Colin D. Funk

Human platelet/erythroleukemia cell prostaglandin G/H synthase: cDNA cloning, expression, and gene chromosomal assignment.

Platelets metabolize arachidonic acid to thromboxane A2, a potent platelet aggregator and vasoconstrictor compound. The first step of this transformation is catalyzed by prostaglandin (PG) G/H synthase, a target site for nonsteroidal antiinflammatory drugs. We have isolated the cDNA for both human platelet and human erythroleukemia cell PGG/H synthase using the polymerase chain reaction and conventional screening procedures. The cDNA encoding the full‐length protein was expressed in COS‐M6 cells. Microsomal fractions from transfected cells produced prostaglandin endoperoxide‐derived products which were inhibited by indomethacin and aspirin. Mutagenesis of the serine residue at position 529, the putative aspirin acetylation site, to an asparagine reduced cyclooxygenase activity to barely detectable levels, an effect observed previously with the expressed sheep vesicular gland enzyme. Platelet‐derived growth factor and phorbol ester differentially regulated the expression of PGG/H synthase mRNA levels in the megakaryocytic/platelet‐like HEL cell line. The PGG/H synthase gene was assigned to chromosome 9 by analysis of a human‐hamster somatic hybrid DNA panel. The availability of platelet PGG/H synthase cDNA should enhance our understanding of the important structure/function domains of this protein and its gene regulation.—Funk, C. D.; Funk, L. B.; Kennedy, M. E.; Pong, A. S.; FitzGerald, G. A. Human platelet/erythroleukemia cell prostaglandin G/H synthase: cDNA cloning, expression, and gene chromosomal assignment. FASEB J. 5: 2304–2312; 1991.

Salt-sensitive hypertension and reduced fertility in mice lacking the prostaglandin EP2 receptor.

Prostaglandins (PGs) are ubiquitous lipid mediators derived from cyclooxygenase metabolism of arachidonic acid that exert a broad range of physiologic activities, including modulation of inflammation, ovulation and arterial blood pressure. PGE2, a chief cyclooxygenase product, modulates blood pressure and fertility, although the specific G protein–coupled receptors mediating these effects remain poorly defined. To evaluate the physiologic role of the PGE2 EP2 receptor subtype, we created mice with targeted disruption of this gene (EP2–/–). EP2–/– mice develop normally but produce small litters and have slightly elevated baseline systolic blood pressure. In EP2–/– mice, the characteristic hypotensive effect of intravenous PGE2 infusion was absent; PGE2 infusion instead produced hypertension. When fed a diet high in salt, the EP2–/– mice developed profound systolic hypertension, whereas wild–type mice showed no change in systolic blood pressure. Analysis of wild–type and EP2–/– mice on day 5 of pregnancy indicated that the reduced litter size of EP2–/– mice is due to a pre–implantation defect. This reduction of implanted embryos could be accounted for by impaired ovulation and dramatic reductions in fertilization observed on day 2 of pregnancy. These data demonstrate that the EP2 receptor mediates arterial dilatation, salt–sensitive hypertension, and also plays an essential part in female fertility.

Molecular biology in the eicosanoid field.

Publisher Summary This chapter describes the molecular biology in the eicosanoid field. Molecular biology made its main entrance to the eicosanoid field, when two groups simultaneously reported the cloning of the cDNA for leukotriene A 4 hydrolase. Subsequently, the field has expanded considerably to include many interesting findings with the lipoxygenases, prostaglandin-synthesizing enzymes, and prostanoid receptors. Availability of the genes for these enzymes should allow targeting strategies to discern potential functions. Clear information on the regulation and significance of leukotriene formation by 5-lipoxygenase, 5-lipoxygenase activating protein, and leukotriene A 4 hydrolase is still lacking. The recent discovery of the second form of cyclooxygenase has reopened a wide interest in the academic and pharmaceutical communities in prostaglandin research. The differential regulation of the two cyclooxygenase forms and the development of selective isoform inhibitors will be the areas of intensive research. Cloning of the different members of the eicosanoid receptor family should facilitate our unravelling of the mechanisms involved in the action of this interesting class of arachidonate metabolites known as eicosanoids.

Structure-function properties of human platelet 12-lipoxygenase: chimeric enzyme and in vitro mutagenesis studies.

Mutant and chimeric lipoxygenases were expressed in human embryonal kidney 293 cells to assess the importance of amino acids and domains for catalytic activity and positional specificity of molecular oxygen insertion. Histidines 360, 365, and 540, when changed to glutamine residues, completely abolished human platelet 12‐lipoxygenase activity. Altered histidines at positions 355, 383, and 392 retained enzymatic activity. The former three histidines could possibly serve as ligands for the catalytically essential non‐heme iron atom. Amino acids adjacent (residues 398‐417) to the five centrally located histidines conserved among all plant and animal lipoxygenases controlled to a limited extent the positional specificity of oxygenation of 12‐lipoxygenase. Variant A417I and the triple variant K416Q/A417I/V418M, designed to introduce 15‐lipoxygenase substitutions, transformed the platelet 12‐lipoxygenase that synthesizes exclusively 12‐hydro(pero)xy‐eicosatetraenoic acid (12‐H(P)ETE) to an enzyme capable of 10‐20% 15‐lipoxygenation. When all amino acids between positions 398‐429 of 12‐lipoxygenase had the corresponding 15‐lipoxygenase sequence, the enzyme made 66% 15‐lipoxygenase products. The latter enzyme had markedly reduced enzyme activity, though, indicating an apparent shift in the optimal alignment of substrate at the active site for hydrogen atom abstraction. The platelet enzyme could not be altered to form 5‐lipoxygenase products by similar manipulations of sequence within this region. Chimeric enzymes consisting of an NH2‐terminal segment from one lipoxygenase and the COOH terminus from another lipoxygenase or large substitutions resulted in nonfunctional enzymes. NH2‐terminal extensions, but not short deletions, could be tolerated functionally. These studies provide some new insights into lipoxygenase structure‐function in the absence of an unresolved three‐dimensional structure.—Chen, X.‐S., Funk, C. D. Structure‐function properties of human platelet 12‐lipoxygenase: chimeric enzyme and in vitro mutagenesis studies. FASEB J. 7: 694‐701; 1993.

Lipoxin generation by human megakaryocyte-induced 12-lipoxygenase

Eicosanoid biosynthesis was examined with a human megakaryocytic cell line (Dami). Megakaryocytes incubated with [1-14C]arachidonic acid and either ionophore A23187 or thrombin generated both thromboxane and 12-hydroxyheptadecatrienoic acid (HHTrE). Exposure to phorbol myristate acetate (PMA) for 1 through 9 days induced differentiation and revealed an increase in the conversion of [1-14C]arachidonate to cyclooxygenase- and lipoxygenase (LO)-derived products. The LO-derived product was identified as 12S-HETE by its physical characteristics including GC/MS and chiral column SP-HPLC. PMA-treated Dami cells did not generate 5-HETE, leukotrienes or lipoxins from exogenous arachidonic acid while they did convert leukotriene A4 (LTA4) to lipoxin A4, lipoxin B4 and their respective all-trans isomers. In addition, COS-M6 cells transfected with a human 12-lipoxygenase cDNA and incubated with either arachidonic acid or LTA4 generated 12-HETE and lipoxins, respectively. The lipoxin profile generated by transfected COS-M6 cells incubated with LTA4 was similar to that generated by the PMA-treated Dami cells. Results indicate that human megakaryocytes can transform arachidonate and LTA4 to bioactive eicosanoids and that the 12-lipoxygenase appears upon further differentiation of these cells. In addition, they indicate that the 12-LO of human megakaryocytes and the 12-LO expressed by transfected COS cells can generate both lipoxins A4 and B4. Together they suggest that the human 12-LO can serve as a model of LX-synthetase activity with LTA4.

Targeted Gene Disruption by Homologous Recombination

Molecular genetic techniques have reached a point where it is now routinely feasible to selectively disrupt the function of any gene. The concept of gene targeting was developed by Mario Capecchi and Oliver Smithies in the early 1980s. and the hallmark experiments were first reported in 1985.’ The success of this early work was founded upon the seminal contributions of Evans and Kaufman2 and Martin3 who developed totipotent, germ-line competent murine embryonic stem (ES) cells. Basically, gene targeting (also referred to as targeted gene disruption or gene “knockout”) involves cloning a gene of interest and insertion of a selectable marker, usually a cassette coding for neomycin resistance, into the exonic sequence to disrupt the gene. The targeting vector also commonly includes a negative selection cassette to reduce the large background of nonhomologously integrated DNA. The linearized targeting vector, containing approximately 510 kb of homologous DNA sequence, is introduced into cultured ES cells by electroporation. After about 10 days of selection, ES cell colonies are screened for the desired targeting event by Southern blot or polymerase chain reaction (PCR) analysis. Targeted cell lines are injected into the blastocoel cavity of 3.5-day postcoital blastocysts, and the injected blastocysts are transferred to pseudopregnant recipient mice. The resultant chimeric offspring can be bred to homozygosity so that both copies of the gene of interest are disrupted. Construction of targeting vectors and the background and methodologies of gene targeting have been reviewed extensively el~ewhere.~-~ Here, recent examples of gene targeting with relevance to blood cell function and vascular biology are reviewed, and experiments involving inactivation of the murine 5-lipoxygenase (SLX) gene are described.

Lipoxygenases of Mice and Men

Seven years have elapsed since the cloning of the cDNA for the first mammalian lipoxygenase and the deduced primary structure (Matsumoto et al., 1988; Dixon et al., 1988). Here, a brief comparative overview of the molecular properties of human and mouse lipoxygenases will be presented. Although each of the human lipoxygenases were isolated and systematically characterized prior to the corresponding murine homologs it became necessary to evaluate their relatedness in view of our present targeted lipoxygenase gene inactivation experiments in mice.