the e pression of numerous genes in a cell type and signal specific manner, and plays a pivotal role in cellu lar proliferation, apoptosis, and embryogenesis. By catalyzing Breast cancer acetylation of histones and transcription fac tors, p300 plays a significant role in epigenetic regula tion. Recent evidence suggests that abnormal p300 function is associated with deregulated target gene e pression, and is implicated in inflammation. This is confirmed by our observation that LPS induced VCAM 1 e pression was reduced by inhibition of p300. Moreover, LPS directly stimulated p300 phosphoryl ation and the formation of ATF2 p300 comple via c Src ROS p38 MAPK. Taken together, we demon strated that LPS could trigger renal inflammation via p300 dependent VCAM 1 induction.
Conclusions In summary, as depicted in Figure 8, our results showed that in HRMCs, LPS induced ROS production through TLR4 MyD88 c Src No 2 or No 4, in turn initiates the activation of p38 MAPK and ATF2. Activated ATF2 was recruited to the promoter Inhibitors,Modulators,Libraries region of VCAM 1 leading to an increase of VCAM 1 promoter activity and the e pres sion of VCAM 1. These results provide new insights into the mechanisms of LPS action on HRMCs to regulate the e pression of VCAM 1 and thus e aggerated the inflam mation responses. Methods Materials Inhibitors,Modulators,Libraries Anti VCAM 1, anti TLR2, anti TLR4, anti MyD88, anti No 2, anti No 4, anti p47pho , anti Gs, Inhibitors,Modulators,Libraries anti c Src, anti B actin, anti p38 MAPK, anti ATF2, and anti p300 antibodies were from Santa Cruz. Anti GAPDH antibody was from Biogenesis.
Anti phospho p38 MAPK, anti phospho p42 p44 MAPK, anti phospho JNK1 2, anti phospho c Src, anti phospho ATF2, and anti phospho p300 antibodies were from Cell Signaling. Diphenyleneiodonium chloride, SP600125, Inhibitors,Modulators,Libraries U0126, SB202190, GR343, and PP1 were from Biomol. 5 chloromethyl 2,7 dichlorodihydrofluorescein diacetate, acetyl ester, 2,7 bis 5 carbo yfluorescein, aceto ymethyl ester, and dihydroethidium were from Molecular Probes. Edaravone was from Tocris Bio science. Anacetrapib Apocynin was purchased from ChromaDe . LPS, enzymes, and other chemicals were from Sigma. Cell culture Human renal mesangial cells were from Scien Cell Research Laboratories. Cells were cultured in DMEM F12 supplemented with 10% FBS and antibiotics at 37 C in a humidified 5% CO2 atmosphere. E periments were performed with cells from passages 4 to 8.
Measurement of intracellular ROS accumulation selleck chem The intracellular H2O2 levels were determined by meas uring fluorescence of DCFH DA, and the O2? levels were determined by measuring the fluorescence of DHE. The fluorescence intensities of DCF and DHE staining were detected at 495 529 and 518 605 nm, respectively, using a fluorescence microscope. In addition, HRMCs were washed with warm HBSS and incubated in HBSS containing 10 uM DCFH DA or DHE at 37 C for 30 min. and then replaced with a fresh medium. HRMCs were incubated with various concen trations of LPS for the indicated time intervals. Cells were washed twice with PBS and detached with trypsin