Regulation of the cytosolic phospholipase A₂ gene

Arachidonic acid (AA) metabolites including platelet activating factor (PAF), prostaglandins (PG), leukotrienes (LT) and thromboxanes are involved in paracrine and autocrine regulation of the inflammatory response. The levels and activities of these lipid mediators have been associated with a number of pathological states including asthma, pulmonary fibrosis, as well as other inflammatory lung diseases. The rate limiting step in eicosanoid biosynthesis is the release of AA from membrane phospholipids, catalyzed by the enzyme phospholipase A₂. The exclusive liberation of AA from the sn-2 position of phospholipids is catalyzed by the cytoplasmic form of this enzyme designated cPLA₂.

Based on the fact that the lung is particularly susceptible to external insults in comparison to most other organs of the body, a properly regulated arachidonic acid cascade and therefore inflammatory response is imperative to lung homeostasis. Controlled inflammatory and fibrotic responses are repair mechanisms employed to counteract an injury, allowing for healing of the damaged tissue.  However, an alteration or loss in the regulation of the arachidonate cascade leads to a chronic inflammatory state, which characterizes a number of pulmonary disorders including emphysema, asthma, and pulmonary fibrosis. The role of cPLA₂ in allergic asthma is dramatically demonstrated by the cPLA₂ knockout mouse (-/-cPLA₂).  Mice deficient in cPLA₂ activity were markedly less sensitive to methacholine challenge than +/+ cPLA₂ mice (26).  The degree of bronchoconstriction in ovalbumin-sensitized +/+ cPLA₂ mice was tremendous compared to -/- cPLA₂ mice (figures below).  When methacholine aerosol was delivered into the lungs of -/- cPLA₂ mice, total lung resistance (R_L) was identical to unsensitized wild type (+/+ cPLA₂) mice treated only with saline, thus demonstrating physiological responses which recapitulate the histology (see the graph below).

Our research and that of others has shown that cPLA₂ is upregulated by proinflammatory cytokines and repressed by glucocorticoids, as shown in the figures below. We have demonstrated that these responses require de novo transcription using nuclear run-on analysis.

The nuclear run on data in the figures below demonstrate that the repression of cPLA₂ by glucocorticoids and the induction by IL-1b is in part a consequence of de novo transcription.

Given the physiological importance of cPLA₂ to the inflammatory process in the lung and the demonstration that cytokine and glucocorticoid regulation is dependent on de novo gene regulation, we initiated studies to characterize the human cPLA₂ genomic locus.  Human P1, PAC, BAC genomic libraries were screened (Genome Systems, St. Louis, MO) using oligonucleotide primer sets generated from cDNA, published 5' noncoding, and exon 1 sequence which corresponded to single copy genomic sequence.  Five overlapping clones were identified one BAC, two P1 and two PAC which hybridize to known cPLA₂ sequence. To delineate the regions of the human cPLA₂ gene that each of these genomic clones encompassed, Southern analysis was performed on restriction enzyme digests of P1, PAC, BAC DNA clones using radiolabeled oligonucleotides corresponding to discrete sequences along the cPLA₂ cDNA. This initial screening technique determined that the five genomic clones overlapped by cumulatively encoding the full length cPLA₂ mRNA, over 20 kb of 5' flanking sequence and a large region of 3' noncoding sequence.

Our future efforts will therefore focus on evaluating  the molecular mechanisms which control basal and regulated cPLA₂  gene expression.