okadaic-acid and sanguinarine

Akt substrate of 160 kDa (AS160) phosphorylation on Thr(642) and Ser(588) by Akt is essential for insulin's full effect on glucose transport. However, protein phosphorylation is determined by the balance of actions by kinases and phosphatases, and the specific phosphatase(s) controlling AS160 dephosphorylation is (are) unknown. Accordingly, we assessed roles of highly expressed skeletal muscle serine/threonine phosphatases (PP1, PP2A, PP2B, and PP2C) on AS160 dephosphorylation. Preliminary screening of candidate phosphatases used an AS160 dephosphorylation assay. Lysates from insulin-stimulated skeletal muscle were treated with pharmacological phosphatase inhibitors and assessed for AS160 Ser(588) and Thr(642) dephosphorylation. AS160 dephosphorylation on both phosphorylation sites was unaltered by PP2B or PP2C inhibitors. Okadaic acid (low dose inhibits PP2A; high dose inhibits PP1) delayed AS160 Ser(588) (both doses) and Thr(642) (high dose only) dephosphorylation concomitant with greater Akt phosphorylation (both doses). AS160 was coimmunoprecipitated with PP1-α but not with PP1-β, PP1-γ1, or PP2A. Recombinant inhibitor-2 protein (a selective PP1 inhibitor) delayed AS160 dephosphorylation on both phosphorylation sites without altering Akt phosphorylation. Furthermore, knockdown of PP1-α but not PP1-β or PP1-γ1 by small interfering RNA caused greater AS160 Ser(588) and Thr(642) phosphorylation concomitant with unaltered Akt phosphorylation. Together, these results identified PP1-α as a regulator of AS160 Thr(642) and Ser(588) dephosphorylation in skeletal muscle.

Topics: Animals; Benzophenanthridines; Cell Line; GTPase-Activating Proteins; Immunoblotting; Immunoprecipitation; Isoquinolines; Male; Muscle, Skeletal; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 1; Protein Phosphatase 2; Rats; Rats, Wistar; Rats, Zucker; RNA, Small Interfering; Serine; Tacrolimus

Pulmonary inflammation is a characteristic of many lung diseases. Increased levels of pro-inflammatory cytokines, such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), have been correlated with lung inflammation. In this study, we demonstrated that various inflammatory agents, including lipopolysaccharide, 12-o-tetradecanoylphorbol-13-acetate, hydrogen peroxide, okadaic acid and ceramide, were able to induce IL-1beta and TNF-alpha productions in human lung epithelial cells (A-549), fibroblasts (HFL1), and lymphoma cells (U-937). Berberine, the protoberberine alkaloid widely distributed in the plant kingdom, was capable of suppressing inflammatory agents-induced cytokine production in lung cells. Inhibition of cytokine production by berberine was dose-dependent and cell type-independent. Moreover, the suppression of berberine on the cytokine production resulted from the inhibition of inhibitory kappaB-alpha phosphorylation and degradation. In conclusion, our findings suggested the potential role of berberine in the treatment of pulmonary inflammation.

Topics: Alkaloids; Anti-Inflammatory Agents; Benzophenanthridines; Berberine; Cell Survival; Ceramides; Dose-Response Relationship, Drug; Epithelial Cells; Fibroblasts; Humans; Hydrogen Peroxide; I-kappa B Proteins; Inflammation; Interleukin-1beta; Isoquinolines; Lipopolysaccharides; Lung; NF-KappaB Inhibitor alpha; Okadaic Acid; Phosphorylation; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha; U937 Cells

The nuclear factor NF-kappaB is a pleiotropic transcription factor whose activation results in inflammation, viral replication, and growth modulation. Due to its role in pathogenesis, NF-kappaB is considered a key target for drug development. In the present report we show that sanguinarine (a benzophenanthridine alkaloid), a known anti-inflammatory agent, is a potent inhibitor of NF-kappaB activation. Treatment of human myeloid ML-1a cells with tumor necrosis factor rapidly activated NF-kappaB, this activation was completely suppressed by sanguinarine in a dose- and time-dependent manner. Sanguinarine did not inhibit the binding of NF-kappaB protein to the DNA but rather inhibited the pathway leading to NF-kappaB activation. The reversal of inhibitory effects of sanguinarine by reducing agents suggests a critical sulfhydryl group is involved in NF-kappaB activation. Sanguinarine blocked the tumor necrosis factor-induced phosphorylation and degradation of IkappaBalpha, an inhibitory subunit of NF-kappaB, and inhibited translocation of p65 subunit to the nucleus. As sanguinarine also inhibited NF-kappaB activation induced by interleukin-1, phorbol ester, and okadaic acid but not that activated by hydrogen peroxide or ceramide, the pathway leading to NF-kappaB activation is likely different for different inducers. Overall, our results demonstrate that sanguinarine is a potent suppressor of NF-kappaB activation and it acts at a step prior to IkappaBalpha phosphorylation.

Topics: Alkaloids; Benzophenanthridines; Cell Compartmentation; Cell Line; Ceramides; Dithiothreitol; DNA-Binding Proteins; Enzyme Inhibitors; Humans; Hydrogen Peroxide; Interleukin-1; Isoquinolines; NF-kappa B; Okadaic Acid; Phosphorylation; Proto-Oncogene Proteins; Signal Transduction; Sodium-Potassium-Exchanging ATPase; Tetradecanoylphorbol Acetate; Transcription Factor RelB; Transcription Factors; Tumor Necrosis Factor-alpha