Comparison of PDGF-AA- and PDGF-BB-induced phosphoinositide formation in human and mouse fibroblasts.

In certain cells, such as human fibroblasts (AG 1523), there is a clear difference in the cell motility response induced by the different isoforms of platelet-derived growth factor (PDGF). PDGF-BB induces extensive actin reorganization and is a potent chemotactic agent, whereas PDGF-AA has a limited effect on actin reorganization and is not chemotactic. In the present study, we wanted to compare these effects on cell motility with the effects of the PDGF isoforms on phosphoinositide (PtdIns) turnover. We find that stimulation of serum-starved AG 1523 cells with PDGF-AA or PDGF-BB caused an initial increase of the phosphatidylinositol phosphate and bisphosphate (PtdInsP and PtdInsP2) pools, suggesting that activation of the phosphoinositide kinases is an initial response to PDGF stimulation. Despite a lower number of PDGF alpha-receptors than beta-receptors on these cells, the initial formation of PtdInsP and PtdInsP2 appears to be stimulated to a similar degree by the two PDGF isoforms. In contrast, PtdInsP2 hydrolysis, indirectly measured as formation of phosphatidic acid, was correlated to the number of receptors. During prolonged exposure to PDGF-BB the stimulated PtdIns turnover remained at a high level, whereas the effect of PDGF-AA appeared more transient. A marked increase in the synthesis of a component migrating as phosphatidylinositol trisphosphate (PtdInsP3) was also detected after stimulation with PDGF-BB for 5 min. With PDGF-AA minor amounts were found, indicating that activation of the PtdIns 3'-kinase occurs also via the PDGF alpha-receptor. Stimulation with PDGF-BB, but not -AA, also induced a 50% decrease in lyso-PtdIns. In murine fibroblasts (Swiss 3T3), where the two PDGF isoforms have a similar effect on cell motility, the two PDGF isoforms also similarly induced PtdIns turnover, PtdInsP3 formation, and a decrease in lyso-PtdIns. Thus, there seems to be a correlation between PDGF-induced PtdIns turnover and PDGF-induced actin reorganization. This is compatible with previous evidence suggesting the microfilament formation is directly linked to an increased turnover of polyphosphoinositides in stimulated cells.

Production, isolation and characterization of human profilin from Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae was used to express human profilin cDNA. The recombinant protein, isolated by affinity chromatography on poly(L-proline)-Sepharose followed by ion exchange chromatography, associates with non-muscle actin and phosphatidylinositol-(4,5)-bisphosphate as authentic profilin.

Polyphosphoinositide synthesis in platelets stimulated with low concentrations of thrombin is enhanced before the activation of phospholipase C.

When platelets, prelabelled with [32P]orthophosphate, were stimulated with thrombin (0.5 U.ml-1) there was an immediate increase in the radioactivity associated with the pools of polyphosphoinositides. Only subsequent to this increase, did the radioactivity of these phospholipid pools decrease as expected from a receptor-mediated activation of phospholipase C (phosphoinositidase). Phosphorylation of diacylglycerol (one of the second messengers formed in the hydrolysis of phosphatidylinositol-bisphosphate) to phosphatidic acid took place with a lag phase of about 3-5 s. Together these experiments suggest that stimulation of kinases phosphorylating phosphatidylinositol and phosphatidylinositol-phosphate may precede or occur in parallel with activation of receptor-linked phosphoinositidase.

Specificity of the interaction between phosphatidylinositol 4,5-bisphosphate and the profilin:actin complex.

Profilactin, the profilin:actin complex, which is present in large amounts in extracts of many types of eukaryotic cells, appears to serve as the precursor of microfilaments. It was reported recently that profilactin interacts specifically with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) (Lassing and Lindberg: Nature 314:472-474, 1985.) The present paper describes in detail the behaviour of profilactin and profilin in the presence of different types of phospholipids and neutral lipids under different conditions. PtdIns(4,5)P2 is the only phospholipid found so far which in the presence of 80 mM KCl and at Ca2+ concentrations below 10(-5) M effectively dissociates profilactin with the resulting polymerization of the actin. Phosphatidylinositol 4-monophosphate exhibits some activity but phosphatidylinositol is inactive. Both calf spleen profilin and profilin from human platelets form stable complexes with PtdIns(4,5)P2 micelles. PtdIns(4,5)P2 is active also when incorporated together with other phospholipids in mixed vesicles.

Protein kinase C-dependent phosphorylation of profilin is specifically stimulated by phosphatidylinositol bisphosphate (PIP2).

Calf spleen profilin is shown to be an in vitro substrate of purified human placental protein kinase C (PKC), with an apparent Km of 4 microM. Phosphatidylinositol bisphosphate (PIP2) was an effective activator of the profilin phosphorylation by PKC and caused a maximum 13-fold increase of Vmax with a half maximal effect at 40 micrograms/ml. The action of PIP2 was not mimicked by phosphatidylserine, phosphatidic acid or phosphatidylinositol, whereas phosphatidylinositol monophosphate was slightly stimulatory. By contrast, protein kinase C-dependent phosphorylation of histone type III-S, myelin basic protein or lipocortin-I was not affected by PIP. It is suggested that PIP2 modifies the nature of the profilin-PKC interactions.

Evidence that the phosphatidylinositol cycle is linked to cell motility.

Transmembrane signaling via specific ligand/receptor interactions induces the immediate polymerization of actin and formation of microfilament assemblies close to the plasma membrane. The profilin:actin complex appears to provide the actin for this filament formation. A clue to the nature of the regulatory mechanism involved was recently found in that phosphatidylinositol 4,5-bisphosphate can bind to profilin, dissociate the profilactin complex, and thus liberate actin for polymerization. This suggests that the phosphatidylinositol (PI) cycle, which plays important roles in cellular regulation, also might control microfilament-based motility. We show here that neomycin, a drug which has a high affinity for phosphoinositides and in vivo interferes with the PI cycle, inhibits the polymerization of actin in platelets induced either by thrombin or by ADP. When ADP was used as agonist (but not in the case of thrombin) the induction of actin polymerization could also be blocked by the addition of aspirin. Introduction of Ca2+ into platelets by the use of the ionophore A23187 or stimulation of protein kinase C (PkC) by the phorbol ester TPA did not induce actin polymerization; neither did the addition of a combination of these two agents. Retinoic acid which inhibits PkC was also without effect on thrombin-induced actin polymerization.

Specific interaction between phosphatidylinositol 4,5-bisphosphate and profilactin.

There is evidence that the polymerization of actin takes place at the plasma membrane, and that profilactin (profilin/actin complex), the unpolymerized form of actin found in extracts of many non-muscle cells, serves as the immediate precursor. Both isolated profilin and profilactin interact with detergent when analysed by charge shift electrophoresis, indicating that they have amphipathic properties and may be able to interact directly with the plasma membrane. We demonstrate here that isolated profilin, as well as the profilactin complex, interacts with anionic phospholipids. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) was found to be the most active phospholipid, causing a rapid and efficient dissociation of profilactin with a concomitant polymerization of the actin in appropriate conditions. These and other observations suggest the possibility of a relationship between the induction of actin filament formation and the increased activity in the phosphatidylinositol cycle seen as a result of ligand-receptor interactions in various systems.

The organization of microfilaments in spreading platelets: a comparison with fibroblasts and glial cells.

Platelets respond to stimulatory agents in general by the formation of long spikelike surface projections built up of tightly bundled microfilaments. During contact stimulation this is followed by a second phase when thin membrane lamellae grow out between the projections. This behaviour resembles that seen for instance in fibroblasts and glial cells, sending out microspikes and lamellipodia as a step in their advancement over solid substrata. Conditions, designed earlier for the preservation and visualization of the fragile organization of microfilaments and microtubules in the peripheral, highly motile parts (leading lamellae) of such cells (Höglund et al. (1980) J. Musc. Res. Cell Motility, 1:127-146), were used here to produce high-resolution images of the ultrastructural organization of platelets spreading on a solid substratum. This revealed an unexpected arrangement of actin filaments running parallel to the advancing edge, and small tufts of microfilaments on the outside of this edge-bundle. Cytochalasin D caused a regression of the spikelike projections as well as of both types of structures in the advancing platelet lamella and led to the appearance of a dense filamentous mat in juxtaposition to the plasma membrane. Analysis of the actin pools using the DNAase inhibition assay showed that the dramatic reorganizations of actin seen during the two phases of contact stimulation was reflected in a shift in the G/F-actin ratio only during the early phase.