u-0126 and Hypertrophy

Secreted factors present in the medium following growth of the periodontal pathogen Porphyromonas gingivalis cause increased cardiomyocyte hypertrophy and apoptosis, whereas secreted factors from Actinobacillus actinomycetemcomitans and Prevotella intermedia have no such effects. The purpose of this study was to clarify the role of mitogen-activated protein kinase (MAPK)/extracellular-regulated protein kinase (ERK) pathways in P. gingivalis medium-induced H9c2 myocardial cell hypertrophy and apoptosis. Cellular morphology, DNA fragmentation, nuclear condensation, total mitogen-activated protein kinase/extracellular-regulated protein kinase-1 (ERK-1), total ERK-1 protein, and phosphorylated ERK-1 protein products in cultured H9c2 myocardial cells were measured by actin immunofluorescence, agarose gel electrophoresis, nuclear condensation, and western blotting following stimulation with P. gingivalis spent growth medium or pre-administration of U0126, a potent MEK-1/2 inhibitor. Components of P. gingivalis spent culture medium not only resulted in increased total MEK-1 and ERK-1 protein products, but also caused increased cellular size, DNA fragmentation, and nuclear condensation in H9c2 cells. These three parameters, and the phosphorylated ERK-1 protein products of H9c2 cells treated with P. gingivalis medium, were all significantly reduced after pre-administration of U0126. The results suggest that P. gingivalis-secreted factors may initiate MEK/ERK signal pathways and lead to myocardial cell hypertrophy and apoptosis.

Topics: Animals; Apoptosis; Blotting, Western; Butadienes; Cell Line; Cell Size; Culture Media, Conditioned; DNA Fragmentation; Electrophoresis, Agar Gel; Enzyme Inhibitors; Fluorescent Antibody Technique; Hypertrophy; Intranuclear Space; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Mitogen-Activated Protein Kinase 3; Myocytes, Cardiac; Nitriles; Porphyromonas gingivalis; Rats; Signal Transduction

We have already reported Gas6 is involved in glomerular hypertrophy observed in diabetic nephropathy. However, the molecular mechanisms involved in glomerular hypertrophy are still unknown, especially in vivo.. In vivo, diabetes was induced in rats and mice by streptozotocin (STZ) and the activation of the Akt/mTOR pathway in glomeruli was examined. In vitro, mesangial hypertrophy was assessed by [(3)H]leucine incorporation and measuring cell areas.. Akt, p70 S6 kinase, and 4E-BP-1 were induced and phosphorylated in rat glomerular lysates after 12 weeks of STZ injection when mesangial and glomerular hypertrophy was observed. We then examined the role of Gas6 by treating STZ-rats with warfarin, and found that warfarin treatment inhibited the phosphorylation of these molecules as well as the hypertrophy. We next examined whether high glucose stimulation can induce the expression of Gas6/Axl in mesangial cells. Stimulation of the cells with 25 mmol/L of glucose increased the expression of Gas6/Axl and mesangial cell size compared with that with 5.6 mmol/L of glucose. This hypertrophic effect was abolished in mesangial cells derived from Gas6 knockout mice. We also found that LY294002 and rapamycin blocked Gas6-induced activation of the Akt/mTOR pathway and mesangial hypertrophy. Furthermore, less phosphorylated Akt-positive or 4E-BP-1-positive areas were found in STZ-treated Gas6 knockout mice than in STZ-treated wild-type mice.. Our study indicates that the Akt/mTOR pathway is a key signaling cascade in Gas6-mediated mesangial and glomerular hypertrophy and revealed a crucial role of Gas6/Axl and the Akt/mTOR pathway in the development of diabetic nephropathy.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antibiotics, Antineoplastic; Butadienes; Carrier Proteins; Cell Cycle Proteins; Cells, Cultured; Chromones; Cyclin-Dependent Kinase Inhibitor p27; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Enzyme Inhibitors; Eukaryotic Initiation Factors; Female; Glomerular Mesangium; Glucose; Hypertrophy; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Morpholines; Nitriles; Phosphoproteins; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; TOR Serine-Threonine Kinases; Tumor Suppressor Proteins