Mechanism of inhibition of adenylate cyclase by phospholipase C-catalyzed hydrolysis of phosphatidylcholine. Involvement of a pertussis toxin-sensitive G protein and protein kinase C.

The phospholipase C-mediated hydrolysis of phosphatidylcholine has been shown recently to be activated by a number of agonists. Muscarinic receptors, which trigger various signal transduction mechanisms including inhibition of adenylate cyclase through Gi, have been shown to be potent stimulants of this novel phospholipid degradative pathway. We demonstrate here, by exogenous addition of Bacillus cereus phosphatidylcholine-hydrolyzing phospholipase C, that phosphatidylcholine breakdown mimics the ability of carbachol to inhibit adenylate cyclase. This effect is sensitive to pertussis toxin and is entirely dependent on the presence of protein kinase C. This kinase is also required for the inhibition by carbachol of adenylate cyclase. These results suggest that the activation of phosphatidylcholine breakdown by phospholipase C may play an important role linking or favoring the coupling muscarinic receptors to Gi. Results presented here also show that phospholipase C-mediated hydrolysis of phosphoinositides by exogenous addition of Bacillus thuringiensis phosphoinositide-hydrolyzing phospholipase C does not affect adenylate cyclase, despite the fact that protein kinase C is translocated to an extent similar to that produced by the hydrolysis of phosphatidylcholine. According to the results shown here, both phospholipases also differ in their ability to down-regulate protein kinase C as well as to phosphorylate p80 and to transmodulate the binding of epidermal growth factor, two well established effects of protein kinase C in Swiss 3T3 fibroblasts. This emphasizes the complexity, from a functional point of view, of protein kinase C activation "in vivo."     

Dysregulated lipid metabolism in hepatocellular carcinoma cancer stem cells

Molecular Biology Reports - According to the stem cell theory for cancer, hepatocellular carcinomas are sustained by a group of cancer stem cells (CSCs) which are responsible for resistance to...     

Kinetic evidence of a rapid activation of phosphatidylcholine hydrolysis by Ki-ras oncogene. Possible involvement in late steps of the mitogenic cascade

A novel phospholipase C specific for phosphatidylcholine has been shown to be activated by several agonists. Also, recent evidence suggests that transformation mediated by the ras oncogene possibly involves the activation of this novel phospholipid degradative pathway which would account for the increased diacylglycerol levels associated with transformation. Here we use a mutant of Ki-ras which is temperature-sensitive for transformation to investigate the kinetics of activation of the phosphodiesterase-mediated turnover of phosphatidylcholine. Upon shift to the permissive temperature, products of the activated phosphatidylcholine-specific phospholipase C were detected by 30 min and reached maximal levels by 1-2 h. These results suggest that the product of the ras oncogene rapidly activates the phosphodiesteratic hydrolysis of phosphatidylcholine. Furthermore, the fact that at least 4 h are required for serum to activate this phospholipase C strongly suggests that the ras oncogene product might be involved in late steps of the mitogenic signaling cascade.     

Involvement of PPARγ in the antitumoral action of cannabinoids on hepatocellular carcinoma

Cannabinoids exert antiproliferative effects in a wide range of tumoral cells, including hepatocellular carcinoma (HCC) cells. In this study, we examined whether the PPARγ-activated pathway contributed to the antitumor effect of two cannabinoids, Δ9-tetrahydrocannabinol (THC) and JWH-015, against HepG2 and HUH-7 HCC cells. Both cannabinoids increased the activity and intracellular level of PPARγ mRNA and protein, which was abolished by the PPARγ inhibitor GW9662. Moreover, genetic ablation with small interfering RNA (siRNA), as well as pharmacological inhibition of PPARγ decreased the cannabinoid-induced cell death and apoptosis. Likewise, GW9662 totally blocked the antitumoral action of cannabinoids in xenograft-induced HCC tumors in mice. In addition, PPARγ knockdown with siRNA caused accumulation of the autophagy markers LC3-II and p62, suggesting that PPARγ is necessary for the autophagy flux promoted by cannabinoids. Interestingly, downregulation of the endoplasmic reticulum stress-related protein tribbles homolog 3 (TRIB3) markedly reduced PPARγ expression and induced p62 accumulation, which was counteracted by overexpression of PPARγ in TRIB3-knocked down cells. Taken together, we demonstrate for the first time that the antiproliferative action of the cannabinoids THC and JWH-015 on HCC, in vitro and in vivo, are modulated by upregulation of PPARγ-dependent pathways.     

Phospholipase C-mediated hydrolysis of phosphatidylcholine is an important step in PDGF-stimulated DNA synthesis

Recent evidence suggests the involvement of phosphatidylcholine (PC) hydrolysis both in the control of normal cell growth and in transformation. We show here that the simple exogenous addition of Bacillus cereus PC-hydrolyzing phospholipase C (PC-PLC) is sufficient to elicit a potent mitogenic response in Swiss 3T3 fibroblasts by a mechanism that is independent of protein kinase C. Our results on the additivity and synergism between B. cereus PC-PLC, PDGF, and insulin in the mitogenic response indicate that this novel phospholipid degradative pathway may be important in the mitogenic signaling cascade activated by PDGF.     

Involvement of cytochrome b5 in the cytotoxic response to Escherichia coli Lipopolysaccharide

Cytotoxic lesions, induced by Gram-negative lipopolysaccharides (LPS), occur mainly in liver where the microsomal compartment of hepatocytes is involved in the detoxification mechanisms as well as in the biosynthesis of different active metabolites.The alterations induced by LPS from E. coli 0111: 134 on cytochrome b₅ and its correlation with cytochrome P450, have been studied using an in vivo reversible endotoxic shock model and 24 h non-replicative hepatocyte monolayers.Results show that cytochrome b₅ is directly affected by LPS that induces also a membrane damage with an active release of lactate dehydrogenase (LDH). The increase of cytochrome b₅ levels may enhance the efficiency of the electron transport, thus facilitating the cytochrome P450-associate oxidations and reactions involved in the repair mechanisms of membranes.     

Evidence for a role of phosphatidylcholine-hydrolysing phospholipase C in the regulation of protein kinase C by ras and src oncogenes.

The products of ras and src oncogenes are thought to be important components in pathways regulating cell proliferation and differentiation. In fibroblasts transformed by these oncogenes, increased diacylglycerol levels have been found which most probably arise from activation of the turnover of phosphatidylcholine. Diacylglycerol is a key activator of protein kinase C whose role in cell growth and transformation has been proposed. We demonstrate here by using immunochemical techniques that transformation by ras or src oncogenes is associated with permanent translocation of protein kinase C to the cytoplasmic membrane. However, no down-regulation of the enzyme is observed despite its permanent activation in these transformants. Importantly, the lack of down-regulation observed in ras and src transformed cell lines is mimicked by chronic treatment of NIH 3T3 fibroblasts with exogenous Bacillus cereus phosphatidylcholine-hydrolysing phospholipase C, but not with phorbol myristate acetate or exogenous Bacillus thuringiensis phosphatidylinositol-hydrolysing phospholipase C. These results strongly suggest that diacylglycerol derived from phosphatidylcholine but not from phosphoinositide turnover is responsible for the atypical regulation of protein kinase C in cell lines transformed by ras and src oncogenes.