dependently by TNF-alpha stimulation at gene and protein levels. The regulation of NO synthesis by integrins or matrix proteins is unclear.


METHODS
We examined whether integrin, especially alphav integrin, regulates NO synthesis in rat MCs and whether OP, an alphav integrin ligand, has an effect on TNF-alpha-induced NO synthesis. Furthermore, OP and inducible NO synthase (iNOS) gene expression was examined by Northern blotting.


RESULTS
TNF-alpha increased NO synthesis in MCs in a time-dependent manner. Synthetic GRGDSP peptide, which is known to inhibit various integrins that interact with RGD-containing extracellular matrices, increased TNF-alpha-induced NO levels in a dose-dependent manner. Cyclical RGD peptide, the specific inhibitor of alphav integrin, also exhibited a dose-dependent effect of increasing NO levels, while GRGESP peptide, which has very low affinity to integrins, had no effect. In addition, NO synthesis was found to be significantly reduced when MCs were plated on OP-coated dishes compared to type I or IV collagen-coated dishes. Furthermore, anti-OP antibody increased NO synthesis in MCs. iNOS mRNA levels were increased by TNF-alpha, and were abruptly diminished after OP mRNA was significantly induced.


CONCLUSIONS
The present study demonstrated the involvement of alphav integrin in TNF-alpha-induced NO synthesis in rat MCs, and the possible role of OP was suggested in the mechanism. TNF-alpha and extracellular matrices can co-operate to regulate the behaviour of MCs at least partly through NO synthesis, which may participate in the course of glomerular diseases.
Nagasaki
T
T
Second Department of Biochemistry, Osaka City University Medical School, Osaka, Japan.
Ishimura
E
E
Koyama
H
H
Shioi
A
A
Jono
S
S
Inaba
M
M
Hasuma
T
T
Yokoyama
M
M
Nishizawa
Y
Y
Morii
H
H
Otani
S
S
D000818:Animals; D015703:Antigens, CD / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D015870:Gene Expression; D005920:Glomerular Mesangium / Q000166:cytology / Q000378:metabolism*; D039564:Integrin alphaV; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D009842:Oligopeptides / Q000494:pharmacology; D053495:Osteopontin; D051381:Rats; D017207:Rats, Sprague-Dawley; D012795:Sialoglycoproteins / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D015703:Antigens, CD; D039564:Integrin alphaV; D009842:Oligopeptides; D012795:Sialoglycoproteins; C506897:Spp1 protein, rat; D014409:Tumor Necrosis Factor-alpha; D053495:Osteopontin; D009569:Nitric Oxide; C058161:glycyl-arginyl-glycyl-aspartyl-seryl-proline; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat