Protein kinase G activation of K(ATP) channels in human-cultured prostatic stromal cells

In this study, we identify and investigate the role of protein kinase G (PKG) in cells cultured from human prostatic stroma. Cells were used for immunocytochemistry, contractility or K(+) fluorescent imaging studies. All cultured prostatic stromal cells showed PKG immunostaining. Phorbol 12,13 diacetate (PDA, 1 microM) elicited contractions from human-cultured prostatic stromal cells that could be blocked by both the L-type Ca(2+) channel blocker, nifedipine (3 microM), and the protein kinase C inhibitor, bisindolylmaleimide (1 microM). The nitric oxide donor, sodium nitroprusside (SNP, molar pIC(50) 5.16+/-0.17) and the cGMP-phosphodiesterase inhibitor, zaprinast (50 microM), inhibited PDA (1 microM)-induced contractions. The PKG activator beta-phenyl-1, N(2)-ethenoguanosine-3',5'-cyclic monophosphate (PET-cGMP, molar pIC(50) 6.96 +/- 0.25) also inhibited PDA (1 microM)-induced contractions. Glibenclamide (10 microM) and Rp-8-Br-cGMPS (5 microM), but not iberiotoxin (100 nM) or Rp-cAMP (5 microM), reversed this inhibition. In human-cultured prostatic stromal cells loaded with the K(+) fluorescent indicator, 1,3-Benzenedicarboxylic acid, 4,4'-[1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diylbis(5-methoxy-6,2-benzofurandiyl)]bis-, tetrakis [(acetyloxy) methyl] ester (PBFI), PET-cGMP (300 nM) caused a reduction in intracellular K(+) that was blocked by glibenclamide (10 microM) and Rp-8-Br-cGMPS (5 microM), but not by iberiotoxin (100 nM). These data are consistent with the hypothesis that, in human-cultured prostatic stromal cells, PKG inhibits contractility through the activation of K(ATP) channels.     

Protein kinase G II-mediated proliferative effects in human cultured prostatic stromal cells

This study investigates the effect of protein kinase G (PKG) activation upon proliferation of human cultured prostatic stromal cells. The PKG II activator (8-pCPT-cGMP; IC50 of 113+/-42 nM) and the phosphodiesterase inhibitor, zaprinast (up to 50 microM), but not the PKG I isoform activators (APT-cGMP and PET-cGMP), reduced foetal calf serum-stimulated proliferation. The effect of 8-pCPT-cGMP (30 microM) was blocked by Rp-8-Br-cGMPS (5 microM) and Rp-8-pCPT-cGMP (5 microM), but not Rp-cAMPS (5 microM). 8-pCPT-cGMP (30 microM) and zaprinast (50 microM), but not PET-cGMP (30 microM), caused a significant increase in atypical nuclei and an increase in annexin-V staining. These data indicate that activation of PKG II induces apoptosis of human cultured prostatic stromal cells.     

Inhibition of DNA synthesis by protein kinase C-activating phorbol esters in NIH/3T3 cells

Fibroblast growth factor (FGF) plus insulin induced DNA synthesis in and proliferation of NIH/3T3 cells. The protein kinase C-activating phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), inhibited both the DNA synthesis and cell proliferation induced by FGF plus insulin. The concentration of TPA required for 50% inhibition of the DNA synthesis was about 5 nM. Phorbol-12,13-dibutyrate, another protein kinase C-activating phorbol ester, also inhibited the DNA synthesis but 4 alpha-phorbol-12,13-didecanoate, known to be inactive for this enzyme, was ineffective. DNA synthesis started at about 12 h after the addition of FGF plus insulin. The inhibitory action of TPA on the DNA synthesis was observed when it was added within 12 h after the addition of FGF plus insulin. These results suggest that phorbol esters exhibit an antiproliferative action through protein kinase C activation in NIH/3T3 cells, and that this action of phorbol esters is due to inhibition of the progression from the late G1 to the S phase of the cell cycle.     

Propranolol stimulates histone phosphorylation by a putative PK-C in partially purified homogenate of rat testicular interstitial cells. A possible mechanism for increased testosterone secretion by propranolol.

The beta-adrenoceptor blocker propranolol stimulated testosterone secretion by rat testicular interstitial cells (Leydig cell-enriched preparation) in vitro at concentrations ranging from 10(-5) M to 10(-4) M. Treatment of these cells with H7 (20 microM), an inhibitor of protein kinase C, reduced the stimulatory effect of L-propranolol on testosterone secretion by about 5-fold. At concentrations ranging from 31.25 microM to 1000 microM, L-propranolol reduced [3H]phorbol 12,13-dibutyrate binding (IC50 = 75 microM) to rat testicular interstitial cells. At similar concentrations, L-propranolol displaced the binding of [3H]phorbol 12,13-dibutyrate to the homogenate of these cells by only 5%. These findings suggest that the effect of L-propranolol on [3H]phorbol 12,13-dibutyrate binding could be indirect, possibly by increasing the concentration of a chemical mediator interacting with the regulatory domain of protein kinase C. At even lower concentrations (10(-9) M to 10(-7) M), propranolol added directly to the reaction mixture with protein kinase C partially purified from rat testicular interstitial cells increases the phosphorylation of histone. This phosphorylation was comparable to that obtained with (25 microg/ml) phosphatidylserine. The D- and L-stereoisomers of propranolol were equally active. A complete reversal of this propranolol effect on histone phosphorylation was achieved with (20 microM) H-7. In the absence of Ca2+, propranolol was not able to phosphorylate the histone. Taken together, these results suggest that protein kinase C could be the putative kinase involved in this reaction and that its activation by propranolol may be due to interaction of the drug with the regulatory domain of the enzyme at a site differing from the site of interaction with phorbol 12,13-dibutyrate. The ability of propranolol to activate the putative protein kinase C could be related to its stimulatory effect on testosterone secretion by Leydig cells.     

The lipophilicity of phorbol esters as a critical factor in determining the pattern of translocation of protein kinase C delta fused to green fluorescent protein

Our previous study showed differential subcellular localization of protein kinase C (PKC) delta by phorbol esters and related ligands, using a green fluorescent protein-tagged construct in living cells. Here we compared the abilities of a series of symmetrically substituted phorbol 12,13-diesters to translocate PKC delta. In vitro, the derivatives bound to PKC with similar potencies but differed in rate of equilibration. In vivo, the phorbol diesters with short, intermediate, and long chain fatty acids induced distinct patterns of translocation. Phorbol 12,13-dioctanoate and phorbol 12,13-nonanoate, the intermediate derivatives and most potent tumor promoters, showed patterns of translocation typical of phorbol 12-myristate 13-acetate, with plasma membrane and subsequent nuclear membrane translocation. The more hydrophilic compounds (phorbol 12,13-dibutyrate and phorbol 12,13-dihexanoate) induced a patchy distribution in the cytoplasm, more prominent nuclear membrane translocation, and little plasma membrane localization at all concentrations examined (100 nM to 10 microM). The highly lipophilic derivatives, phorbol 12,13-didecanoate and phorbol 12, 13-diundecanoate, at 1 microM caused either plasma membrane translocation only or no translocation at incubation times up to 60 min. Our results indicate that lipophilicity of phorbol esters is a critical factor contributing to differential PKC delta localization and thereby potentially to their different biological activities.     

Differential roles of PKCalpha and PKCepsilon in controlling the gene expression of Nox4 in human endothelial cells

NADPH oxidases are major sources of superoxide in the vascular wall. This study investigates the role of protein kinase C (PKC) in regulating gene expression of NADPH oxidases. Treatment of human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 endothelial cells with phorbol 12-myristate 13-acetate (PMA) or phorbol 12,13-dibutyrate led to a PKC-dependent biphasic expression of the gp91phox homolog Nox4. A downregulation of Nox4 was observed at 6 h and an upregulation at 48 h after phorbol ester treatment. The early Nox4 downregulation was associated with a reduced superoxide production, whereas the late Nox4 upregulation was accompanied by a clear enhancement of superoxide. PMA activated the PKC isoforms alpha and epsilon in HUVEC and EA.hy 926 cells. Knockdown of PKCepsilon by siRNA prevented the early downregulation of Nox4, whereas knockdown of PKCalpha selectively abolished the late Nox4 upregulation. Vascular endothelial growth factor (VEGF), which activates PKCalpha but not PKCepsilon in HUVEC, increased Nox4 expression without the initial downregulation. VEGF-induced Nox4 upregulation was associated with an enhanced proliferation and angiogenesis of HUVEC. Both effects could be reduced by inhibition of NADPH oxidase. Thus, a selective inhibition/knockdown of PKCalpha may represent a novel therapeutic strategy for vascular disease.     

Ca2+/calmodulin mediated pathways regulate the uptake of L-DOPA in mouse neuroblastoma neuro 2A cells

The present study examined the involvement of protein kinase A (PKA), protein kinase G (PKG), protein kinase C (PKC), protein tyrosine kinase (PTK) and Ca2+/calmodulin mediated pathways on the uptake of L-DOPA through the L-type amino acid transporter in Neuro 2A cells, an in vitro model of neuronal cells. Non-linear analysis of the saturation curve for L-DOPA revealed a Km value (in microM) of 54+/-2 and a Vmax value (in nmol mg protein/6 min) of 34+/-1. L-DOPA uptake was a sodium-independent process and insensitive to N-(methylamino)-isobutyric acid (MeAIB, 1 mM), but sensitive to 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC, IC50=82 microM). The Ca2+/calmodulin inhibitors calmidazolium and trifluoperazine inhibited L-DOPA (2.5 microM) uptake with IC50's of 33 and 105 microM, respectively. The inhibitory effect of BHC on the accumulation of L-DOPA was of the competitive type, whereas that of calmidazolium and trifluoperazine was of the non-competitive type. Modulators of PKA (cyclic AMP, forskolin, isobutylmethylxanthine and cholera toxin), PKG (cyclic GMP, zaprinast, LY 83583 and sodium nitroprusside), PKC (phorbol 12,13-dibutirate, phorbol 12-myristate 13-acetate and chelerythrine) and PTK (genistein and tyrphostin 25) failed to affect the accumulation of a non-saturating (2.5 microM) concentration of L-DOPA. It is concluded that L-DOPA uptake in Neuro 2A cells is promoted through the L-type amino acid transporter and appears to be under the control of Ca2+/calmodulin mediated pathways.     

Protein kinase Calpha is required for endothelin-1-induced proliferation of human myometrial cells.

The role of protein kinase C (PKC)-alpha in endothelin-1 (ET-1)-induced proliferation of human myometrial cells was investigated. Inhibition of conventional PKC with G? 6976 eliminated the proliferative effect of ET-1. Treatment of myometrial cells with an antisense oligonucleotide against PKCalpha efficiently reduced PKCalpha protein expression without effect on other PKC isoforms and resulted in the loss of ET-1-induced cell growth. Immunocytochemistry using an antibody against PKCalpha revealed that there was no PKCalpha immunoreactivity in the nuclei of quiescent nonconfluent untreated cells, whereas it is evenly distributed throughout the cytoplasm. Exposure of myometrial cells to ET-1 for 15 min caused the PKCalpha to shift towards the perinuclear area, and incubation for 60 min caused a shift towards the nucleus. These results reveal that PKCalpha is required for ET-1-induced human myometrial cell growth and suggest that targeting of PKCalpha by antisense nucleotides might be an important approach for the development of anticancer treatments.     

Tumor-promoting phorbol esters stimulate the proliferation of interleukin-3 dependent cells

To determine whether activation of protein kinase C is involved in the proliferation of interleukin-3 (IL-3) -dependent cells, we examined the effect of tumor-promoting phorbol esters on the in vitro proliferation of the IL-3-dependent cell lines FD and DA-1. The viability of FD and DA-1 cells cultured for 24 hours in 100 nM phorbol myristate acetate (PMA) and 10% FCS was similar to that of cells cultured in 25% WEHI-3 conditioned medium as a source of IL-3, and 10% FCS. FD cells failed to proliferate in concentrations of FCS of up to 50%, while DA-1 cell proliferation was not markedly influenced by FCS. By contrast, PMA promoted the proliferation of FD and DA-1 cells in the absence of FCS and enhanced their proliferation in the presence of 10% FCS, 60- and 20-fold, respectively. Stimulation of proliferation was achieved with as little as 10 nM PMA and was maximal at 100 nM PMA. Low concentrations (0.05-0.1%) of WEHI-3 CM promoted the proliferative response of FD and DA-1 cells to PMA, but at concentrations of WEHI-3 CM greater than 0.8%, no further increment in proliferation was obtained with PMA. As little as 1/2 hour of exposure to phorbol esters was sufficient to cause translocation of protein kinase C from the cytosol to the membranes of DA-1 cells, and 1 hour of exposure to phorbol esters was sufficient to stimulate DNA synthesis. A protein kinase C inhibitor, H-7, at a concentration of 10 microM inhibited phorbol ester-induced stimulation of DA-1 cell proliferation. When DA-1 cells were exposed to the calcium ionophore A23187 in addition to both a phorbol ester and IL-3, their proliferation was enhanced over that stimulated by only the phorbol ester and IL-3. The data indicate that stimulation of proliferation of IL-3-dependent cells involves the activation of protein kinase C.     

Regulation of osteoprotegerin secretion from primary cultures of human bone marrow stromal cells

Osteoprotegerin (OPG) is a soluble receptor for receptor activator of NF kappa B-ligand, a factor required for osteoclastogenesis. OPG secreted from bone marrow stromal cells is believed to inhibit osteoclast differentiation and several agents known to influence bone resorption have been demonstrated to regulate mRNA levels of OPG. In this report we have investigated the secretion of OPG protein from primary cultures of human bone marrow stromal cells. An ELISA was developed for measuring the concentration of OPG in culture medium. OPG secretion was decreased by 50% when the human bone marrow stromal cells were treated with 1 microM of prostaglandin E(2), possibly through activation of the protein kinase A-pathway since stimulation of protein kinase A by forskolin also inhibited OPG secretion. Treatment with phorbol 12,13 di butyrate, an activator of the protein kinase C-pathway, potently stimulated the secretion of OPG from human bone marrow stromal cells. The cells were also stimulated with inflammatory mediators and glucocorticoids. Treatment with interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF-alpha) stimulated OPG secretion to 500% and 400% of control whereas dexamethasone decreased OPG production by 40%. In conclusion, an ELISA measuring OPG in cell culture media was developed. Using this ELISA, the amount of OPG secreted from human bone marrow stromal cells was clearly detectable, and the secretion of OPG-protein was potently regulated by prostaglandin E(2), forskolin, phorbol 12,13 di butyrate, IL-1 alpha, TNF-alpha, and dexamethasone.     

Down regulation of phorbol diester receptors by proteolytic degradation of protein kinase C in a cultured cell line of fetal rat skin keratinocytes

Down regulation of phorbol diester receptors was studied with respect to proteolysis of protein kinase C, which is activated by Ca2+, phospholipids, and diacylglycerols and which binds to phorbol diesters. We used FRSK cells, a cell line derived from fetal rat skin keratinocytes, because in these cells specific binding of phorbol 12,13-dibutyrate decreased rapidly (50% decrease in 30 min). This decrease (down regulation) was inhibited by some protease inhibitors, such as N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), N-p-tosyl-L-lysine chloromethyl ketone (TLCK), and leupeptin, but not by inhibitors of lysosomal hydrolases. On treatment with 12-O-tetradecanoylphorbol 13-acetate, protein kinase C was rapidly translocated from the cytosol to the membranes and then decreased. This decrease in protein kinase C was also inhibited by TPCK, TLCK, and leupeptin. The decrease in membrane activity of protein kinase C was associated with increase in cytosolic activity of a protein kinase that was smaller in molecular weight (Mr 40,000-60,000) than protein kinase C, did not depend on Ca2+/phosphatidylserine/diacylglycerol, and did not bind to phorbol 12,13-dibutyrate. These results indicate that down regulation of phorbol diester receptors is probably caused by nonlysosomal proteolysis of protein kinase C. The kinase formed by cleavage may be an active catalytic site of protein kinase C.     

Protein kinase C activators suppress stimulation of capillary endothelial cell growth by angiogenic endothelial mitogens

The intracellular events regulating endothelial cell proliferation and organization into formalized capillaries are not known. We report that the protein kinase C activator beta-phorbol 12,13-dibutyrate (PDBu) suppresses bovine capillary endothelial (BCE) cell proliferation (K50 = 6 +/- 4 nM) and DNA synthesis in response to human hepatoma-derived growth factor, an angiogenic endothelial mitogen. In contrast, PDBu has no effect on the proliferation of bovine aortic endothelial cells and is mitogenic for bovine aortic smooth muscle and BALB/c 3T3 cells. Several observations indicate that the inhibition of human hepatoma-derived growth factor-stimulated BCE cell growth by PDBu is mediated through protein kinase C. Different phorbol compounds inhibit BCE cell growth according to their potencies as protein kinase C activators (12-O-tetradecanoylphorbol 13-acetate greater than PDBu much greater than beta-phorbol 12,13-diacetate much much greater than beta-phorbol; alpha-phorbol 12,13-dibutyrate; alpha-phorbol 12,13-didecanoate). PDBu binds to a single class of specific, saturable sites on the BCE cell with an apparent Kd of 8 nM, in agreement with reported affinities of PDBu for protein kinase C in other systems. Specific binding of PDBu to BCE cells is displaced by sn-1,2-dioctanoylglycerol, a protein kinase C activator and an analog of the putative second messenger activating this kinase in vivo. The weak protein kinase C activator, sn-1,2-dibutyrylglycerol, does not affect PDBu binding. A cytosolic extract from BCE cells contains a calcium/phosphatidylserine-dependent protein kinase that is activated by sn-1,2-dioctanoylglycerol and PDBu, but not by beta-phorbol. These findings indicate that protein kinase C activation can cause capillary endothelial cells to become desensitized to angiogenic endothelial mitogens. This intracellular regulatory mechanism might be invoked during certain phases of angiogenesis, for example when proliferating endothelial cells become differentiated to organize into nongrowing tubes.     

Phorbol esters stimulate phosphate accumulation synergistically with A23187 in cultured renal tubular cells

The effects of phorbol esters and diacylglycerol on phosphate accumulation in the cultured mouse kidney cells were investigated to assess the possible role of Ca2+-activated, phospholipid dependent protein kinase (protein kinase C) on the renal phosphate handling. 12-O-tetradecanoyl phorbol-13-acetate (TPA) stimulated phosphate accumulation dose-dependently. TPA-induced phosphate accumulation was synergistically enhanced with A23187. 4 alpha-phorbol 12,13-didecanoate did not stimulate the phosphate accumulation, while 4 beta-phorbol 12,13-didecanoate stimulated it. Additionally, 1-oleoyl-2-acetyl-glycerol exhibited a stimulatory effect on phosphate accumulation. These data indicated that protein kinase C is one of possible regulators of phosphate transport at the renal tubules.     

Human umbilical vein and dermal microvascular endothelial cells show heterogeneity in response to PKC activation

Changes in endothelial cell (EC) phenotype are central to thefunction of endothelium in inflammation. Although these events mainlyoccur in the microvasculature, previous studies have predominantly usedlarge-vessel EC. Using enzyme-linked immunosorbent and flow cytometricassays, we compared the responses of human umbilical vein endothelialcells (HUVEC) and dermal microvascular endothelial cells (DMEC) to theactivation of protein kinase C (PKC). Stimulation with phorbol12,13-dibutyrate and more selective PKC agonists, including12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA), inducedmorphological changes and proliferation in both EC types. PKCactivation induced a marked increase in Thy-1 expression on DMEC andonly a moderate rise on HUVEC. Furthermore, heterogeneity in theinduction of the adhesion molecules intercellular adhesion molecule 1, vascular cell adhesion molecule 1 (VCAM-1), and E-selectin between thetwo EC types following activation of PKC was demonstrated. Inparticular, E-selectin and VCAM-1 were significantly upregulated onHUVEC but not DMEC. The data indicate that the PKC pathway is unlikelyto be important for E-selectin and VCAM-1 expression in themicrovasculature but are consistent with a role for PKC inangiogenesis. This diversity in signaling in response to PKC activationmay depend on differential utilization of PKC isozymes and mayfacilitate specialized endothelial responses.

     

Inhibition of DNA synthesis by phorbol esters through protein kinase C in cultured rabbit aortic smooth muscle cells

In cultured rabbit aortic smooth muscle cells (SMC), 12-O-tetradecanoylphorbol-13-acetate (TPA) induced DNA synthesis in the presence of plasma-derived serum to a small extent, but inhibited markedly the rabbit whole blood serum (WBS)-, platelet-derived growth factor (PDGF)- and epidermal growth factor-induced DNA synthesis. Phorbol-12,13-dibutyrate (PDBu) mimicked this antiproliferative action of TPA, but 4 alpha-phorbol-12,13-didecanoate was inactive in this capacity. Prolonged treatment of the cells with PDBu caused the partial down-regulation of protein kinase C. In these protein kinase C-reduced cells, WBS still induced DNA synthesis, but TPA did not inhibit the WBS-induced DNA synthesis. We have previously shown that protein kinase C is involved at least partially in the PDGF-induced DNA synthesis in rabbit aortic SMC. The present results together with this earlier observation suggest that protein kinase C has not only a proliferative but also an antiproliferative action in rabbit aortic SMC.     

Phorbol ester modulates serotonin-stimulated phosphoinositide breakdown in cultured vascular smooth muscle cells

Stimulation of cultured rabbit aortic vascular smooth muscle cells (VSMC) with serotonin (5HT) induced a rapid generation of inositol phosphates from receptor-mediated hydrolysis of inositol phospholipids. Pretreatment of these cells with 500ng/ml of pertussis toxin for 24h prior to addition of 5HT reduced 5HT-induced formation of inositol phosphates. Phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA) or phorbol-12,13-dibutyrate (PDBu), are known to activate protein kinase C (PKC), but their role on cultured VSMC stimulated by 5HT has not been defined. TPA exhibited a rapid inhibition of 5HT-stimulated phosphoinositide breakdown, although 4 alpha-phorbol-12,13-didecanoate (4 alpha PDD), an inactive phorbol ester, did not inhibit it. These data suggest that a guanine nucleotide inhibitory (Gi) protein couples 5HT receptor to phospholipase C and TPA modulates 5HT-stimulated hydrolysis of inositol phospholipids in cultured VSMC through activation of PKC.     

Phorbol ester-induced G1 phase arrest selectively mediated by protein kinase Cdelta-dependent induction of p21

Although protein kinase C (PKC) has been widely implicated in the positive and negative control of proliferation, the underlying cell cycle mechanisms regulated by individual PKC isozymes are only partially understood. In this report, we show that PKCdelta mediates phorbol ester-induced G1 arrest in lung adenocarcinoma cells and establish an essential role for this novel PKC in controlling the expression of the cell cycle inhibitor p21. Activation of PKC with phorbol 12-myristate 13-acetate (PMA) in early G1 phase impaired progression of lung adenocarcinoma cells into S phase, an effect that was completely abolished by specific depletion of PKCdelta, but not PKCalpha. Although the PKC effect was unrelated to the inhibition of cyclin D1 expression, PKC activation significantly up-regulated p21 and down-regulated Rb hyperphosphorylation and cyclin A expression. Elevations in p21 mRNA and protein by PMA were mediated by PKCdelta but not PKCalpha. Studies using luciferase reporters also revealed an essential role for PKCdelta in the PMA-induced inhibition of Rb-dependent cyclin A promoter activity. Finally, we showed that the cell cycle inhibitory effect of PKCdelta is greatly attenuated by RNA interference-mediated knock-down of p21. Our results identify a novel link between PKCdelta and G1 arrest via p21 up-regulation and highlight the complexities in the downstream effectors of PKC isozymes in the context of cell cycle progression and proliferation.     

Angiotensin II activates extracellular signal regulated kinases via protein kinase C and epidermal growth factor receptor in breast cancer cells

Angiotensin II (Ang II) induces, through AT1, intracellular Ca(2+) increase in both normal and cancerous breast cells in primary culture (Greco et al., 2002 Cell Calcium 2:1-10). We here show that Ang II stimulated, in a dose-dependent manner, the 24 h-proliferation of breast cancer cells in primary culture, induced translocation of protein kinase C (PKC)-alpha, -beta1/2, and delta (but not -epsilon, -eta, -theta, -zeta, and -iota), and phosphorylated extracellular-regulated kinases 1 and 2 (ERK1/2). The proliferative effects of Ang II were blocked by the AT1 antagonist, losartan. Also epidermal growth factor (EGF) had mitogenic effects on serum-starved breast cancer cells since induced cell proliferation after 24 h and phosphorylation of ERK1/2. The Ang II-induced proliferation of breast cancer cells was reduced by (a) G?6976, an inhibitor of conventional PKC-alpha and -beta1, (b) AG1478, an inhibitor of the tyrosine kinase of the EGF receptor (EGFR), and (c) downregulation of 1,2-diacylglycerol-sensitive PKCs achieved by phorbol 12-myristate 13-acetate (PMA). A complete inhibition of the Ang II-induced cell proliferation was achieved using the inhibitor of the mitogen activated protein kinase kinase (MAPKK or MEK), PD098059, or using G?6976 together with AG1478. These results indicate that in human primary cultured breast cancer cells AT1 regulates mitogenic signaling pathways by two simultaneous mechanisms, one involving conventional PKCs and the other EGFR transactivation.