PPARgamma Inhibits Inflammation through the Suppression of ERK1/2 Kinase Activity in Human Gingival Fibroblasts

Periodontal disease is a major oral disorder and comprises a group of infections that lead to inflammation of the gingiva and the destruction of periodontal tissues. PPARgamma plays an important role in the regulation of several metabolic pathways and has recently been implicated in inflammatory response pathways. However, its effects on periodontal inflammation have yet to be clarified. In our current study, we evaluated the anti-inflammatory effects of PPARgamma on periodontal disease. Human gingival fibroblasts (HGFs) treated with lipopolysaccharide (LPS) showed high levels of intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), matrix metalloproteinase-2 (MMP-2), and -9 (MMP-9). Moreover, these cells also showed upregulated activities for extracellular signal regulated kinase (ERK1/2), inducible nitric oxide synthase (iNOS) and cyclooxygnase-2. However, cells treated with Ad/PPARgamma and rosiglitazone in same culture system showed reduced ICAM-1, VCAM-1, MMP-2, -9 and COX-2. Finally, the anti-inflammatory effects of PPARgamma appear to be mediated via the suppression of the ERK1/2 pathway and consequent inhibition of NF-kB translocation. Our present findings thus suggest that PPARgamma indeed has a pivotal role in gingival inflammation and may be a putative molecular target for future therapeutic strategies to control chronic periodontal disease.

Role of Ca(2+) in diallyI disulfide-induced apoptotic cell death of HCT-15 cells

Diallyl disulfide (DADS) induced apoptosis through the caspase-3 dependent pathway in leukemia cells was earlier reported from this laboratory. In this study, we investigated the involvement of Ca(2+) in DADS-induced apoptotic cell death of HCT-15, human colon cancer cell line. DADS induced the elevation of cytosolic Ca(2+) by biphasic pattern; rapid Ca(2+) peak at 3 min and following slow and sustained elevation till 3 h after the addition of DADS. Production of H(2)O(2) was also observed with its peak value at 4 h. Apoptotic pathways including the sequence of caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and DNA fragmentation by DADS were completely blocked by various inhibitors such as specific caspase-3 inhibitor, free radical scavenger, and intracellular Ca(2+) chelator. N-acetylcystein and catalase treatment prevented the accumulation of H2O2 and later caspase-3 dependent apoptotic pathway. However, these radical scavengers did not block the elevation of intracellular Ca(2+). Treatment of cells with 1, 2-bis (2-aminophenoxyethane)-N, N, N-tetraacetic acid tetrakis -acetoxymethyl ester (BAPTA-AM), cellular Ca(2+) chelator, resulted in a complete blockage of the caspase-3 dependent apoptotic pathway of HCT-15 cells. It abolished the elevation of intracellular Ca(2+), and furthermore, completely inhibited the production of H(2)O(2). These results indicate that cytosolic Ca(2+) elevation is an earlier signaling event in apoptosis of HCT-15 cells. Collectively, our data demonstrate that DADS can induce apoptosis in HCT-15 cells through the sequential mechanism of Ca(2+) homeostasis disruption, accumulation of H(2)O(2), and resulting caspase-3 activation.

Retinoic acid inhibits inducible nitric oxide synthase expression in 3T3-L1 adipocytes

The present study was undertaken to explore whether retinoids, which are known to have immunomodulatory actions, could attenuate tumor necrosis factor-alpha (TNF)-stimulated inducible nitric oxide synthase (iNOS) expression in 3T3-L1 adipocytes. Adipocytes incubated with TNF induced dose- and time-dependent accumulation of nitrite in the culture medium through the iNOS induction as confirmed by Western blotting. Treatment of cells with TNF in the presence of all-trans-retinoic acid (RA) significantly decreased their ability to produce nitrite and iNOS induction. Both 13-cis- and all- trans-RA-induced suppression was dose-dependent, and all-trans-RA was somewhat potent than 13-cis-RA. The inhibitory effect of RA on TNF-induced iNOS induction was reversible, completely recovered after 2 days, and was exerted through the inhibition of NF-kappaB activation. TNF also suppressed the lipoprotein lipase (LPL) activity of 3T3-L1 adipocytes. RA could not reverse the TNF- induced LPL suppression at RA levels causing near complete inhibition of the TNF-induced NO production. These results indicate that RAs attenuate iNOS expression reversibly in TNF-stimulated 3T3-L1 adipocytes, and that the TNF- induced LPL suppression is not the result of NO overproduction.

A variant of ornithine aminotransferase from mouse small intestine

The ornithine aminotransferase (OAT) activity of mouse was found to be highest in the small intestine. The mitochondrial OAT from mouse small intestine was purified to homogeneity by the procedures including heart treatment, ammonium sulfate fractionation, octyl-Sepharose chromatography, and Sephadex G-150 gel filtration. Comparing to the amino acid sequence of mouse hepatic OAT, six N-terminal amino acid residues have been deleted in intestinal OAT. However, the subsequent sequence was identical with that of hepatic OAT. The molecular weights of both intestinal and hepatic OAT were estimated as 46 kDa by SDS-gel electrophoresis and as 92 kDa by gel filtration, indicating that both native OATs are dimeric. Biochemical properties of intestinal OAT, such as molecular weight, pH optimum and K(m) values for L-ornithine and alpha-ketoglutarate, were similar to those of hepatic OAT. However, intestinal OAT was more labile than hepatic OAT to tryptic digestion.