Abstract Breast cancer (BC) is a worldwide health issue as it represents the leading cause of cancer in women and the second leading cause of cancer-related mortality in women, with an increasing incidence. In the US, African American (AA) women have a significantly higher rate of mortality due to BC compared to Caucasian American (CA) women. It is becoming increasingly apparent that racial disparity in cancer exists due to molecular differences in tumor biology as well as, or in addition to, socioeconomic and standard of care issues. Sparse information exists regarding the molecular mechanisms that promote BC health disparity therefore, a greater understanding of the risk factors and biological links associated with BC, will significantly impact AA women. Our studies have identified a race specific mechanistic link between microRNA-204 (miR-204) and the Insulin-like Growth Factor-2 Receptor. MicroRNAs are small non-coding RNAs that function to inhibit gene expression through translational repression. Our published studies identified miR-204 as a novel oncomir and we recently identified IGF2R as a direct target. The IGF2R has been proposed to be a tumor suppressor gene in several cancers including breast cancer. We show that over-expression of miR-204 results in a decrease in IGF2R protein levels. We propose that the inhibition of IGF2R allows IGF-2 to bind the IGF1R leading to hyperactivation of this pathway which results in increased proliferation, migration and invasion, processes that are required for tumor progression. Indeed, we show that miR-204 expression results in the activation of the IGF1R/IRS-1/ERK pathway together with an increase in proliferation, migration and invasion. Published studies have shown that exogenous expression of activated IGF1R increases migration in non-transformed breast cells. We have shown that exogenous expression of miR-204 also increases migration in these cells. However, when miR-204 and IGF1R are both exogenously expressed no additional increase in migration was observed, suggesting that mIR-204 mediated increase in migration is through activation of the IGF1R pathway. Reduced IGF2R expression correlates with poor patient prognosis in BC patients and a recent study showed significantly higher levels of IGF2R in CA compared to AA tumor samples, suggesting that decreased IGF2R expression may contribute to BC disparity. We examined serum from BC patients and found elevated levels of miR-204 in AA compared to CA women. Circulating miRNAs can serve as accessible biomarkers for diagnosis & prognosis and may define a novel area of potential therapeutic intervention to reduce BC disparity. Our data also suggest that miR-204 mediated inhibition of IGF2R could be a potential biological mechanism driving BC disparity. A major hurdle to the identification of biological mechanisms conferring cancer health disparity is a lack of suitable experimental models with which to investigate race specific differences in tumor biology. We have developed a unique inducible miR-204 transgenic mouse model to define in vivo the oncogenic potential of miR-204 and the mechanism and functional consequences of IGF2R loss. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-07-02.

Two new cloned human cDNAs encode paralogs of the 85-kDa cytosolic phospholipase A2 (cPLA2). We propose to call these cPLA2beta (114 kDa) and cPLA2gamma (61 kDa), giving the name cPLA2alpha to the well known 85-kDa enzyme. cPLA2beta mRNA is expressed more highly in cerebellum and pancreas and cPLA2gamma more highly in cardiac and skeletal muscle. Sequence-tagged site mapping places cPLA2beta on chromosome 15 in a region near a phosphoinositol bisphosphate phosphatase. The mRNA for cPLA2beta is spliced only at a very low level, and Northern blots in 24 tissues show exclusively the unspliced form. cPLA2beta has much lower activity on 2-arachidonoyl-phosphatidylcholine liposomes than either of the other two enzymes. Its sequence contains a histidine motif characteristic of the catalytic center of caspase proteases of the apoptotic cascade but no region characteristic of the catalytic cysteine. Sequence-tagged site mapping places cPLA2gamma on chromosome 19 near calmodulin. cPLA2gamma lacks the C2 domain, which gives cPLA2alpha its Ca2+ sensitivity, and accordingly cPLA2gamma has no dependence upon calcium, although cPLA2beta does. cPLA2gamma contains a prenyl group-binding site motif and appears to be largely membrane-bound. cPLA2alpha residues activated by phosphorylation do not appear to be well conserved in either new enzyme. In contrast, all three previously known catalytic residues, as well as one additional essential arginine, Arg-566 in cPLA2alpha, are conserved in both new enzyme sequences. Mutagenesis shows strong dependence on these residues for catalytic activity of all three enzymes.

The present study identifies the phosphorylation sites of the 85-kDa cytosolic phospholipase A2 (cPLA2) in human platelets and HeLa cells. Tryptic digests of 32P-phosphorylated and -immunoprecipitated cPLA2 were analyzed by microbore high performance liquid chromatography and two-dimensional phosphopeptide mapping against synthetic phosphopeptide standards. Thrombin stimulated significant phosphorylation of platelet cPLA2 at two sites, Ser-505 and Ser-727. Exclusive phosphorylation on these two sites was also seen in collagen-stimulated platelets and HeLa cells stimulated with interferon-alpha or arsenite; no tyrosine phosphorylation was detected. The inhibitor of the 38-kDa stress-activated protein kinase (p38(mapk)), SB 203580, reduced phosphorylation of both Ser-505 and Ser-727 by 50 and 60%, respectively, in thrombin-stimulated platelets. An additional p38(mapk) inhibitor SB 202190 also partially (60%) inhibited the phosphorylation of cPLA2 in arsenite-stimulated HeLa cells. These studies extend the previous work on the identification of multiple phosphorylation sites on cPLA2 expressed in a baculovirus/insect cell system to cPLA2 in mammalian cells stimulated with physiological agonists. They also underscore the necessity of high resolution phosphopeptide mapping combined with microbore high performance liquid chromatography for quantification of phosphorylation levels, which has lead to the conclusion that Ser-505 and Ser-727 are common phosphorylation sites on cPLA2 in different mammalian cells stimulated with multiple agonists.

Phosphorylation and activation of cytosolic phospholipase A2 (PLA2) can occur independently of the activation of 42/44-kDa mitogen-activated protein (MAP) kinase in human platelets. We have investigated the hypothesis that the stress-activated p38 MAP kinase plays a role in the regulation of cytosolic PLA2. The specific inhibitor of p38 MAP kinase, SB 203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl) imidazole], completely blocked the collagen-stimulated phosphorylation of cytosolic PLA2 in the presence of a cyclooxygenase blocker, and reduced the release of [3H]arachidonic acid by low concentrations of collagen. Stimulation of platelets with collagen (100 microg/ml) enhanced in vitro PLA2 activity of platelet lysates twofold over basal levels. In vitro PLA2 activity was reduced to basal levels when platelets were stimulated in the presence of SB 203580, but not in the presence of an inhibitor of the kinase that activates p42/p44 MAP kinase. SB 203580 only partially inhibited phosphorylation of cytosolic PLA2 in platelets that had not been treated with a cyclooxygenase blocker indicating that secondary stimulation by thromboxane A2 induces cytosolic PLA2 phosphorylation, by kinase(s) other than p38 MAP kinase. Under these conditions, inhibition of p42/p44 MAP kinase did not result in a reduction of cytosolic PLA2 phosphorylation, which is in agreement with the results obtained in the presence of cyclooxygenase blockers. In contrast to collagen, both p38 MAP kinase and p42/p44 MAP kinase participated in the phosphorylation of cytosolic PLA2 in platelets stimulated by cross-linking of the low-affinity receptor for immune complexes, Fc gammaRIIA. The present results demonstrate an important role for p38 MAP kinase in the regulation of cytosolic PLA2 activity in collagen-stimulated human platelets.

Incubation of rat glomerular mesangial cells with potent proinflammatory cytokines like interleukin 1beta, (IL- 1beta) triggers the expression of a non-pancreatic secretory phospholipase A2 (sPLA2) and increases the formation of prostaglandin E2. We show here that sPLA2 acts in an autocrine fashion on mesangial cells and induces a rapid activation of protein kinase C (PKC) isoenzymes delta and epsilon and of p42 mitogen-activated protein kinase (MAPK), two putative activators of cytosolic phospholipase A2 (cPLA2). sPLA2 also activates Raf-1 kinase in mesangial cells which integrates the signals coming from PKC for further processing along the MAPK cascade. Subsequently a phosphorylation and activation of cPLA2 is observed, thus arguing for a cross-talk between the two classes of PLA2. Pretreatment of cells with either the highly specific PKC inhibitor Ro-318220 or the highly specific MAPK kinase (MEK) inhibitor PD 98059 completely blocked the sPLA2-induced cPLA2 activation, indicating that both kinases are essential for the cross-talk between the two types of PLA2. The effect of sPLA2 is mimicked by lysophosphatidylcholine (LPC), a reaction product of sPLA2 activity. LPC stimulates PKC-epsilon, Raf-1 kinase and MAPK activation as well as cPLA2 activation with a subsequent increase in arachidonic acid release from mesangial cells. These data suggest that sPLA2 by cleaving membrane phospholipids and generating LPC and other lysophospholipids activates cPLA2 via the PKC/Raf-1/MAPK signalling pathway. Hence a network of interactions between different PLA2s is operative in mesangial cells and may contribute to the progression of glomerular inflammatory processes.

Recombinant human group II phospholipase A2 (sPLA2) added to human platelets in the low microg/ml range induced platelet activation, as demonstrated by measurement of platelet aggregation, thromboxane A2 generation and influx of intracellular free Ca2+ concentration and by detection of time-dependent tyrosine phosphorylation of platelet proteins. The presence of Ca2+ at low millimolar concentrations is a prerequisite for the activation of platelets by sPLA2. Mg2+ cannot replace Ca2+. Mg2+, given in addition to the necessary Ca2+, inhibits sPLA2-induced platelet activation. Pre-exposure to sPLA2 completely blocked the aggregating effect of a second dose of sPLA2. Albumin or indomethacin inhibited sPLA2-induced aggregation, similarly to the inhibition of arachidonic acid-induced aggregation. Platelets pre-treated with heparitinase or phosphatidylinositol-specific phospholipase C lost their ability to aggregate in response to sPLA2, although they still responded to other agonists. This suggests that a glycophosphatidylinositol-anchored platelet-membrane heparan sulphate proteoglycan is the binding site for sPLA2 on platelets. Previous reports have stated that sPLA2 is unable to activate platelets. The inhibitory effect of albumin and Mg2+, frequently used in aggregation studies, and the fact that isolated platelets lose their responsiveness to sPLA2 relatively quickly, may explain why the platelet-activating effects of sPLA2 have not been reported earlier.

Mitogen-activated protein kinases (MAPKs), a family of protein serine/threonine kinases regulating cell growth and differentiation, are activated by a dual-specificity kinase through phosphorylation at threonine and tyrosine. We used a recently described selective inhibitor of the p42/p44mapk-activating enzyme, PD 98059 [2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one], to investigate the role of the p42/p44mapk pathway in human platelets. PD 98059 inhibited p42/p44mapk activation in thrombin-, collagen- and phorbol esterstimulated platelets, as determined from in-gel renaturation kinase assays, with an IC50 of approx. 5 microM (thrombin stimulation). It also prevented activation of MAPK kinase, which was measured in whole-cell lysates with glutathione S-transferase/p42mapk fusion protein (GST-MAPK) as substrate. Inhibition of p42/p44mapk did not affect platelet responses to thrombin or collagen such as aggregation, 5-hydroxytryptamine release and protein kinase C activation. In addition, PD 98059 did not interfere with release of arachidonic acid, a response mediated by cytosolic phospholipase A2 (cPLA2), or with cPLA2 phosphorylation. This suggests that platelet cPLA2 is not regulated by p42/p44mapk after stimulation with physiological agonists. In contrast, phorbol ester-induced phosphorylation of cPLA2 and potentiation of arachidonic acid release stimulated by Ca2+ ionophore A23187 were inhibited by PD 98059, indicating that p42/p44mapk phosphorylates cPLA2 after activation of protein kinase C by the non-physiological tumour promoter.

Cytosolic phospholipase A2 (cPLA2) hydrolyzes the sn-2-acyl ester bond of phospholipids and shows a preference for arachidonic acid-containing substrates. We found previously that Ser-228 is essential for enzyme activity and is likely to function as a nucleophile in the catalytic center of the enzyme (Sharp, J. D., White, D. L., Chiou, X. G., Goodson, T., Gamboa, G. C., McClure, D., Burgett, S., Hoskins, J., Skatrud, P. L., Sportsman, J. R., Becker, G. W., Kang, L. H., Roberts, E. F., and Kramer, R. M. (1991) J. Biol. Chem. 266, 14850-14853). cPLA2 contains a catalytic aspartic acid motif common to the subtilisin family of serine proteases. Substitution within this motif of Ala for Asp-549 completely inactivated the enzyme, and substitutions with either glutamic acid or asparagine reduced activity 2000- and 300-fold, respectively. Additionally, using mutants with cysteine replaced by alanine, we found that Cys-331 is responsible for the enzyme's sensitivity to N-ethylmaleimide. Surprisingly, substituting alanine for any of the 19 histidines did not produce inactive enzyme, demonstrating that a classical serine-histidine-aspartate mechanism does not operate in this hydrolase. We found that substituting alanine or histidine for Arg-200 did produce inactive enzyme, while substituting lysine reduced activity 200-fold. Results obtained with the lysine mutant (R200K) and a coumarin ester substrate suggest no specific interaction between Arg-200 and the phosphoryl group of the phospholipid substrate. Arg-200, Ser-228, and Asp-549 are conserved in cPLA2 from six species and also in four nonmammalian phospholipase B enzymes. Our results, supported by circular dichroism, provide evidence that Asp-549 and Arg-200 are critical to the enzyme's function and suggest that the cPLA2 catalytic center is novel.

The Ca2+-sensitive 85-kDa cytosolic phospholipase A2 (cPLA2) is responsible for thrombin-stimulated mobilization of arachidonic acid for the synthesis of thromboxane A2 in human platelets. We have previously shown that thrombin activates p38 kinase, a recently discovered new member of the mitogen-activated protein kinase family (Kramer, R. M., Roberts, E. F., Strifler, B. A., and Johnstone, E. M. (1995) J. Biol. Chem. 270, 27395-27398) and also induces phosphorylation of cPLA2, thereby increasing its intrinsic catalytic activity. In the present study we have examined the role of p38 kinase in the phosphorylation and activation of cPLA2 in stimulated platelets. We have observed that activation of p38 kinase accompanies receptor-mediated events in platelets and coincides with cPLA2 phosphorylation. Furthermore, in the presence of inhibitors of p38 kinase, the proline-directed phosphorylation of cPLA2 was completely blocked in platelets stimulated with the thrombin receptor agonist peptide SFLLRN and was suppressed during the early (up to 2 min) phase of platelet stimulation caused by thrombin. Unexpectedly, we found that prevention of proline-directed phosphorylation of cPLA2 in stimulated platelets did not attenuate its ability to release arachidonic acid from platelet phospholipids. We conclude that: 1) cPLA2 is a physiological target of p38 kinase; 2) p38 kinase is involved in the early phosphorylation of cPLA2 in stimulated platelets; and 3) proline-directed phosphorylation of cPLA2 is not required for its receptor-mediated activation.

In human platelets a proline-directed kinase distinct from the ERK MAP kinases is stimulated by both thrombin and the thrombin receptor agonist peptide SFLLRN and may be involved in the activation of Ca2+-dependent cytosolic phospholipase Az (Kramer, R. M., Roberts, E. F., Hyslop, P. A., Utterback, B. G., Hui, K. Y., and Jakubowski, J. A. (1995) J. Biol. Chem. 270, 14816-14823). Here we show that this kinase is identical with or closely related to p38 (the mammalian homolog of HOG1 from yeast), a recently discovered protein kinase typically activated by inflammatory cytokines and environ mental stress. Further, we demonstrate that activation of this kinase by thrombin is transient (with maximal stimulation at 1 min), is accompanied by tyrosine phosphorylation, and precedes the activation of the ERK kinases, This is the first report to show that p38 kinase is activated by thrombin and to suggest a role for this MAP kinase in the thrombin-mediated signaling events during platelet activation.

The thrombin receptor agonist peptide SFLLRN was less effective than thrombin in eliciting the liberation of arachidonic acid and the generation of thromboxane A(2) by human platelets, We found that while SFLLRN evokes an initial transient increase in cytosolic free calcium concentration ([Ca2+](i)) of similar magnitude as that caused by thrombin, the SFLLRN-induced elevation of [Ca2+](i) declines more rapidly to near resting levels than that evoked by thrombin, suggesting that disparate levels of [Ca2+](i) may contribute to the attenuated arachidonic acid release. Furthermore, we observed that SFLLRN is less effective than thrombin in mediating the ''activating'' phosphorylation of cytosolic phospholipase A(2) (cPLA(2)). Both thrombin and SFLLRN rapidly and transiently activated kinases that phosphorylate the 21-residue synthetic peptide Thr(669) derived from the epidermal growth factor receptor, but the maximal activation of proline-directed kinases by SFLLRN was less pronounced than that by thrombin. MonoQ chromatography and immunoblot analysis of extracts from stimulated platelets revealed that while thrombin induced a prominent activation of the mitogen-activated protein kinases ERK1 and ERK2, SFLLRN completely failed to do so. On the other hand, SFLLRN, like thrombin, stimulated the activity of a proline-directed kinase distinct from ERK1/2, but the activation of this kinase was less pronounced following stimulation of platelets with SFLLRN compared with thrombin. We conclude 1) that the partial activation of cPLA(2) and the subsequent attenuated mobilization of arachidonic acid in response to SFLLRN may be the consequence of a less prolonged elevation of [Ca2+](i) and insufficient activation of proline-directed kinase(s) by SFLLRN and 2) that the ability of SFLLRN to mediate the activating phosphorylation of cPLA(2) in the absence of ERK1/2 stimulation suggests that, at least in human platelets, proline-directed kinases other than ERK1/2 may phosphorylate and activate cPLA(2).

The 85-kDa cytosolic PLA2 (cPLA2) is present in most cells and tissues and its structural and functional properties have been described. Different agonists, growth factors and cytokines activate cPLA2 to hydrolyze cellular phospholipids thereby providing the precursor substrates for the biosynthesis of eicosanoids and platelet-activating factor (PAF), the well-known mediators of inflammatory and allergic reactions. Recent studies discussed here suggest that cPLA2 is a receptor-regulated enzyme involved in the inflammatory response. Therefore, inhibitors of cPLA2 may be useful as therapeutic agents in the treatment of inflammatory diseases.

Calcium-sensitive cytosolic phospholipase A(2) (cPLA(2)) is responsible for receptor-mediated liberation of arachidonic acid, and thus plays an important role in the initiation of the inflammatory lipid-mediator cascade generating eicosanoids and platelet-activating factor. In this study we have investigated the cellular distribution of cPLA(2) in brain using a monoclonal antibody raised against cPLA(2) to immunostain tissue sections of human cerebral cortex. We have localized cPLA(2) in astrocytes of the gray matter. Colocalization with glial fibrillary acidic protein (GFAP) confirmed that cPLA(2) is associated predominantly with protoplasmic astrocytes. Astrocytes of the white matter, on the other hand, were not immunoreactive. In experiments using different human astrocytoma cell lines we found that cPLA(2) can be immunochemically localized in UC-11 MG cells, but cannot be detected in U-373 MG cells. This finding is consistent with the observation that cPLA(2) mRNA as well as cPLA(2) enzymatic activity can be readily measured in UC-11 MG astrocytoma cells, yet cannot be detected in U-373 MG cells. Our data suggest that the astrocyte is a primary source of cPLA(2) in the brain and provide further evidence for the importance of this cell type in inflammatory processes in the brain.

The Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2) displays both a phospholipase A2 and a lysophospholipase activity. Numerous hydrolases, including lipases, catalyze the hydrolysis of ester bonds by means of an active site triad of amino acids that includes a serine or a cysteine residue. We have examined whether human cPLA2 belongs to this class of enzymes by using site-directed mutagenesis. Although chemical inactivation of cPLA2 by the sulfhydryl reagent N-ethylmaleimide made it appear that cysteine(s) may be essential for catalysis, all 9 cysteine residues of cPLA2 proved dispensable, allowing near-normal enzyme activity when substituted by alanine. We noted that cPLA2 contains a 110-amino-acid region with sequence homology to phospholipase B (PLB) from Penicillium notatum. Interestingly, one of the conserved serines of cPLA2, Ser-228, within this domain aligns with the lipase consensus sequence Gly-X(Leu)-Ser(137)-X(Gly)-Gly of PLB. Replacement of Ser-228 by alanine (or threonine or cysteine) yielded catalytically inactive cPLA2, even though the native conformation was maintained as determined by CD spectroscopy. Likewise, the lysophospholipase activity was completely abolished by the Ser-228 mutations. In contrast, substitution by alanine of three different serines of cPLA2 (Ser-195, Ser-215, or Ser-577) that also aligned with the PLB sequence allowed for substantial enzymatic activity of cPLA2. Our findings provide evidence that 1) Ser-228 participates in the catalytic mechanism of cPLA2 and that 2) both the phospholipase A2 and the lysophospholipase activities of cPLA2 are catalyzed by the same active site residue(s).

The 85-kD cytosolic phospholipase A2 (cPLA2) is a novel receptor-regulated phospholipase that is thought to initiate the production of inflammatory lipid mediators. Since cPLA2 is present only in minute amounts (less than 0.01% of total cellular protein) in various cells and tissues, we have used the baculovirus expression system to produce sufficient quantities of cPLA2 for structural and functional analysis. The cDNA for cPLA2 was cloned into a baculovirus expression vector and, upon infection of Spodoptera frugiperda Sf-21 cells with the recombinant virus, cPLA2 was produced at high levels (9% of total cellular soluble protein). Gel electrophoresis and immunoblot analysis demonstrated that the recombinant protein has properties indistinguishable from cPLA2 present in human monocytic U937 cells. Structural analysis of recombinant cPLA2, using electrospray mass spectrometry in conjunction with automated sequence analysis, confirmed the expected sequence and revealed two post-translational modifications of the protein, phosphorylation on at least one site, and acetylation of the N-terminal serine residue after removal of the initiating methionine. In spite of the presence of six potential N-glycosylation sites, there is no evidence that any of them is glycosylated. The baculovirus expression system should prove useful for production of cPLA2, and electrospray mass spectrometry is a rapid and accurate method for the analysis of post-translational modifications.

The effect of pancreatic group I phospholipase A2 (PLA2-I) on receptor-mediated expression of arthritic group II phospholipase A2 (PLA2-II) and its correlation with prostaglandin E2 (PGE2) synthesis were examined in cultured rat mesangial cells. Scatchard analysis using 125I-PLA2-I revealed the existence of a single class of specific binding sites for PLA2-I in rat mesangial cells with an equilibrium dissociation constant (Kd) of 1.6 nM and a maximum binding capacity of 10.1 fmol/10(6) cells. The mammalian mature type of PLA2-I specifically recognized this binding site, whereas its inactive zymogen and mammalian PLA2-II showed much lower affinities. PLA2-I markedly increased PLA2-II mRNA levels as well as PLA2-II secretion from the cells in a time- and dose-dependent manner that was closely correlated with PGE2 production. Both PLA2-II expression and PGE2 synthesis were completely suppressed by pretreatment of the cells with actinomycin D, cycloheximide, or dexamethasone. These results strongly suggest that there may be crosstalk between PLA2-I and PLA2-II via the specific PLA2-I receptor that elicits PGE2 synthesis.