Advertisement
Research ArticleArticle

Corticotropin-Releasing Hormone Induces Vascular Endothelial Growth Factor Release from Human Mast Cells via the cAMP/Protein Kinase A/p38 Mitogen-Activated Protein Kinase Pathway

Jing Cao, Curtis L. Cetrulo and Theoharis C. Theoharides
Molecular Pharmacology March 2006, 69 (3) 998-1006; DOI: https://doi.org/10.1124/mol.105.019539

Abstract

Mast cells are involved in allergic reactions but also in innate immunity and inflammation. Corticotropin-releasing hormone (CRH), the key regulator of the hypothalamic-pituitary-adrenal axis, also has proinflammatory effects, apparently through mast cells. We showed recently that CRH selectively stimulates human leukemic mast cells and human umbilical cord blood-derived mast cells to release newly synthesized vascular endothelial growth factor (VEGF) without release of either preformed mediators or cytokines. This effect was mediated through the activation of CRH receptor-1 and adenylate cyclase with increased intracellular cAMP. However, the precise mechanism by which CRH induces VEGF secretion has not yet been defined. Here, we show that CRH-induced VEGF release was dose-dependently inhibited by the specific protein kinase A inhibitor N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89) or the p38 mitogen-activated protein kinase (MAPK) inhibitor 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580) but not by the specific inhibitor 2′-amino-3′-methoxyflavone (PD98059) of mitogen-activated protein kinase kinase, the upstream kinase of the extracellular signal-regulated protein kinase (ERK) or the c-Jun N-terminal kinase (JNK) inhibitor 1,9-pyrazoloanthrone anthra-(1,9-cd)pyrazol-6(2H)-one (SP600125). Furthermore, CRH significantly increased protein kinase A activity, which could be mimicked by the cell-permeable cAMP analog 8-bromo-cAMP, and was blocked by H89 or the adenylate cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purine-6-amine (SQ22536). CRH also induced rapid phosphorylation of p38 MAPK, which was mimicked by 8-bromo-cAMP and was inhibited by H89 or SB203580. CRH did not stimulate ERK or JNK phosphorylation and did not increase intracellular calcium levels. These results indicate that CRH induces VEGF release in human mast cells via selective activation of the cAMP/protein kinase A/p38 MAPK signaling pathway, thereby providing further insight into the molecular mechanism of how CRH affects the release of a key proinflammatory mediator.

Corticotropin-releasing hormone (CRH), mainly produced in hypothalamus, is a 41 amino acid peptide that regulates endocrine and behavioral responses to stress through the activation of the hypothalamic-pituitary-adrenal axis, which down-regulates immune responses (Karalis et al., 1997). CRH, however, is also secreted peripherally and has proinflammatory effects possibly mediated through stimulation of mast cells (Theoharides et al., 1998). The cellular effects of CRH are initiated by the binding and activation of two main types of receptors, CRH-R1 and CRH-R2, which belong to G protein-coupled seven-transmembrane receptors (Grammatopoulos and Chrousos, 2002; Wiesner et al., 2003). We showed recently that mast cells express CRH-R1, the stimulation of which by CRH leads to the selective release of vascular endothelial growth factor (VEGF) through the activation of adenylate cyclase and increased cAMP (Cao et al., 2005). The exact mechanisms of VEGF release, however, have not yet been fully clarified.

CRH receptors are linked to a number of intracellular signaling pathways, including ligand-dependent increase of intracellular cAMP and calcium. In most cells, binding of CRH to CRH-R1 leads to the activation of adenylate cyclase and increased protein kinase A activity, which in turn phosphorylates and activates its downstream targets (Grammatopoulos and Chrousos, 2002). It was shown in several cell types that cAMP/protein kinase A pathway plays a key role in VEGF production (Hoper et al., 1997; Cheng et al., 1998; Amano et al., 2001). In addition, CRH receptor-mediated activation of mitogen-activated protein kinase (MAPK) signal transduction pathways has also been reported (Dermitzaki et al., 2002; Kovalovsky et al., 2002). The release of inflammatory mediators is believed to be mediated through signaling pathways involving MAPKs. The family of MAPKs consists of at least three different subgroups, including extracellular signal-regulated protein kinase (ERK1/2 or p44/p42), the p38 MAPK, the c-Jun N-terminal kinase (JNK1/2 or p46/p54), and some more distantly related kinases (Johnson and Lapadat, 2002). The p38 MAPK regulates the expression of many cytokines. In addition to its recognized role in IL-1 and tumor necrosis factor-α production (Lee et al., 1994), p38 MAPK is also involved in the production of IL-4 (Schafer et al., 1999), IL-6 (Wery-Zennaro et al., 2000), IL-8 (Marie et al., 1999), IL-12 (Lu et al., 1999), and VEGF (Tokuda et al., 2003b). In addition, ERK and JNK have also been shown to be involved in VEGF production in some cell types (Wang et al., 2002; Tanabe et al., 2003). The present study was under-taken to elucidate the role of protein kinase A and MAPK signaling pathways in CRH-mediated VEGF release in human mast cells.

We report that CRH triggers the activation of protein kinase A and p38 MAPK in human leukemic mast cells (HMC-1) and human umbilical cord blood-derived mast cells (hCBMCs). Using highly selective inhibitors of adenylate cyclase, protein kinase A, and p38 MAPK, we found that stimulation of the cAMP/protein kinase A/p38 MAPK pathway is crucial in the mechanism of CRH-mediated VEGF release in human mast cells.

Materials and Methods

Reagents. CRH was purchased from Phoenix Pharmaceuticals. Inc. (Belmont, CA) and was dissolved in deionized H2O on the same day of the experiments. SQ22536, H89, PD98059, SB203580, and SP600125 were purchased from Calbiochem-Novabiochem Corp. (La Jolla, CA) and were dissolved in dimethyl sulfoxide on the day of the experiments.

Culture of Human Mast Cells. HMC-1 cells were kindly supplied by Dr. J. H. Butterfield (Mayo Clinic, Rochester, MN) and were cultured in Iscove's modified Dulbecco's medium (Invitrogen, Carlsbad, CA) supplemented with 10% (v/v) bovine calf serum (Hyclone, Logan, UT), 100 U/ml penicillin/streptomycin, and 2 μM α-thioglycerol. Cells were kept in 5% CO2-balanced air at 37°C.

Human cord blood was obtained from human placentas from normal vaginal deliveries in accordance with established institutional guidelines. hCBMCs were derived by the culture of CD34+ progenitor cells as described previously (Kempuraj et al., 1999) with minor modifications. In brief, mononuclear cells were isolated by layering heparin-treated cord blood onto lymphocyte separation medium (MP Biomedicals, Irvine, CA). CD34+ progenitor cells were isolated from mononuclear cells by the selection of cells positive for the AC133 Ag (CD133+/CD34+) by magnetic cell-sorting (Miltenyi Biotec, Auburn, CA). For the first 4 weeks, CD34+ cells were cultured in Iscove's modified Dulbecco's medium supplemented with 0.55 μM 2-methoxyestradiol, 100 mg/l insulin-transferrin-selenium, 0.1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO), 100 U/ml penicillin/streptomycin, 100 ng/ml human recombinant stem cell factor (Amgen, Thousand Oaks, CA), and 50 ng/ml IL-6 (Chemicon Inc., Temecula, CA) at 37°C in 5% CO2-balanced air. After 4 weeks of culture, bovine serum albumin and insulin-transferrin-selenium in the culture medium were substituted with 10% FBS (Invitrogen).

VEGF Release Assay. For stimulation of VEGF production, HMC-1 cells (2 × 105 cells/200 μl) and hCBMCs (105 cells/200 μl) were distributed to 96-well microtiter assay plates in duplicate or triplicate and stimulated in complete culture medium with the indicated concentrations of CRH. Optimal concentrations were determined from the dose-response experiments. VEGF was determined in cell-free supernatants with a commercial ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer's directions. VEGF secretion data are expressed as picograms per 106 cells.

Measurement of Protein Kinase A Activity. Cells were plated in 48-well plates (3 × 105 cells/300 μl) in each well and serum-starved overnight. Next day, cells were stimulated with CRH (100 nM for HMC-1 cells or 1 μM for hCBMCs) for 3 min. Stimulation was terminated by the addition of ice-cold PBS. The cells were collected and sonicated on ice in cold sample preparation buffer (50 mM Tris-HCl, 50 mM β-mercaptoethanol, 10 mM EGTA, 5 mM EDTA, 1 mM PMSF, and 10 mM benzamidine, pH 7.5). Cell lysates were prepared after centrifugation of the sonicated samples at 100,000g for 1 h at 4°C. Protein concentration of the supernatant was determined by the Bio-Rad protein assay (Bio-Rad, Hercules, CA). The protein kinase A activities were measured by ELISA (Calbiochem-Novabiochem) according to the manufacturer's instructions.

Western Blot Analysis of MAPK Phosphorylation. HMC-1 cells were plated in six-well plates (2 × 106 cells/2 ml/well) in serum-free medium and serum-starved overnight. The next day, cells were stimulated with CRH (100 nM) for the indicated time points, or were stimulated with PMA (10 nM) for 5 min as a positive control for ERK activation or IL-1 (10 ng/ml) for 10 min as a positive control for JNK activation. Stimulation was terminated by the addition of ice-cold PBS and cells were washed once with PBS. Cells were then lysed in lysis buffer containing 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mM Na3VO4. Cell lysates were cleared by centrifugation, and supernatants were subjected to 10% SDS-PAGE with antibodies against the phosphorylated form of p38 MAPK, ERK, or JNK at a dilution of 1:1000. Horseradish peroxidase-conjugated antibody against rabbit IgG was used at 1:2000 dilution. The reaction was detected using the enhanced chemiluminescence reagent (Amersham, Little Chalfont, Buckinghamshire, UK). The membrane was then stripped and reprobed with the antibody that reacts with total p38 MAPK, total ERK, or total JNK.

ELISA of Phosphorylated p38 MAPK. HMC-1 cells and hCB-MCs were washed twice in PBS, distributed to 48-well plates (105 cells/200 μl/well) in duplicate in serum-free culture medium, and serum-starved overnight. The next day, hCBMCs were stimulated with 8-bromo-cAMP (1 mM) for 5 min or with CRH (1 μM) for indicated times; HMC-1 cells were stimulated with 8-bromo-cAMP (1 mM) for 5 min. Stimulation was terminated by the addition of ice-cold PBS. Cells were washed twice in PBS and lysed in 50 μl of cell extraction buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, 1 mM PMSF, and protease inhibitor cocktail) (Sigma) for 30 min on ice. Activation of p38 MAPK was determined by quantifying the amounts of phosphorylated and total form of kinase using ELISA (Biosource International, Camarillo, CA). Samples were diluted at least 1:10 for the assay. Data in each sample was normalized against the total amount of kinase and expressed as -fold induction.

Fig. 1.
Fig. 1.

Effect of H89 on CRH-induced VEGF release in HMC-1 cells and hCBMCs. HMC-1 cells (a) and hCBMCs (b) were plated in 96-well plates and preincubated with protein kinase A inhibitor H89 at the indicated concentrations for 30 min and were then stimulated with CRH (100 nM for HMC-1 or 1 μM for hCBMCs) for another 6 (HMC-1) or 24 h (hCB-MCs) in the presence or absence of H89 in complete culture media. Data are the mean ± S.E.M. (n = 4). *, p < 0.05 versus control; **, p < 0.05 versus CRH.

Fig. 2.
Fig. 2.

Effect of SB203580 on CRH-induced VEGF release in HMC-1 cells and hCBMCs. HMC-1 cells (a) and hCBMCs (b) were plated in 96-well plates and preincubated with the p38 MAPK inhibitor SB203580 at the indicated concentrations for 30 min and were then stimulated with CRH (100 nM for HMC-1 or 1 μM for hCBMCs) for another 6 (HMC-1) or 24 h (hCBMCs) in the presence or absence of SB203580 in complete culture media. SB, SB203580. Data are the mean ± S.E.M. (n = 4). *, p < 0.05 versus control; **, p < 0.05 versus CRH.

Measurement of Intracellular Calcium. Cells (2 × 106 cells/ml) were resuspended in Tyrode's buffer (133 mM NaCl, 4 mM KCl, 0.64 mM KH2PO4, 10 mM HEPES, 1 g/l glucose, 1 mM CaCl2, 0.6 mM MgCl2, and 0.03% human serum albumin, pH 7.2) containing 2.5 mM probenecid and 2 μM cell-permeant fluorescent calcium indicator Calcium Green-1 acetoxymethyl ester (Molecular Probes, Eugene, OR) for 30 min at room temperature. Cells were then washed and resuspended in fresh Tyrode's buffer (106 cells/ml). After two washes, 2 × 106 cells were placed in a cuvette with constant stirring at 37°C, maintained by a closed-circuit temperature-regulated water pump. Samples were excited at 506 nm, and fluorescence was recorded at 530 nm using a fluorescence spectrophotometer (LS-5B; PerkinElmer Life and Analytical Sciences, Boston, MA). Cells were stimulated with CRH, anti-IgE (Chemicon) or Br-A23187 (Sigma). For anti-IgE stimulation, hCBMCs were resuspended (106 cells/ml) and passively sensitized by incubation with human myeloma IgE (2 μg/ml/106 cells; Chemicon) for 48 h before stimulation with anti-IgE. Data are presented in arbitrary fluorescence units.

Statistical Analysis. Data represent the mean ± S.E.M. from three or more experiments, each in duplicate or triplicate; for hCB-MCs, each experiment was done with a culture from a different donor. Data were analyzed with the Student's t test, Mann-Whitney test, and analysis of variance, followed by the Holm-Sidak test or Newman-Keuls test as appropriate. Statistical significance was considered at p < 0.05.

Results

Inhibition of CRH-Stimulated VEGF Release by the Protein Kinase A Inhibitor H89. Because protein kinase A is a direct target of cAMP in many cells, we examined whether protein kinase A is involved in CRH-stimulated VEGF production. The specific protein kinase A inhibitor H89 significantly blocked CRH-induced VEGF release in a dose-dependent manner in HMC-1 cells and in hCBMCs (Fig. 1, a and b). H89, when added to cell cultures for 30 min before the addition of CRH, completely inhibited CRH-induced VEGF release at a concentration of 10 μM in HMC-1 cells (Fig. 1a) and 1 μM in hCBMCs (Fig. 1b). H89 alone at the concentrations used did not alter the basal level of VEGF production and did not affect cell viability (data not shown). This result suggests that protein kinase A is essential for CRH-mediated VEGF release.

Inhibition of CRH-Stimulated VEGF Release by the p38 MAPK Inhibitor SB203580. To test whether MAPK signaling pathways are involved in the effect of CRH on VEGF release, HMC-1 cells and hCBMCs were treated with the specific p38 MAPK inhibitor SB203580, the upstream inhibitor of ERK, PD98059, or the JNK inhibitor SP600125 at various concentrations for 30 min before treatment with CRH. IL-1 (10 ng/ml) was used as a positive stimulus for MAPK activation and VEGF release. Both PD98059 (50 μM) and SP600125 (1 μM) partially blocked IL-1-induced VEGF release in both cells but had no effect on CRH-induced VEGF release; other concentrations of both inhibitors yielded similar results (data not shown). The p38 MAPK inhibitor SB203580 dose-dependently inhibited CRH-induced VEGF release (Fig. 2, a and b). SB203580 alone at the concentrations used did not alter the basal level of VEGF release and did not affect cell viability (data not shown). These results indicate that ERK and JNK are not involved in CRH-induced VEGF production, whereas p38 MAPK is critical in this process.

Fig. 3.
Fig. 3.

CRH induces the activation of protein kinase A in HMC-1 cells and hCBMCs. HMC-1 cells (a) and hCBMCs (b) were plated in 48-well plates and serum-starved overnight. The next day, cells were stimulated with CRH (100 nM for HMC-1 or 1 μM for hCBMCs) for 3 min. Cell lysates were prepared as described under Materials and Methods. The protein kinase A activities were measured by ELISA according to the manufacturer's instructions. Data are the mean ± S.E.M. (n = 3). *, p < 0.05 versus control.

Fig. 4.
Fig. 4.

Effect of H89 on CRH-stimulated activation of protein kinase A in HMC-1 cells and hCBMCs. HMC-1 cells (a) and hCBMCs (b) were plated in 48-well plates and serum-starved overnight. The next day, cells were preincubated with the protein kinase A inhibitor H89 at the indicated concentrations for 30 min before stimulation with CRH (100 nM for HMC-1 cells or 1 μM for hCBMCs) for 3 min in the presence or absence of H89 in serum-free culture media. Cell lysates were prepared as described under Materials and Methods. The protein kinase A activities were measured by ELISA according to the manufacturer's instruction. Data are the mean ± S.E.M. (n = 3). *, p < 0.05 versus control.

CRH Induces Activation of Protein Kinase A and p38 MAPK. To further investigate the role of protein kinase A in CRH-induced VEGF release, protein kinase A activity was measured in both cells. Incubation of HMC-1 cells with CRH (100 nM) or hCBMCs with CRH (1 μM) increased protein kinase A activity significantly (n = 3, p < 0.05) by 2.5 ± 0.6- and 1.8 ± 0.3-fold, respectively (Fig. 3, a and b). The protein kinase A activity in both cells was inhibited by H89 in a dose-dependent manner (Fig. 4, a and b). H89 alone at the concentrations used did not alter the basal level of protein kinase A activity. To further study the role of p38 MAPK in CRH-induced VEGF release, HMC-1 cells were treated with CRH (100 nM) for different time periods, and the phosphorylation status of p38 MAPK was analyzed by Western blotting using a commercially available antibody specific for the phosphorylated form of p38 MAPK. The maximal activation of p38 was observed within 5 to 15 min and returned to basal level at 30 to 60 min (Fig. 5a). We failed to detect any ERK or JNK phosphorylation in response to CRH stimulation by Western blot analysis in HMC-1 cells (Fig. 5, b and c). Because of the shortage of hCBMCs, p38 MAPK activation was analyzed using phosphospecific p38 MAPK ELISA. Phosphorylation of p38 MAPK was enhanced significantly above baseline (n = 3, p < 0.05) from 5 to 30 min and decreased to basal level within 60 min (Fig. 5d). The phosphorylation of p38 MAPK in HMC-1 cells and hCBMCs was dose-dependently inhibited by SB203580 (Fig. 6, a and b). SB203580 alone at the concentrations used did not alter the basal level of p38 MAPK phosphorylation and did not affect cell viability (data not shown). Our results strongly suggest that protein kinase A and p38 MAPK activation is critical for CRH-mediated VEGF release in human mast cells.

CRH-Induced VEGF Release Is Mediated through cAMP/Protein Kinase A/p38 MAPK Signaling Pathway. We investigated whether protein kinase A is a downstream target of cAMP in HMC-1 and hCBMCs and whether CRH-stimulated p38 MAPK activation requires protein kinase A activity. HMC-1 cells and hCBMCs were preincubated with SQ22536 for 30 min before stimulation with CRH (100 nM for HMC-1 cells or 1 μM for hCBMCs), and protein kinase A activity was measured at 3 min. SQ22536 at a concentration of 10 mM completely abrogated protein kinase A activity in both cells (Fig. 7, a and b). SQ22536 alone at the concentrations used did not alter the basal level of protein kinase A activity and did not affect cell viability (data not shown). Moreover, the effects of CRH on protein kinase A activation and p38 MAPK phosphorylation were mimicked by the cAMP analog 8-bromo-cAMP (1 mM) in both cells (Figs. 8 and 9), suggesting protein kinase A and p38 MAPK as downstream targets of cAMP. Next, we showed that H89 dose-dependently inhibited p38 MAPK phosphorylation in both cells (Fig. 10). H89 alone at the concentrations used did not alter the basal level of p38 MAPK phosphorylation and did not affect cell viability (data not shown). Taken together, these results suggest that the cAMP/protein kinase A/p38 MAPK signaling pathway is key in CRH-induced VEGF release in human mast cells.

Fig. 5.
Fig. 5.

CRH induces p38 MAPK phosphorylation in HMC-1 cells and hCBMCs. HMC-1 cells were plated in six-well plates and serum-starved overnight. The next day, cells were stimulated with CRH for indicated times (100 nM), or they were stimulated with PMA (10 nM) for 5 min as a positive control for ERK activation or IL-1 (10 ng/ml) for 10 min as a positive control for JNK activation. Cell lysates were subjected to 10% SDS-PAGE and immunoblotted with phospho-p38 (Thr180/Tyr182) (a, top), phospho-ERK (Thr202/Tyr204) (b, top; +, PMA) or phospho-JNK (Thr183/Tyr185) (c, top; +, IL-1)-specific antibody. Membranes were stripped and reprobed with total p38 (a, bottom), total ERK (b, bottom), or total JNK (c, bottom) antibody. The results are representative of at least three independent experiments. d, hCBMCs were distributed to 48-well plates and serum-starved overnight. The next day, cells were stimulated for the indicated times with CRH (1 μM). Cell lysates were then prepared, and activation of p38 MAPK was determined by quantifying the amounts of the phosphorylated and total form of kinase using ELISA. Data in each sample were normalized against the total amount of kinase and were expressed as -fold induction. Data are the mean ± S.E.M. (n = 3). *, p < 0.05 versus control.

Fig. 6.
Fig. 6.

Effect of SB203580 on CRH-induced p38 MAPK phosphorylation in HMC-1 and hCBMCs. HMC-1 cells were plated in a six-well plate (a), and hCBMCs (b) were distributed to 48-well plates and serum-starved overnight. The next day, cells were pretreated with SB203580 at the indicated concentrations for 30 min before stimulation with CRH (100 nM for HMC-1 cells or 1 μM for hCBMCs) in the presence or absence of SB203580 for another 5 min. Cell lysates were subjected to 10% SDS-PAGE (HMC-1) or ELISA (hCBMCs). SB, SB203580. Data are the mean ± S.E.M. (n = 3). *, p < 0.05 versus control, **, p < 0.05 versus CRH.

Effects of CRH on Intracellular Calcium Levels in HMC-1 and hCBMCs. In addition to increased production of cAMP, activation of CRH has also been shown to increase the hydrolysis of phosphatidylinositol 4,5-bisphosphate (Xiong et al., 1995; Kiang, 1997), leading to elevated levels of intracellular calcium (Fazal et al., 1998; Wiesner et al., 2003) that were also reported to be involved in VEGF production (Tokuda et al., 2000). Therefore, we examined whether CRH could increase calcium concentrations in human mast cells. HMC-1 cells were stimulated with 100 nM CRH followed by 0.5 μg/ml Br-A23187. Only Br-A23187 resulted in an increase of calcium (Fig. 11a). Sensitized hCBMCs were stimulated by CRH (1 μM), followed by anti-IgE (10 μg/ml). Only anti-IgE resulted in a sustained increase in calcium level (Fig. 11b). These results were confirmed using different concentrations of CRH at different time points (data not shown). Results are presented as arbitrary fluorescence units.

Discussion

We recently demonstrated that CRH selectively stimulates HMC-1 cells and hCBMCs to release newly synthesized VEGF without the release of preformed mediators or other newly synthesized cytokines. This effect was mediated through the activation of CRH-R1 and adenylate cyclase with increased intracellular cAMP (Cao et al., 2005). In the present study, we showed that activation of protein kinase A and p38 MAPK are essential steps in CRH-induced VEGF release; the cAMP/protein kinase A/p38 MAPK pathway is the key signaling pathway without a concomitant increase of intracellular calcium levels. To our knowledge, this is the first report clarifying the molecular mechanism of CRH-induced VEGF release in human mast cells. What is particularly unique in these findings is the apparent ability of CRH to stimulate only one signaling pathway without involving other pathways necessary for degranulation and cytokine production.

Fig. 7.
Fig. 7.

Effect of SQ22536 on CRH-stimulated protein kinase A activation in HMC-1 and hCBMCs. HMC-1 cells (a) and hCBMCs (b) were plated in 48-well plates and serum-starved overnight. The next day, cells were preincubated with SQ22536 at the indicated concentrations for 30 min before stimulation with CRH (100 nM for HMC-1 or 1 μM for hCBMC) for 3 min in the presence or absence of SQ22536 in serum-free culture media. Cell lysates were then prepared and assayed for protein kinase A activity using ELISA. SQ, SQ22536. Data are the mean ± S.E.M. (n = 3). *, p < 0.05 versus control; **, p < 0.05 versus CRH.

Fig. 8.
Fig. 8.

8-Bromo-cAMP induces activation of protein kinase A in HMC-1 and hCBMCs. HMC-1 cells (a) and hCBMCs (b) were plated in 48-well plates and serum-starved overnight. The next day, cells were stimulated with 8-bromo-cAMP (1 mM) for 3 min in the serum-free culture media. Cell lysates were then prepared and assayed for protein kinase A activity. Data are the mean ± S.E.M. (n = 3). *, p < 0.05 versus control.

The effect of CRH on cytokine release has been reported in various cell types. CRH could stimulate IL-1, IL-2, and IL-6 production in human mononuclear cells (Singh and Leu, 1990; Salas et al., 1997) and IL-6 and IL-11 release from human HaCaT keratinocytes (Zbytek et al., 2002). CRH binding to CRH-R1 typically activates adenylate cyclase, which leads to increased intracellular concentrations of cAMP and activation of protein kinase A (Grammatopoulos and Chrousos, 2002). For instance, the antiproliferative effects of CRH in human endometrial adenocarcinoma cells is via CRH-R1-mediated activation of the cAMP-protein kinase A pathway (Graziani et al., 2002). CRH regulates the pro-opiomelanocortin gene expression in AtT20 cells (Aoki et al., 1997) and NURR1 gene expression in primary microvascular endothelial cells (McEvoy et al., 2002) through the cAMP-protein kinase A pathway. Our results showed, for the first time, that CRH induces protein kinase A activation in human mast cells, and the specific protein kinase A inhibitor H89 significantly inhibited CRH-induced VEGF release. Moreover, the activation of protein kinase A could be blocked by the adenylate cyclase inhibitor SQ22536 or mimicked by the cell-permeable cAMP analog 8-bromo-cAMP, supporting the involvement of a cAMP-protein kinase A pathway in VEGF release. Indeed, the role of cAMP-protein kinase A pathway in VEGF production has been reported in other cell types. Prostaglandin E2 induced VEGF expression in a human monocytic cell line (Hoper et al., 1997) and in cultured rat Muller cells (Cheng et al., 1998) via the cAMP-protein kinase A pathway. Leydig cells produced VEGF through protein kinase A activation by a process under gonadotropic control (Anand and Hickey, 1992).

In addition to protein kinase A, it has been demonstrated that CRH can activate MAPK-dependent signaling pathways. CRH was reported to induce the proliferation and release of tumor necrosis factor-α in cultured rat microglia by the activation of ERK and p38 MAPK (Wang et al., 2003). CRH down-regulated IL-18 expression in human HaCaT keratinocytes by the activation of p38 MAPK pathway (Park et al., 2005). Our findings showed for the first time that CRH-induced VEGF release in human mast cells could be abolished by the specific p38 MAPK inhibitor SB203580 but not by the specific inhibitor of mitogen-activated protein kinase kinase, PD98059, the upstream kinase of ERK, or the JNK inhibitor SP600125. SB203580 was able to inhibit CRH-induced rapid phosphorylation of p38 MAPK, suggesting a critical role of p38 MAPK in CRH-mediated VEGF release. Indeed, other groups have reported the involvement of the p38 MAPK signaling pathway in VEGF production in other cell types, including human ovarian carcinoma cells in response to hypoxia (Xu et al., 2004), as well as osteoblasts in response to transforming growth factor-β (Tokuda et al., 2003a) or bone morphogenetic protein-4 (Tokuda et al., 2003b). The p38 MAPK pathway controls the expression of many genes by phosphorylating and enhancing the activity of multiple transcription factors, such as activating transcription factor-2, cAMP response element-binding protein, Sp-1, and nuclear factor-κB (Kyriakis and Avruch, 2001). Multiple regulatory elements have been found in the promoter region of human VEGF gene, including the binding sites for transcription factors Sp-1, activator protein-1, activator protein-2, nuclear factor-κB, and cAMP response element-binding protein (Loureiro and D'Amore, 2005). Therefore, p38 MAPK could regulate VEGF gene expression at the transcriptional level by activation of one or some of these transcription factors. VEGF can also be regulated at the level of mRNA stability (Dibbens et al., 1999). It was shown that activation of the p38 MAPK increased the stability of the VEGF mRNA in some cell types, an action presumably mediated through the AU-rich region of its 3′ untranslated region (Pages et al., 2000). Therefore, p38 MAPK could also regulate VEGF gene expression by increasing VEGF mRNA stability in human mast cells. Further investigation is undergoing in our laboratory to address these questions.

Fig. 9.
Fig. 9.

8-Bromo-cAMP induces p38 MAPK phosphorylation in HMC-1 cells and hCBMCs. HMC-1 cells (a) and hCBMCs (b) were plated in 48-well plates and serum-starved overnight. The next day, cells were stimulated with 8-bromo-cAMP (1 mM) for 5 min in the serum-free culture media. Cell lysates were then prepared, and activation of p38 MAPK was determined by quantifying the amounts of the phosphorylated and total forms of kinase using ELISA. Data in each sample were normalized against the total amount of kinase and expressed as -fold induction. Data are the mean ± S.E.M. (n = 3). *, p < 0.05 versus control.

Fig. 10.
Fig. 10.

The effect of H89 on CRH-induced p38 MAPK phosphorylation in HMC-1 and hCBMCs. HMC-1 cells were in a six-well plate (a) and hCBMCs (b) were distributed to 48-well plates and serum-starved overnight. The next day, cells were pretreated with H89 at the indicated concentrations for 30 min before stimulation with CRH (100 nM for HMC-1 cells or 1 μM for hCBMCs) in the presence or absence of H89 for another 5 min. Cell lysates were then prepared and subjected to 10% SDS-PAGE (HMC-1 cells) or ELISA (hCBMCs). Data are the mean ± S.E.M. (n = 3). *, p < 0.05 versus control.

Fig. 11.
Fig. 11.

The effect of CRH on intracellular calcium levels. a, HMC-1 cells were stimulated with 100 nM CRH followed by 0.5 μg/ml Br-A23187. b, hCBMCs were stimulated by CRH (1 μM) followed by anti-IgE (10 μg/ml). Results are presented as arbitrary fluorescence units.

MAPKs have been shown to be substrates of protein kinase A in osteoblast-like cells (Kakita et al., 2004) and PC12 cells (Park et al., 2000). In this study, we extended the findings to human mast cells and showed that protein kinase A is required for CRH-induced p38 MAPK activation, and CRH-induced VEGF release is mediated through the cAMP/protein kinase A/p38 MAPK signaling pathway. It has been reported that CRH could also increase intracellular levels of calcium in some cell types (Fazal et al., 1998; Wiesner et al., 2003). However, we failed to detect any increase of calcium in both HMC-1 cells and hCBMCs. The selective activation of p38 MAPK without the involvement of ERK, JNK, or calcium by CRH differs from the mast cell signaling mediated by the high-affinity IgE receptor; in the latter case, there is broad activation of the ERK, JNK, and p38 MAPK, as well as increased levels of intracellular calcium that leads to mast cell degranulation, as well as de novo synthesis and release of both cytokines and arachidonic acid metabolites (Siraganian, 2003). Therefore, our results could explain, at least in part, why CRH selectively stimulates VEGF production in human mast cells without degranulation and other cytokine production. The potential mechanism for the selective activation of p38 MAPK could involve activation by CRH of other CRH receptor isoforms that inhibit other signaling pathways. For instance, a soluble CRH-R1 isoform, CRH-R1e, was shown to attenuate CRH-R1α-coupled cAMP production stimulated by CRH in COS cells (Pisarchik and Slominski, 2004). A soluble splice variant of CRH-R2α (sCRH-R2α) dose-dependently inhibited the cAMP response to CRH and its structurally related peptide urocortin in human embryonic kidney 293T cells transfected with mouse CRH-R2α (Chen et al., 2005). Moreover, sCRH-R2α inhibited the activation by urocortin of ERK in CATH.a cells, which endogenously express CRH-R1 and CRH-R2α (Chen et al., 2005).

It should be noted that CRH induced a relative modest but significant increase (∼30% increase) of VEGF release from hCBMCs compared with that from HMC-1 cells (∼100% increase). The extent of VEGF increase in this study is in agreement with the previous report showing that immunological stimulation of hCBMCs through FcϵRI cross-linking by anti-IgE resulted in a small but significant induction of VEGF secretion compared with untreated controls (Boesiger et al., 1998). Hence, CRH stimulation of hCBMCs for VEGF release parallels that of immunological stimulus. In addition, our recent findings show that forskolin and 8-bromo-cAMP mimic the effect of CRH on VEGF release in hCBMCs by inducing a modest increase of VEGF secretion (∼40% increase), whereas both agents induce a much higher increase of VEGF release (∼10-fold) from HMC-1 cells (Cao et al., 2005). These observations indicate that the VEGF production by neoplastic mast cell lines in response to some stimuli can greatly exceed those produced by the corresponding nonneo-plastic cells. This property of neoplastic mast cells could explain why patients with mastocytosis are more susceptible to developing vascular abnormalities, which can be worse during inflammation (Escribano et al., 2002). The VEGF release from other cell types and in response to other stimuli have been documented. Human eosinophils also spontaneously release VEGF that is up-regulated (∼100% increase) by granulocyte-macrophage colony-stimulating factor and IL-5 (Horiuchi and Weller, 1997). Macrophage colony-stimulating factor induces freshly isolated normal human monocytes to produce and release VEGF in a dose-dependent manner, with a maximum of 5-fold more VEGF release at 5 days in culture (Eubank et al., 2003). UV radiation and prostaglandin E2 can increase VEGF release from cultured human dermal fibroblasts by 3-fold after treatment for 24 h (Trompezinski et al., 2001). The extent of the increase of VEGF release varies from cell to cell and depends on a specific stimulus.

This is the first report showing that cAMP/protein kinase A/p38 MAPK pathway is the key signaling pathway mediating the effect of CRH on selective VEGF release in human mast cells, thereby providing insight into the molecular mechanism of how CRH affects the release of a key proinflammatory mediator. There has been increasing evidence indicating that mast cells are involved in inflammatory diseases worsened by stress in which degranulation is not necessarily present (Bienenstock, 2002; Theoharides and Cochrane, 2004), many of which are associated with increased VEGF expression. For instance, increased and activated mast cells have been documented in the bladder of patients with interstitial cystitis (Theoharides et al., 2001), an inflammatory condition worsened by stress recently associated with increased bladder VEGF expression (Tamaki et al., 2004). Mast cells are also increased in psoriasis, the symptoms of which worsen by stress (Katsarou-Katsari et al., 1999). There is increased VEGF expression in psoriatic skin (Detmar et al., 1994), and VEGF levels from psoriatic plaques correlated with the extent of clinical symptoms (Bhushan et al., 1999). Moreover, overexpression of VEGF in transgenic mice led to a skin condition resembling psoriasis (Xia et al., 2003). Our findings, therefore, provide the molecular basis for explaining and possibly developing new approaches for the treatment of inflammatory diseases, especially those affected by stress (Theoharides et al., 2004).

Acknowledgments

We thank Dr. J. H. Butterfield (Mayo Clinic, Rochester, MN) for kindly providing the HMC-1 cells and Amgen Inc. for the human recombinant stem cell factor. We are grateful to the nurses of the Labor and Delivery Division of Tufts-New England Medical Center (Boston, MA) for the umbilical cord blood collection. We also thank Jessica Christian for patience and word processing skills.

Footnotes

  • This work was supported in part by National Institutes of Health grant AR47652 (to T.C.T.).

  • ABBREVIATIONS: CRH, corticotropin-releasing hormone; HMC-1, human leukemic mast cell line; hCBMC, human umbilical cord blood-derived mast cell; VEGF, vascular endothelial growth factor; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated protein kinase; IL, interleukin; JNK, c-Jun N-terminal kinase; PMSF, phenylmethylsulfonyl fluoride; ELISA, enzyme-linked immunosorbent assay; Br-A23187, 4-bromo-calcimycin; PMA, phorbol 12-myristate 13-acetate; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; H89, N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; PD98059, 2′-amino-3′-methoxyflavone; SP600125, 1,9-pyrazoloanthrone anthra(1,9-cd)pyrazol-6(2H)-one; SQ22536, 9-(tetrahydro-2-furanyl)-9H-purine-6-amine.

  • Received October 18, 2005.
  • Accepted December 5, 2005.

References

  1. ↵
    Amano H, Ando K, Minamida S, Hayashi I, Ogino M, Yamashina S, Yoshimura H, and Majima M (2001) Adenylate cyclase/protein kinase A signaling pathway enhances angiogenesis through induction of vascular endothelial growth factor in vivo. Jpn J Pharmacol 87: 181-188.
    OpenUrlCrossRefPubMed
  2. ↵
    Anand KJS and Hickey PR (1992) Halothane-morphine compared with high-dose sufentanil for anesthesia and postoperative analgesia in neonatal cardiac surgery. N Engl J Med 326: 1-9.
    OpenUrlCrossRefPubMed
  3. ↵
    Aoki Y, Iwasaki Y, Katahira M, Oiso Y, and Saito H (1997) Regulation of the rat proopiomelanocortin gene expression in AtT-20 cells. I: effects of the common secretagogues. Endocrinology 138: 1923-1929.
    OpenUrlCrossRefPubMed
  4. ↵
    Bhushan M, McLaughlin B, Weiss JB, and Griffiths CE (1999) Levels of endothelial cell stimulating angiogenesis factor and vascular endothelial growth factor are elevated in psoriasis. Br J Dermatol 141: 1054-1060.
    OpenUrlCrossRefPubMed
  5. ↵
    Bienenstock J (2002) Stress and asthma: the plot thickens. Am J Respir Crit Care Med 165: 1034-1035.
    OpenUrlPubMed
  6. ↵
    Boesiger J, Tsai M, Maurer M, Yamaguchi M, Brown LF, Claffey KP, Dvorak HF, and Galli SJ (1998) Mast cells can secrete vascular permeability factor/vascular endothelial cell growth factor and exhibit enhanced release after immunoglobulin E-dependent upregulation of Fcϵ receptor I expression. J Exp Med 188: 1135-1145.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Cao J, Papadopoulou N, Kempuraj D, Boucher WS, Sugimoto K, Cetrulo CL, and Theoharides TC (2005) Human mast cells express corticotropin-releasing hormone (CRH) receptors and CRH leads to selective secretion of vascular endothelial growth factor (VEGF). J Immunol 174: 7665-7675.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    Chen AM, Perrin MH, Digruccio MR, Vaughan JM, Brar BK, Arias CM, Lewis KA, Rivier JE, Sawchenko PE, and Vale WW (2005) A soluble mouse brain splice variant of type 2α corticotropin-releasing factor (CRF) receptor binds ligands and modulates their activity. Proc Natl Acad Sci USA 102: 2620-2625.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    Cheng T, Cao W, Wen R, Steinberg RH, and LaVail MM (1998) Prostaglandin E2 induces vascular endothelial growth factor and basic fibroblast growth factor mRNA expression in cultured rat Muller cells. Investig Ophthalmol Vis Sci 39: 581-591.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Dermitzaki E, Tsatsanis C, Gravanis A, and Margioris AN (2002) Corticotropin-releasing hormone induces fas ligand production and apoptosis in PC12 cells via activation of P38 mitogen-activated protein kinase. J Biol Chem 277: 12280-12287.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    Detmar M, Brown LF, Claffey KP, Yeo KT, Kocher O, Jackman RW, Berse B, and Dvorak HF (1994) Overexpression of vascular permeability factor/vascular endothelial growth factor and its receptors in psoriasis. J Exp Med 180: 1141-1146.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    Dibbens JA, Miller DL, Damert A, Risau W, Vadas MA, and Goodall GJ (1999) Hypoxic regulation of vascular endothelial growth factor mRNA stability requires the cooperation of multiple RNA elements. Mol Biol Cell 10: 907-919.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    Escribano L, Akin C, Castells M, Orfao A, and Metcalfe DD (2002) Mastocytosis: current concepts in diagnosis and treatment. Ann Hematol 81: 677-690.
    OpenUrlCrossRefPubMed
  14. ↵
    Eubank TD, Galloway M, Montague CM, Waldman WJ, and Marsh CB (2003) M-CSF induces vascular endothelial growth factor production and angiogenic activity from human monocytes. J Immunol 171: 2637-2643.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    Fazal N, Slominski A, Choudhry MA, Wei ET, and Sayeed MM (1998) Effect of CRF and related peptides on calcium signaling in human and rodent melanoma cells. FEBS Lett 435: 187-190.
    OpenUrlCrossRefPubMed
  16. ↵
    Grammatopoulos DK and Chrousos GP (2002) Functional characteristics of CRH receptors and potential clinical applications of CRH-receptor antagonists. Trends Endocrinol Metab 13: 436-444.
    OpenUrlCrossRefPubMed
  17. ↵
    Graziani G, Tentori L, Portarena I, Barbarino M, Tringali G, Pozzoli G, and Navarra P (2002) CRH inhibits cell growth of human endometrial adenocarcinoma cells via CRH-receptor 1-mediated activation of CAMP-PKA pathway. Endocrinology 143: 807-813.
    OpenUrlCrossRefPubMed
  18. ↵
    Hoper MM, Voelkel NF, Bates TO, Allard JD, Horan M, Shepherd D, and Tuder RM (1997) Prostaglandins induce vascular endothelial growth factor in a human monocytic cell line and rat lungs via cAMP. Am J Respir Cell Mol Biol 17: 748-756.
    OpenUrlCrossRefPubMed
  19. ↵
    Horiuchi T and Weller PF (1997) Expression of vascular endothelial growth factor by human eosinophils: upregulation by granulocyte macrophage colony-stimulating factor and interleukin-5. Am J Respir Cell Mol Biol 17: 70-77.
    OpenUrlCrossRefPubMed
  20. ↵
    Johnson GL and Lapadat R (2002) Mitogen-activated protein kinase pathways mediated by ERK, JNK and P38 protein kinases. Science (Wash DC) 298: 1911-1912.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Kakita A, Suzuki A, Ono Y, Miura Y, Itoh M, and Oiso Y (2004) Possible involvement of P38 MAP kinase in prostaglandin E1-induced ALP activity in osteoblast-like cells. Prostaglandins Leukot Essent Fatty Acids 70: 469-474.
    OpenUrlCrossRefPubMed
  22. ↵
    Karalis K, Louis JM, Bae D, Hilderbrand H, and Majzoub JA (1997) CRH and the immune system. J Neuroimmunol 72: 131-136.
    OpenUrlCrossRefPubMed
  23. ↵
    Katsarou-Katsari A, Filippou A, and Theoharides TC (1999) Effect of stress and other psychological factors on the pathophysiology and treatment of dermatoses. Int J Immunopathol Pharmacol 12: 7-11.
    OpenUrlPubMed
  24. ↵
    Kempuraj D, Saito H, Kaneko A, Fukagawa K, Nakayama M, Toru H, Tomikawa M, Tachimoto H, Ebisawa M, Akasawa A, et al. (1999) Characterization of mast cell-committed progenitors present in human umbilical cord blood. Blood 93: 3338-3346.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Kiang JG (1997) Corticotropin-releasing factor-like peptides increase cytosolic [Ca2+] in human epidermoid A-431 cells. Eur J Pharmacol 329: 237-244.
    OpenUrlCrossRefPubMed
  26. ↵
    Kovalovsky D, Refojo D, Liberman AC, Hochbaum D, Pereda MP, Coso OA, Stalla GK, Holsboer F, and Arzt E (2002) Activation and induction of NUR77/NURR1 in corticotrophs by CRH/cAMP: involvement of calcium, protein kinase A and MAPK pathways. Mol Endocrinol 16: 1638-1651.
    OpenUrlCrossRefPubMed
  27. ↵
    Kyriakis JM and Avruch J (2001) Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev 81: 807-869.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D, McNulty D, Blumenthal MJ, Heys JR, Landvatter SW, et al. (1994) A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature (Lond) 372: 739-746.
    OpenUrlCrossRefPubMed
  29. ↵
    Loureiro RM and D'Amore PA (2005) Transcriptional regulation of vascular endothelial growth factor in cancer. Cytokine Growth Factor Rev 16: 77-89.
    OpenUrlCrossRefPubMed
  30. ↵
    Lu HT, Yang DD, Wysk M, Gatti E, Mellman I, Davis RJ, and Flavell RA (1999) Defective IL-12 production in mitogen-activated protein (MAP) kinase kinase 3 (Mkk3)-deficient mice. EMBO (Eur Mol Biol Organ) J 18: 1845-1857.
    OpenUrlAbstract
  31. ↵
    Marie C, Roman-Roman S, and Rawadi G (1999) Involvement of mitogen-activated protein kinase pathways in interleukin-8 production by human monocytes and polymorphonuclear cells stimulated with lipopolysaccharide or mycoplasma fermentans membrane lipoproteins. Infect Immun 67: 688-693.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    McEvoy AN, Bresnihan B, FitzGerald O, and Murphy EP (2002) Corticotropin-releasing hormone signaling in synovial tissue vascular endothelium is mediated through the cAMP/CREB pathway. Ann NY Acad Sci 966: 119-130.
    OpenUrlCrossRefPubMed
  33. ↵
    Pages G, Berra E, Milanini J, Levy AP, and Pouyssegur J (2000) Stress-activated protein kinases (JNK and P38/HOG) are essential for vascular endothelial growth factor mRNA stability. J Biol Chem 275: 26484-26491.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Park HJ, Kim HJ, Lee JH, Lee JY, Cho BK, Kang JS, Kang H, Yang Y, and Cho DH (2005) Corticotropin-releasing hormone (CRH) downregulates interleukin-18 expression in human HaCaT keratinocytes by activation of P38 mitogen-activated protein kinase (MAPK) pathway. J Investig Dermatol 124: 751-755.
    OpenUrlCrossRefPubMed
  35. ↵
    Park JH, Park JK, Bae KW, and Park HT (2000) Protein kinase A activity is required for depolarization-induced proline-rich tyrosine kinase 2 and mitogen-activated protein kinase activation in PC12 cells. Neurosci Lett 290: 25-28.
    OpenUrlCrossRefPubMed
  36. ↵
    Pisarchik A and Slominski A (2004) Molecular and functional characterization of novel CRFR1 isoforms from the skin. Eur J Biochem 271: 2821-2830.
    OpenUrlPubMed
  37. ↵
    Salas MA, Brown OA, Perone MJ, Castro MG, and Goya RG (1997) Effect of the corticotropin releasing hormone precursor on interleukin-6 release by human mononuclear cells. Clin Immunol Immunopathol 85: 35-39.
    OpenUrlCrossRefPubMed
  38. ↵
    Schafer PH, Wadsworth SA, Wang L, and Siekierka JJ (1999) P38 alpha mitogen-activated protein kinase is activated by CD28-mediated signaling and is required for IL-4 production by human CD4+CD45RO+ T cells and Th2 effector cells. J Immunol 162: 7110-7119.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    Singh VK and Leu CSJ (1990) Enhancing effect of corticotropin-releasing neurohormone on the production of interleukin-1 and interleukin-2. Neurosci Lett 120: 151-154.
    OpenUrlCrossRefPubMed
  40. ↵
    Siraganian RP (2003) Mast cell signal transduction from the high-affinity IgE receptor. Curr Opin Immunol 15: 639-646.
    OpenUrlCrossRefPubMed
  41. ↵
    Tamaki M, Saito R, Ogawa O, Yoshimura N, and Ueda T (2004) Possible mechanisms inducing glomerulations in interstitial cystitis: relationship between endoscopic findings and expression of angiogenic growth factors. J Urol 172: 945-948.
    OpenUrlCrossRefPubMed
  42. ↵
    Tanabe K, Dohi S, Matsuno H, Hirade K, and Kozawa O (2003) Midazolam stimulates vascular endothelial growth factor release in aortic smooth muscle cells: role of the mitogen-activated protein kinase superfamily. Anesthesiology 98: 1147-1154.
    OpenUrlCrossRefPubMed
  43. ↵
    Theoharides TC and Cochrane DE (2004) Critical role of mast cells in inflammatory diseases and the effect of acute stress. J Neuroimmunol 146: 1-12.
    OpenUrlCrossRefPubMed
  44. ↵
    Theoharides TC, Donelan JM, Papadopoulou N, Cao J, Kempuraj D, and Conti P (2004) Mast cells as targets of corticotropin-releasing factor and related peptides. Trends Pharmacol Sci 25: 563-568.
    OpenUrlCrossRefPubMed
  45. ↵
    Theoharides TC, Kempuraj D, and Sant GR (2001) Mast cell involvement in interstitial cystitis: a review of human and experimental evidence. Urology 57: 47-55.
    OpenUrlCrossRefPubMed
  46. ↵
    Theoharides TC, Singh LK, Boucher W, Pang X, Letourneau R, Webster E, and Chrousos G (1998) Corticotropin-releasing hormone induces skin mast cell degranulation and increased vascular permeability, a possible explanation for its proinflammatory effects. Endocrinology 139: 403-413.
    OpenUrlCrossRefPubMed
  47. ↵
    Tokuda H, Hatakeyama D, Akamatsu S, Tanabe K, Yoshida M, Shibata T, and Kozawa O (2003a) Involvement of MAP kinases in TGF-beta-stimulated vascular endothelial growth factor synthesis in osteoblasts. Arch Biochem Biophys 415: 117-125.
    OpenUrlCrossRefPubMed
  48. ↵
    Tokuda H, Hatakeyama D, Shibata T, Akamatsu S, Oiso Y, and Kozawa O (2003b) P38 MAP kinase regulates BMP-4-stimulated VEGF synthesis via P70 S6 kinase in osteoblasts. Am J Physiol 284: E1202-E1209.
    OpenUrl
  49. ↵
    Tokuda H, Kozawa O, and Uematsu T (2000) Basic fibroblast growth factor stimulates vascular endothelial growth factor release in osteoblasts: divergent regulation by P42/P44 mitogen-activated protein kinase and P38 mitogen-activated protein kinase. J Bone Miner Res 15: 2371-2379.
    OpenUrlCrossRefPubMed
  50. ↵
    Trompezinski S, Pernet I, Schmitt D, and Viac J (2001) UV radiation and prostaglandin E2 up-regulate vascular endothelial growth factor (VEGF) in cultured human fibroblasts. Inflamm Res 50: 422-427.
    OpenUrlCrossRefPubMed
  51. ↵
    Wang W, Ji P, and Dow KE (2003) Corticotropin-releasing hormone induces proliferation and TNF-alpha release in cultured rat microglia via MAP kinase signalling pathways. J Neurochem 84: 189-195.
    OpenUrlCrossRefPubMed
  52. ↵
    Wang X, Tokuda H, Hirade K, and Kozawa O (2002) Stress-activated protein kinase/c-Jun N-terminal kinase (JNK) plays a part in endothelin-1-induced vascular endothelial growth factor synthesis in osteoblasts. J Cell Biochem 87: 417-423.
    OpenUrlCrossRefPubMed
  53. ↵
    Wery-Zennaro S, Zugaza JL, Letourneur M, Bertoglio J, and Pierre J (2000) IL-4 regulation of IL-6 production involves Rac/Cdc42- and P38 MAPK-dependent pathways in keratinocytes. Oncogene 19: 1596-1604.
    OpenUrlCrossRefPubMed
  54. ↵
    Wiesner B, Roloff B, Fechner K, and Slominski A (2003) Intracellular calcium measurements of single human skin cells after stimulation with corticotropin-releasing factor and urocortin using confocal laser scanning microscopy. J Cell Sci 116: 1261-1268.
    OpenUrlAbstract/FREE Full Text
  55. ↵
    Xia YP, Li B, Hylton D, Detmar M, Yancopoulos GD, and Rudge JS (2003) Transgenic delivery of VEGF to mouse skin leads to an inflammatory condition resembling human psoriasis. Blood 102: 161-168.
    OpenUrlAbstract/FREE Full Text
  56. ↵
    Xiong Y, Xie LY, and bou-Samra AB (1995) Signaling properties of mouse and human corticotropin-releasing factor (CRF) receptors: decreased coupling efficiency of human type II CRF receptor. Endocrinology 136: 1828-1834.
    OpenUrlCrossRefPubMed
  57. ↵
    Xu L, Pathak PS, and Fukumura D (2004) Hypoxia-induced activation of P38 mitogen-activated protein kinase and phosphatidylinositol 3′-kinase signaling pathways contributes to expression of interleukin 8 in human ovarian carcinoma cells. Clin Cancer Res 10: 701-707.
    OpenUrlAbstract/FREE Full Text
  58. ↵
    Zbytek B, Mysliwski A, Slominski A, Wortsman J, Wei ET, and Mysliwska J (2002) Corticotropin-releasing hormone affects cytokine production in human HaCaT keratinocytes. Life Sci 70: 1013-1021.
    OpenUrlCrossRefPubMed
Back to top

In this issue

Download PDF
Article Alerts
Email Article
Citation Tools

Research ArticleArticle

Corticotropin-Releasing Hormone Induces Vascular Endothelial Growth Factor Release from Human Mast Cells via the cAMP/Protein Kinase A/p38 Mitogen-Activated Protein Kinase Pathway

Jing Cao, Curtis L. Cetrulo and Theoharis C. Theoharides
Molecular Pharmacology March 1, 2006, 69 (3) 998-1006; DOI: https://doi.org/10.1124/mol.105.019539
del.icio.us logo Digg logo Reddit logo Twitter logo Facebook logo Google logo Mendeley logo

Jump to section

Advertisement