Interleukin 4 receptor signaling in human monocytes and U937 cells involves the activation of a phosphatidylcholine-specific phospholipase C: a comparison with chemotactic peptide, FMLP, phospholipase D, and sphingomyelinase.

Abstract

Interleukin 4 (IL-4) diminishes cytokine activation of human macrophage. IL-4 binding to monocyte IL-4R is associated with protein kinase C (PKC) translocation to a nuclear fraction. The cleavage of diacyglycerol (DAG), an activator of PKC, from membrane phospholipids was investigated to define the proximal events of IL-4R signaling. IL-4 induced a statistically significant time-and dose-dependent generation of DAG. The IL-4-triggered production of DAG was not derived from phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis, since neither cytosolic calcium flux nor liberation of inositol phosphates was detected in response to IL-4. Experiments were performed using [14C- methyl]choline-labeled U937 cells and monocytes to determine whether IL- 4R activated phospholipase C (PLC), PLD, or PLA2 to use membrane phosphatidylcholine (PC) to form DAG. IL-4 induced a time- and dose- dependent increase of phosphocholine (pchol) with concomitant degradation of membrane PC (p < 0.05 compared with control). The finding that the peak reduction of PC was equivalent to peak production of pchol suggested that IL-4R signaling involved the activation of a PC- specific PLC. Changes in choline (chol) or lyso-PC and glycerolphosphocholine, the respective products of PC cleavage by PLD or PLA2, were not detected in IL-4-treated cells. In contrast, exogenous PLD induced an increase in chol and concomitant loss of membrane PC. Additional investigation suggested that IL-4R signaling does not involve PLD. In cells labeled with L-lyso-3-PC 1-[1- 14C]palmitoyl, PLD but not IL-4, increased the production of phosphatidic acid (PA) and phosphatidyl-ethanol when pretreated with ethanol. Propranolol, an inhibitor of phosphatidate phosphohydrolase, and calyculin A, a phosphatase 1 and 2A inhibitor, blocked DAG production in response to FMLP but not to IL-4. In propranolol pretreated cells, PMA but not IL-4 triggered the production of PA and lowered the amount of DAG. Evidence that PLA2 is not coupled to IL-4R is the detection of arachidonate production in response to FMLP but not to IL-4. Furthermore, IL-4R is not coupled to sphingomyelinase (SMase) since IL-4, unlike exogenous SMase, did not generate ceramide but induced the hydrolysis of PC to pchol that was comparable to exogenous PLC. In summary, IL-4R signaling in monocytes and U937 cells involves PLC and not PLD, PLA2, or SMase, and it uses PC and not PIP2 to form DAG.

Phosphatidylcholine-Specific Phospholipase C Activity Is Necessary for the Activation of STAT6

“…Therefore, these data strongly suggest that the activation of PC-PLC, but not other phospholipases, is involved in IL-4-induced STAT6 activation. Our data are also in agreement with previous studies showing the ability of IL-4 to induce PC-PLC, but not PI-PLC, PLD, or phospholipase A activation (29).…”

“…D609 is a xanthogenate compound that has been shown to inhibit a PC-PLC (28,35). IL-4 has been reported to promote the activation of a PC-PLC, although its role in IL-4 signaling has not been defined (29). Because the concentration of D609 required to inhibit STAT6 activation corresponded with those reported to block PC-PLC (28,35), our data suggested that PC-PLC is involved in the activation of STAT6 by IL-4.…”

“…IL-4 has previously been shown to exclusively activate a PC-specific phospholipase (29). However, the role for this lipase in IL-4 signaling has not been defined.…”

“…2, A and B). Similarly, propranolol, which inhibits phosphatidic acid phosphohydrolase blocking 1,2-diacylglycerol (DAG) formation through PLD (29,39), had little effect on the activation of STAT6 by IL-4 (Fig. 2C).…”

“…PC-PLC has been shown to be necessary for the activation of NF-B induced by several agents including TNF-␣ (28). It was previously shown that IL-4 can induce PC-PLC activation (29); however, the role of this lipase in IL-4 signaling has not yet been defined. In this study, we have found that the activation of PC-PLC by IL-4 is required not only to signal STAT6 activation but also to regulate the activation of JAK1 and JAK3.…”