Arachidonic acid release from membrane phospholipids is involved in a myriad of physiologic and pathophysiologic cell functions and is under stringent regulation. Phospholipase A2 (PLA2), which hydrolyzes the sn-2 bond of membrane phospholipids, is the enzyme controlling arachidonic acid release. The renal mesangial cell is capable of a diverse array of cell functions associated with arachidonic acid release. These include the synthesis and secretion of bioactive agents, phagocytosis, contraction, and proliferative responses, all of which have a profound impact on glomerular function. In these cells, PLA2 regulation is multi- faceted and of central importance to our understanding of these complex processes. We have identified and purified a novel Ca2+-dependent high- molecular weight form of PLA2 (cPLA2), which is regulated by hormones and growth factors in rat renal mesangial cells. This enzyme is activated by physiologic concentrations of Ca2+, and associates with membranes in Ca2+-dependent manner. The enzyme is also regulated by protein phosphorylation. We have demonstrated that cPLA2 is phosphorylated and activated in vitro by two growth-factor regulated protein kinases: protein kinase C and p42 MAP kinase. We have raised antibodies which immunoprecipitate cPLA2, and obtained the cDNA for the human homolog of the enzyme, which when expressed in COS cells, results in induction of PLA2 activity.

Fatty acid release also occurs in response to ischemic and other forms of cell injury in many tissues. We have identified a second form of PLA2 which is activated during liver regeneration, a classic model of cell injury and proliferation, as well as in renal proximal tubules during hypoxia. This enzyme, which we have designated hPLA2 is distinct from cPLA2, as well as from other well-characterized isoforms of PLA2. It is likely that these distinct isoforms of PLA2 will have separate physiologic roles. In particular, we hypothesize that cPLA2 is involved in proliferation, whereas hPLA2 participates in response to cell injury. The proposed studies have two major objectives.

1) To elucidate the mechanisms of cPLA2 regulation in the mesangial cell. Regulation of cPLA2 by phosphorylation will be examined in detail. Using transient expression in COS cells, functional domains of the enzyme will be defined by mutagenizing the cPLA2 cDNA. The specific association of cPLA2 with the membrane and with putative modulatory proteins will be explored.

2) To purify hPLA2 and develop antibodies against the enzyme. These reagents will be used to examine the regulation of this enzyme in regenerating liver, and hypoxic tubules. It is anticipated that these studies will lead to a greater understanding of the role of arachidonic acid release in mesangial cell function, and of the mechanisms of cell signaling by hormones and growth factors. It is hoped that characterization of these distinct forms of PLA2 and their regulation will to isoform-specific inhibitors to selectively modulate pathophysiologic responses associated with PLA2.

R01DK039902

1987-09-30

2002-11-30