Background: High glucose (HG) environments activate several protein kinase pathways in mesangial cells, including the mitogen-activated protein kinase (MAPK) pathway, ERK. The p38 MAPK pathway is activated by events that occur in the setting of diabetes, such as protein kinase C (PKC) up-regulation and cellular stresses (osmotic stress and redox changes). Substrates of activated p38 MAPK include transcription factors that are involved in the microvascular complications of diabetes. This current study investigated the mechanisms of HG-mediated activation of p38 MAPK in cultured human mesangial cells (HMCs) and the effects of p38 MAPK activation on the transcription factor activator protein-1 (AP-1).
Methods: HMCs were cultured in 5 mmol/L D-glucose [normal glucose (NG)] or 30 mmol/L D-glucose (HG) for seven days. Cells were also treated with HG for brief periods of time (0.5 to 4 hours) to assess the acute effects of HG on p38 MAPK. Using Western blotting of HMC lysates, changes in the tyrosine and threonine phosphorylation of p38 MAPK were measured. The kinase activity of immunoprecipitated p38 MAPK was determined by an in vitro assay that measured the phosphorylation and activation of MAPKAP kinase-2, an intermediary signaling protein downstream of p38 MAPK. To investigate the role of osmotic stress in HG activation of p38 MAPK, cells were acutely treated with mannitol (25 to 250 mOsm/L x 5 to 60 min) or were grown seven days in media supplemented with mannitol at concentrations iso-osmotic to HG media. To investigate the role of PKC in HG-mediated p38 MAPK activation, HMCs were treated with the PKC inhibitors GF 109203X, Ro 32-0432, or rottlerin during the last several hours of HG treatment. HG conditioned cells were also treated with the antioxidants L-N-acetylcysteine (L-NAC) or diphenyliodonium (DPI) prior to harvest. To determine a functional significance of HG-mediated p38 MAPK activation, the DNA binding of the transcription factor complex AP-1 was measured by electrophoretic mobility shift assay.
Results: The p38 MAPK pathway was not activated by the acute addition of HG to the HMCs. However, activation of p38 MAPK in HMCs grown seven days in HG was demonstrated by increased tyrosine and threonine phosphorylation of p38 MAPK proteins and increased kinase activity of immunoprecipitated p38 MAPK. As assessed by a kinase assay, p38 MAPK activity in cells grown in HG for seven days exceeded that of NG cells by more than 250%. This difference was not due to differences in the amount of p38 MAPK protein between the treatment groups. Acute osmotic activation of p38 MAPK occurred at extremely high mannitol concentrations (250 mOsm/L) that exceeded the osmotic stress of acute HG. Furthermore, in cells grown for seven days in mannitol at concentrations similar to HG, p38 MAPK activity was similar to control values. Phorbol ester (PMA) treatment stimulated a twofold increase in p38 MAPK activity. The addition of GFX or Ro 32-0432 to HG cells, at concentrations that inhibited PMA activation of p38 MAPK, did not inhibit the glucose-mediated p38 MAPK activation. Rottlerin, a PKC delta inhibitor, also failed to reverse the HG-mediated p38 MAPK activation. Treatment of HG cells with L-NAC or DPI inhibited the HG-mediated p38 MAPK phosphorylation. As we have previously shown, DNA binding of the transcription factor complex AP-1 was increased in HG cells. This binding was reversed by treatment of the HG cells with the p38 MAPK inhibitor SB 203580.
Conclusions: Chronic exposure of HMC to HG concentrations activates the p38 MAPK pathway. This activation appears to be independent of changes in the amount of total p38 MAPK produced by the cells, independent of chronic osmotic stress and independent of PKC activation. The reversal of p38 MAPK by L-NAC and DPI suggests the glucose-mediated p38 MAPK activation may occur via reactive oxygen species. The activity of AP-1, a transcription factor complex that regulates several genes involved in diabetic nephropathy, is reversed when the p38 MAPK pathway is inhibited. These findings suggest the p38 MAPK pathway may be an important pathway involved in diabetic complications.