Protein Kinase Cα Is a Major Mediator of the Stimulatory Effect of Phorbol Ester on Phospholipase D-mediated Hydrolysis of Phosphatidylethanolamine

Abstract

Stimulation of phospholipase D (PLD)-mediated hydrolysis of phosphatidylcholine (PtdCho) by phorbol 12-myristate 13-acetate (PMA) has been shown to be mediated by the ␣-and ␤I-isoforms of protein kinase C (PKC). To determine the role of various PKC isozymes in the regulation of PLD-mediated phosphatidylethanolamine (PtdEtn) hydrolysis, MCF-7 human breast carcinoma cells overexpressing the ␣-and -isoforms, and R6 rat fibroblasts overexpressing the ␣-, ␤I-, and ⑀-isoforms were used. In the vector control MCF-7 cells, which contain low levels of PKC-␣, PMA (100 nM) had only small effects on the hydrolysis of PtdEtn (1.1-1.35-fold) and PtdCho (1.15-1.6-fold). Stable expression of PKC-␣ in MCF-7 cells, which was accompanied by increased levels of the ␤I-and -isoforms as well, greatly enhanced both PMA-induced PLD-mediated formation of phosphatidylethanol (ϳ5-fold) and the hydrolysis of PtdEtn (2.5-2.9-fold) and PtdCho (5.5-7.2-fold). The effects of PMA on the hydrolysis of PtdEtn (and PtdCho) in MCF-7/PKC-␣ cells were significantly inhibited by 0.5-3 M concentrations of Gö 6976, a selective inhibitor of the conventional PKC subfamily. Stable expression of PKC-␣ in R6 fibroblasts enhanced, at a shorter (10 min) incubation time, the effects of PMA on the hydrolysis of both PtdEtn and, to a lesser extent, PtdCho. In contrast, stable expression of PKC-␤I in R6 fibroblasts, which originally did not contain this enzyme, enhanced the effects of PMA only on PtdCho, but not PtdEtn, hydrolysis. Overexpression of either PKC-in MCF-7 cells or PKC-⑀ in R6 and NIH 3T3 fibroblasts had no detectable effects on PMA-induced hydrolysis of PtdEtn. Collectively, the results suggest that PKC-␣ has a major role in the mediation of phorbol ester action on PtdEtn hydrolysis, while PtdCho hydrolysis may be regulated by both the ␣ and ␤I isoforms.In many cell lines activators of phospholipase D (PLD), 1 including the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA), appear to stimulate only the hydrolysis of phosphatidylcholine (PtdCho) (1, 2). In MadinDarby canine kidney cells (3) and in Swiss/3T3 cells (4) PKC-␣ was shown to be a major regulator of PtdCho hydrolysis. However, expression of PKC-␤I (5) or addition of PKC-␤I to isolated membranes (6, 7) also enhanced the effect of PMA on PtdCho hydrolysis. Interestingly, while PKC-␣ was a more effective mediator of PMA effect in lung fibroblast membranes than PKC-␤I (6), a reversed order of potency was observed in neutrophil membranes (7). Collectively, these data suggest that if expressed, both PKC-␣ and PKC-␤I may be able to regulate PtdCho hydrolysis.The role of increased PtdCho hydrolysis in the mediation of cellular actions of PKC activators is unknown. Recently, we presented evidence showing that in NIH 3T3 fibroblasts PtdCho hydrolysis is unlikely to play a major role in the mediation of mitogenic effects of PMA (8). In fact, choline phosphate, which in agonist-treated cells can be formed by the sequential actions of PLD and choline kinase, and PMA were found to sti...

Calcium and Protein Kinase C (PKC)-Related Kinase Mediate α1A-Adrenergic Receptor-Stimulated Activation of Phospholipase D in Rat-1 Cells, Independent of PKC

“…Rat-1 cells express only PKC isoforms ␣, ␦, ⑀, , and but not ␤1 and ␤2, ␥, , or . PKC␣ and ␤1/2 are the only isoforms that have been reported to activate PLD in vitro (Mukherjee et al, 1996;Ohguchi et al, 1996). Therefore, the absence of effect of Gö 6976,, and PKC␣ AS on PLD activity, the lack of effect of PKC␣ depletion on PKC␤1/2 and PKC␦ or PKC⑀ depletion on PKC␣ level, and the inability of PHE to activate classical PKCs rules out PKC␣ involvement in PLD activation.…”

“…Although only classical PKC subtypes have been implicated in PLD activation in vitro (Ohguchi et al, 1996) or in cells overexpressing classical PKC (Mukherjee et al, 1996), there are reports indicating receptor-mediated PLD activation that is independent of PKC (Yang et al, 1998;Muthalif et al, 2000). PLD activation by classical PKCs does not involve a phosphorylation mechanism in vitro (Singer et al, 1996).…”

Cell Density Sensing Alters TGF-β Signaling in a Cell-Type-Specific Manner, Independent from Hippo Pathway Activation

“…Indeed, in the early days of TGF-β research, the use of dense, confluent cell cultures was often preferred to better understand the broad capacity of TGF-β to regulate gene expression and/or differentiation. For example, confluent skin, gingival or lung fibroblasts (Chung et al, 1996; Daniels et al, 2004; Mauviel et al, 1994; Wrana et al, 1991), keratinocytes (Kon et al, 1999; Mauviel et al, 1996), chondrocytes (Redini et al, 1991), and/or calvarial bone cells (Wrana et al, 1988), to cite a few, all respond to TGF-β with increased or decreased expression of fibrillar and non-fibrillar collagens (Chung et al, 1996; Kon et al, 1999; Mauviel et al, 1994), fibronectin (Daniels et al, 2004; Ignotz et al, 1989), SPARC (Wrana et al, 1991), laminins (Korang et al, 1995), integrins (Heino and Massagué, 1989), proteoglycans (Redini et al, 1991), metalloproteinases and protease inhibitors such as plasminogen activator inhibitor 1 (PAI-1) or tissue inhibitor of metal-loproteases (Mauviel et al, 1996; Overall et al, 1989). Likewise, early-response genes are efficiently induced by TGF-β in confluent skin fibroblasts and keratinocytes (Mauviel et al, 1993, 1996).…”

MAPK/AP-1 activation mediates TLR2 agonist-induced SPLUNC1 expression in human lung epithelial cells

“…By searching TRANFAC database (http://www.cbrc.jp/research/db/TFSEARCH.html) and other publications (Chung et al, 1996; Kyo et al, 1997; Lee et al, 1987), several putative AP-1/c-Jun binding motifs (Fig 4A) were identified in the 5′ flanking region (−5kb) from the SPLUNC1 transcription start site. To further investigate the role of c-Jun in Pam 3 CSK 4 -mediated SPLUNC1 gene regulation, we determined c-Jun binding to the 5′ flanking DNA region (up to −2.5kb) of SPLUNC1 gene including the sequence of our SPLUNC1 promoter reporter construct (−943/+47 base).…”