Modulation of Cell-Substrate Adhesion by Arachidonic Acid: Lipoxygenase Regulates Cell Spreading and ERK1/2-inducible Cyclooxygenase Regulates Cell Migration in NIH-3T3 Fibroblasts

Abstract

Adhesion of cells to an extracellular matrix is characterized by several discrete morphological and functional stages beginning with cell-substrate attachment, followed by cell spreading, migration, and immobilization. We find that although arachidonic acid release is rate-limiting in the overall process of adhesion, its oxidation by lipoxygenase and cyclooxygenases regulates, respectively, the cell spreading and cell migration stages. During the adhesion of NIH-3T3 cells to fibronectin, two functionally and kinetically distinct phases of arachidonic acid release take place. An initial transient arachidonate release occurs during cell attachment to fibronectin, and is sufficient to signal the cell spreading stage after its oxidation by 5-lipoxygenase to leukotrienes. A later sustained arachidonate release occurs during and after spreading, and signals the subsequent migration stage through its oxidation to prostaglandins by newly synthesized cyclooxygenase-2. In signaling migration, constitutively expressed cyclooxygenase-1 appears to contribute ϳ25% of prostaglandins synthesized compared with the inducible cyclooxygenase-2. Both the second sustained arachidonate release, and cyclooxygenase-2 protein induction and synthesis, appear to be regulated by the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK)1/2. The initial cell attachment-induced transient arachidonic acid release that signals spreading through lipoxygenase oxidation is not sensitive to ERK1/2 inhibition by PD98059, whereas PD98059 produces both a reduction in the larger second arachidonate release and a blockade of induced cyclooxygenase-2 protein expression with concomitant reduction of prostaglandin synthesis. The second arachidonate release, and cyclooxygenase-2 expression and activity, both appear to be required for cell migration but not for the preceding stages of attachment and spreading. These data suggest a bifurcation in the arachidonic acid adhesion-signaling pathway, wherein lipoxygenase oxidation generates leukotriene metabolites regulating the spreading stage of cell adhesion, whereas ERK 1/2-induced cyclooxygenase synthesis results in oxidation of a later release, generating prostaglandin metabolites regulating the later migration stage. INTRODUCTIONCell adhesion to the extracellular matrix (ECM) is a sequential process of discrete temporal stages. Detached cells, e.g., leukocytes, metastasized cancer cells, or suspension-cultured cells, initially attach to an ECM protein substrate by means of plasma membrane receptors. Attached cells then undergo spreading, which results in flattening and the formation of focal adhesions. Subsequently, some cell types undergo migration on the ECM, whereas others remain stationary. Each of these stages of adhesion, i.e., attachment, spreading, migration, and immobilization, involves changes in morphology and cytoskeletal structure that are regulated by incompletely defined protein kinase and lipid second messenger pathways (reviewed in Huttenlocher et al., 1995;Heidemann an...

5-Lipoxygenase and cyclooxygenase regulate wound closure in NIH/3T3 fibroblast monolayers

“…We (32) and others (23) also showed that when cells are stimulated by either mechanical wounding and/or adhesion to the ECM, AA production is also stimulated. We can therefore infer that in this case, wounding, and more specifically, wounded marginal cells, are responsible for the LTB 4 and PGE 2 production described in these assays, because little activity was observed in the absence of wounding (Fig.…”

“…In previous studies, we (32) showed that the production of LTB 4 and PGE 2 in these cells during the course of cell spreading and migration is dependent on the production of AA via cytosolic PLA 2 activation. Our preliminary work using pan and specific inhibitors of PLA 2 also suggested that calciumdependent secretory PLA 2 may be required for wound healing (unpublished observations).…”

“…In the case of cell-ECM adhesion, sequential spreading and migration are initiated by the cell attachment stage (32), whereas in wound closure, the spreading and migration stages are initiated by wounding of the cell monolayer. Each stage of cell adhesion involves changes in overall morphology and cytoskeletal structure that are regulated inter alia by the oxidative enzymes of the AA cascade: LOX and COX.…”

Cytosolic Phospholipase A2 Alpha is Required for Human Smooth Muscle Cell Proliferation and Not Migration to Platelet Derived Growth Factor BB

“…It was reported that arachidonic acid and its eicosanoid metabolites play a role in cell migration [28][29][30][31]. This study investigates the role of cPLA2 and iPLA2 through pharmacologic inhibition in migration of HASMC to PDGF-BB in vitro and further the investigation of targeted suppression of cPLA2α in multiple migration assays to PDGF-BB.…”

Interaction of brain fatty acid-binding protein with the polyunsaturated fatty acid environment as a potential determinant of poor prognosis in malignant glioma

“…In NIH3T3 mouse fibroblasts, the initial transient burst in AA resulting from cPLA 2 activation is responsible for cell adhesion, with the AA metabolites leukotrienes and PGs regulating cell spreading and cell migration, respectively [93][94][95]. Other reports have shown that cPLA 2 α and its Ser-505-phosphorylated form are rapidly and specifically recruited at sites of actin polymerization such as membrane ruffles and leading edges [92].…”