Overexpression of protein kinase C beta 1 enhances phospholipase D activity and diacylglycerol formation in phorbol ester-stimulated rat fibroblasts.

Pai, Jin-Keon; Pachter, Jonathan A.; Weinstein, I. Bernard; Bishop, W. Robert

doi:10.1073/pnas.88.2.598

Cited by 70 publications

(28 citation statements)

References 40 publications

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“…First, PKC may directly regulate PLD activity by phosphorylation, as suggested by enhanced phorbol ester stimulation of PLD in R6-PKC3 fibroblasts (Pai et al, 1991). In support of this, MacNulty et aL (1990) found that ETstimulated choline release was abolished on PKC down-regulation.…”

Section: Discussionmentioning

confidence: 55%

“…As we proposed previously (Pai et al, 1991), enhanced PLD activity can generate an enhanced DAG signal by the PLD-PA phosphohydrolase pathway, and this DAG may lead to further PKC activation, thus perpetuating the signal through a feed-forward loop of cell stimulation (Pai et al, 1991). A role for sustained DAG formation in the mitogenic response of fibroblasts has previously been suggested (Muldoon et aL, 1990;Wright et aL, 1990 (Martin et al, 1990;Leach et al, 1991) argue against such a model.…”

Section: Discussionmentioning

confidence: 85%

“…For example, the in vivo pressor effects of ET are long-lasting, and ET causes a slowly developing and sustained contraction of vascular smooth muscle strips . Phorbol esters also induce sustained activation of PLD in various cell types (Billah and Anthes, 1990;Pai et al, 1991) and, similar to ET, they induce sustained contraction of vascular smooth muscle (Rasmussen et al, 1984). It is possible that the mitogenic response elicited by ET and phorbol esters in R6-PKC3 cells and the persistent nature of vascular smooth muscle contraction induced by these agents can be attributed to the prolonged activation of PLD.…”

Section: Discussionmentioning

confidence: 99%

“…These observations along with our results emphasize the potential importance of PLD activation in the mitogenic response. Maintenance of cultures R6-C1 and R6-PKC3 were grown to confluency on 1 00-mm dishes in Dulbecco's modified Eagle's medium (DMEM) containing 100% fetal calf serum and G418 (50 ,g/ml) as described previously Pai et al, 1991). They were then serum-deprived for 24 h in DMEM containing 0.5% fetal bovine serum.…”

Section: Discussionmentioning

confidence: 99%

“…We recently reported that overexpression of PKC,1 in Rat-6 fibroblasts enhances PLD activation in response to phorbol-1 2-myristate-1 3-acetate (PMA) (Pai et al, 1991). These PKC overexpressing cells exhibit multiple growth abnormalities , display an enhanced mitogenic response to growth factors (Hoshina etal., 1990), and are more susceptible to transformation by activated Ha-ras oncogenes .…”

Section: Introductionmentioning

confidence: 99%

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Endothelin-1 activates phospholipase D and thymidine incorporation in fibroblasts overexpressing protein kinase C beta 1.

Pai¹,

Dobek²,

Bishop³

1991

Cell Regul

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[H]DAG from PC by the sequential action of PLD and PA phosphohydrolase. Phorbol esters are known to stimulate thymidine incorporation and PLD activity to a greater extent in PKC overexpressing cells than in control cells. ET-1 also stimulates thymidine incorporation to a greater extent in the PKC overexpressing cells. The effect of ET-1 on thymidine incorporation into DNA in the overexpressing cells was also dose-dependent with a half-maximal effect at 0.3 x 10-9 M. Enhanced PLD activity induced by ET-1 in the overexpressing cells may contribute to the mitogenic response, especially in light of a possible role of the PLD product, PA, in regulation of cell growth.

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Section: Discussionmentioning

confidence: 55%

Section: Discussionmentioning

confidence: 85%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Endothelin-1 activates phospholipase D and thymidine incorporation in fibroblasts overexpressing protein kinase C beta 1.

Pai¹,

Dobek²,

Bishop³

1991

Cell Regul

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Protein kinase C β modulates thrombin-induced Ca2+ signaling and endothelial permeability increase

et al. 1998

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We investigated the function of the Ca2+-dependent protein kinase C (PKC) beta1 in the regulation of endothelial barrier property. Human dermal microvascular endothelial cells (HMEC-1) were transduced with full-length PKCbeta1 antisense (AS) cDNA or control pLNCX vector to generate stable cell lines (HMEC-AS and HMEC-pLNCX, respectively). Analyses indicated that HMEC-AS expressed the antisense PKCbeta1 transcript with decreased PKCbeta protein level (without a change in PKCalpha or PKCepsilon). The baseline transendothelial 125I-albumin clearance rates of HMEC-1, HMEC-pLNCX, and HMEC-AS were 5.0+/-0.5 x 10(-2), 6.8+/-0.4 x 10(-2), and 6.9+/-0.6 x 10(-2) microl/min, respectively. Activation of HMEC-1 and HMEC-pLNCX with phorbol 12-myristate 13-acetate (PMA) increased the rates to the respective 14.5+/-1.7 x 10(-2) microl/min and 16.9+/-2.8 x 10(-2) microl/min (corresponding to 191% and 149% increases over baseline). However, in HMEC-AS, PMA increased the rate to 9.8+/-1.0 x 10(-2) microl/min (42%). When HMEC-1 and HMEC-pLNCX were activated with thrombin, the rates increased to 10.8+/-1.4 x 10(-2) and 14.0+/-1.9 x 10(-2) microl/min, respectively (116% and 106%). In contrast, thrombin stimulation of HMEC-AS more than doubled the increase to 27.2+/-3.5 x 10(-2) microl/min (294%). Furthermore, the thrombin-induced peak increase in the [Ca2+]i in HMEC-AS was greater than in control cells. Fluorescence-activated cell sorter analysis of thrombin receptor expression indicated that the augmented thrombin-induced responses were not attributable to altered receptor density in HMEC-AS. These results indicate that PKCbeta functions in a negative feedback manner to inactivate thrombin-generated signals and thereby modulates the endothelial permeability increase. Because decreased PKCbeta expression significantly reduced the PMA-induced permeability increase, PKCbeta may downregulate thrombin receptor function upstream of PKC activation (i.e., Ca2+).

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Role of protein kinase Cα, arf, and cytoplasmic calcium transients in phospholipase D activation by sodium fluoride in osteoblast-like cells

Bourgoin

Harbour

Poubelle

1996

Journal of Bone and Mineral Research

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The effect of fluoride on phospholipase D (PLD) activation was studied using in vitro culture of Saos-2, MG-63 osteosarcoma cells, and normal osteoblast-like cells derived from human bone explants. Millimolar concentrations of NaF induced a significant accumulation of phosphatidylethanol (PEt) in Saos-2 cells but not in MG-63 and normal osteoblast-like cells. PLD activation was evident at 15 mM and concentration-dependent up to 50 mM. This stimulation was inhibited by deferoxamine, a chelator of Al3+, suggesting that PLD activation involves fluoride-sensitive G proteins. A good correlation was found between the levels of intracellular free Ca2+ and the activation of PLD. The time courses of the two responses were nearly identical. The ability of NaF to induce both responses was largely dependent on the presence of extracellular calcium. The calcium ionophore A23187 reproduced the effect of NaF, and this effect was antagonized by EGTA, suggesting that PLD activation was, at least in part, a calcium-regulated event. Phorbol 12-myristate 13-acetate (PMA) also stimulated PLD activity in human bone cells. Protein kinase C alpha (PKC alpha) and epsilon were expressed in Saos-2 cells. Acute pretreatment of cells with PMA reduced concomitantly the amounts of PKC alpha, but not of PKC epsilon, and the subsequent activation of PLD elicited by PKC activators. The PLD response to NaF was not attenuated but rather enhanced by down-regulation of PKC alpha. Therefore, PKC-alpha-induced PLD activation is unlikely to mediate the effect of NaF. Moreover, PMA and NaF showed a supraadditive effect on PLD activation in Saos-2 cells. This stimulation, in contrast to NaF alone, was not reduced by EGTA. Hence, mobilization of calcium by NaF cannot account for the enhanced PLD activation in response to PMA stimulation. Membrane Arf and RhoA contents were assessed by Western immunoblot analyses. Membranes derived from NaF-stimulated Saos-2 cells contained more Arf and RhoA when compared with membranes derived from control or PMA-stimulated cells. Translocation of the small GTPases was calcium-independent. We conclude that PLD activation by NaF in Saos-2 cells includes a fluoride-sensitive G protein, increases in the levels of intracellular calcium, and Arf/RhoA redistribution to membranes. The results also indicate that the NaF-induced Arf/RhoA translocation exerts in concert with PMA-activated PKC alpha a synergistic effect on the activation of PLD in Saos-2 cells.

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Overexpression of protein kinase C beta 1 enhances phospholipase D activity and diacylglycerol formation in phorbol ester-stimulated rat fibroblasts.

Abstract: We are using a Rat-6 fibroblast cell line that stably overexpresses the P1 isozyme of protein kinase C (PKC)to study regulation of phospholipid hydrolysis by PKC.

Cited by 70 publications

References 40 publications

Endothelin-1 activates phospholipase D and thymidine incorporation in fibroblasts overexpressing protein kinase C beta 1.

Endothelin-1 activates phospholipase D and thymidine incorporation in fibroblasts overexpressing protein kinase C beta 1.

Protein kinase C β modulates thrombin-induced Ca2+ signaling and endothelial permeability increase

Role of protein kinase Cα, arf, and cytoplasmic calcium transients in phospholipase D activation by sodium fluoride in osteoblast-like cells

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