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Pancreatic phospholipase A₂ via its receptor regulates expression of key enzymes of phospholipid and sphingolipid metabolism¹

Section on Developmental Genetics,
^* Section on Cellular Differentiation, Heritable Disorders Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-1830, USA

²Correspondence: NIH, Bldg. 10, Rm. 9S241, Bethesda, MD 20892-1830, USA. E-mail: mukherja{at}exchange.nih.gov

SPECIFIC AIMS

Although theenzymatic properties of secretory phospholipase A₂ have been studied thoroughly, its receptor-mediated functions are not clearly understood. In the present investigation, we sought to determine the receptor-mediated functions of pancreatic group IB secretory phospholipase A₂ (sPLA₂IB) and the signaling pathway by which it regulates expression of genes involved in the generation of various lipid mediators.

PRINCIPAL FINDINGS

1. sPLA₂IB stimulates mRNA expression of key enzymes for phospholipid and sphingolipid metabolism
Recent studies have identified sPLA₂IB as a dual-function enzyme as it has both catalytic and receptor-mediated functions that include the release of lipid mediators. To determine whether sPLA₂IB via its receptor regulates expression of genes involved in the generation of lipid mediators, we treated mouse fibroblast cells (NIH 3T3) with varying doses of porcine pancreatic sPLA₂IB for varying lengths of time. We then analyzed total RNA isolated from treated and untreated cells by Northern blotting using the cDNA probes of key phospholipid- and sphingolipid-metabolizing enzymes. We found that sPLA₂IB not only stimulates mRNA expression of cytosolic phospholipase A₂ (cPLA₂) and cyclooxygenase-2 (COX-2), but also stimulates mRNA expression of key sphingolipid-metabolizing enzymes such as Mg²⁺-dependent neutral sphingomyelinase (NSMase) and lysosomal acid ceramidase (AC) in a dose (Fig. 1A ) and time (Fig. 1B ) -dependent manner. Induction of COX-2 mRNA expression by sPLA₂IB is biphasic (Fig. 1A ). The levels of both cPLA₂ (85 kDa) and COX-2 (72 kDa) proteins were elevated by sPLA₂IB treatment in a time-dependent manner (Fig. 1C ). cPLA₂ was also phosphorylated to its active form upon sPLA₂IB treatment of the cells (Fig. 1D ). Taken together, it appears that sPLA₂IB may have important roles in the regulation of at least two key enzymes of phospholipid and sphingolipid metabolism, respectively.

View larger version (74K): [in this window] [in a new window]   Figure 1. A) Stimulation of cPLA2, COX-2, NSMase, and AC-mRNA expression by sPLA2IB in NIH 3T3 cells. Forty micrograms of total RNA were subjected to Northern blot analysis using the indicated murine cDNA probes. B) Time course of stimulation of cPLA2, COX-2, NSMase, and AC-mRNA expression by sPLA2IB in NIH 3T3 cells. C) Elevation of cPLA2 and COX-2 proteins by sPLA2IB treatment. Semiconfluent NIH3T3 cells were treated with 75 nM porcine pancreatic sPLA2IB for the indicated intervals of time and equal amounts of protein from each cell lysate were subjected to Western blot analysis using mouse monoclonal anti-cPLA2 antibody and goat polyclonal anti-COX-2 antibody, respectively. D) Phosphorylation-dependent activation of cPLA2 by sPLA2IB. Semiconfluent NIH3T3 cells were treated with 75 nM porcine pancreatic sPLA2IB for the indicated intervals and equal amounts of protein from each cell lysate were subjected to Western blot analysis using mouse monoclonal anti-cPLA2 antibody to detect the level of phosphorylated (cPLA2-P) and nonphosphorylated cPLA2.

2. sPLA₂IB stimulates expression of cPLA₂, COX-2, NSMase, and AC-mRNAs via its receptor
To determine whether the observed stimulation of cPLA₂-, COX-2, Mg²⁺-dependent NSMase-, and AC-mRNA expression by sPLA₂IB is receptor mediated and independent of its catalytic activity, we first determined expression of sPLA₂IB receptor mRNA and protein in NIH3T3 cells by RT-PCR analysis and affinity cross-linking assays. The results show these cells express both the sPLA₂IB receptor mRNA and protein (data not shown). We then treated these cells with sPLA₂IB, which is inactivated by boiling or chemical means, using a specific inhibitor, MJ33. We found that the inactivated sPLA₂IB is fully capable of stimulating mRNA expression of cPLA₂, COX-2, Mg²⁺-dependent NSMase, and AC (Fig. 2A ). MJ33 alone had no effect on mRNA expression of these enzymes (data not shown). Thus, the effects sPLA₂IB on mRNA expression of these enzymes appear to be receptor mediated.

View larger version (53K): [in this window] [in a new window]   Figure 2. sPLA2IB receptor-mediated stimulation of mRNA expression for phosholipid- and sphingolipid-metabolizing enzymes in NIH 3T3 cells. A) Cells were treated for 7 h with either active or inactivated sPLA2IB in the presence or absence of bisindolyl maleimide III (BMIII), a PKC inhibitor. Total RNA were subjected to Northern blot analysis using the indicated cDNA probes. B) Cells were treated for 7 h with 75 µM sPLA2IB in the presence or absence of the ERK inhibitor PD98059 or the p38 MAPK inhibitor SB203580, which were dissolved in DMSO. Northern analysis was performed using the indicated cDNA probes. C) Activation of p38 MAPK by sPLA2IB treatment of NIH3T3 cells. Semiconfluent NIH3T3 cells were treated with 75 nM porcine pancreatic sPLA2IB for the intervals indicated and equal amounts of protein from each cell lysate were subjected to Western blot analysis using rabbit polyclonal anti-phospho p38 MAPK antibody. D) Effects of sPLA2IB and various growth factors or mitogens on expression of cPLA2, COX-2, NSMase, and AC-mRNA in the NIH 3T3 cells.

It has been reported that sPLA₂IB stimulates expression of cPLA₂and COX-2, but this receptor-mediated pathway is not clearly defined. Our understanding of the regulation of expression of NSMase and AC genes by sPLA₂IB is not clear either. Since signal transduction via the sPLA₂ receptor involves activation of several kinases, we sought to determine the specific kinases that might be involved in stimulating expression of the enzymes in the phospholipid- and sphingolipid-metabolizing pathway. Accordingly, we treated NIH3T3 cells with sPLA₂IB in presence and absence of inhibitors of specific kinases in some of the well-characterized signaling pathways. Total RNA isolated from cells cultured in the absence or presence of these inhibitors was subjected to Northern blot analyses. We found that the sPLA₂IB receptor-mediated stimulation of cPLA₂-mRNA expression is blocked by a very specific and potent protein kinase C (PKC) inhibitor, bisindolyl maleimide III (BMIII). This suggests that the sPLA₂IB receptor-mediated stimulation of cPLA₂ mRNA expression occurs via the PKC pathway (Fig. 2A ). However, the sPLA₂IB receptor-mediated stimulation of COX-2 expression was found to be via the p38-MAPK pathway, as the stimulation of COX-2 mRNA expression was blocked by the selective p38-MAPK inhibitor SB203580 (Fig. 2B ). There was no inhibitory effect on the sPLA₂IB-mediated stimulation of mRNA expression when the ERK inhibitor PD98059 was used to treat the cells (Fig. 2B ). An important finding in this study is that sPLA₂IB stimulated phosphorylation of p38 MAPK and consequent activation in NIH3T3 cells (Fig. 2C ). Further, stimulation of Mg²⁺-dependent NSMase and AC mRNA expression via the sPLA₂IB receptor was not affected when PKC, p38-MAPK, or ERK inhibitors were used.

3. Similarity between receptor-mediated regulation of gene expression by sPLA₂IB and some cytokines
It has been reported that sPLA₂IB stimulates cell proliferation, activates cPLA₂, and induces sPLA₂IIA and COX-2 expression via its receptor. These effects of sPLA₂IB at the cellular level are reminiscent of those of some cytokines. Thus, we sought to detect similarities, if any, between the effects of sPLA₂IB and the cytokines on NIH 3T3 cells with respect to the induction of mRNA expression of key enzymes of both phospholipid and sphingolipid metabolism. Accordingly, we treated the NIH3T3 cells with sPLA₂IB, TNF, LPS, IL-1ß, FGF, or HGF and determined the expression pattern of cPLA₂-, COX-2-, NSMase-, and AC-mRNAs by Northern blotting. The results show that IL-1ß, FGF, and HGF, like sPLA₂IB, also stimulate expression of cPLA₂-, COX-2, NSMase-, and AC-mRNAs (Fig. 2 D). These results raise the possibility that under our experimental conditions, sPLA₂IB may function like a cytokine or growth factor via its receptor.

CONCLUSIONS

There is compelling evidence to suggest that several important functions of sPLA₂s are mediated via its receptor. These functions include the stimulation of cell proliferation, airway contraction, extracellular matrix invasion, and chemokinesis.

sPLA₂IB has been reported to activate p38 MAP kinase, p42/44 MAP kinase, and c-Jun kinase by phosphorylation in a human astrocytoma cell line. This enzyme has also been reported to induce expression of the inflammatory sPLA₂IIA and the activation of cPLA₂. Activation of cPLA₂ requires phosphorylation via p42/p44 MAP kinase in the human astrocytoma cell line and bone marrow-derived murine mast cells and the PKC/Raf-1/MAPK pathway in rat renal mesangial cells. Moreover, the growth-dependent alteration in arachidonic acid release from endothelial cells is mediated via PKC activation. The induction of COX-2 expression by sPLA₂IB has also been reported but the signaling pathway and the specific transcription mechanism have remained unknown. Recently, we have shown that in a mouse osteoblast cell line, the sPLA₂IB receptor-mediated induction of COX-2 expression is mediated via the transcription factor C/EBPß. In the present study, we discovered that, via its receptor, both active and enzymatically inactive sPLA₂IB stimulates expression of cPLA₂, and COX-2 mRNAs, and proteins in a dose- and time-dependent manner. Bisindolyl maleimide III, a highly selective inhibitor of PKC, blocks sPLA₂-IB receptor-mediated stimulation of the cPLA₂ mRNA expression, suggesting a possible role for PKC in this pathway. It has been demonstrated that sPLA₂-stimulated selective release of AA is mediated via its receptor. Our results are intriguing in that sPLA₂IB not only activates the cPLA₂ by stimulating its phosphorylation, but also regulates expression of cPLA₂-mRNA. sPLA₂IB receptor-mediated induction of COX-2 expression is blocked by a very selective p38 MAP kinase inhibitor, SB203580, but not by an ERK pathway inhibitor or PKC inhibitor, suggesting a possible role of p38 MAP kinase in the sPLA₂IB-induced expression of COX-2 mRNA. The p38 MAP kinase has been shown to play important roles in the regulation of COX-2 expression in human monocytes and J774 macrophages. We also found that in addition to stimulating expression of cPLA₂ and COX-2 mRNAs, sPLA₂IB also stimulates mRNA expression of Mg²⁺-dependent NSMase and AC. The sPLA₂IB-induced stimulation of expression of NSMase and AC-mRNAs was not inhibited by the PKC inhibitor nor by p38 MAPK/ERK signaling pathway-specific inhibitors. These results suggest that like IL-1ß, FGF, and HGF, sPLA₂IB via its receptor plays a pivotal role in the generation of lipid mediators (e.g., eicosanoids, platelet-activating factor, ceramide, and sphingosine) by regulating phospholipid and sphingolipid metabolism; perhaps there is a cross talk among these pathways (Fig. 3 ).

View larger version (28K): [in this window] [in a new window]   Figure 3. A possible mechanism by which sPLA2IB, via its receptor, regulates the production of proinflammatory lipid mediators. Arrows indicate stimulation and (?) indicates the pathways that are yet to be characterized. CM, cell membrane; cPLA2, cytosolic phospholipase A2; cPLA2-P, the phosphorylated form (active) of cPLA2; PKC, protein kinase C; MAPK, mitogen-activated kinase; COX, cyclooxygenase; NSMase, Mg2+-dependent neutral sphingomyelinase; AC, acid ceramidase; AA, arachidonic acid; PGs, prostaglandins.

In summary, we have uncovered for the first time that sPLA₂IB via its receptor pathway regulates mRNA expression of key enzymes involved in the phospholipid and sphingolipid metabolism and that there may be cross talk among these metabolic pathways.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0831fje ; to cite this article, use FASEB J. (June 27, 2001) 10.1096/fj.00-0831fje

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