Expression of p300-truncated fragments results in the modulation of apoptosis in rat mesangial cells.
BACKGROUND
Mesangial cell proliferation, apoptosis, and matrix deposition have pivotal roles in the pathogenesis of renal diseases such as diabetic nephropathy and glomerulonephritis. The behavior of mesangial cells depends on the integration of intracellular signals elicited by hormones and cytokines. We hypothesized that p300 is primarily involved in the integration of signal transduction pathways in rat mesangial cells (RMCs) and that interference with p300 function will alter apoptotic signals.


METHODS
We established an RMC cell line expressing the Tet-activator (tTA). RMC-tTA cells were transiently transfected with vectors coding for either the N-terminal third or the C-terminal third of p300. Expression was induced by the addition of doxycycline [Dox; 1 microg/mL; 5% fetal bovine serum (FBS)]. The percentage of apoptosis was determined using the TUNEL technique. Specific protein-protein interactions were determined by Western blot analysis of immunoprecipitated complexes. Cells were treated with 5% FBS or with H2O2 (500 micromol/L, 1 h) with and without Dox.


RESULTS
The expression of p300-C resulted in increased susceptibility to low serum-induced (20.0 +/- 4.6 vs. 3.0 +/- 1.7%) and to H2O2-induced apoptosis (75.3 +/- 13.3 vs. 50.8 +/- 6.5%) compared with controls. Immunoprecipitation of p300-C showed an interaction with the transcription factor c-Fos, which was enhanced by H2O2 treatment. Expression of the p300-N resulted in a rescue (34.8 +/- 6. 4 vs. 50.8 +/- 6.5%) from H2O2-induced apoptosis compared with controls. P300-N was shown to form a complex with the transcription factor nuclear factor-kappaB (NF-kappaB).


CONCLUSIONS
The data indicate that endogenous p300 is involved in apoptosis in mesangial cells. We propose that interference or enhancement of endogenous p300 function, by expression of exogenous fragments, can alter interactions with c-Fos or NF-kappaB and modulate signals during cellular stress.
Segelmark
M
M
Division of Nephrology and Hypertension, Department of Medicine, University of North Carolina at Chapel Hill, 27599, USA.
Barrett
C
C
Pendergraft
W
W
Falk
R
R
Preston
G
G
D000818:Animals; D017209:Apoptosis; D050882:CREB-Binding Protein; D002460:Cell Line; D005920:Glomerular Mesangium / Q000473:pathology*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D051379:Mice; D016328:NF-kappa B / Q000502:physiology; D009687:Nuclear Proteins / Q000502:physiology*; D016760:Proto-Oncogene Proteins c-fos / Q000502:physiology; D051381:Rats; D015534:Trans-Activators / Q000502:physiology*
D016328:NF-kappa B; D009687:Nuclear Proteins; D016760:Proto-Oncogene Proteins c-fos; D015534:Trans-Activators; D006861:Hydrogen Peroxide; D050882:CREB-Binding Protein; C494364:Crebbp protein, mouse; C494365:Crebbp protein, rat