Role Of C-Jun N-terminal Kinase In Transforming Growth Factor-beta1 Induced Overproduction Of Fibronectin In Mesangial Cells

PrefaceThe incidence rate of diabetic mellitus is increasing with sublimation of living standard and acceleration of population aging. Diabetic Nephropathy is a leading cause of mortality among complications of diabetes. The basic pathologic transform are kidney cells multiplication and excess ECM deposition early, glo-merular sclerosis in advanced stage. It's very necessary to investigate the patho-genesis of DN to improve patients living quality and delay kidney functional lesion. TGF - β₁ is correlated intimately with DN ,it stimulates excess ECM deposition specially. In recent years the role of TGF - β₁ regulating cell cyclic by Smad family signal pathway was demonstrated clearly , but the signal pathway that TGF - β₁ regulated ECM composition is not identified. Mitogen activated protein kinase signal pathway was focus recently, including :ERK,p38MAPK and JNK pathways. Many trails identify that MAPK pathway mediate TGF - β₁ inducing excess ECM deposition, but the role of JNK pathway is not very clear. Our study's aim is to detect the transform of JNK activation and FN secretion induced by TGF - β₁ in mesangial cells,and to discuss the role of JNK pathway in this progress in order to study the pathogenesis and treatment of DN.Materials and MethodsMesangial cells were cultured in the MEM culture medium in which contains the serum of fetal cattle at a concentration of 10% . All experiments were performed using cells between the subculturing 3^th and5^th passages. Transplantmesangial cells (1 x 107/bottle) in the logarithmic grow period into the culture utensil with the diameter of 6cm. When the cells grow to 80% amalgamation, change the culture fluid ( contains MEM culture fluid with 0. 5%FCS) and continue to culture for 48 hours. Groups;Group 1: use TGF - (3, (2ng/ml) to stimulate for 0,2,15 ,30min ,1,2,6,16,24h. Group 2: add different concentration of TGF - (3, (0,0. 5,1. 0,2. 0,4. Ong/ml) to stimulate for 30min. Group 3 : deal with different concentration SP600125 ( 10|xmol/l, 15|xmol/l, 20ujnol/l) for 30min,then add TGF - (3j (2ng/ml) to stimulate for 30min contrasted with the none stimulated ones. Every group has 4 parallel culture utensils. At the end of experimentation, wash samples by the pre - cooled PBS twice, scrape off the cells and centrifugate for 5min at 2000rpm/min. Put away the upper clear fluid, add cell fission fluid 100jxl( contains 1%NP-4O, 0.5% Sodium deoxycholate, 0. l%SDS,l%PBS,lmM PMSF,l|xg/ml aproptin ,l|xg/ml leupeptin) in the deposit, to be incubated on ice for 30min, -10°C and reserved in case of use. Before electrophoresis, measure the concentration of extracted fluid with Braford method to make sure that the sample of each hole is the same. The protein sample was put into the 10% SDS - PAGE and electrophorese for 90min , converted tto PVDF membrane at 4°C for a whole night, incubated with phosphorylated JNK antibody for 2 hours and another 2 hours with HRP marked sheep - anti - rabbit antibody, then DAB coloration. FN was detected by ELISA.ResultsTGF - pjcan induce JNK activition. Mesangial cells in the normal comparison group had low level Lactivation of JNK. The activity of JNK was obviously up - regulated in 2 min, reached the peak at 30min, recovered to the normal level at lh, and increased again after 6h, It can keep higher than normal for 24h. With the increasing of the concentration of TGF - (3, ,the activity of JNK increases too.With the increasing of the concentration of SP600125. The activation of JNK by stimulation of mesangial cells with TGF - (3j was gradually inhibited.When the concentration of SP600125 reached 20u,mol/l, the activation of JNK was obviously inhibited.After the stimulation of mesangial cells by TGF - (3j, the secretion of FN obviously increases and displays dose relying on effect.After the stimulation of mesangial cells by TGF - Pj , the secretion of FN obviously increases and displays time relying on effect.SP600125can inhibit the secretion of FN in mesangial cells induced by TGF ³i- With the concentration increasing, the inhibiting effect increases. When the concentration reach 20|xmol/l, the inhibiting effect is the highest. However, the concentration of FN is different with the normal contrast group(P <0. 05). There is no obvious significance comparing the single SP600125 group with the normal contrast group( P > 0.05 )DiscussionThere are three isoforms in mammals;TGF - p, ,TGF - p2and TGF - p3. TGF — Pi increase most in diabetic nephropathy. Isono' s study has demonstrated that TGF - p! activated ERK and p38MAPK but not JNK signal pathway . While Hocevar' study demonstrated that TGF - Pj induced overproduction of FN in cells by JNK signal pathway and cAMP - response element activation inBA cells. The study of human mesangial cells confirmed TGF — (3, activated ERK and JNK but not p38MAPK signal pathway. In short,the role of JNK in DN is unclear. The results of research show us that TGF - pt can quickly induce JNK in the mesangial cells. About 2min after stimulated by TGF - pj, the activity of JNK increased obviously;reached the peak at 30min and recovered to the normal level after 1 h;JNK increased again after 6h;activity of JNK was still higher than normal untill 24 h. It is also found that with the increase of the concentration of TGF -Pj ,the activity of JNK also increased. We considered this result as that TGF -pi activated JNK through some mechanism, and the JNK also can increase the TGF - p, created by mesangial cells themselves, which makes the persistent increase of JNK. This research confirms that TGF — Pj stimulates mesangial cells tosecrete FN relying on time and dose. SP600125 can inhibit it, and with the increase of concentration of SP600125, the inhibition increases, but it can not reach the level of the contrast group, which shows that JNK is not the only pathway in the increase of FN induced by TGF - Bj. This conclusion is consistent with previous study about MAPK pathway. The research from Wahab NA inden-tified that TGF - Bj induced phosphorylation of serin 133 in cAMP - response element binding protein (CBP) by extracellular signal -regulated kinase(ERKl/ ERK2) and p38MAPK,then induced FN overexpression .ConclusionTGF - Bj stimulates mesangial cells to secrete FN relying on time and dose. Through the activity of kinase and production, we confirmed that JNK pathway took part in FN overexpression in mesangial cells induced by TGF — Bt, inhibiting this signal pathway can decrease FN secretion. . The results indicate JNK pathway induced by TGF - Bj regulated overexpression of FN in mesangial cells .