Preliminary evidence of effectiveness of TECAR in lymphedema.

Lymphedema of the lower limbs often contributes to the mobility impairment of morbidly obese patients. Defining novel costeffective protocols is important for reducing treatment costs. The study aimed to assess if Capacitive and Resistive Energy Transfer (TECAR) can reduce edema and the minimum number of sessions needed to observe volume reduction. Forty-eight severely obese subjects (age range: 46-78 years; BMI >40 kg/m2) with bilateral lower limb lymphedema were divided into three groups undergoing either manual lymphatic drainage, pressure therapy, or TECAR, in addition to a multidisciplinary rehabilitation program. They were compared to a control group composed by 12 women (age: 67.4 ± 8.9 years, BMI: 44.6 ± 4.1 Kg/m2) undergoing only the rehabilitation program. A handheld laser scanner 3D system was used for volume measurements. In addition, patients were evaluated with a Timed Up and Go (TUG) test and pain/heaviness of the lower limbs with a Visual Analog Scale (VAS). A significant volume reduction was observed after 6 sessions of TECAR: specifically, in the whole limb (PRE: 9.7+2.8 dm3; POST: 9.4+2.8 dm3; p<0.05) and in the thigh (PRE: 3.5+1.3 dm3; POST: 3.3+1.2 dm3; p<0.05). The TUG and VAS for pain showed a significant improvement in all groups. Our preliminary results suggest that TECAR can provide a relatively early reduction of lower limb edema with improvement of patients' function and pain.

Crucial role of c-Jun NH2-terminal kinase 1 (JNK1) in cold-restraint stress-induced gastric lesions in mice.

c-Jun NH2-terminal kinase 1 /JNK1, is activated in response to a broad array of cellular stresses. We investigated the role of JNK1 in the pathophysiology of cold-restraint stress-induced gastric lesions in mice. B6/129, wild type (WT) mice, or mutant mice deficient in Jnk1 (Jnk1-/- mice) were exposed to cold-restraint stress for different time periods. Gastric lesions were identified and quantitated by morphometric analysis. JNK1 activity in mucosal homogenates was quantitated by immunoprecipitation and in-vitro kinase assays. JNK1 expression and Akt activation were assessed by Western blots with anti-JNK1 and anti-phospho Akt antibodies, respectively. Gastric mucosal homogenates from Jnk1-/- mice exhibited no significant expression of JNK1 and no detectable level of JNK1 activation. Exposure of WT mice to cold-restraint stress led to the development of significant gastric lesions and to a greater than three-fold induction in JNK1 activity, while no lesions were detected in the gastric mucosa of Jnk1-/- mice. Since cold-restraint stress-induced gastric lesions involve the activation of cholinergic pathways, we tested the effect of atropine on both the development of gastric lesions and JNK1 activation. Pretreatment of WT mice with atropine completely inhibited both cold-restraint stress-induced lesions and JNK1 activation. Cold-restraint stress induced protein kinase B/Akt to a similar level in the gastric mucosa of both WT and Jnk1-/- mice indicating the integrity of other signaling pathways. JNK1 plays a key role in the development of cold-restraint stress-induced gastric lesions in mice through the activation of cholinergic, atropine sensitive pathways.

Accuracy of stool antigen test in posteradication assessment of Helicobacter pylori infection.

Our aim was to evaluate the accuracy of HpSA test in the diagnosis of Helicobacter pylori infection after the end of eradication therapy. In all 106 H. pylori-positive patients (55 men and 51 women, mean age 51 years, range 19-82) were treated with a course of eradicating regimen. [13C]Urea breath test (UBT) and HpSA were performed four weeks after stopping the treatment. The diagnostic accuracy of HpSA was evaluated in comparison with the results of [13C]UBT. In 90 patients (85%) H. pylori was eradicated according to [13C]urea breath test. After eradication, sensitivity of HpSA was 87.5%, specificity 95.5%, positive predictive value 77.8%, negative predictive value 97.7%, and diagnostic accuracy 94.3%. HpSA is a valuable test in the posteradication assessment of H. pylori infection.

TNF-alpha and interleukin 1 activate gastrin gene expression via MAPK- and PKC-dependent mechanisms.

Helicobacter pylori and proinflammatory cytokines have a direct stimulatory effect on gastrin release from isolated G cells, but little is known about the mechanism by which these factors regulate gastrin gene expression. We explored whether tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 directly regulate gastrin gene expression and, if so, by what mechanism. TNF-alpha and IL-1 significantly increased gastrin mRNA in canine G cells to 181 +/- 18% and 187 +/- 28% of control, respectively, after 24 h of treatment. TNF-alpha and IL-1 stimulated gastrin promoter activity to a maximal level of 285 +/- 12% and 415 +/- 26% of control. PD-98059 (a mitogen-activated protein kinase kinase inhibitor), SB-202190 (a p38 kinase inhibitor), and GF-109203 (a protein kinase C inhibitor) inhibited the stimulatory action of both cytokines on the gastrin promoter. In conclusion, both cytokines can directly regulate gastrin gene expression via a mitogen-activated protein kinase- and protein kinase C-dependent mechanism. These data suggest that TNF-alpha and IL-1 may play a direct role in Helicobacter pylori-induced hypergastrinemia.

Functional role of protein kinase B/Akt in gastric acid secretion.

Epidermal growth factor (EGF) stimulates gastric acid secretion and H(+)/K(+)-ATPase alpha-subunit gene expression. Because EGF activates the serine-threonine protein kinase Akt, we explored the role of Akt in gastric acid secretion. Akt phosphorylation and activation were measured by kinase assays and by Western blots with an anti-phospho-Akt antibody, using lysates of purified (>95%) canine gastric parietal cells in primary culture. EGF induced Akt phosphorylation and activation, whereas carbachol had no effect. LY294002, an inhibitor of phosphoinositide 3-kinase, completely blocked EGF induction of Akt phosphorylation, whereas the MEK1 inhibitor PD98059 and the protein kinase C inhibitor GF109203X had no effect. We examined the role of Akt in H(+)/K(+)-ATPase gene expression by Northern blotting using a canine H(+)/K(+)-ATPase alpha-subunit cDNA probe. The parietal cells were transduced with a multiplicity of infection of 100 of the adenoviral vector Ad.Myr-Akt, which overexpresses a constitutively active Akt gene, or with the control vector Ad.CMV-beta-gal, which expresses beta-galactosidase. Ad.Myr-Akt induced H(+)/K(+)-ATPase alpha-subunit gene expression 3-fold, whereas it failed to stimulate the gene cyclooxygenase-2, which was potently induced by carbachol in the same parietal cells. Ad.Myr-Akt induced aminopyrine uptake 4-fold, and it potentiated the stimulatory action of carbachol 3-fold. In contrast, Ad.Myr-Akt failed to induce changes in either parietal cell actin content, measured by Western blots with an anti-actin antibody or in the organization of the actin cellular cytoskeleton, visualized by fluorescein phalloidin staining and confocal microscopy. Transduction of the parietal cells with a multiplicity of infection of 100 of the adenoviral vector Ad.dom.neg.Akt, which overexpresses an inhibitor of Akt, blocked the stimulatory effect of EGF on both aminopyrine uptake and H(+)/K(+)-ATPase production, measured by Western blots with an anti-H(+)/K(+)-ATPase alpha-subunit antibody. Thus, EGF induces a cascade of events in the parietal cells that results in the activation of Akt. The functional role of Akt appears to be stimulation of gastric acid secretion through induction of H(+)/K(+)-ATPase expression.

Molecular mechanisms for the antiapoptotic action of gastrin.

Gastrin (G17) has a CCK-B receptor-mediated growth-promoting effect on the AR42J rat acinar cell line. We examined whether G17 inhibits apoptosis induced by serum withdrawal of AR42J cells and CHO-K1 cells stably expressing CCK-B receptors (CHO-K1/CCK-B cells). Cellular apoptosis was measured by flow cytometry and the terminal deoxynucleotidyltransferase-mediated dUTP-FITC nick end-labeling method. Serum withdrawal induced AR42J and CHO-K1/CCK-B cell apoptosis. Addition of 10 nM G17 reversed these effects. We examined the action of G17 (10 nM) on phosphorylation and activation of protein kinase B/Akt, a kinase known to promote cell survival. Akt phosphorylation and activation were measured by kinase assays and Western blots with an anti-phospho-Akt antibody. G17 stimulated Akt phosphorylation and activation. G17 induction of Akt phosphorylation was inhibited by the phosphoinositide 3-kinase (PI 3-kinase) inhibitors LY-294002 (10 microM) and wortmannin (200 nM) but not by the mitogen-activated protein kinase kinase 1 inhibitor PD-98059 (50 microM). To study the role of p38 kinase in G17 signaling to Akt, we examined the effect of G17 on p38 kinase activation and phosphorylation using kinase assays and Western blots with an anti-phospho-p38 kinase antibody. G17 induced p38 kinase activity at doses and with kinetics similar to those observed for Akt induction. The p38 kinase inhibitor SB-203580 inhibited G17 induction of Akt phosphorylation and activation at a concentration (10 microM) 10-fold higher than necessary to block p38 kinase (1 microM), suggesting the possible involvement of kinase activities other than p38 kinase. Transduction of AR42J cells with the adenoviral vector Adeno-dn Akt, which overexpresses an inhibitor of Akt, reversed the antiapoptotic action of G17. In conclusion, G17 promotes AR42J cell survival through the induction of Akt via PI 3-kinase and SB-203580-sensitive kinase activities.

Accuracy of the stool antigen test in the diagnosis of Helicobacter pylori infection before treatment and in patients on omeprazole therapy.

AIM: To evaluate the Helicobacter pylori stool antigen (HpSA) test in the assessment of H. pylori infection and the effect of omeprazole treatment on its accuracy. METHODS: Study 1: 140 dyspeptic patients were enrolled in the study and defined as H. pylori positive if histology and rapid urease test, or culture alone were positive. HpSA was performed on all patients and 13C-urea breath test (UBT) on 87. Study 2: 75 patients testing positive using both UBT and HpSA, were given omeprazole 20 mg for 2 weeks (Group A) or omeprazole 40 mg for 2 weeks (Group B), or OAC for 1 week (group C). A Helicobacter pylori stool antigen test was performed on all patients on days 3, 5, 7 and 14 during treatment, and also on days 7 and 14 post-treatment in groups A and B. UBT was performed in groups A and B on days 7 and 14 during treatment, and days 7 and 14 post-treatment. RESULTS: 80/140 patients were H. pylori positive. The sensitivity and specificity of HpSA were 93.8 and 90%, similar to UBT (93.9 and 92.1%). Omeprazole significantly reduced both HpSA and UBT values, resulting in a decreased accuracy. Of 25 patients receiving 20 mg omeprazole, HpSA gave 5 and 6 false negatives after 7 and 14 days treatment respectively, while UBT gave 4 and 7 false negatives after 7 and 14 days treatment. Of 25 patients receiving 40 mg omeprazole, HpSA gave 7 and 9 false negatives after 7 and 14 days of treatment, while UBT gave 8 and 9 false negatives after 7 and 14 days of treatment. Two weeks after stopping omeprazole treatment, the HpSA and UBT were positive in all cases. CONCLUSIONS: The Helicobacter pylori stool antigen test is valuable in the assessment of H. pylori infection. Short-term omeprazole treatment decreases the accuracy of both HpSA and UBT in a similar manner.

Antibody response to Helicobacter pylori CagA and heat-shock proteins in determining the risk of gastric cancer development.

OBJECTIVE: To investigate whether the systemic antibody response to Helicobacter pylori heat shock protein B can be considered, in addition to anti cytotoxin-associated protein [CagA) antibody determination, a further serological marker of increased risk of gastric cancer development. METHODS: A total of 98 Giemsa positive Helicobacter pylori patients (28 with gastric cancer, 30 with duodenal ulcer and 40 with nonulcer dyspepsia) were studied. Serum samples obtained from all patients were tested for IgG antibodies to CagA (116 kDa), VacA [89kDa) and heat skock protein B (54 kDa) antigens of Helicobacter pylori by the Western blot technique. RESULTS: 26/28 patients [(92.9% with gastric carcinoma, 29/30 patients [96.7%) with duodenal ulcer and 30/40 patients (75.0%) with non-ulcer dyspepsia were seropositive for CagA protein. The prevalence of serum IgG antibody to CagA in the cancer patients was not significantly higher than in duodenal ulcer and non-ulcer dyspepsia patients. The prevalence of antibodies to VacA was not significantly different between gastric carcinoma and non-ulcer dyspepsia patients. In contrast the prevalence of systemic antibodies to heat skock protein B was significantly higher in gastric cancer patients (78.6%) than in duodenal ulcer (36.7%, p=0.002) or nonulcer dyspepsia patients (52.5%, p=0.029). CONCLUSIONS: The detection of antibodies to heat shock protein B is proposed as an additional test which, in association with the determination of serum antibodies to CagA, could help in determining the risk of developing severe gastroduodenal disease, and gastric cancer, in particular.

The human histamine H(2) receptor regulates c-jun and c-fos in a differential manner.

Previously, we demonstrated that activation of the human H(2) receptor (hH(2)R) leads to an increase in c-fos transcription and cell proliferation. The purpose of these studies was to examine whether hH(2)R regulates c-jun expression and, if so, explore the mechanisms by which it does so. Histamine induced an increase in c-jun mRNA in human embryonic kidney cells stably transfected with the hH(2)R (maximal effect: 554.6 +/- 86.8% of control). The protein kinase C (PKC) inhibitors staurosporine (10(-6) M) and GF-109203X (10(-6) M) significantly inhibited histamine-stimulated c-fos mRNA while not altering c-jun expression. The protein kinase A (PKA) pathway inhibitors Rp-cAMP and protein kinase inhibitor did not affect the action of histamine on c-jun or c-fos mRNA. Histamine (10(-4) M) stimulated extracellularly regulated kinase 2 tyrosine phosphorylation. The specific inhibitor of the mitogen-activated protein (MAP) kinase pathway, PD-98059 (5 x 10(-5) M), significantly inhibited histamine-induced c-fos and c-jun mRNA. Of interest, the p70 S6 kinase inhibitor rapamycin (10(-6) M) but not wortmannin decreased histamine-stimulated c-jun mRNA by 58.5 +/- 12% (mean +/- SE, n = 4) while not significantly altering c-fos message. Histamine (10(-4) M) also led to an approximately 4.5-fold increase in Jun NH(2)-terminal kinase activity in a PKC-, PKA-, and MAP kinase-independent but rapamycin-sensitive manner. Our findings suggest that histamine stimulates both c-fos and c-jun mRNA in a differential manner. PKC is involved in histamine-mediated c-fos activation, whereas p70 S6 kinase is important for linkage of this receptor to c-jun.

Regulation and function of p38 protein kinase in isolated canine gastric parietal cells.

We examined the regulation and functional role of p38 kinase in gastric acid secretion. p38 kinase was immunoprecipitated from cell lysates of highly purified gastric parietal cells in primary culture, and its activity was quantitated by in vitro kinase assay. Carbachol effects were dose- and time-dependent, with a maximal 10-fold stimulatory effect detected after 30 min of incubation. SB-203580, a highly selective inhibitor of p38 kinase, blocked carbachol induction of p38 kinase activity, with maximal inhibition at 10 microM. Stimulation by carbachol was unaffected by preincubation of parietal cells with the intracellular Ca(2+) chelator BAPTA-AM, but incubation of cells in Ca(2+)-free medium led to a 50% inhibition of carbachol induction of p38 kinase activity. Because some of the effects of carbachol are mediated by the small GTP-binding protein Rho, we examined the role of Rho in carbachol induction of p38 kinase activity. We tested the effect of exoenzyme C3 from Clostridium botulinum (C3), a toxin known to ADP-ribosylate and specifically inactivate Rho. C3 led to complete ADP-ribosylation of Rho, and it inhibited carbachol induction of p38 kinase by 50%. We then tested the effect of SB-203580 and C3 on carbachol-stimulated uptake of [(14)C]aminopyrine (AP). Inhibition of p38 kinase by SB-203580 led to a dose-dependent increase in AP uptake induced by carbachol, with maximal (threefold) effect at 10 microM SB-203580. Similarly, preincubation of parietal cells with C3 led to a twofold increase in AP uptake induced by carbachol. Thus carbachol induces a cascade of events in parietal cells that results in activation of p38 kinase through signaling pathways that are at least in part dependent on Rho activation and on the presence of extracellular Ca(2+). p38 kinase appears to inhibit gastric acid secretion.

EGF stimulates gastrin promoter through activation of Sp1 kinase activity.

Epidermal growth factor (EGF) receptor activation stimulates gastrin gene expression through a GC-rich element called gastrin EGF response element (gERE). This element is bound by Sp1 family members and is a target of the ras-extracellular signal-regulated kinase (Erk) signal transduction cascade. This raised the possibility that Sp1 may be phosphorylated by kinases of this signaling pathway. Erk is capable of phosphorylating other mitogen-inducible transcription factors, e.g., Elk and Sap, suggesting that Erk may also mediate EGF-dependent phosphorylation of Sp1. This possibility was tested by studying Sp1-dependent kinase activity in extracts prepared from EGF-activated AGS cells by use of solid-phase kinase assays and immunoprecipitation of metabolically labeled Sp1. The results revealed that Sp1 kinase activity (like gastrin promoter activation) is inhibited by PD-98059 and, therefore, is dependent on mitogen-activated protein kinase kinase 1 (Mek 1). However, EGF-dependent activation of endogenous Erk did not account for most of the Sp1 kinase activity, since Erk and additional Sp1 kinase activity analyzed in a solid-phase kinase assay eluted from an ion-exchange column in different fractions. Phosphoamino acid analysis of in vivo radiolabeled Sp1 demonstrated that the kinase phosphorylates Sp1 on Ser and Thr in response to EGF. Therefore, most EGF-stimulated Sp1 kinase activity is Mek 1 dependent and distinct from Erk.

Helicobacter pylori stool antigen test: clinical evaluation and cost analysis of a new enzyme immunoassay.

Noninvasive tests for Helicobacter pylori are increasingly used. Recently, an enzyme immunoassay for H. pylori detection in feces has been put on the market. Aim of this multicenter study was to evaluate the usefulness of this novel test as a predictor of H. pylori status in the pretreatment setting. Three hundred consecutive patients were enrolled. None of the patients had received any eradicating treatment in the last 12 months, and all underwent gastroscopy with biopsies of the antrum and body for histology (H) and rapid urease test (RUT). H. pylori status was defined positive (or negative) if both H and RUT were positive (or negative). When H and RUT gave conflicting results, the patients were classified as H. pylori-indeterminate. A stool specimen was collected for each patient and tested by using a novel enzyme immunoassay for H. pylori detection (HpSAT). Sensitivity, specificity, and diagnostic accuracy of the test were calculated, as was the cost of each assay. H. pylori status was positive in 159 patients, negative in 131, and indeterminate in 10. HpSAT gave evaluable results (positive or negative) in 293 patients, and doubtful results in 7 (2.3%). Sensitivity, specificity, and diagnostic accuracy of HpSAT were 96.8%, 89.7%, and 93.6% respectively. Considering the H. pylori-indeterminate patients as positive, the percentages were 95.8%, 98.7%, and 93.2% respectively. The cost for each assay was about US $27. These results suggest that HpSAT is a noninvasive, simple, reliable, fast, and cheap method for evaluating H. pylori status in the pretreatment setting.

Carbachol activates IkappaB kinase in isolated canine gastric parietal cells.

IkappaB kinase (IKK) is a recently discovered kinase complex composed of the kinases IKKalpha and beta, which plays a crucial role in the activation of NF-kappaB. In this study we examined the regulation of IKK by carbachol in isolated gastric parietal cells. IKKalpha and beta activities were measured by immune complex kinase assay. Carbachol induced both IKK alpha and beta in a time-dependent fashion, with a maximal stimulatory effect detected after 5 min of incubation. The action of carbachol was inhibited by the intracellular Ca(++) chelator BAPTA-AM, the PKC inhibitor GF109203X, and the NF-kappaB inhibitor PDTC. Carbachol also induced degradation of IkappaBalpha, which was reversed by addition of both GF109203X and PDTC and stimulated the activity of a NF-kappaB-luciferase reporter gene plasmid in COS-7 cells stably expressing the human M3 muscarinic receptor. In conclusion, carbachol induces IKK in the parietal cells via intracellular Ca(++)- and PKC-dependent signaling pathways. This observation represents a novel mechanism for the regulation of NF-kappaB through the activation of seven transmembrane G-protein-coupled receptors.

Glycine-extended gastrin regulates HEK cell growth.

Posttranslational processing of progastrin to a carboxy terminally amidated form (G-NH(2)) is essential for its effect on gastric acid secretion and other biological effects mediated by gastrin/CCK-B receptors. The immediate biosynthetic precursor of G-NH(2), glycine-extended gastrin (G-Gly), does not stimulate gastric acid secretion at physiological concentrations but is found in high concentrations during development. G-NH(2) and G-Gly have potent growth stimulatory effects on gastrointestinal tissues, and G-NH(2) can stimulate proliferation of human kidney cells. Thus we sought to explore the actions of G-NH(2) and G-Gly on the human embryonic kidney cell line HEK 293. HEK 293 cells showed specific binding sites for (125)I-labeled Leu(15)-G17-NH(2) and (125)I-Leu(15)-G(2-17)-Gly. Both G-NH(2) and G-Gly induced a dose-dependent increase in [(3)H]thymidine incorporation, and both peptides together significantly increased [(3)H]thymidine incorporation above the level of either peptide alone. G-NH(2) and G-Gly were detected by radioimmunoassay in serum-free conditioned media. Antibodies directed against G-NH(2) and G-Gly lead to a significant reduction in [(3)H]thymidine incorporation. G-NH(2) but not G-Gly increased intracellular Ca(2+) concentration. We conclude that G-NH(2) and G-Gly act cooperatively via distinct receptors to stimulate the growth of a nongastrointestinal cell line (HEK 293) in an autocrine fashion.

Glycine-extended gastrin exerts growth-promoting effects on human colon cancer cells.

BACKGROUND: Since human colon cancers often contain significant quantities of progastrin-processing intermediates, we sought to explore the possibility that the biosynthetic precursor of fully processed amidated gastrin, glycine-extended gastrin, may exert trophic effects on human colonic cancer cells. MATERIALS AND METHODS: Binding of radiolabeled glycine-extended and amidated gastrins was assessed on five human cancer cell lines: LoVo, HT 29, HCT 116, Colo 320DM, and T 84. Trophic actions of the peptides were assessed by increases in [3H]thymidine incorporation and cell number. Gastrin expression was determined by northern blot and radioimmunoassay. RESULTS: Amidated gastrin did not bind to or stimulate the growth of any of the five cell lines. In contrast, saturable binding of radiolabeled glycine-extended gastrin was seen on LoVo and HT 29 cells that was not inhibited by amidated gastrin (10(-6) M) nor by a gastrin/CCKB receptor antagonist (PD 134308). Glycine-extended gastrin induced a dose-dependent increase in [3H]thymidine uptake in LoVo (143 +/- 8% versus control at 10(-10) M) and HT 29 (151 +/- 11% versus control at 10(-10) M) cells that was not inhibited by PD 134308 or by a mitogen-activated protein (MAP) or ERK kinase (MEK) inhibitor (PD 98509). Glycine-extended gastrin did stimulate jun-kinase activity in LoVo and HT 29 cells. The two cell lines expressed the gastrin gene at low levels and secreted small amounts of amidated gastrin and glycine-extended gastrin into the media. CONCLUSIONS: Glycine-extended gastrin receptors are present on human colon cancer cells that mediate glycine-extended gastrin's trophic effects via a MEK-independent mechanism. This suggests that glycine-extended gastrin and its novel receptors may play a role in colon cancer cell growth.

Cell type-specific requirement of the MAPK pathway for the growth factor action of gastrin.

Gastrin (G17) has a CCKB receptor-mediated growth-promoting effect on the AR42J rat acinar cell line that is linked to induction of both mitogen-activated protein kinase (MAPK) and c-fos gene expression. We investigated the mechanisms that regulate the growth factor action of G17 on the rat pituitary adenoma cell line GH3. Both AR42J and GH3 cells displayed equal levels of CCKB receptor expression and similar binding kinetics of 125I-labeled G17. G17 stimulation of cell proliferation was identical in both cell lines. G17 stimulation of GH3 cell proliferation was completely blocked by the CCKB receptor antagonist D2 but not by the MEK inhibitor PD-98059 or the protein kinase C inhibitor GF-109203X, which completely inhibited G17 induction of AR42J cell proliferation. G17 induced a c-fos SRE-luciferase reporter gene plasmid more than fourfold in the AR42J cells, whereas it had no effect in the GH3 cells. In contrast to what we observed in the AR42J cells, G17 failed to stimulate MAPK activation and Shc tyrosyl phosphorylation and association with the adapter protein Grb2. Epidermal growth factor induced the MAPK pathway in the GH3 cells, demonstrating the integrity of this signaling system. G17 induced Ca2+ mobilization in both the GH3 and AR42J cells. The calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide inhibited AR42J cell proliferation by 20%, whereas it completely blocked G17 induction of GH3 cell growth. The Ca2+ ionophore ionomycin stimulated GH3 cell proliferation to a level similar to that observed in response to G17, but it had no effect on AR42J cell proliferation. Thus there are cell type specific differences in the requirement of the MAPK pathway for the growth factor action of G17. Whereas in the AR42J cells G17 stimulates cell growth through activation of MAPK and c-fos gene expression, in the GH3 cells, G17 fails to activate MAPK, and it induces cell proliferation through Ca2+-dependent signaling pathways. Furthermore, induction of Ca2+ mobilization in the AR42J cells appears not to be sufficient to sustain cell proliferation.

Carbachol activates ERK2 in isolated gastric parietal cells via multiple signaling pathways.

We previously reported that both carbachol and epidermal growth factor (EGF) are potent inducers of the extracellular signal-regulated protein kinases (ERKs) in isolated gastric canine parietal cells and that induction of these kinases leads to acute inhibitory and chronic stimulatory effects on gastric acid secretion. In this study we investigated the molecular mechanisms responsible for these effects. Both carbachol (100 microM) and EGF (10 nM) induced Ras activation. The role of Ras in ERK2 induction was examined by transfecting parietal cells with a vector expressing hemoagglutinin (HA)-tagged ERK2 (HA-ERK2) together with a dominantly expressed mutant (inactive) ras gene. HA-ERK2 activity was quantitated by in-gel kinase assays. Dominant negative Ras reduced carbachol induction of HA-ERK2 activity by 60% and completely inhibited the stimulatory effect of EGF. Since Ras activation requires the assembly of a multiprotein complex, we examined the effect of carbachol and EGF on tyrosyl phosphorylation of Shc and its association with Grb2 and the guanine nucleotide exchange factor Sos. Western blot analysis of anti-Shc immunoprecipitates with an anti-phosphotyrosine antibody demonstrated that both carbachol and EGF induced tyrosyl phosphorylation of a major 52-kDa shc isoform. Grb2 association with Shc was demonstrated by blotting Grb2 immunoprecipitates with an anti-Shc antibody. Probing of anti-Sos immunoprecipitates with an anti-Grb2 antibody revealed that Sos was constitutively bound to Grb2. To examine the functional role of Sos in ERK2 activation, we transfected parietal cells with the HA-ERK2 vector together with a dominantly expressed mutant (inactive) sos gene. Dominant negative Sos did not affect carbachol stimulation of HA-ERK2 but inhibited the stimulatory effect of EGF by 60%. We then investigated the role of betagamma-subunits in carbachol induction of HA-ERK2. Parietal cells were transfected with the HA-ERK2 vector together with a vector expressing the carboxy terminus of the beta-adrenergic receptor kinase 1, known to block signaling mediated by betagamma-subunits. In the presence of this vector, carbachol induction of HA-ERK2 was inhibited by 40%. Together these data suggest that, in the gastric parietal cells, carbachol activates the ERKs through Ras- and betagamma-dependent mechanisms that require guanine nucleotide exchange factors other than Sos.

Gastrin induces c-fos gene transcription via multiple signaling pathways.

We previously observed that the trophic actions of gastrin (G17) on the AR42J rat acinar cell line are mediated by mitogen-activated protein kinase (MAPK)-induced c-fos gene transcription via protein kinase C (PKC)-dependent and -independent pathways. In this study, we further investigated the signaling pathways that target c-fos in response to G17. G17 led to a sixfold induction in luciferase activity in cells transfected with plasmids containing the -356+109 sequence of the murine c-fos promoter, which includes the Sis-inducible element (SIE), serum response element (SRE), and the Ca2+/cAMP response element (CRE) regulatory elements. Addition of either the selective PKC inhibitor GF-109203X or the MAPK/extracellular signal-regulated kinase inhibitor PD-98059 resulted in an 80% reduction in luciferase activity. G17 induced the transcriptional activity of both Elk-1 and Sap-1a, transcription factors that bind to the E26 transformation specific (Ets) DNA sequence of the SRE, and this effect was inhibited by both GF-109203X and PD-98059. Point mutations in the Ets sequence led to a 4-fold induction of c-fos transcription stimulated by G17 and to a 1.3-fold induction in response to epidermal growth factor (EGF). In contrast, mutations in the CA rich G (CArG) sequence of the SRE prevented transcriptional activation by both G17 and EGF. G17 induction of the Ets mutant construct was unaffected by either GF-109203X or PD-98059. Because activation of the SRE involves the small GTP-binding protein Rho A, we examined the role of Rho A in G17 induction of c-fos transcription. Inactivation of Rho A by either the specific inhibitor C3 or by expression of a dominant negative Rho A gene inhibited G17 induction of both the wild-type and the Ets mutant constructs by 60%. C3 also inhibited G17-stimulated AR42J cell proliferation. Thus G17 targets the c-fos promoter CArG sequence via Rho A-dependent pathways, and Rho A appears to play an important role in the regulation of the trophic action of G17.

Sp1 phosphorylation by Erk 2 stimulates DNA binding.

EGF stimulates gene expression through a variety of signal transduction pathways that include the ras-Erk pathway. We have shown previously that EGF receptor activation stimulates gastrin gene expression through a GC-rich element called gERE. This element binds Sp1 family members and raises the possibility that the ras-Erk signal transduction cascade may target this novel EGF responsive element. Moreover, it is known that Erk 2 is capable of phosphorylating other mitogen-inducible transcription factors, e.g., Elk, Sap suggesting that Erk may also inducibly phosphorylate Sp1. To test this hypothesis directly using cotransfection experiments, we show that ras and Erk 2 activation indeed target the gERE element. The Mek 1 kinase inhibitor, PD98059, blocks 50% of EGF-inducible gastrin promoter activity. Pretreatment of the extracts with recombinant Erk2 stimulated Sp1 binding; whereas dephosphorylation reduced but did not eliminate Sp1 binding. Together, these studies demonstrate the novel finding that inducible binding of Sp1 is regulated by its state of phosphorylation. Further, gastrin promoter activation is mediated in part by the ras-Erk signaling cascade that targets Sp1.