Interleukin-3 Regulates Development of the 5-Lipoxygenase/Leukotriene C4 Synthase Pathway in Mouse Mast Cells

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129

160

Type II secretory phospholipase A 2 (sPLA 2 ) has been shown to be induced by a variety of proinflammatory stimuli and, therefore, has been implicated in the inflammatory process. In order to determine whether association of sPLA 2 with cell surfaces via heparan sulfate proteoglycan is important for its effects on cellular functions, we have identified the critical domain in sPLA 2 for heparin and cell surface binding and examined its role in cellular prostaglandin (PG) biosynthesis. Replacement of several conserved Lys residues in the Cterminal region of mouse and rat sPLA 2 s by Glu resulted in a marked reduction of their capacities to bind to heparin and mammalian cell surfaces without affecting their enzymatic activities toward dispersed phospholipid as a substrate. CHO cells stably transfected with wild-type sPLA 2 released about twice as much arachidonic acid (AA) during culture for 10 h with fetal calf serum and interleukin-1␤ than cells transfected with vector alone, whereas the ability to enhance AA release was impaired in sPLA 2 mutants incapable of binding to cell surfaces. AA released by wild-type sPLA 2 -transfected CHO cells was metabolized to prostaglandin E 2 via prostaglandin endoperoxide H synthase (PGHS)-2 after IL-1␤ stimulation, revealing a particular functional linkage of sPLA 2 to PGHS-2. In contrast, A23187-initiated immediate AA release over 30 min was not affected by sPLA 2 overexpression. Taken together, these results suggest that sPLA 2 expressed endogenously and anchored on cell surfaces via its C-terminal heparinbinding domain is involved in the PGHS-2-dependent delayed PG biosynthesis initiated by growth factors and cytokines during long term culture.

Section: Discussionmentioning

confidence: 93%

Type II Secretory Phospholipase A2 Associated with Cell Surfaces via C-terminal Heparin-binding Lysine Residues Augments Stimulus-initiated Delayed Prostaglandin Generation

Murakami

Nakatani

Kudo

1996

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129

160

“…Isolation and Characterization of the mMCP-9 Transcript in Different Tissues-The mMCP-9 transcript was sought in various tissues of normal BALB/c mice (The Jackson Lab., Bar Harbor, ME), BALB/c mice that had been infected with Trichinella spiralis (20,21), BALB/c mice that had a systemic mastocytosis following the adoptive transfer of the V3 mast cell line (22), and in in vitro derived BALB/c mast cells (23,24). RNA was isolated according to the method of Chomczynski and Sacchi (25) from liver, spleen, ear, intestine, and uterus of normal BALB/c mice and from liver and spleen of V3 mastocytosis mice.…”

Section: Methodsmentioning

confidence: 99%

“…V3 mastocytosis mice were used 2-4 weeks after the adoptive transfer of ϳ10 6 v-ablimmortalized V3 mast cells into the tail vein of each 6-week-old BALB/c mouse (22). RNA also was isolated from mouse bone marrow-derived mast cells (mBMMC) that were developed for 3 weeks with recombinant mouse interleukin-3 (IL) (100 units/ml) alone (23) or 5 weeks with both recombinant mouse c-kit ligand (KL) (100 ng/ml) and IL-10 (100 units/ ml) (24). Lastly, BALB/c mice were infected with ϳ500 T. spiralis larvae (20,21), and then total RNA was isolated from the intestine on day 10 after infection.…”

Section: Methodsmentioning

confidence: 99%

Mouse Mast Cell Protease 9, a Novel Member of the Chromosome 14 Family of Serine Proteases that is Selectively Expressed in Uterine Mast Cells

Hunt

Friend

Gurish

et al. 1997

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Mouse mast cell protease (mMCP) 1, mMCP-2, mMCP-4, and mMCP-5 are members of a family of related serine proteases whose genes reside within an ϳ850 kilobase (kb) complex on chromosome 14 that does not readily undergo crossover events. While mapping the mMCP-1 gene, we isolated a novel gene that encodes a homologous serine protease designated mMCP-9. The mMCP-9 and mMCP-1 genes are only ϳ7 kb apart on the chromosome and are oriented back to back. The proximity of the mMCP-1 and mMCP-9 genes now suggests that the low recombination frequency of the complex is due to the closeness of some of its genes. The mMCP-9 transcript and protein were observed in the jejunal submucosa of Trichinella spiralis-infected BALB/c mice. However, in normal BALB/c mice, mMCP-9 transcript and protein were found only in those mast cells that reside in the uterus. Thus, the expression of mMCP-9 differs from that of all other chymases. The observation that BALB/c mouse bone marrow-derived mast cells developed with interleukin (IL) 10 and c-kit ligand contain mMCP-9 transcript, whereas those developed with IL-3 do not, indicates that the expression of this particular chymase is regulated by the cytokine microenvironment. Comparative protein structure modeling revealed that mMCP-9 is the only known granule protease with three positively charged regions on its surface. This property may allow mMCP-9 to form multimeric complexes with serglycin proteoglycans and other negatively charged proteins inside the granule. Although mMCP-9 exhibits a >50% overall amino acid sequence identity with its homologous chymases, it has a unique substrate-binding cleft. This finding suggests that each member of the chromosome 14 family of serine proteases evolved to degrade a distinct group of proteins.

“…De novo induced LTC 4 S transcript, protein, and function have been documented during differentiation and maturation of human eosinophils from cord blood progenitors with interleukin (IL)-3 and IL-5 in the presence of Matrigel over 2-4 weeks (11). Furthermore, mouse bone marrow-derived immature mast cells obtained with stem cell factor and IL-10 respond to stimulation with IL-3 in the presence of stem cell factor and IL-10 with marked up-regulation of LTC 4 S transcript, protein, and function over a 2-week period (12). Although full regulation of LTC 4 S expression requires consideration of gene transcription, post-transcriptional mRNA stability (13), and post-translational protein phosphorylation (14,15), the developmental expression of LTC 4 S in in vitro studies of the human eosinophil and the mouse mast cell prompts an initial focus on transcriptional regulation of this selectively expressed gene.…”

mentioning

confidence: 99%

Cell-specific Transcription of Leukotriene C4Synthase Involves a Kruppel-like Transcription Factor and Sp1

Zhao

Austen

Lam

2000

Leukotriene C 4 synthase (LTC 4 S) is responsible for the biosynthesis of cysteinyl leukotrienes that participate in allergic and asthmatic inflammation. We analyzed 2.1 kilobases of the 5-flanking region of the human LTC 4 S gene, which contains three DNase I hypersensitivity sites, for its transcriptional activity when fused to a promoterless and enhancerless luciferase gene. Deletion analysis revealed a nonspecific basal promoter region between nucleotides ؊122 and ؊56 upstream of the translation start site which contains a consensus Sp1 binding site and a putative initiator element (Inr) and cell-specific enhancer regions further upstream. A single mutation of either the Sp1 binding site between nucleotides ؊120 and ؊115 or the Inr (CA-GAC) between nucleotides ؊66 and ؊62 reduced the expression of the reporter gene by ϳ60%, whereas double mutations decreased the expression by ϳ80%. The incubation of nuclear extracts from THP-1 and K562 cells with a 32 P-labeled oligonucleotide containing the Sp1 site or the Inr sequence gave gel-shifted complexes that were blocked by their respective cold oligonucleotides, and antisera specific for Sp1 and Sp3 provided supershifts for the former. Linker-scanning mutations of a cell-specific regulatory region revealed that mutations from nucleotides ؊165 to ؊125 reduced reporter activity. This region contains a tandem CACCC repeat (at nucleotides ؊149 to ؊145 and ؊139 to ؊135). An oligonucleotide containing the distal CACCC motif was gel shifted by THP-1 cell nuclear extract and was supershifted by antisera to Sp1 and Sp3. Cotransfection of an Sp1 expression plasmid into Drosophila SL2 cells with a ؊228 to ؊3 LTC 4 S reporter construct transactivated the reporter gene, whereas mutations at the CACCC repeat region reduced Sp1 transactivation by ϳ66%. Similarly, the Kruppel-like factor Zf9/CPBP (core promoter-binding protein) transactivated the ؊228 construct in COS cells but not its CACCC mutant construct. These findings indicate the involvement of Sp1 and an Inr in noncell-specific regulation and a Kruppel-like transcription factor and Sp1 in the cell-specific regulation of the LTC 4 S gene. These are the first such analyses of a member of a newly recognized superfamily of membraneassociated proteins involved in eicosanoid and glutathione metabolism, which contains key proteins involved in the generation of both prostanoids and cysteinyl leukotrienes.LTC 4 synthase (LTC 4 S) 1 catalyzes the conjugation of leukotriene A 4 and glutathione (GSH) to form LTC 4 . LTC 4 is implicated in the pathobiology of bronchial asthma by the efficacy of inhibitors of its biosynthesis and of receptor blockers for its constrictor metabolites in the treatment of this condition. LTC 4 S is an 18-kDa perinuclear membrane protein that functions as a dimer (1, 2) and is expressed predominantly in mast cells, basophils, and eosinophils (3-5). LTC 4 S was the first catalytic protein identified in a newly recognized superfamily of membrane-associated proteins involved in eicosanoid and GSH metabolism (M...