Signal transduction during HeLa cell adhesion to a collagen substratum: Role of second messenger formation

HeLa cells, a transformed human epithelial cell line, attach to a variety of substrata but spread only on collagen or gelatin. Spreading is dependent on collagen receptor upregulation, clustering, and binding to the cytoskeleton. The purpose of this research is to examine whether collagen receptor interaction with gelatin induces formation of a second messenger(s) which turns on cell functions leading to spreading. Levels of arachidonic acid (AA) are increased upon attachment and prior to spreading of HeLa cells on a gelatin substratum. Inhibition of phospholipase A2 (PLA2) blocks AA release and cell spreading. Among the inhibitors of AA metabolic pathways, only inhibitors of lipoxygenase (LOX) block cell spreading indicating that a LOX metabolite(s) of AA is a second messenger(s) which initiates HeLa cell spreading. AA appears to be released from phosphatidylcholine (PC) since levels of PC which contain AA decrease during cell spreading. Also, lysophosphatidylcholine is the only lysophospholipid which is detected during cell spreading. AA is released upon clustering of collagen receptors. Receptor occupancy is not sufficient to release AA. The formation of a LOX metabolite(s) appears to induce production of diacylglycerol (DG) which is correlated with activation of protein kinase C (PKC). PKC is activated upon attachment and prior to spreading. Inhibition of PKC blocks cell spreading, and the inhibition is not reversed by addition of AA. However, inhibition of cell spreading by inhibitors of PLA2 or LOX is overcome by PKC activation. This indicates that PLA2 activation occurs prior to PKC activation, and that direct activation of PKC bypasses the requirement for AA release and LOX metabolite formation. Activation of PKC with phorbol ester also enhances cell spreading. The ability of PKC to induce cell spreading appears to be mediated by the modulation of F-actin formation. Cell spreading is accompanied by an increase in F-actin content. Inhibition of PKC blocks the relative increase in F-actin content. By contrast, treating cells with PKC activator prior to spreading assays not only enhances cell spreading but also increases the relative F-actin content. Cell spreading is an amplification process centered on the production of AA. AA production is amplified by hydrolysis of DG and activation of PKC