Nagasaki T, Ishimura E, Koyama H, Shioi A, Jono S, Inaba M, Hasuma T, Yokoyama M, Nishizawa Y, Morii H, Otani S
Second Department of Biochemistry, Osaka City University Medical School, Osaka, Japan.
Nephrol Dial Transplant. 1999 Aug;14(8):1861-6. doi: 10.1093/ndt/14.8.1861.
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.
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.
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.
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.
肿瘤坏死因子-α（TNF-α）可诱导大鼠系膜细胞（MCs）合成一氧化氮（NO）。我们之前证实，骨桥蛋白（OP）是一种主要与αv整合素家族相互作用的基质蛋白，在基因和蛋白水平上，其受TNF-α刺激后呈时间依赖性增加。整合素或基质蛋白对NO合成的调节尚不清楚。
我们研究了整合素，尤其是αv整合素，是否调节大鼠MCs中的NO合成，以及αv整合素配体OP是否对TNF-α诱导的NO合成有影响。此外，通过Northern印迹法检测OP和诱导型一氧化氮合酶（iNOS）基因的表达。
TNF-α以时间依赖性方式增加MCs中NO的合成。已知能抑制与含RGD的细胞外基质相互作用的各种整合素的合成GRGDSP肽，以剂量依赖性方式增加TNF-α诱导的NO水平。αv整合素的特异性抑制剂环状RGD肽也表现出增加NO水平的剂量依赖性效应，而对整合素亲和力极低的GRGESP肽则无作用。此外，与I型或IV型胶原包被的培养皿相比，当MCs接种在OP包被的培养皿上时，发现NO合成显著减少。此外，抗OP抗体增加了MCs中NO的合成。TNF-α使iNOS mRNA水平升高，在OP mRNA被显著诱导后，iNOS mRNA水平突然下降。
本研究证明αv整合素参与了TNF-α诱导的大鼠MCs中NO的合成，并提示了OP在该机制中的可能作用。TNF-α和细胞外基质至少部分可通过NO合成协同调节MCs的行为，这可能参与了肾小球疾病的病程。
