Activation of extracellular signal-regulated kinase mediates bombesin-induced mitogenic responses in prostate cancer cells

Bombesin and its mammalian homologue gastrin-releasing peptide have been shown to be highly expressed and secreted by neuroendocrine cells in prostate cancer, and are thought to be related to the carcinogenesis and progression of this disease. We found, in this study, bombesin specifically induced mitogen-activated protein (MAP) kinase activation as shown by increased extracellular regulated kinase (ERK) phosphorylation and epidermal growth factor (EGF) receptor transactivation in prostate cancer cells, which express functional gastrin-releasing peptide receptor. The transactivation of EGF receptor was required for bombesin-induced ERK phosphorylation. Furthermore, non-receptor tyrosine kinase Src and cellular Ca2+ were shown to be involved in bombesin-induced EGF receptor transactivation and ERK phosphorylation. Inhibition of either EGF receptor transactivation or ERK activation blocked bombesin-induced DNA synthesis in these cells. Taken together, these data suggest bombesin may act as a mitogen in prostate cancer by activating MAP kinase pathway via EGFR transactivation.     

EGF receptor function is required in late G(1) for cell cycle progression induced by bombesin and bradykinin

We examined therole of epidermal growth factor (EGF) receptor (EGFR) tyrosine kinaseactivation in G protein-coupled receptor (GPCR) agonist-inducedmitogenesis in Swiss 3T3 and Rat-1 cells. Addition of EGFR tyrosinekinase inhibitors (e.g., tyrphostin AG-1478) abrogated bombesin-inducedextracellular signal-regulated kinase (ERK) activation in Rat-1 cellsbut not in Swiss 3T3 cells, indicating the importance of cell contextin determining the role of EGFR in ERK activation. In strikingcontrast, treatment with tyrphostin AG-1478 markedly (~70%)inhibited DNA synthesis induced by bombesin in both Swiss 3T3 and Rat-1cells. Similar inhibition of bombesin-induced DNA synthesis in Swiss3T3 cells was obtained using four structurally different inhibitors ofEGFR tyrosine kinase. Furthermore, kinetic analysis indicates that EGFRfunction is necessary for bombesin-induced mitogenesis in mid-lateG₁ in both Swiss 3T3 and Rat-1 cells. Our results indicatethat EGFR kinase activity is necessary in mid-late G₁ forpromoting the accumulation of cyclins D1 and E and implicate EGFRfunction in the coupling of GPCR signaling to the activation of thecell cycle.

     

Protein kinase Calpha mediates feedback inhibition of EGF receptor transactivation induced by Gq-coupled receptor agonists

While a great deal of attention has been focused on G-protein-coupled receptor (GPCR)-induced epidermal growth factor receptor (EGFR) transactivation, it has been known for many years that the tyrosine kinase activity of the EGFR is inhibited in cells treated with tumor-promoting phorbol esters, a process termed EGFR transmodulation. Because many GPCR agonists that elicit EGFR transactivation also stimulate the G_q/phospholipase C (PLC)/protein kinase C (PKC) pathway, we hypothesized that PKC-mediated inhibition of EGFR transactivation operates physiologically as a feedback loop that regulates the intensity and/or duration of GPCR-elicited EGFR transactivation. In support of this hypothesis, we found that treatment of intestinal epithelial IEC-18 cells with the PKC inhibitors GF 109203X or Ro 31-8220 or chronic exposure of these cells to phorbol-12,13-dibutyrate (PDB) to downregulate PKCs, markedly enhanced the increase in EGFR tyrosine phosphorylation induced by angiotensin II or vasopressin in these cells. Similarly, PKC inhibition enhanced EGFR transactivation in human colonic epithelial T84 cells stimulated with carbachol, as well as in bombesin-stimulated Rat-1 fibroblasts stably transfected with the bombesin receptor. Furthermore, cell treatment with inhibitors with greater specificity towards PKCα,  including Gö6976, Ro 31-7549 or Ro 32-0432, also increased GPCR-induced EGFR transactivation in IEC-18, T84 and Rat-1 cells. Transfection of siRNAs targeting PKCα  also enhanced bombesin-induced EGFR tyrosine phosphorylation in Rat-1 cells. Thus, multiple lines of evidence support the hypothesis that conventional PKC isoforms, especially PKCα, mediate feedback inhibition of GPCR-induced EGFR transactivation.     

Galardin (GM 6001), a broad-spectrum matrix metalloproteinase inhibitor, blocks bombesin- and LPA-induced EGF receptor transactivation and DNA synthesis in rat-1 cells

Matrix metalloproteinases (MMPs) have been implicated in the transactivation of the epidermal growth factor receptor (EGFR) induced by G-protein coupled receptor (GPCR) agonists. Although EGFR phosphorylation and downstream signaling have been shown to be dependent on MMP activity in many systems, a role for MMPs in GPCR-induced DNA synthesis has not been studied in any detail. In this study we utilized the broad-spectrum matrix metalloproteinase inhibitor, galardin (Ilomastat, GM 6001), to study the mechanism of bombesin- or LPA-induced EGFR transactivation and the role of MMPs in early and late response mitogenic signaling in Rat-1 cells stably transfected with the bombesin/GRP receptor (BoR-15 cells). Addition of galardin to cells stimulated with bombesin or LPA specifically inhibited total EGFR phosphorylation, as well as site-specific phosphorylation of tyrosine 845, a putative Src phosphorylation site, and tyrosine 1068, a typical autophosphorylation site. Galardin treatment also inhibited extracellular signal-regulated kinase (ERK) activation induced by bombesin or LPA, but not by EGF. In addition, galardin inhibited bombesin- or LPA-induced DNA synthesis in a dose dependent manner, when stimulated by increasing concentrations of bombesin, and when added after bombesin stimulation. Furthermore, addition of galardin post-bombesin stimulation indicated that by 3 h sufficient accumulation of EGFR ligands had occurred to continue to induce transactivation despite an inhibition of MMP activity. Taken together, our results suggest that MMPs act as early as 5 min, and up to around 3 h, to mediate GPCR-induced EGFR transactivation, ERK activation, and stimulation of DNA synthesis.     

IL-8 promotes cell proliferation and migration through metalloproteinase-cleavage proHB-EGF in human colon carcinoma cells

Interleukin-8 (IL-8) has been reported to promote tumor cell growth in colon cancer cells after binding to its receptors, which are members of the G-protein coupled receptor (GPCR) family. Recent studies demonstrated that stimulation of GPCR can induce shedding of epidermal growth factor (EGF) ligands via activation of a disintegrin and metalloprotease (ADAM), with subsequent transactivation of the EGF receptor (EGFR). In this study, we investigated mechanisms of cell proliferation and migration stimulated by IL-8 in a human colon carcinoma cell line (Caco2). IL-8 increased DNA synthesis of Caco2 in a dose dependent manner and this was inhibited by ADAM, EGFR kinase, and MEK inhibitors. IL-8 transiently induced EGFR tyrosine phosphorylation after 5-90 min and this was completely inhibited by ADAM inhibitor. Neutralizing antibody against HB-EGF as a key ligand for EGFR also blocked transactivation of EGFR and cell proliferation by IL-8. Since IL-8-induced cell migration was further suppressed by the ADAM inhibitor and the HB-EGF neutralizing antibody, our data indicate that IL-8 induces cell proliferation and migration by an ADAM-dependent pathway, and that HB-EGF plays an important role as the major ligand for this pathway.     

CCK(B)/gastrin receptor mediates synergistic stimulation of DNA synthesis and cyclin D1, D3, and E expression in Swiss 3T3 cells.

In order to develop a model system for identifying signaling pathways and cell cycle events involved in gastrin-mediated mitogenesis, we have used high efficiency retroviral-mediated transfection of cholecystokinin (CCK)(B)/gastrin receptor into Swiss 3T3 cells. The retrovirally-transfected CCK(B)/gastrin receptor binds 125I-CCK-8 with high affinity (Kd = 1.1 nM) and is functionally coupled to intracellular signaling pathways including rapid and transient increase in Ca2+ fluxes, protein kinase C-dependent protein kinase D activation, and MEK-dependent ERK1/2 activation. In the presence of insulin, CCK-8 or gastrin induced a 66.5 +/- 8.8-fold (mean +/- SEM, n = 24 in eight independent experiments) increase in cellular DNA synthesis, reaching a level similar to that achieved by stimulation with a saturating concentration of fresh serum, and much greater than the response to each agonist added alone. CCK-8 also induced a striking increase in the expression of cyclins D1, D3, and E and hyperphosphorylation of Rb acting synergistically with insulin. Similar effects were observed when CCK(B)/gastrin receptor was activated in the presence of EGF or bombesin. Our results demonstrate that activation of CCK(B)/gastrin receptor retrovirally-transfected into Swiss 3T3 induces a potent synergistic effect on DNA synthesis, accumulation of cyclins D1, D3, and E and hyperphosphorylation of Rb in combination with insulin, EGF, or bombesin. Thus, the CCK(B)/gastrin receptor transfected into Swiss 3T3 cells provides a novel model system to elucidate mitogenic signal transduction pathways and cell cycle events activated via this receptor.     

Activation of muscarinic acetylcholine receptors inhibits cell cycle progression of small cell lung carcinoma.

We previously reported that activation of muscarinic acetylcholine receptors (mAChR) of M3 subtype causes hydrolysis of phosphoinositides and inhibits voltage-gated Ca2+ channel activity in small cell lung carcinoma (SCLC) cells. We now report that mAChR activation causes exponentially growing SCLC cells to arrest in S and G2/M phases of the cell cycle, concomitant with a decrease in DNA synthesis. Cell cycle progression and DNA synthesis resume when mAChR are down-regulated. In serum-starved SCLC cells, mAChR activation inhibits DNA synthesis induced by serum, bombesin, insulin, or insulin-like growth factor-I. The finding that DNA synthesis is inhibited even when mAChR are activated after exposure of cells to growth factors indicates that decreased signal transduction by growth factor receptors is not the mechanism of mAChR-mediated growth inhibition. Our data suggest that mAChR activation disrupts a common event that is induced by different growth factors and is fundamental for cell cycle progression.     

Inhibition of bombesin-induced mitogenesis by pertussis toxin: dissociation from phospholipase C pathway

Prior incubation of quiescent cultures of Swiss 3T3 cells with pertussis toxin selectively inhibited the stimulation of DNA synthesis induced by peptides of the bombesin family. While pertussis toxin blocked mitogenesis at an early stage in the action of the peptide, the toxin did not impair the rapid stimulation of polyphosphoinositide breakdown, Ca2+ mobilization or activation of protein kinase C promoted by bombesin. Thus, inhibition of bombesin-induced mitogenesis by pertussis toxin can be dissociated from inactivation of the phospholipase C signalling pathway.     

Epidermal growth factor receptor transactivation mediates substance P-induced mitogenic responses in U-373 MG cells

Ligand-induced activation of G protein-coupled receptors is emerging as an important pathway leading to the activation of certain receptors with intrinsic tyrosine kinase activity, such as the epidermal growth factor receptor (EGFR). Substance P (SP) exerts many effects via activation of its G protein-coupled receptor (neurokinin-1, NK-1). SP participates in acute inflammation and activates key proteins involved in mitogenic pathways, such mitogen-activated protein kinases (MAPKs), stimulating DNA synthesis. We tested the hypothesis that SP-induced MAPK activation and DNA synthesis require activation of the EGFR. In U-373 MG cells, which express functional NK-1, SP induced tyrosine phosphorylation of several proteins including EGFR. SP induced formation of an activated EGFR complex containing the adapter proteins SHC and Grb2, but not c-Src. SP activated the MAPK pathway as shown by increased Erk2 kinase activity. SP induced Erk2 activation, and DNA synthesis was inhibited in cells transfected with a dominant negative EGFR plasmid lacking kinase activity, as well as in cells treated with a specific EGFR inhibitor. In addition, pertussis toxin, an inhibitor of Galpha(iota) protein subunits, prevented SP-induced EGFR transactivation and subsequent DNA synthesis. Our results implicate EGFR as an essential regulator in SP/NK-1-induced activation of the MAPK pathway and cell proliferation in U-373 MG cells, and these events are mediated by a pertussis toxin-sensitive Galpha protein. We suggest that this mechanism by which SP controls cell proliferation is an important pathway in tissue restoration and healing.     

Bombesin induction of c-fos and c-myc proto-oncogenes in Swiss 3T3 cells: significance for the mitogenic response

Bombesin is a potent mitogen for Swiss 3T3 cells and acts synergistically with insulin and other growth factors. We show here that addition of bombesin to quiescent Swiss 3T3 cells causes a striking increase in the levels of c-fos and c-myc mRNAs. Enhanced expression of c-fos (122 +/- 14-fold) occurred within minutes of peptide addition followed by increased expression of c-myc (82 +/- 16-fold). The concentrations of peptide required for half-maximal increase in the levels of c-fos and c-myc mRNAs were 1.0 and 0.9 nM, respectively. The peptide [D-Arg1, D-Pro2, D-Trp7,9, Leu11] substance P which inhibits the binding of bombesin to its receptor and bombesin-stimulated DNA synthesis in Swiss 3T3 cells blocked the increase in c-fos and c-myc mRNA levels promoted by bombesin. Down-regulation of protein kinase C by long-term exposure to phorbol esters prevented c-fos and c-myc induction by bombesin. This and other results indicate that the induction of these proto-oncogenes by bombesin could be mediated by the coordinated effects of protein kinase C activation and Ca2+ mobilization. The marked synergistic effect between bombesin and insulin was used to assess whether the increase in the induction of c-fos and c-myc is an obligatory event in cell activation. In the presence of insulin, bombesin stimulated DNA synthesis at subnanomolar concentrations but had only a small effect on c-fos and c-myc mRNA levels. This apparent dissociation of mitogenesis from proto-oncogene induction was even more dramatic in 3T3 cells with down-regulated protein kinase C. In these cells bombesin stimulated DNA synthesis in the presence of insulin but failed to enhance c-fos and c-myc mRNA levels at comparable concentrations. Thus, the induction of c-fos and c-myc may be a necessary step in the mitogenic response initiated by ligands that act through activation of protein kinase C but the expression of these proto-oncogenes may not be an obligatory event in the stimulation of mitogenesis in 3T3 cells by mitogens that utilise other signalling pathways.     

Effects of Endogenous Epidermal Growth Factor Receptor Signaling on DNA Synthesis and ERK Activation in a Cytokine-Dependent Hematopoietic Cell Line

Epidermal growth factor (EGF) is commonly thought to affect the proliferation of many cells, especially epithelial cells. Aberrant expression of the receptor for EGF, (EGFR) or members of the EGFR family is often implicated in the etiology of many cancers. Ligation of the EGFR results in the activation of many downstream signaling pathways which have profound effects on cell cycle progression and the prevention of apoptosis. In general, the EGFR is thought to be either not expressed or expressed at low levels in hematopoietic cells. We determined that the EGFR was expressed at a low level in the murine cytokine-dependent hematopoietic cell line FDC-P1 but not in an additional murine IL-3 dependent cell line FL5.12. EGF induced a mild effect on DNA synthesis and ERK activation in EGFR positive FDC-P1 cells but not EGFR negative FL5.12 cells. Addition of suboptimal concentrations of IL-3 synergized with EGF in stimulating DNA synthesis in EGFR-positive FDC-P1 cells. Likewise, the EGFR inhibitor AG1478 induced apoptosis in EGFR positive FDC-P1 cells but not EGFR negative FL5.12 cells. Both cell lines can be directly transformed to cytokine independence by activated EGFR (v-ERBB) expression in the absence of autocrine growth factors indicating that they are poised to fully utilize EGFR mediated signal transduction pathways as a means for proliferation. These results document the functional importance of endogenous EGFR signaling pathway in some hematopoietic 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.     

Bradykinin-mediated cell proliferation depends on transactivation of EGF receptor in corneal fibroblasts

In previous studies, bradykinin (BK) has been shown to induce cell proliferation through BK B2 receptor (B2R) via p42/p44 MAPK in Statens Seruminstitut Rabbit Corneal Cells (SIRCs). In addition to this pathway, EGFR transactivation pathway has been implicated in linking a variety of G-protein coupled receptors to MAPK cascades. Here, we further investigate whether these transactivation mechanisms participating in BK-induced cell proliferation in SIRCs. Using an immunofluorescence staining and RT-PCR, we initially characterize that SIRCs were corneal fibroblasts and predominantly expressed B2R by BK. Inhibition of p42/p44 MAPK by the inhibitors of Src, EGFR, and Akt or transfection with respective siRNAs prevents BK-induced DNA synthesis in SIRCs. The mechanisms underlying these responses were mediated through phosphorylation of Src and EGFR via the formation of Src/EGFR complex which was attenuated by PP1 and AG1478. Moreover, BK-induced p42/p44 MAPK and Akt activation was mediated through EGFR transactivation, which was diminished by the inhibitors of MMP-2/9 and heparin-binding EGF-like factor (HB-EGF). Finally, increased nuclear translocation of Akt and p42/p44 MAPK turns on early gene expression leading to cell proliferation. These results suggest that BK-induced cell proliferation is mediated through c-Src-dependent transactivation of EGFR via MMP2/9-dependent pro-HB-EGF shedding linking to activation of Akt and p42/p44 MAPK in corneal fibroblasts.     

Interferon gamma-mediated growth regulation of epithelial cells

Interferon gamma (IFNgamma) is known to inhibit proliferation of certain transformed cell lines. Recently, we have demonstrated the transactivation of the epidermal growth factor receptor (EGFR) in response to IFNgamma (Burova et al., 2007) and provided direct evidence for the dependence of IFNgamma-induced EGFR transactivation upon EGFR expression level in epithelial cells (Gonchar et al., 2008). This study examines an antiproliferative effect of IFNgamma on human epithelial cells lines A431 and HeLa which express high levels of EGFR, as well as HEK293, which expresses low levels of EGFR. We characterized the IFNgamma-induced changes in these cells by studying cell growth, the cell cycle and induction of apoptosis. The response to IFNgamma differed in the tested cell lines: cell growth was inhibited in both A431 and HeLa cells, but not in HEK293 cells, as shown by cell counts and MTT. The cell cycle phases analyzed by flow cytometry were disturbed in A431 and HeLa cells in response to IFNgamma. In contrast, IFNgamma treatment did not alter distribution by cell cycle phases in HEK293. Our results indicate that IFNgamma exhibit an antiproliferative effect depending on the increased expression of EGFR in A431 and HeLa cells. Further, it was demonstrated that IFNgamma induced the caspase 3 activation in A431 cells, suggesting an involvement of active caspase 3 in IFNgamma-induced apoptosis.     

Beta-adrenergic receptor-mediated DNA synthesis in cardiac fibroblasts is dependent on transactivation of the epidermal growth factor receptor and subsequent activation of extracellular signal-regulated kinases

Cardiac hypertrophy often leads to heart failure and is associated with abnormal myocardial adrenergic signaling. This enlargement of myocardial mass can involve not only an increase in cardiomyocyte size, but increased proliferation of cardiac fibroblasts. A potential key player in the cardiac hypertrophic response is the ERK family of MAPKs. To gain mechanistic insight into adrenergic regulation of myocardial mitogenic signaling, we examined beta-adrenergic receptor (beta-AR) stimulation of ERK activation and DNA synthesis in cultured adult rat cardiac fibroblasts, including the involvement of tyrosine kinases in this signaling pathway. Addition of the beta-AR agonist isoproterenol (ISO) to serum-starved cells induced DNA synthesis in a dose-dependent manner, and this was inhibited by selective inhibitors of the epidermal growth factor receptor (EGFR). Importantly and in agreement with the involvement of MAPKs and the EGFR in this response in cardiac fibroblasts, the EGFR inhibitor AG1478 attenuated ISO-induced ERK phosphorylation. Moreover, pretreatment with PP2, a selective inhibitor of the Src tyrosine kinase, attenuated both ISO-mediated EGFR phosphorylation and ERK activation. Furthermore, studies in these cardiac fibroblasts showed that phosphatidylinositol 3-kinase contributed to beta-AR-mediated ERK activation, but not to EGFR activation. Finally, studies using selective inhibitors of matrix metalloproteases indicated that they and heparin-bound EGF shedding were involved in beta-AR-induced ERK activation and subsequent DNA synthesis in cardiac fibroblasts. Because these cells primarily express the beta(2)-AR subtype, our findings indicate that beta(2)-AR-mediated EGFR transactivation of intracellular tyrosine kinase signaling pathways is the major signaling pathway responsible for the adrenergic stimulation of mitogenesis of cardiac fibroblasts.     

EGFR inhibition by pentacyclic triterpenes exhibit cell cycle and growth arrest in breast cancer cells

Aims

Pentacyclic triterpenes are a group of molecules with promising anticancer potential, although their precise molecular target remains elusive. The current work aims to investigate the antiproliferative and associated mechanisms of triterpenes in breast cancer cells in vitro.

Main methods

Effect of triterpenes on cell cycle distribution, ROS and key regulatory proteins were analyzed in three breast cancer cells in vitro. Growth inhibition, new DNA synthesis, colony formation assays and Western blot analysis were performed to assess the EGFR inhibitory effect of triterpenes. Molecular docking was performed to study the interaction between EGFR and triterpenes.

Key findings

We have demonstrated the ability of dimethyl melaleucate (DMM), a pentacyclic triterpene to exhibit cell cycle arrest at G0/G1 phase by down-regulation of cyclin D1 through PI3K/AKT inhibition. Further, to identify the upstream target of DMM, potential EGFR inhibitory activity of DMM and three structurally related pentacyclic triterpenes, ursolic acid, 18α-glycyrrhetinic acid and carbenoxolone was investigated. Interestingly, pentacyclic triterpenes limit EGF mediated breast cancer proliferation through sustained inhibition of EGFR and its downstream effectors STAT3 and cyclin D1 in breast cancer lines. We also show pentacyclic triterpenes to bind at the ATP binding pocket of tyrosine kinase domain of EGFR leading to the hypothesis that pentacyclic triterpenes could be a novel class of EGFR inhibitors. In conclusion, pentacyclic triterpenes inhibit EGFR activation through binding with tyrosine kinase domain thereby suppressing breast cancer proliferation.

Significance

Pentacyclic triterpenes may serve as a potential platform for development of novel drugs against breast cancer.     

Cyclin D2 induces proliferation of cardiac myocytes and represses hypertrophy

The myocytes of the adult mammalian heart are considered unable to divide. Instead, mitogens induce cardiomyocyte hypertrophy. We have investigated the effect of adenoviral overexpression of cyclin D2 on myocyte proliferation and morphology. Cardiomyocytes in culture were identified by established markers. Cyclin D2 induced DNA synthesis and proliferation of cardiomyocytes and impaired hypertrophy induced by angiotensin II and serum. At the molecular level, cyclin D2 activated CDK4/6 and lead to pRB phosphorylation and downregulation of the cell cycle inhibitors p21Waf1/Cip1 and p27Kip1. Expression of the CDK4/6 inhibitor p16 inhibited proliferation and cyclin D2 overexpressing myocytes became hypertrophic under such conditions. Inhibition of hypertrophy by cyclin D2 correlated with downregulation of p27Kip1. These data show that hypertrophy and proliferation are highly related processes and suggest that cardiomyocyte hypertrophy is due to low amounts of cell cycle activators unable to overcome the block imposed by cell cycle inhibitors. Cell cycle entry upon hypertrophy may be converted to cell division by increased expression of activators such as cyclin D2.     

5-HT2A receptor induces ERK phosphorylation and proliferation through ADAM-17 tumor necrosis factor-alpha-converting enzyme (TACE) activation and heparin-bound epidermal growth factor-like growth factor (HB-EGF) shedding in mesangial cells

In this study, we present multiple lines of evidence to support a critical role for heparin-bound EGF (epidermal growth factor)-like growth factor (HB-EGF) and tumor necrosis factor-alpha-converting enzyme (TACE) (ADAM17) in the transactivation of EGF receptor (EGFR), ERK phosphorylation, and cellular proliferation induced by the 5-HT(2A) receptor in renal mesangial cells. 5-hydroxy-tryptamine (5-HT) resulted in rapid activation of TACE, HB-EGF shedding, EGFR activation, ERK phosphorylation, and longer term increases in DNA content in mesangial cells. ERK phosphorylation was attenuated by 1) neutralizing EGFR antibodies and the EGFR kinase inhibitor, AG1478, 2) neutralizing HB-EGF, but not amphiregulin, antibodies, heparin, or CM197, and 3) pharmacological inhibitors of matrix-degrading metalloproteinases or TACE small interfering RNA. Exogenously administered HB-EGF stimulated ERK phosphorylation. Additionally, TACE was co-immunoprecipitated with HB-EGF. Small interfering RNA against TACE also blocked 5-HT-induced increases in ERK phosphorylation, HB-EGF shedding, and DNA content. In aggregate, this work supports a pathway map that can be depicted as follows: 5-HT --> 5-HT(2A) receptor --> TACE --> HB-EGF shedding --> EGFR --> ERK --> increased DNA content. To our knowledge, this is the first time that TACE has been implicated in 5-HT-induced EGFR transactivation or in proliferation induced by a G protein-coupled receptor in native cells in culture.     

Protein kinase D potentiates DNA synthesis and cell proliferation induced by bombesin, vasopressin, or phorbol esters in Swiss 3T3 cells

We examined whether protein kinase D (PKD) overexpression in Swiss 3T3 cells potentiates the proliferative response to either the G protein-coupled receptor agonists bombesin and vasopressin or the biologically active phorbol ester phorbol 12,13-dibutyrate (PDBu). In order to generate Swiss 3T3 cells stably overexpressing PKD, cultures of these cells were infected with retrovirus encoding murine PKD and green fluorescent protein (GFP) expressed as two separate proteins translated from the same mRNA. GFP was used as a marker for selection of PKD-positive cells. PKD overexpressed in Swiss 3T3 cells was dramatically activated by cell treatment with bombesin or PDBu as judged by in vitro kinase autophosphorylation assays and exogenous substrate phosphorylation. Concomitantly, these stimuli induced PKD phosphorylation at Ser(744), Ser(748), and Ser(916). PKD activation and phosphorylation were prevented by exposure of the cells to protein kinase C-specific inhibitors. Addition of bombesin, vasopressin, or PDBu to cultures of Swiss 3T3 cells overexpressing PKD induced a striking increase in DNA synthesis and cell number compared with cultures of Swiss 3T3-GFP cells. In contrast, stimulation of DNA synthesis in response to epidermal growth factor, which acts via protein kinase C/PKD-independent pathways, was not enhanced. Our results demonstrate that overexpression of PKD selectively potentiates mitogenesis induced by bombesin, vasopressin, or PDBu in Swiss 3T3 cells.     

Role of Ca2+ influx in bombesin-induced mitogenesis in Swiss 3T3 fibroblasts

In Swiss 3T3 fibroblasts a peptide mitogen bombesin, which acts through the phospholipase C-protein kinase C signaling pathway, stimulates DNA synthesis in a manner strictly dependent on the medium calcium concentration: [3H]thymidine incorporation into DNA in the presence of a saturating concentration of bombesin (10(-8) M) is 4-fold greater at 3.0 mM extracellular calcium as compared with a value obtained at 0.03 mM calcium. In the present study we attempted to identify the site and the mechanism of action of Ca2+ influx along the bombesin-induced mitogenic signaling pathway, by comparing bombesin effects at 0.03 and 3.0 mM of medium calcium. Bombesin induces the same extent of increases in [3H]inositol phosphates after 1 min, and comparable sustained increases in the cellular content of 1,2-diacylglycerol for up to 4 h, at either 0.03 or 3.0 mM calcium. Bombesin induces the same extent of phosphorylation of MARCKS protein, the major cellular substrate for protein kinase C, irrespective of the medium calcium concentration for at least 4 h. Moreover, diverse cellular responses elicited by bombesin, including c-fos expression, activation of microtubule-associated protein 2 kinase and S6 kinase, glucose uptake, and protein synthesis but not the release of arachidonic acid and its metabolites, are induced similarly at either 0.03 or 3.0 mM calcium. Down-regulation of cellular protein kinase C nearly completely abolishes bombesin effects on c-fos expression, S6 kinase activation, glucose uptake, and DNA synthesis. These results suggest that the target of Ca2+ influx in bombesin-induced mitogenic signaling pathway is not located along the phospholipase C-protein kinase C signal transduction system including cellular events in early G1 phase that exist downstream to protein kinase C action.