Differential signaling during macropinocytosis in response to M-CSF and PMA in macrophages

Abstract

The cellular movements that construct a macropinosome have a corresponding sequence of chemical transitions in the cup-shaped region of plasma membrane that becomes the macropinosome. To determine the relative positions of type I phosphatidylinositol 3-kinase (PI3K) and phospholipase C (PLC) in this pathway, we analyzed macropinocytosis in macrophages stimulated by the growth factor macrophage-colony-stimulating factor (M-CSF) and by the diacylglycerol (DAG) analog phorbol 12-myristate 13-acetate (PMA). In cells stimulated with M-CSF, microscopic imaging of fluorescent probes for intracellular lipids indicated that the PI3K product phosphatidylinositol (3,4,5)-trisphosphate (PIP3) appeared in cups just prior to DAG. We then tested the hypothesis that PMA and DAG function after PI3K and prior to Ras and protein kinase C (PKC) during macropinosome formation in macrophages. Although the PI3K target Akt was activated by M-CSF, the Akt inhibitor MK-2206 did not inhibit macropinocytosis. The phospholipase C (PLC) inhibitor U73122 blocked macropinocytosis by M-CSF but not PMA. Macropinocytosis in response to M-CSF and PMA was inhibited by the Ras inhibitor farnesyl thiosalicylate (FTS), by the PKC inhibitor Calphostin C and by the broad specificity inhibitor rottlerin. These studies support a model in which M-CSF stimulates PI3K in macropinocytic cups, and the resulting increase in PIP3 activates PLC, which in turn generates DAG necessary for activation of PKC, Ras and the late stages of macropinosome closure.

Drinking problems: mechanisms of macropinosome formation and maturation

“…However, the authors also found that the subsequent deactivation of Rac1 is equally important as it allows ruffle collapse and closure, which are essential for successful fluid capture. This is supported by quantitative microscopy studies that demonstrate that Rac1 is physiologically deactivated just prior to closure .…”

Regulation mechanism of PDK1 on macrophage metabolism and function

“…However, M‐CSF induced macropinocytosis in macrophages are not inhibited by MK‐2206, one of Akt inhibitor. PI3K triggered PLCγ activates DAG, PKC, Ras, and finally closes the macropinosome . Other studies suggest that PDK1 activates PLCγ1 on residue Tyr783 in EGF induced MDA‐MB‐231 cells.…”

The cell surface environment for pathogen recognition and entry

“…Phosphoinositide transitions initiated in ruffles carry through to the macropinosomes. The transition of PI(4,5)P2 to PI(3,4,5)P3 to PI(3,4)P2 and then to PI(3)P is conserved during the processes of macropinosome closure and maturation from C.elegans to mammalian cells 45 , 47 , 48 . PI3kinases are crucially involved in macropinocytosis and the broad spectrum PI3Kinase inhibitors, LY294002 and wortmannin blocks macropinosome formation, and specifically macropinosome closure, without necessarily affecting the preceding ruffling 42 , 49 , 50 .…”

“…PI3kinases are crucially involved in macropinocytosis and the broad spectrum PI3Kinase inhibitors, LY294002 and wortmannin blocks macropinosome formation, and specifically macropinosome closure, without necessarily affecting the preceding ruffling 42 , 49 , 50 . The transient appearance of PI(3,4,5)P3 is accompanied by Rac1 activation and diacylglycerol production, and this is followed by the appearance of PI(3,4)P2 during macropinosome closure 48 . Lipid probes variably show accumulation of PI(3,4,5)P3 on the macropinosome membranes before or just after closure.…”

“…Lipid probes variably show accumulation of PI(3,4,5)P3 on the macropinosome membranes before or just after closure. PI(3,4,5)P3 can then activate phospholipase C and diacylglycerol, as well as protein kinase C and Ras during macropinosome maturation 48 . Accordingly, inhibitors for these enzymes variably block macropinocytosis (Table 1).…”