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Cell-Specific Regulation of Type II Phospholipase A2 Expression in Rat Mesangial Cells

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Cell-Specific Regulation of Type II Phospholipase A2 Expression in Rat Mesangial Cells Cell-specific regulation of type II phospholipase A2 expression in rat mesangial cells. M Konieczkowski, J R Sedor J Clin Invest. 1993;92(5):2524-2532. https://doi.org/10.1172/JCI116861. Research Article IL-1 stimulates mesangial cells to synthesize specific proteins, including a non-pancreatic (Type II) phospholipase A2 (PLA2). We have studied the regulation of PLA2 by proinflammatory mediators, implicated in the pathogenesis of glomerulonephritis, and have assessed whether the activation of second messenger systems modulates or mimics PLA2 gene expression by cytokines. IL-1 alpha and beta, TNF alpha, and LPS, but not serum, IL-2, or PDGF, potently induce PLA2 mRNA, and enzyme expression. IL-1-stimulated mesangial cells express a 1.0 kB PLA2 mRNA transcript that is induced in a dose- and time-dependent manner. IL-1-stimulated increases in steady-state PLA2 mRNA abundance result from a moderate increase in PLA2 transcription rate that is amplified by the prolonged persistence of the transcript. Forskolin and dibutyryl cAMP potentiate IL-1-induced PLA2 mRNA and enzyme expression, but have no effect in the absence of cytokine. 12-tetradecanoyl phorbol 13-acetate, sn-1, 2-dioctanoyl glycerol or 1-oleoyl-2-acetyl-sn-glycerol fail to induce PLA2 expression or to alter the effect of IL-1 when coincubated with the cytokine. In contrast, serum deprivation for 24 h specifically enhances IL-1-stimulated PLA2. Genistein potentiates PLA2 mRNA expression in cells exposed to both IL-1 and serum. The inhibitory effect of serum on IL-1-induced PLA2 mRNA abundance is reproduced by PDGF but not dexamethasone. These data demonstrate that the signaling pathways directly engaged by IL-1 to induce PLA2 expression in mesangial cells […] Find the latest version: https://jci.me/116861/pdf Cell-specific Regulation of Type 11 Phospholipase A2 Expression in Rat Mesangial Cells M. Konieczkowski and J. R. Sedor* Departments ofMedicine and Physiology and *Biophysics, School ofMedicine, Case Western Reserve University and Divisions ofNephrology, MetroHealth Medical Center and University Hospitals, Cleveland, Ohio 44109 Abstract tion of glomerular filtration rate. However, in vitro and in vivo evidence suggests that the mesangial cell both produces and IL-1 stimulates mesangial cells to synthesize specific proteins, responds to biological modifiers and participates in the destruc- including a non-pancreatic (Type II) phospholipase A2 tive and restorative processes that follow the initiation of glo- (PLA2). We have studied the regulation of PLA2 by proinflam- merular injury ( 1, 2). Intraglomerular expression of the cyto- matory mediators, implicated in the pathogenesis of glomerulo- kines, IL- 1 and TNF a l has been demonstrated in both human nephritis, and have assessed whether the activation of second and experimental glomerulonephritis (3, 4). IL- 1 and TNF messenger systems modulates or mimics PLA2 gene expression activate the mesangial cell in culture to express new structural by cytokines. IL-la and ,B, TNFa, and LPS, but not serum, and functional characteristics in part by inducing expression of IL-2, or PDGF, potently induce PLA2 mRNA, and enzyme ex- specific genes (3, 4). IL- 1 a, IL- I 3, and TNF stimulate release pression. IL-l-stimulated mesangial cells express a 1.0 kB of the neutrophil chemotactic intercrine, IL-8 (5-7), and IL-6 PLA2 mRNA transcript that is induced in a dose- and time-de- (6, 7) and regulate expression of genes controlling extracellular pendent manner. IL-i-stimulated increases in steady-state matrix synthesis and turnover (3, 4, 8). We have demonstrated PLA2 mRNA abundance result from a moderate increase in in rat mesangial cells that IL- 1 a directly stimulates prostaglan- PLA2 transcription rate that is amplified by the prolonged per- din synthesis by increasing phospholipase A2 (PLA2)l activity sistence of the transcript. Forskolin and dibutyryl cAMP po- through synthesis of a nonpancreatic (Type II) PLA2 (9). The tentiate IL-i-induced PLA2 mRNA and enzyme expression, increases in PLA2 activity and PLA2 mRNA expression are but have no effect in the absence of cytokine. 12-tetradecanoyl coordinate and follow the same time course as cytokine-stimu- phorbol 13-acetate, sn-i, 2-dioctanoyl glycerol or 1-oleoyl-2- lated PGE2 synthesis. acetyl-sn-glycerol fail to induce PLA2 expression or to alter the As a result of these and other studies, many investigators effect of IL-1 when coincubated with the cytokine. In contrast, have hypothesized that TNF- and IL-1-stimulated mesangial serum deprivation for 24 h specifically enhances IL-i-stimu- cells express an altered phenotype that contributes to glomeru- lated PLA2. Genistein potentiates PLA2 mRNA expression in lar injury. The intracellular events that mediate the cytokine- cells exposed to both IL-1 and serum. The inhibitory effect of induced transition of mesangial function are poorly under- serum on IL-i-induced PLA2 mRNA abundance is reproduced stood. This process involves induction ofa specific gene set by a by PDGF but not dexamethasone. These data demonstrate that network of short-term signals generated by interaction of the the signaling pathways directly engaged by IL-1 to induce cytokine with the binding domain of its receptor. The cytosolic PLA2 expression in mesangial cells interact with several sec- signals generated by IL- 1 and TNF in the mesangial cell have a manner. We ond messenger systems in cell-specific speculate been fairly well defined. We and others have shown that IL- 1 a that IL-1 induces specialized changes in mesangial cell struc- rapidly stimulates 1, 2-diacylglycerol and phosphatidic acid ture and function through direct activation of a transcription formation ( 10, 11 ) but does not induce rapid changes in cysto- factor(s), that result in induction of a specific gene set. (J. lic Ca>2 or pHi (10). Both IL- 1 and TNF cause phosphoryla- Clin. Invest. 1993. 92:2524-2532.) Key words: cell phenotype* tion of mesangial cell proteins, but neither cytokine increases cell differentiation * interleukin-1 * glomerulonephritis - the phosphorylation of the putative 80 kD substrate of protein cytokine kinase C (3, 12). Pfeilschifter shows no increase in cAMP accu- mulation in response to IL- 1 ( 13), and TNF and IL- 1 only Introduction stimulate cGMP formation after 4 to 6 h ( 14, 15). The mesangial cell occupies a central anatomical position We have used the induction of PLA2 mRNA expression by within the glomerulus ( 1, 2). Historically this cell has been IL- 1 as a model system to define whether these early signals are regarded as unifunctional, only being important in the regula- converted to long-term changes in cell phenotype through in- duction of specific genes. Our studies suggest that the signalling pathways directly engaged by IL- I in the mesangial cells inter- This work was presented in part at the annual meeting of the American act with several second in a Society of Nephrology, 2-5 December 1990, Washington, DC. messenger systems cell-specific Address correspondence to Dr. John R. Sedor, Division ofNephrol- manner. ogy, MetroHealth Medical Center, 2500 MetroHealth Drive, Office Methods R-338, Cleveland, OH 44109-1998. l Receivedfor publication 23 June 1992 and in revisedform 16 July Materials. Recombinant human IL- a (rIL- 1 ), and TNF were kindly 1993. provided by Drs. Peter Lomedico (Hoffmann-LaRoche, Nutley, NJ) J. Clin. Invest. 1. Abbreviations used in this paper: DiOG, sn- 1, 2-dioctanoylglycerol; © The American Society for Clinical Investigation, Inc. OAG, l-oleoyl-2-acetyl-sn-glycerol; PA, phosphatidic acid; PLA2, 0021-9738/93/011/2524/09 $2.00 phospholipase A2; rIL- 1, recombinant human IL- la; TPA, tetradeca- Volume 92, November 1993, 2524-2532 noyl phorbol 13-acetate. 2524 M. Konieczkowski and J. R. Sedor and Leo Linn (Cetus Corporation, Emeryville, CA). Specific activities known fragment sizes (Bethesda Research Laboratories, Gaithersburg, and endotoxin contents have previously been reported (9, 10, 16). MD). The filters were stripped and rehybridized with a 32P-labeled Recombinant human interleukin-l 3 (2 X I07 U/mg) was a gift from chicken a-tubulin cDNA probe. IL-6 mRNA expression was also as- Dr. Nigel Staite (UpJohn, Kalamazoo, MI), and endotoxin concentra- sayed using the hybridization conditions described above. A 32P-la- tion determined by limulus lysate assay was 1.8 ng/mg protein. Stock beled 650 bp cDNA fragment from murine IL-6 (gift from Dr. Steven cytokines were diluted 1:50,000 before use with pyrogen-free saline Clark, Genetics Institute, Cambridge, MA) was used as a probe in some containing 2 mg/ml lipopolysaccharide-free BSA (Calbiochem-Nova- studies. biochem Corp., San Diego, CA). Recombinant human platelet derived PLA2 mRNA abundance was assessed by RNA-RNA solution hy- growth factor (PDGF, BB homodimer) was obtained from Boehringer bridization as we have previously described in detail (9). Briefly, T7 Mannheim Biochemicals, Indianapolis, IN. Tetradecanoyl phorbol RNA polymerase was used to synthesize a [32PlcRNA probe using a 13-acetate (TPA), forskolin, genistein, l-oleoyl-2-acetyl-sn-glycerol Pvu Il-linearized DNA template that contained a cDNA sequence (bp (OAG) and sn-l, 2-dioctanoylglycerol (diOG) were from Calbiochem- 381-758) for rat splenic, nonpancreatic (Type II) PLA2. A heteroge- Novabiochem Corp. LPS, dibutyryl cAMP and arachidonic acid nous nucleotide residue found in position 461 of the mature PLA2 were supplied by Sigma Chemical Co. (St. Louis, MO). L-a-l- RNA transcript produces protected transcripts of both 377 nucleotide palmitoyl, 2-arachidonyl[arachidonyl-l- "4C]-phosphatidylethanola- and 297 nucleotide (9). For this study, we have constructed, by stan- mine (60 mCi/mmol), and uridine 5'-triphosphate, [a-32p] (800 Ci/ dard methodologies, an additional DNA template that contains bp mmol) were purchased from DuPont-New England Nuclear (Boston, 666-759 of the PLA2 cDNA. T7 RNA polymerase was used to synthe- MA).
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