PKCalpha regulates phosphorylation and enzymatic activity of cPLA2 in vitro and in activated human monocytes

Phospholipases A(2) (PLA(2)) are potent regulators of the inflammatory response. We have observed that Group IV cPLA(2) activity is required for the production of superoxide anion (O(2)(-)) in human monocytes [Li Q., Cathcart M.K. J. Biol. Chem. 272 (4) (1997) 2404-2411.]. We have previously identified PKCalpha as a kinase pathway required for monocyte O(2)(-) production [Li Q., Cathcart M.K. J. Biol. Chem. 269 (26) (1994) 17508-17515.]. We therefore investigated the potential interaction between PKCalpha and cPLA(2) by evaluating the requirement for specific PKC isoenzymes in the process of activating cPLA(2) enzymatic activity and protein phosphorylation upon monocyte activation. We first showed that general PKC inhibitors and antisense oligodeoxyribonucleotides (ODN) to the cPKC group of PKC enzymes inhibited cPLA(2) activity. To distinguish between PKCalpha and PKCbeta isoenzymes in regulating cPLA(2) protein phosphorylation and enzymatic activity, we employed our previously characterized PKCalpha or PKCbeta isoenzyme-specific antisense ODN [Li Q., Subbulakshmi V., Fields A.P., Murray, N.R., Cathcart M.K., J. Biol. Chem. 274 (6) (1999) 3764-3771]. Suppression of PKCalpha expression, but not PKCbeta expression, inhibited cPLA(2) protein phosphorylation and enzymatic activity. Additional studies ruled out a contribution by Erk1/2 to cPLA(2) phosphorylation and activation. We also found that cPLA(2) co-immunoprecipitated with PKCalpha and vice versa. In vitro studies demonstrated that PKCalpha could directly phosphorylate cPLA(2).and enhance enzymatic activity. Finally, we showed that addition of arachidonic acid restored the production of O(2)(-) in monocytes defective in either PKCalpha or cPLA(2) expression. Taken together, our data suggest that PKCalpha, but not PKCbeta, is the predominant cPKC isoenzyme required for cPLA(2) protein phosphorylation and maximal induction of cPLA(2) enzymatic activity upon activation of human monocytes. Our data also support the concept that the requirements for PKCalpha and cPLA(2) in O(2)(-) generation are solely due to their seminal role in generating arachidonic acid.     

Specific physiological roles of cytosolic phospholipase A(2) as defined by gene knockouts

The cytosolic 85 kDa phospholipase A(2) (cPLA(2)) is a unique member of the phospholipase A(2) (PLA(2)) superfamily. Because PLA(2) activity and eicosanoid production are important in normal and pathophysiological states we and the laboratory of Shimizu created a mouse deficient in cPLA(2) (cPLA(2)(-/-) mouse). cPLA(2)(-/-) mice develop normally but the females have severe reproductive defects. cPLA(2)(-/-) mice suffer smaller infarcts and fewer neurological deficits after transient occlusion of the middle cerebral artery and have less injury after administration of a dopaminergic selective neurotoxin. cPLA(2)(-/-) mice have a more rapid recovery from allergen-induced bronchoconstriction and have no airway hyperresponsiveness. Peritoneal macrophages from cPLA(2)(-/-) mice fail to produce prostaglandins, leukotriene B(4) and cysteinyl leukotrienes after stimulation. Bone marrow-derived mast cells from cPLA(2)(-/-) mice fail to produce eicosanoids in either immediate or delayed phase responses. Thus the cPLA(2) knockout mouse has revealed important roles of cPLA(2) in normal fertility, generation of eicosanoids from inflammatory cells, brain injuries and allergic responses. Furthermore the cPLA(2)(-/-) mouse reveals that the many other forms of PLA(2) cannot replace many functions of cPLA(2). The importance of cPLA(2) in inflammation and tissue injury suggests that pharmacological targeting of this enzyme may have important therapeutic benefits.     

PLIP, a Novel Splice Variant of Tip60, Interacts with Group IV Cytosolic Phospholipase A2, Induces Apoptosis, and Potentiates Prostaglandin Production

The group IV cytosolic phospholipase A(2) (cPLA(2)) has been localized to the nucleus (M. R. Sierra-Honigmann, J. R. Bradley, and J. S. Pober, Lab. Investig. 74:684-695, 1996) and is known to translocate from the cytosolic compartment to the nuclear membrane (S. Glover, M. S. de Carvalho, T. Bayburt, M. Jonas, E. Chi, C. C. Leslie, and M. H. Gelb, J. Biol. Chem. 270:15359-15367, 1995; A. R. Schievella, M. K. Regier, W. L. Smith, and L. L. Lin, J. Biol. Chem. 270:30749-30754, 1995). We hypothesized that nuclear proteins interact with cPLA(2) and participate in the functional effects of this translocation. We have identified a nuclear protein, cPLA(2)-interacting protein (PLIP), a splice variant of human Tip60, which interacts with the amino terminal region of cPLA(2). Like Tip60, PLIP cDNA includes the MYST domain containing a C2HC zinc finger and well-conserved similarities to acetyltransferases. Both PLIP and Tip60 coimmunoprecipitate and colocalize with cPLA(2) within the nuclei of transfected COS cells. A polyclonal antibody raised to PLIP recognizes both PLIP and Tip60. Endogenous Tip60 and/or PLIP in rat mesangial cells is localized to the nucleus in response to serum deprivation. Nuclear localization coincides temporally with apoptosis. PLIP expression, mediated by adenoviral gene transfer, potentiates serum deprivation-induced prostaglandin E(2) (PGE(2)) production and apoptosis in mouse mesangial cells from cPLA(2)(+/+) mice but not in mesangial cells derived from cPLA(2)(-/-) mice. Thus PLIP, a splice variant of Tip60, interacts with cPLA(2) and potentiates cPLA(2)-mediated PGE(2) production and apoptosis.     

Inhibition of intestinal polyposis with reduced angiogenesis in ApcMin/+ mice due to decreases in c-Myc expression

The c-myc oncogene plays an important role in tumorigenesis and is frequently deregulated in many human cancers, including gastrointestinal cancers. In humans, mutations of the adenomatous polyposis coli (Apc) tumor suppressor gene occur in most colorectal cancers. Mutation of Apc leads to stabilization of beta-catenin and increases in beta-catenin target gene expression (c-myc and cyclin D1), whose precise functional significance has not been examined using genetic approaches. Apc(Min/+) mice are a model of familial adenomatous polyposis and are heterozygous for an Apc truncation mutation. We have developed a model for examining the role of c-Myc in Apc-mediated tumorigenesis. We crossed c-myc(+/-) mice to Apc(Min/+) to generate Apc(Min/+) c-myc(+/-) animals. The compound Apc(Min/+) c-myc(+/-) mice were used to evaluate the effect of c-myc haploinsufficiency on the Apc(Min/+) phenotype. We observed a significant reduction in tumor numbers in the small intestine of Apc(Min/+) c-myc(+/-) mice compared with control Apc(Min/+) c-myc(+/+) mice. In addition, we observed one to three polyps per colon in Apc(Min/+) c-myc(+/+) mice, whereas only two lesions were observed in the colons of Apc(Min/+) mice that were haploinsufficient for c-myc. Moreover, reduction in c-myc levels resulted in a significant increase in the survival of these animals. Finally, we observed marked decreases in vascular endothelial growth factor, EphA2, and ephrin-B2 expression as well as marked decreases in angiogenesis in intestinal polyps in Apc(Min/+) c-myc(+/-) mice. This study shows that c-Myc is critical for Apc-dependent intestinal tumorigenesis in mice and provides a potential therapeutic target in the treatment of colorectal cancer.     

Inhibition of Epstein-Barr virus (EBV) reactivation by short interfering RNAs targeting p38 mitogen-activated protein kinase or c-myc in EBV-positive epithelial cells

Latent Epstein-Barr virus (EBV) is reactivated by 12-O-tetradecanoylphorbol-13-acetate (TPA) in EBV-infected cells. In this study, we found that TPA up-regulated phosphorylation of p38, a mitogen-activated protein kinase, and activated c-myc mRNA in EBV-positive epithelial GT38 cells. The EBV immediate-early gene BZLF1 mRNA and its product ZEBRA protein were induced following TPA treatment. Protein kinase C inhibitors, 1-(5-isoquinolinesulphonyl)-2, 5-dimethylpiperazine (H7) and staurosporine, inhibited the induction of p38 phosphorylation and the activation of c-Myc by TPA. The p38 inhibitor SB203580 blocked both p38 phosphorylation and ZEBRA expression by TPA. Pretreatment of GT38 cells with the nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine inhibited p38 phosphorylation and c-Myc activation by TPA, suggesting that NO may inhibit EBV reactivation via both p38 and c-Myc. By using short interfering RNA (siRNA) targeting either p38 or c-myc, we found that p38 or c-myc siRNA specifically inhibited expression of the respective gene and also suppressed the induction of ZEBRA and EBV early antigen. The interferon (IFN)-responsive gene expression tests ruled out the possibility that the antiviral effect of siRNA is dependent on IFN. Our present study demonstrates for the first time that either p38 or c-myc siRNA can efficiently inhibit TPA-induced EBV reactivation in GT38 cells, indicating that p38- and/or c-myc-associated signaling pathways may play critical roles in the disruption of EBV latency by TPA.     

Lipopolysaccharide-induced release of arachidonic acid and prostaglandins in liver macrophages: regulation by Group IV cytosolic phospholipase A2, but not by Group V and Group IIA secretory phospholipase A2.

Lipopolysaccharide (LPS) induces a delayed release (lag phase of 2-4 h) of arachidonic acid (AA) and prostaglandin (PG) D2 in rat liver macrophages. Group IV cytosolic phospholipase A2 (cPLA2) becomes phosphorylated within minutes after the addition of LPS. The phosphorylated form of cPLA2 shows an enhanced in vitro activity. The Ca2+ dependence of cPLA2 activity is not affected by phosphorylation of the enzyme. In addition, LPS induces an enhanced expression of cPLA2 mRNA (after 2-4 h) and an enhanced expression of cPLA2 protein (after 8 h). The cellular cPLA2 activity is enhanced about twofold 24 h after LPS treatment. Liver macrophages constitutively express mRNAs encoding Groups V and IIA secretory PLA2 (sPLA2). LPS has no effect on the levels of Groups V and IIA sPLA2 mRNA expression. Despite mRNA expression, Groups V and IIA sPLA2 protein and sPLA2 activity are not detectable in unstimulated or LPS-stimulated liver macrophages. Collectively, these and earlier [Mediators Inflammation 8 (1999) 295.] results suggest that in liver macrophages the LPS-induced delayed release of AA and prostanoids is mediated by phosphorylation and an enhanced expression of cPLA2, a de novo expression of cyclooxygenase (COX)-2, but not by the actions of Group V or Group IIA sPLA2.     

Roles for c-Myc in self-renewal of hematopoietic stem cells

Notch and HOXB4 have been reported to expand hematopoietic stem cells (HSCs) in vitro. However, their critical effector molecules remain undetermined. We found that the expression of c-myc, cyclin D2, cyclin D3, cyclin E, and E2F1 was induced or enhanced during Notch1- or HOXB4-induced self-renewal of murine HSCs. Since c-Myc can act as a primary regulator of G(1)/S transition, we examined whether c-Myc alone can induce self-renewal of HSCs. In culture with stem cell factor, FLT3 ligand, and IL-6, a 4-hydroxytamoxifen-inducible form of c-Myc (Myc/ERT) enabled murine Lin(-)Sca-1(+) HSCs to proliferate with the surface phenotype compatible with HSCs for more than 28 days. c-Myc activated by 4-hydroxytamoxifen augmented telomerase activities and increased the number of CFU-Mix about 2-fold in colony assays. Also, in reconstitution assays, HSCs expanded by c-Myc could reconstitute hematopoiesis for more than 6 months. As for the mechanism of c-myc induction by Notch1, we found that activated forms of Notch1 (NotchIC) and its downstream effector recombination signal-binding protein-J kappa (RBP-VP16) can activate the c-myc promoter through the element between -195 bp and -161 bp by inducing the DNA-binding complex. Together, these results suggest that c-Myc can support self-renewal of HSCs as a downstream mediator of Notch and HOXB4.     

The glucocorticoid receptor is increased in Atm-/- thymocytes and in Atm-/- thymic lymphoma cells, and its nuclear translocation counteracts c-myc expression

We have previously demonstrated that spontaneous DNA synthesis in immature thymocytes of Atm-/- mice is elevated, and that treatment with the glucocorticoid dexamethasone (Dex) attenuates this increased DNA synthesis and prevents the development of thymic lymphomas. Deregulation of c-myc may drive the uncontrolled proliferation of Atm-/- thymocytes, since upregulation of c-myc parallels the elevated DNA synthesis in the cells. In this study, we show that the glucocorticoid receptor (GR) is expressed at high levels in Atm-/- thymocytes and in Atm-/- thymic lymphoma cells, although serum glucocorticoid (GC) levels in Atm-/- mice are similar to those in Atm+/+ mice. In cultured Atm-/- thymic lymphoma cells treated with Dex, GR nuclear translocation occurs, resulting in suppression of DNA synthesis and c-myc expression at both the mRNA and protein levels. Interestingly, the GR antagonist RU486 also causes GR nuclear translocation, but does not affect DNA synthesis and c-myc expression in Atm-/- thymic lymphoma cells. As expected, RU486 reverses the suppressive effects of Dex on DNA synthesis and c-myc expression. Administration of Dex to Atm-/- mice decreases the elevated c-Myc protein levels in their thymocytes. These findings suggest that GC/GR signaling plays an important role in regulating c-myc expression in Atm-/- thymocytes and thymic lymphoma cells.