Pituitary tumor transforming gene (PTTG) stimulates thyroid cell proliferation via a vascular endothelial growth factor/kinase insert domain receptor/inhibitor of DNA binding-3 autocrine pathway

CONTEXT: Vascular endothelial growth factor (VEGF) exerts its biological effects by binding to the tyrosine kinase receptors VEGF receptor type 1 (VEGFR1/Flt-1) and VEGFR2 (Flk-1/KDR). Kinase insert domain receptor (KDR) is the critical receptor controlling proliferation and migration of endothelial cells and has been shown to be expressed in some nonendothelial cells. We recently reported that the proangiogenic pituitary tumor transforming gene (PTTG) stimulates VEGF and up-regulates inhibitor of DNA binding-3 (ID3), an important gene in VEGF-dependent angiogenesis. OBJECTIVE: Our objective was to test whether VEGF, ID3, and KDR confer a PTTG-mediated effect on thyroid cell growth. DESIGN: Gene expression, MAPK stimulation, and cell proliferation were assessed in follicular thyroid cancer FTC133 cells. Gene expression and clinical associations were determined in 21 normal and 38 tumorous thyroid specimens (nine follicular and 29 papillary). RESULTS: ID3 correlated with VEGF mRNA expression in our series of thyroid cancers, which also showed up-regulated KDR mRNA. Stimulation of FTC133 cells with exogenous VEGF enhanced ID3 expression, which could be abrogated by the KDR-specific inhibitor ZM323881, suggesting that VEGF regulation of ID3 is KDR dependent. PTTG significantly correlated with KDR mRNA expression in our thyroid cancer cohort and up-regulated KDR and VEGF expression in FTC133 cells. Finally, cells transfected with PTTG demonstrated increased cell proliferation and phosphorylation of MAPK, which was abrogated by ZM323881. CONCLUSIONS: We report the presence of a VEGF/KDR/ID3-dependent autocrine pathway in FTC133 thyroid cells. By up-regulating both VEGF and KDR expression, we propose a novel PTTG-mediated proliferative pathway that may be critical to thyroid cancer growth and progression.     

Pseudomonas aeruginosa induces MUC5AC production via epidermal growth factor receptor.

Hypersecretory disease associated with Pseudomonas aeruginosa (PA) infections is characterised by increased goblet cells and increased mucin production. Recently, an epidermal growth factor receptor (EGFR) signalling cascade was shown to be a common pathway through which many stimuli induce mucin MUC5AC expression in airways by differentiation to a goblet cell phenotype. This study looked at whether PA products induce EGFR expression and activation and thus result in mucin MUC5AC production. Human airway epithelial (NCI-H292) cells were stimulated with PA culture supernatant (Sup). MUC5AC protein production, MUC5AC and EGFR messenger ribonucleic acid (mRNA) expression, and phosphorylated EGFR and phosphorylated p44/42 mitogen-activated protein kinase (MAPK) were all examined using enzyme-linked immunosorbent assay, by in situ hybridisation and by immunoblotting. PA Sup induced MUC5AC mRNA and subsequent protein expression, EGFR and p44/42 MAPK phosphorylation and EGFR mRNA expression. Induction of MUC5AC mRNA and protein expression and EGFR and p44/42 MAPK phosphorylation were inhibited completely by pretreatment with a selective EGFR tyrosine kinase inhibitor. Pretreatment with a selective inhibitor of MAPK kinase prevented MUC5AC production and p44/42 MAPK phosphorylation but not EGFR phosphorylation. The authors conclude that PA products induce mucin MUC5AC production in human airway epithelial cells via the expression and activation of epidermal growth factor receptor.     

Role of epidermal growth factor in pathogenesis of uterine leiomyomas

Objective:To investigate the role of epidermal growth factor(EGF) in the pathogenesis of uterine leiomyomas.Methods:Human myometrial smooth muscle cells(HM-SMCs) and smooth muscle cells of human uterine leiomyomas(HL-SMCs) were separated from patients' specimens and cultured.After processed by EGF or PD98059(inhibitor of MKK/MEK) +EGF,the proliferation rate of both SMCs was detected by BrdU method and the phosphorylation level of p44/42 mitogen-activated protein kinase(MAPK) was determined by Western-blot.After different processing time by EGF,the phosphorylation levels of p44/42 MAPK and AKT and p27 expression level in both SMCs were detected by Western-blot.Results:EGF could significantly promote HL-SMCs proliferation and PD98059 could inhibit this effect(P0.05);besides,PD98059 could inhibit the increase of the phosphorylation level of p44/42 MAPK in both SMCs induced by EGF.When the processing time by EGF was over 15 min,the phosphorylation levels of p44/42 MAPK and AKT in both SMCs decreased sharply and were close to zero:p27 expression in HM-SMCs raised significantly while the upregulation in HL-SMCs was little.Conclusions:EGF could not cause activation of EGFR because of the dephosphorylation of p44/42 MAPK and AKT in HL-SMCs,which caused p27 expression insufficiently and cell cycle dysregulation.     

Phosphatidylethanol in high density lipoproteins increases the vascular endothelial growth factor in smooth muscle cells

To study whether qualitative changes in high density lipoprotein (HDL) phospholipids mediate part of the advantageous effects of ethanol on atherosclerosis, we investigated whether HDL associated phosphatidylethanol (PEth) affects the secretion of vascular endothelial growth factor (VEGF) from cultured human smooth muscle cells. Serum-starved human umbilical vein HUVS-112D smooth muscle cells were incubated in the presence of PEth–HDL, HDL, or buffer. The phosphorylation of protein kinase C (PKC) and mitogen activated protein kinase (p44/42 MAPK) was determined by specific antibodies against phosphorylated and total proteins. VEGF concentrations were measured from cell culture medium of the cells. PEth increased the secretion of VEGF into the culture medium of HUVS cells. PEth–HDL increased the PKC phosphorylation by 2.1-fold and p44/42 MAPK phosphorylation by 3.3-fold compared with HDL, indicating that PEth-containing HDL particles influence vascular smooth muscle cells by PKC and p44/42 MAPK signalling. This may mediate the effects of ethanol on vascular wall by increasing the VEGF secretion from smooth muscle cells. The secreted VEGF may inhibit the formation of neointima and in doing so helps prevent atherosclerosis.     

p38MAPK inhibition enhances basal and norepinephrine-stimulated p42/44MAPK phosphorylation in rat pinealocytes

Interaction between p38MAPK and p42/44MAPK in rat pinealocytes was examined by determining the effects of p38MAPK inhibitors on the phosphorylation of p42/44MAPK using Western blot analysis. Treatment with SB202190, a specific inhibitor of p38MAPK, increased p42/44MAPK phosphorylation in a concentration-dependent manner. SB202190 also enhanced the magnitude and the duration of norepinephrine-activated p42/44MAPK phosphorylation. The effect of SB202190 on p42/44MAPK phosphorylation was abolished by PD98059 or UO126, inhibitors of MEK, suggesting that SB202190 is acting upstream of MEK in activating p42/44MAPK. The SB202190-induced phosphorylation of p42/44MAPK was not blocked by inhibitors of cGMP-dependent kinase (KT5823), protein kinase C (calphostin C) or Ca2+/calmodulin dependent kinase (KN93) suggesting that these pathways may not be involved in the effect of SB202190. SB202190 further increased p42/44MAPK phosphorylation that was stimulated by 8-bromo-cGMP, 4beta phorbol 12-myristate 13-acetate, or ionomycin. In contrast, inhibition of p42/44MAPK phosphorylation by dibutyryl-cAMP persisted when p42/44MAPK phosphorylation was increased by SB202190. Furthermore, inhibition of p42/44MAPK phosphorylation had no effect on p38MAPK activation. These results suggest that inhibition of p38MAPK causes activation of p42/44MAPK and acts synergistically with norepinephrine in the regulation of p42/44MAPK activation in rat pinealocytes.     

Advanced glycation end product (AGE) receptor 1 suppresses cell oxidant stress and activation signaling via EGF receptor

Advanced glycation end product receptors (AGERs) play distinct functional roles in both the toxicity and disposal of advanced glycation end products (AGEs), substances that are linked to diabetes and aging. Overexpression of AGER1 in murine mesangial cells (MCs) (MC-R1) inhibited AGE-induced MAPK1,2 phosphorylation and NF-kappaB activity and also increased AGE degradation. The mechanism of the inhibitory effects of AGER1, upstream of MAPK, was explored in MCs and HEK293 AGER1-expressing cells. AGE-induced Ras activation was found to be linked to Shc/Grb2 complex formation and Shc phosphorylation in MCs, responses that were markedly reduced in MC-R1 cells. AGE responses also included EGF receptor (EGFR) phosphorylation in MCs or HEK293 cells, but this link was blocked in both MC-R1 and HEK293-R1 cells. Coexpression of AGER1 and EGFR in HEK293 cells decreased AGE-mediated EGFR and p44/p42 phosphorylation but not EGF-induced p44/p42 activation. AGE, S100/calgranulin, or H(2)O(2) promoted MAPK phosphorylation in EGFR(+) cells in a manner that was inhibitable by an EGFR inhibitor, AG1478. Also, in AGER1 cells, AGE-induced H(2)O(2) formation and AGE- or S100-induced p44/p42 phosphorylation were suppressed, and these effects were restored by R1 siRNA. These data confirm that R1 negatively regulates AGE-mediated oxidant stress-dependent signaling via the EGFR and Shc/Grb2/Ras pathway. AGER1 could serve as a model for developing therapeutic targets against vascular and kidney disorders related to diabetes and aging.     

VEGFR2 signalling contributes to increased endothelial susceptibility to TNF-α under chronic non-uniform shear stress

Objectives

Vascular endothelial growth factor receptor 2 (VEGFR2), a tyrosine kinase receptor activated by VEGF and shear stress, is critically involved in endothelial mechanotransduction. We investigated the role of VEGFR2 in non-uniform shear stress-induced endothelial susceptibility to inflammatory stimuli.

Methods

Endothelial cells (ECs) were exposed to non-uniform shear stress, followed by stimulation with TNF-α. ECs were transfected with siRNAs against VEGFR2. Alternatively, ECs were treated with blocking antibody against VEGFR2, or with inhibitors of VEGFR2 (ZM 323881), PI3K (LY 294002), or Src-kinase (PP2). THP-1 monocytes were used for dynamic adhesion assays. Endothelial protein expression was determined by immunofluorescence.

Results

siRNA against VEGFR2 decreased VEGFR2 protein expression by 40% as determined by Western blotting. In endothelial cells exposed to non-uniform shear stress, VEGFR2 knockdown inhibited TNF-α-induced NF-κB translocation to the nucleus, and the upregulation of VCAM-1 and E-selectin. Consequently, monocytic cell recruitment to endothelium under non-uniform shear stress conditions was reduced. Similar effects were observed by blocking VEGFR2 activity using a specific inhibitor ZM 323881, or an antibody against VEGFR2 before TNF-α stimulation. Inhibition of PI3K with LY 294002 significantly reduced non-uniform shear stress-induced endothelial susceptibility to TNF-α, whereas blocking Src-kinase with PP2 was ineffective.

Conclusion

VEGFR2 is critically involved in adhesion molecule induction and monocytic cell recruitment to endothelium in response to non-uniform shear stress and TNF-α. Targeting the mechanosensory cascade can prevent endothelial activation in atherosclerosis-prone regions.     

Phorbol esters stimulate growth and inhibit differentiation in cultured thyroid cells

The potent tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) has biological effects on cell growth and differentiation similar to the effects of epidermal growth factor (EGF) on a variety of cells. Since EGF has been shown recently to stimulate thyroid cell proliferation and inhibit iodine metabolism, we examined the effects of phorbol esters on primary ovine thyroid cultures. TPA stimulated cell growth in a manner similar to EGF. The growth effects of EGF and TPA in combination were not additive. In contrast, TPA (1.6 X 10(-7) M) was a more potent inhibitor of iodine uptake and incorporation than EGF (10(-9) M) at their maximally effective concentrations. The inhibitory effects of TPA were also more rapid and less reversible than those of EGF. TPA and EGF in combination inhibited iodine metabolism more than either agent alone at its maximally effective concentration. Both TPA and EGF reduced the accumulation of cAMP in TSH-stimulated cells, but (Bu)2cAMP and stimulators of adenylate cyclase failed to overcome TPA's inhibition of iodine metabolism. TPA interacted with EGF by reducing the affinity of membrane receptors for [125I]iodo-EGF. Although the alteration in EGF-receptor interaction induced by TPA may play a role in mediating TPA's biological effects, the additive effects of TPA and EGF on iodine metabolism suggest that TPA does not act solely through the EGF receptor-effector system. Agents other than TSH, including phorbol esters and EGF, are potent modulators of thyroid growth and differentiated function. Despite several similarities in biological activity, TPA and EGF do not modulate differentiated function in an identical manner. Both factors act at least partially through a non-cAMP-dependent pathway, providing indirect evidence of another second messenger(s) in the control of thyroid function.     

Parathyroid hormone activates mitogen-activated protein kinase in opossum kidney cells

Many G protein-coupled receptor agonists activate p42/p44 mitogen-activated protein kinase (MAPK), using signaling pathways that are a function of receptor, G protein-coupled, and effector complement. In opossum kidney (OK) cells, activation of endogenous PTH receptors caused a time- (peak within 15-30 min, sustained for approximately 2 h) and dose-dependent (EC50 approximately 3 x 10(-10) M) activation of MAPK. Immunoblot analysis with an activation- specific MAPK antibody indicated that PTH activated both p42 and p44 MAPK. Epidermal growth factor (EGF) also activated p42 and p44MAPK in a time- (peak at 5 min, return to basal within 2 h) and dose-dependent (EC50 approximately 3 ng/ml) fashion. PTH-dependent MAPK activation was mimicked by the protein kinase C activator (PKC) phorbol myristate acetate (PMA), and the protein kinase A activators 8 bromo-cAMP (8-Br-cAMP) and forskolin but was not affected by pertussis toxin pretreatment. PMA or 8-Br-cAMP pretreatment blocked MAPK activation by reexposure to each kinase activator but caused no significant reduction in MAPK activation by PTH. MAPK activation by PTH, EGF, and 8-Br-cAMP was inhibited by the MAPK kinase inhibitor PD98059 and an EGF receptor (EGFR)-selective inhibitor tyrphostin AG1478. AG1478 also blocked MAPK activation by insulin-like growth factor-1 and platelet-derived growth factor. EGF and PTH caused time- and AG1478-sensitive phosphorylation of the EGFR, but EGFR desensitization did not affect MAPK activation by PTH. EGF, PMA, and low doses of PTH (10(12) to 10(-9) M) stimulated while 8-Br-cAMP and high doses of PTH (10(-8) to 10(-6) M) inhibited [3H]thymidine uptake. These data demonstrate that PTH activates MAPK and suggest that PKC, protein kinase A, and the EGFR play roles in PTH signaling. The biphasic effect of PTH on DNA synthesis suggests that MAPK activation by the hormone leads to distinct cellular responses.     

Adipocyte-derived plasma protein adiponectin acts as a platelet-derived growth factor-BB-binding protein and regulates growth factor-induced common postreceptor signal in vascular smooth muscle cell

BACKGROUND: Vascular smooth muscle cell proliferation plays an important role in the development of atherosclerosis. We previously reported that adiponectin, an adipocyte-specific plasma protein, accumulated in the human injured artery and suppressed endothelial inflammatory response as well as macrophage-to-foam cell transformation. The present study investigated the effects of adiponectin on proliferation and migration of human aortic smooth muscle cells (HASMCs). Methods and Results- HASMC proliferation was estimated by [(3)H] thymidine uptake and cell number. Cell migration assay was performed using a Boyden chamber. Physiological concentrations of adiponectin significantly suppressed both proliferation and migration of HASMCs stimulated with platelet-derived growth factor (PDGF)-BB. Adiponectin specifically bound to (125)I-PDGF-BB and significantly inhibited the association of (125)I-PDGF-BB with HASMCs, but no effects were observed on the binding of (125)I-PDGF-AA or (125)I-heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) to HASMCs. Adiponectin strongly and dose-dependently suppressed PDGF-BB-induced p42/44 extracellular signal-related kinase (ERK) phosphorylation and PDGF beta-receptor autophosphorylation analyzed by immunoblot. Adiponectin also reduced PDGF-AA-stimulated or HB-EGF-stimulated ERK phosphorylation in a dose-dependent manner without affecting autophosphorylation of PDGF alpha-receptor or EGF receptor. CONCLUSIONS: The adipocyte-derived plasma protein adiponectin strongly suppressed HASMC proliferation and migration through direct binding with PDGF-BB and generally inhibited growth factor-stimulated ERK signal in HASMCs, suggesting that adiponectin acts as a modulator for vascular remodeling.     

The thyrotropin receptor is not involved in the activation of p42/p44 mitogen-activated protein kinases by thyrotropin preparations in Chinese hamster ovary cells expressing the human thyrotropin receptor.

We studied whether bovine pituitary thyrotropin (bTSH) or human recombinant thyrotropin (rhTSH) stimulated p42/p44 mitogen-activated protein kinases (MAPKs) in Chinese hamster ovary cells expressing human thyrotropin receptor (CHO-hTSHR cells). We show that p42/p44 MAPK phosphorylation was induced by both TSH preparations at similar levels in CHO-hTSHR cells and in wild-type CHO cells. In contrast, cyclic adenosine monophosphate (cAMP) production was stimulated by TSH only in CHO-hTSHR cells, demonstrating that p42/p44 MAPK stimulation was independent of the TSH receptor. Moreover, similar results were obtained with two other cell lines: the FRTL-5 thyroid cell line and the CCL39 fibroblast cell line. Maximal stimulation of p42/p44 MAPK phosphorylation was observed after a 5- to 10-minute incubation with bTSH and rhTSH preparations. At this time, the phosphorylation of GST-Elk1 was also increased in a time- and concentration-dependent manner by bTSH preparations. The phosphorylation of p42/p44 MAPKs was abolished by PD 98059 and GF 109203X, indicating the involvement of MAPK kinases (MEK 1/2) and protein kinase C. In contrast, the activation of p42/p44 MAPKs was insensitive to H89, to cholera toxin and to pertussis toxin. These data suggest that the protein kinase A pathway was not implicated in p42/p44 MAPK activation by TSH preparations. Moreover, Gs or Gi/Go proteins do not appear to participate in p42/p44 MAPK activation. We also showed that these TSH preparations failed to induce activation of c-Jun NH2 terminal kinase. We therefore conclude that the commercial TSH preparations used in this study contained factor(s) responsible for the specific activation of p42/p44 MAPKs by a TSH receptor-independent mechanism.     

Specific contribution of estrogen receptors on mitogen-activated protein kinase pathways and vascular cell activation

Randomized clinical trials have not provided conclusive data that hormone replacement therapy confers cardioprotection against coronary artery disease in postmenopausal women. However, other studies have shown that estrogens can induce beneficial effects on the vasculature. Nevertheless, the specific contribution of estrogen receptors (ERs) alpha and beta on vascular cells is not well characterized. Therefore, we used an antisense gene therapy approach to investigate the contribution of ERalpha and ERbeta on p38 and p42/44 mitogen-activated protein kinase (MAPK) activation and on vascular cell responsiveness. Treatment of porcine smooth muscle cells (PSMCs) with platelet-derived growth factor-BB induced p38 and p42/44 MAPK activation and their migration and proliferation. These effects were prevented by pretreatment with 17beta-estradiol (17betaE). The inhibitory effects of 17betaE on PSMCs were abrogated by the downregulation of ERbeta protein expression with selective ERbeta mRNA antisense oligomers, whereas the downregulation of ERalpha had no effect. However, treatment of porcine aortic endothelial cells with 17betaE promoted p38 and p42/44 MAPK phosphorylation and their migration and proliferation. These effects were ERalpha dependent, as defined by antisense gene therapy. These results suggest that in PSMCs, 17betaE reduces p42/44 and p38 MAPK activity through ERbeta stimulation, whereas in contrast, in porcine aortic endothelial cells, 17betaE induces p42/44 and p38 MAPK through ERalpha activation. 17betaE may contribute to the vascular healing process and to the prevention of restenosis by improving reendothelialization through ERalpha activation and by decreasing smooth muscle cell migration and proliferation through ERbeta stimulation.     

Role of cyclooxygenase 2, p38 and p42/44 MAPK in the secretion of prostacyclin induced by epidermal growth factor,endothelin-1 and angiotensin II in rat ventricular cardiomyocytes

We studied the respective roles of cyclooxygenases (COX) isoforms as well as the p38 and p42/44 MAP kinase cascades in angiotensin II (AngII)-, endothelin-1 (ET-1)- and epidermal growth factor (EGF)-induced prostacyclin (PGI(2)) secretion in neonatal rat ventricular cardiomyocytes. Exposure of these cells for 1 h to 100 nM AngII, ET-1 or EGF resulted in an increase in prostacyclin formation which was abolished by the COX-2 specific inhibitor NS-398 (1 microM), while the COX-1 inhibitor valeryl salicylate (5 microM) had no effect. Agonist-induced prostacyclin secretion was also abolished in the presence of cycloheximide (10 microg/ml), indicating that newly synthesized proteins are necessary for this response. In this context, the COX-2 protein amount was significantly increased following 1 h incubation of cardiomyocytes, with AngII, ET-1 and EGF. These results indicate that in cardiomyocytes AngII, ET-1 and EGF induce both the synthesis and the activity of COX-2. Investigating the role of MAPK in the stimulation of prostacyclin induced by these three agonists, we found that both the p42/44 MAPK inhibitor PD 98059 (50 microM) and the p38 MAPK blocker SB 203580 (5 microM) prevented agonist-induced PGI(2) secretion without affecting COX-2 activity or synthesis. Our results show that p42/44 and p38 MAPK activation is at the basis of AngII-, ET-1- and EGF-induced prostacyclin secretion in cardiomyocytes. They further suggest that these MAPK act on a target(s) located upstream of COX-2.     

Differential phosphorylation of p38 induced by apoptotic and anti-apoptotic stimuli in murine hepatocytes

AIM: To investigate the differential phosphorylation and activation of p38 in hepatocytes by pro-apoptotic Transforming Growth Factor-βi (TGF-β1), pro-survival factors Epidermal Growth Factor (EGF) and 12-0- tetradecanoylphorbol-13-acetate (TPA) and the potential mechanisms. METHODS: The phosphorylation and activation of p38 were determined by immunoblotting. Apoptosis was analyzed by morphological staining and observation, FACS analysis of sub-G1 content and DNA fragmentation assay. To quantitatively determine caspase activation, caspase activity assay was performed in vitro. RESULTS: TGF-β1-induced apoptosis was associated with the phosphorylation of p38, and SB202190, a specific inhibitor of p38, which was able to inhibit TGF-β1-induced caspase activation and apoptosis. TPA and EGF also blocked apoptosis induced by TGF-β1. Both of them induced the phosphorylation of p38. The results showed SB202190 had no effect on TGF-β1-induced phosphorylation of p38, but effectively inhibited both EGF and TPA-induced phosphorylation of p38. CONCLUSION: Pro-apoptotic TGF-β1, anti-apoptotic TPA and EGF induce the phosphorylation of p38 through different mechanisms that can be distinguished by SB202190. The data suggest that TPA and EGF-induced p38 phosphorylation is through an autophosphorylation-dependent mechanism. Since p38 phosphorylation induced by TGF-β1 plays an important role in caspase activation and apoptosis, TPA and EGF-induced p38 phosphorylation may not be requisite for their anti-apoptotic function.     

Comitogenic effect of catecholamines on rat cardiac fibroblasts in culture

OBJECTIVE: We studied the ability of norepinephrine and of other catecholamines to affect the proliferation of cardiac fibroblasts isolated from adult rat hearts. Furthermore, we investigated the possible adrenergic receptor involved in this process. METHODS: Norepinephrine (NE), phenylephrine (PE), isoproterenol (ISO), forskolin (FO), epidermal growth factor (EGF), platelet-derived growth factor AA (PDGF-AA) and specific inhibitors of the alpha(1)-, alpha(2)-, beta(1)- and beta(2)-adrenoceptors and of the protein kinase A (PKA) were applied to cardiac fibroblasts in culture. Cell number was measured by use of a Coulter Counter. Activation of the cAMP response element binding protein (CREB) was measured by Western blotting and subsequent use of a phospho-specific antibody. Activation of the p42- and the p44-mitogen activated protein kinase (p42/p44(MAPK)) was assessed by detection of phosphorylation shifts and by incorporation of 32P-labelled phosphate into myelin basic protein. RESULTS: Fibroblasts isolated from hearts of adult rats were grown in 10% serum-containing media which induced an increase in cell number by 94%. After 48 h, treatment with 10 microM NE caused an even greater increase in cell number by 222%, i.e. another 128% (comitogenic effect). In contrast, NE alone had no effect on the growth of serum-deprived cells. EGF and PDGF-AA did not replace serum as the basic mitogen. After addition of NE to proliferating cells under serum conditions, there was a rapid, time-dependent significant activation of the p42/p44(MAPK) and of CREB for up to 60 and 120 min, respectively. In both cases, the maximum of activation was reached after 5 min. Application of FO (0.1-20 microM) caused a strong activation of CREB, while no increase in the phosphorylation of the p42/p44(MAPK) was detected. Treatment with 20 microM FO led to an identical increase in cell number as application of NE. Specific blockade of PKA with RpcAMPS prevented the activation of CREB and also the comitogenic effect of FO as well as of NE. The alpha- and beta-adrenergic receptor blocker carvedilol (10 microM) normalized all NE-induced effects. Prazosin and yohimbine, inhibitors of alpha(1)- and alpha(2)-adrenoceptor activation, respectively, did not influence the NE-evoked increase in cell number. In contrast, the non-selective beta-adrenoceptor blocker propranolol (1 microM) completely suppressed the comitogenic effect of NE. A similar effect was obtained with the specific beta(2)-adrenoceptor blocker ICI 118,551 (5 microM), while the beta(1)-adrenoceptor blocker metoprolol did not influence the increase in cell number. CONCLUSIONS: NE elicits a comitogenic effect on cultured rat cardiac fibroblasts which is prevented by beta(2)-adrenergic blockade. The activation of CREB contributes to the increase in proliferation. The p42/p44(MAPK) which was also found to be activated by NE might as well be involved in the regulation of the comitogenic effect.     

Diurnal variation in p42/44 mitogen-activated protein kinase in the rat pineal gland

In this study, we investigated whether there was a diurnal difference in mitogen-activated protein kinase (p42/44(MAPK)) phosphorylation in the rat pineal gland. Under a lighting regimen with 12h of darkness, there was a two- to four-fold increase in phosphorylated levels of MAPK kinase 1/2 (MEK1/2) and p42/44(MAPK) 2h after onset of darkness, an increase that was sustained for 8h. The increases in phosphorylated levels of MEK1/2 and p42/44(MAPK) occurred without increases in MEK1/2 and p42/44(MAPK) proteins. When rats were treated with propranolol 1h before onset of darkness or subjected to continuous light exposure during the dark phase, the nocturnal increase in MEK1/2 and p42/44(MAPK) phosphorylation was reduced. Acute light exposure during darkness caused a decline in MEK1/2 and p42/44(MAPK) phosphorylation within 30 min of light exposure. These results indicate the presence of a diurnal difference in MEK1/2 and p42/44(MAPK) phosphorylation in the rat pineal gland that is under adrenergic control.     

The role of protein kinases A and C pathways in the regulation of mitogen-activated protein kinase activation in response to gonadotropin-releasing hormone receptor activation

There is convincing evidence that mitogen-activated protein kinase (MAPK) activation is coupled to both receptor tyrosine kinase and G protein-coupled receptors. The presence of the epidermal growth factor (EGF) receptor and the GnRH receptor on the surface of GGH(3)1' cells makes this cell line a good model for the assessment of MAPK activation by receptor tyrosine kinases and G protein-coupled receptors. In this study, to assess the activated and total (i.e. activated plus inactivated) MAPK, the phosphorylation state of p44 and p42 MAPKs was examined using antisera that distinguish phospho-p44/42 MAPK (Thr202/Tyr204) from p44/42 MAPK (phosphorylation state independent). The data show that both EGF (200 ng/ml) and Buserelin (a GnRH agonist; 10 ng/ml) provoke rapid activation of MAPK (within 5 and 15 min, respectively) after binding to their receptors. The role of protein kinase A (PKA) and protein kinase C (PKC) signal transduction pathways in mediating MAPK activation was also assessed. Both phorbol ester (phorbol 12-myristate 13-acetate; 10 ng/ml) and (Bu)2cAMP (1 mM) trigger the phosphorylation of MAPK, suggesting potential roles for PKC and PKA signaling events in MAPK activation in GGH(3)1' cells. Treatment of PKC-depleted cells with Buserelin activated MAPK, suggesting involvement of PKC-independent signal transduction pathways in MAPK activation in response to GnRH. Similarly, treatment of PKC-depleted cells with forskolin (50 microM) or cholera toxin (100 ng/ml) stimulated MAPK activation, whereas pertussis toxin (100 ng/ml) had no measurable effect. To further assess the role of PKA in response to EGF and Buserelin, cells were treated with EGF (200 ng/ml) for 3 min or with Buserelin (10 ng/ml) for 10 min after pretreatment with 3-isobutyl-1-methylxanthine (0.5 mM), forskolin (50 microM), or (Bu)2cAMP (1 mM) for 15 min. The results show that MAPK can be activated in a PKA-dependent manner in GGH(3)1' cells. Consistent with previous reports, the current data support the view that MAPK activation can be achieved via both PKC- and PKA-dependent signaling pathways triggered by the GnRH receptor that couples to G(q/11) and Gs alpha-subunit proteins. In contrast, G(i/o)alpha does not appear to participate in MAPK activation in GGH(3)1' cells.     

Role of p42/p44 mitogen-activated-protein kinase and p21waf1/cip1 in the regulation of vascular smooth muscle cell proliferation by nitric oxide

The purpose of this study was to determine the involvement of the p42/p44 mitogen-activated protein kinase (MAPK) pathway and induction of p21(waf1/cip1) in the antiproliferative effects of nitric oxide (NO) on rat aortic smooth muscle cells (RASMC). NO, like alpha-difluoromethylornithine (DFMO), interferes with cell proliferation by inhibiting ornithine decarboxylase (ODC) and, therefore, polyamine synthesis. S-nitroso-N-acetylpenicillamine or (Z)-1-[N-(2-aminoethyl)-N-(2-aminoethyl)-amino]-diazen-1-ium-1,2-diolate inhibited RASMC growth at concentrations as low as 3 microM, and DFMO elicited effects at concentrations of 100 microM or greater. The cytostatic effect of NO and DFMO was prevented by the MAPK kinase 1/2 inhibitors PD 098,059 or U0126. This finding suggests that the p42/p44 MAPK pathway is involved in the inhibition of RASMC proliferation by NO. Western blot analysis revealed that treatment of RASMC with NO or DFMO leads to activation of p42/p44 MAPK and induction of p21(waf1/cip1). This effect was prevented by MAPK kinase 1/2 inhibitors, suggesting that induction of p21(waf1/cip1) depended on activation of p42/p44. Moreover, activation of p42/p44 and induction of p21(waf1/cip1) were prevented by exogenous putrescine but not ornithine, suggesting this effect was due to the inhibition of ODC by NO or DFMO. Finally, activation of p42/p44 MAPK and induction of p21(waf1/cip1) were cGMP-independent. Neither 1H-(1,2,4)oxadiazolo[4,3-alpha]quinoxalin-1-one nor zaprinast influenced the cytostatic effect of NO or DFMO or their ability to activate these signal transduction pathways. These observations suggest that inhibition of ODC and accompanying putrescine production are the underlying mechanisms by which NO and DFMO activate the MAPK pathway to promote induction of p21(waf1/cip1) and consequent inhibition of cell proliferation.     

雌激素膜快速效应对兔血管平滑肌细胞iNOS快速激活的影响

目的 探讨17β-雌二醇（17β-estradiol,E2）通过快速激活诱导型一氧化氮合成酶（iNOS）的活性,调节细胞外信号调节激酶（mitogen-activated protein kinase,p42/44 MAPK）表达抑制血管平滑肌细胞（vascular smooth muscle cells,VSMCs）增殖的机制。方法 在培养的VSMCs的基础上,采用放射免疫测定法(RIA)和Western blot检测E2预处理前后胎牛血清（fetal calf serum,FCS）对VSMCs中诱导型iNOS的活性及磷酸化p42/44 MAPK蛋白和p42/44 MAPK总蛋白表达的影响。采用比色法检测E2预处理后VSMCs中NO含量的变化。结果 E2预处理5 min,即可逆转FCS诱导的iNOS活性下降,此效应在E2预处理15 min时达到高峰,处理30 min后此效应减弱。基因转录抑制剂放线菌素D（actinomycin D,Act D）预处理后对此过程无影响,而三苯氧胺预处理可明显逆转E2诱导的iNOS活性增加。比色法检测上清液中NO的含量显示,E2预处理15 min,NO的含量明显增加。Western blot的结果显示,E2预处理15 min,可明显抑制磷酸化p42/44 MAPK蛋白的表达,而对总p42/44 MAPK蛋白的表达无影响。NO合酶(NOS)抑制剂L-硝基左旋精氨酸甲酯(L-NAME)预处理,可逆转E2诱导的磷酸化p42/44 MAPK的表达下降。结论 E2可通过快速激活iNOS增加NO释放,下调磷酸化p42/44 MAPK的活性,抑制VSMCs增殖。