1Geriatric Research, Education and Clinical Center and 2Research Service, Audie L. Murphy Division, South Texas Veterans Health Care System; and Departments of 3Dental Diagnostic Science, 4Surgery, 5Community Dentistry, and 6Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas
Submitted 29 July 2004 ; accepted in final form 27 January 2005
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ABSTRACT |
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mitogen-activated protein kinase; CD44
MAPK pathways constitute an evolutionarily conserved series of serine/threonine kinases traditionally linked to receptor tyrosine kinases such as receptors for EGF and PDGF. The ERKs are one class of MAPKs that are regulated by a three-tiered cascade composed of a MAPK kinase kinase (MAPKKK; e.g., c-Raf1 or B-Raf), a MAPK kinase (e.g., MEK1 and MEK2) and a MAPK (e.g., ERK1 and ERK2) (23). In the past several years, it has been demonstrated that numerous GPCR-stimulated cellular functions are mediated through the MAPK cascades. In a number of systems, -adrenergic receptor-induced activation of ERKs proceeds through PKA-, G
i-, and G
-mediated signaling events and may be attributable to the transactivation of the EGF receptor (19). Transactivation of receptor tyrosine kinases after GPCR stimulation has been documented for EGF, PDGF, and insulin-like growth factor receptors and is the subject of several recent reviews (7, 13, 22). However, the mechanisms by which
-adrenergic receptors and other GPCRs activate the ERK/MAPK cascade are cell type-, ligand-, and G protein-specific and may be dependent on the physiological conditions of the cell (18).
In salivary gland tissues, activation of -adrenergic receptors plays an important role in protein secretion, secretory protein expression, and postnatal development and growth (2). Chronic treatment of rodents with the
-adrenergic receptor agonist isoproterenol has long been known to cause salivary gland enlargement without the development of neoplasm (4, 28). There is some evidence suggesting that MAPKs are activated during isoproterenol treatment. One study showed that Ras guanine nucleotide exchange activity, which triggers the MAPKKK-MEK-MAPK cascade, was increased in parotid cell homogenates from isoproterenol-treated rats compared with untreated rats (31). The same group of investigators reported that in parotid gland lysates from isoproterenol-treated rats, levels of phosphorylated ERK2 increased for up to 12 h of treatment, whereas phosphorylated ERK1 levels decreased gradually over time; the activation of ERKs by isoproterenol was apparently not affected by cAMP accumulation or PKA activity (5). Other studies suggested that EGF receptor signaling cascades may be involved in mediating isoproterenol effects on the salivary gland in rodents (14, 26). Despite these earlier studies, the involvement of ERK signaling pathway(s) and downstream targets as mediators of salivary cell responses to
-adrenergic receptor activation has yet to be defined clearly. Among potential targets downstream of ERK signaling in salivary cells is CD44, an adhesion molecule thought to regulate salivary gland growth and development (8, 24).
In the present study we examined the cellular signals linking -adrenergic receptors with the activation of ERKs in a human salivary cell line (HSY). Our results suggest that isoproterenol activates ERKs via two different signaling pathways: on the one hand, activation of ERKs is induced by transactivation of the EGF receptor, whereas an additional activation pathway possibly mediated by cAMP signaling appears to be at least partly independent of receptor tyrosine kinase. Furthermore, we have identified CD44 as a possible downstream effector of
-adrenergic receptor-induced ERK activation in salivary gland cells.
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MATERIALS AND METHODS |
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Cell culture.
The HSY cell line, which was originally established by Yanagawa et al. (33), was kindly provided by Dr. James Turner [National Institute of Dental and Craniofacial Research (NIDCR), National Institutes of Health, Bethesda, MD]. Cells were plated at a density of 2 x104 cells/cm2 in 100-mm culture plates and cultured in DMEM supplemented with 10% FBS and penicillin (100 U/ml)-streptomycin (100 µg/ml) at 37°C in a humidified 5% CO2-atmosphere incubator. Unless otherwise specified cells were grown to near confluence before use. The cells were treated with reagents such as isoproterenol or EGF for various time periods and then prepared for immunoblot or immunoprecipitation analysis.
Transfection. A dominant-negative c-Src (K295R/Y527F) construct (DnSrc) was kindly provided by Dr. Joan S. Brugge (Harvard University, Cambridge, MA). HSY cells were transiently transfected with 1 µg of DnSrc plasmid or empty vector (control) with LipofectAMINE PLUS reagent (Invitrogen, Carlsbad, CA) and cotransfected with 100 ng of green fluorescent protein (GFP) gene vector (phrGFP-N1) in each 100-mm culture dish as described previously (10). Cells were incubated with the DNA-LipofectAMINE mix for 34 h, after which the cells were washed with PBS and allowed to recover in culture medium for 24 h. Cells were then trypsinized, and GFP-expressing cells were sorted and collected using flow cytometry with FACSCalibur (BD Biosciences Immunocytometry Systems, San Jose, CA). Sorted cells were continued in culture and used for experiments at 48 h posttransfection.
Western blot analysis.
Immunoblot (Western blot) analysis was performed as described previously, with minor modification (35). HSY cells were washed three times with cold PBS, scraped, and lysed in a buffer containing 50 mM Tris·HCl (pH 7.4), 150 mM NaCl, 0.1% NP-40, 3 µg/ml leupeptin, 3 µg/ml aprotinin, 3 µg/ml pepstatin A, 3 µg/ml phenanthroline, 30 µM benzamidine HCl, 0.3 mM PMSF, 20 mM -glycerol phosphate, 10 mM sodium fluoride, and 1 mM Na3VO4 at 4°C for 30 min. After centrifugation of the cell lysates at 10,000 g for 2 min at 4°C, supernatant protein samples (50 µg) were added to 15 µl of 4x sample buffer (150 mM Tris·HCl, pH 8.8, 1% SDS, 40% glycerol) and
-mercaptoethanol and then diluted with lysis buffer (without protease and phosphatase inhibitors) to a total volume of 60 µl. The protein samples were separated on 10% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (Schleicher & Schuell, Keene, NH). The membranes were immunoblotted with primary antibody [1:500 dilution for p44/p42MAPK, phospho-p44/p42MAPK, and EGF receptor; 1:250 dilution for phospho-EGF receptor (Y1068)] and a secondary horseradish peroxidase-conjugated antibody (1:10,000). MAPKs/ERKs and EGF receptor were visualized using an enhanced chemiluminescence system (SuperSignal West Pico Chemiluminescent Substrate; Pierce Biotechnology, Rockford, IL), and immunoblots were quantified with ImageQuant computer software (version 5; Molecular Dynamics, Sunnyvale, CA).
Immunoprecipitation assay of EGF receptor. Phosphorylation of the EGF receptor was detected by immunoprecipitating of the protein from cell lysates, followed by immunoblotting with anti-phosphotyrosine antibodies as described elsewhere (11). Briefly, whole cell lysates (500 µg protein) were precleared by mixing them with 25 µl of 50% protein A-Sepharose beads in 400 µl of lysis buffer containing 1 µg/ml BSA for 1 h (11). The supernatants were incubated with EGF receptor antibody (1 µg/sample) overnight. Next, 20 µl of protein A-Sepharose was added for 1 h, and the immunocomplexes were washed three times with lysis buffer. Samples were separated using SDS-PAGE, and proteins were detected by performing Western blot analysis using anti-phosphotyrosine monoclonal antibody (pY; 1:500).
Flow cytometric analyses.
Cells subjected to various treatments were harvested from culture plates with trypsin-EDTA and then washed with cold PBS. One million cells in balanced salt solution (pH 7.2) containing 0.02% NaN3 were incubated with 0.5 µg/ml of mouse anti-human CD44 antibody for 1 h at 4°C. After three washes with balanced salt solution, cells were incubated with 0.2 µg/ml of phycoerythrin-conjugated rat anti-mouse -chain antibody for 1 h at 4°C. Cells were again washed and fixed in 2% formaldehyde, and fluorescence was measured using flow cytometry with FACSCalibur. Cells incubated with the secondary antibody alone were used as negative controls.
Data analysis. The densities of Western blots were quantified with ImageQuant. Phosphorylation of p44/p42MAPK, EGF receptor, or tyrosine induced by isoproterenol, EGF, or other reagents was normalized to the total immunoreactive protein of interest for each sample and expressed as the fold increase relative to the normalized value in untreated (control) cells. Bar graphs show the untransformed mean values ± SE or range (if n = 2). For statistical analysis, single and multiple comparisons were conducted using Student's t-test and Bonferroni analysis, respectively, with logarithmically transformed values.
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RESULTS |
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PKA is thought to be involved in -adrenergic receptor-mediated activation of ERKs in a variety of cell types (19). However, in HSY cells, the stimulatory effects of isoproterenol and cholera toxin on phosphorylation of ERK1/2 were not affected by pretreatment (2 h) with the PKA inhibitor H-89 (5 x 106 M; Fig. 5). Likewise, prevention of cAMP degradation with the phosphodiesterase inhibitor IBMX also increased ERK phosphorylation, whereas the stimulatory effect of IBMX was not influenced by pretreatment with H-89 (Fig. 5). Activation of ERKs by cAMP-elevating agents was not reduced by increasing concentrations of H-89 up to 2.5 x 105 M (data not shown). On the basis of these results, activation of PKA has no apparent role in mediating
-adrenergic receptor-, G
s-, or cAMP-responsive ERK activation in HSY cells.
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DISCUSSION |
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Activation of MAPKs by GPCRs such as -adrenergic receptors has been demonstrated to be mediated by the transactivation of EGF receptors in other cell types, e.g., cardiac fibroblasts and COS-7 cells (12, 15, 20). Our results show that isoproterenol activates ERK1/2 in HSY cells by a signal transduction mechanism involving the transactivation of EGF receptors. Isoproterenol treatment was found to induce phosphorylation of the EGF receptor at Y1068, a major site for receptor autophosphorylation. Furthermore, blockade of EGF receptor phosphorylation with the specific inhibitor AG-1478 diminished isoproterenol-induced activation of ERK1/2 (Fig. 2). Together, these data demonstrate interreceptor signaling "cross-talk" between
-adrenergic and EGF receptors in HSY cells.
In this study we performed experiments to elucidate the molecular mechanism by which isoproterenol induces EGF receptor transactivation in HSY cells. Previous work suggests that in a number of tissues, -adrenergic receptor-induced activation of ERKs is sensitive to the G
i inhibitor pertussis toxin. In these systems Gi-dependent ERK activation appears to be mediated through the G
subunit and may involve EGF receptor transactivation (16, 18). Moreover, Gi-dependent activation of ERKs by the
-adrenergic receptor is thought to result from a PKA-induced phosphorylation of the receptor, which causes a "switch" in the coupling of the receptor from Gs to Gi (18). We found that pretreatment of HSY cells with pertussis toxin attenuated isoproterenol-induced ERK activation (Fig. 3); in preliminary work isoproterenol-responsive phosphorylation of the EGF receptor was also reduced by pertussis toxin (data not shown). Short-term activation of G
s with cholera toxin was observed to increase phosphorylation of ERK1/2, whereas long-term toxin treatment, which downregulates G
s in several tissues (5, 21, 29), blocked ERK activation by isoproterenol (Fig. 4B). These findings, implicating the involvement of both Gi and Gs in the isoproterenol response, are consistent with a switch in G protein coupling by the
-adrenergic receptor in HSY cells. Interestingly, treatment of HSY cells with the PKA inhibitor H-89 had no effect on isoproterenol-induced ERK activation (Fig. 5A). Thus, in marked contrast to other cell types in which pertussis-sensitive isoproterenol responses were profoundly inhibited by H-89 (6), HSY cells appear to undergo a switch in
-adrenergic receptor-G protein coupling without obvious involvement of PKA. It should be noted that the interpretation of data from experiments using H-89 could be limited by direct PKA inhibition of c-Raf1, an activator of ERKs downstream of the EGF receptor and the GTPase Ras (18). Indeed, basal levels of ERK phosphorylation in HSY cells tended to increase in the presence of higher concentrations (>5 x 106 M) of H-89, even though the isoproterenol response remained unaffected (data not shown). Although our studies do not entirely exclude a possible role for PKA in Gs-to-Gi switching in HSY cells, in preliminary experiments isoproterenol-induced phosphorylation of the EGF receptor (Y1068) was unaffected by H-89 (not shown).
Earlier reports have proposed that, in contrast to the Gi-dependent pathway, a distinct, pertussis toxin-insensitive pathway involving cAMP signaling may also mediate -adrenergic receptor-induced activation of ERKs (9, 18). In HSY cells pertussis toxin and AG-1478 produced only partial inhibition of ERK activation by the
-adrenergic agonist (Figs. 2 and 3). These findings suggested that isoproterenol activates ERK1/2 in HSY cells in part by a signaling mechanism other than the Gi-dependent pathway involving transactivation of the EGF receptor. Like isoproterenol, the cAMP analog CPT-cAMP and cAMP-elevating agents (cholera toxin and IBMX) also activated ERK1/2 in HSY cells (Figs. 4 and 5). In preliminary experiments (not shown) the activating effect of CPT-cAMP on ERK phosphorylation was not blocked by the EGF receptor inhibitor AG-1478. These results suggest the presence in HSY cells of a cAMP-mediated pathway of isoproterenol-induced ERK activation independent of the Gi-mediated cascade. The determination of whether the two distinct pathways coexist within individual HSY cells or are present in different cell subpopulations requires further investigation. Of note, partial pertussis toxin sensitivity of isoproterenol-induced ERK activation has also been reported in some (but not all) isolates of HEK-293 cells (18); this observation, which is analogous to our own finding in HSY cells (Fig. 3), is consistent with coexisting Gi-dependent and -independent pathways within individual HEK-293 cells but could also reflect heterogeneity of signaling pathways in different subpopulations of the same cell line. Interestingly, in a recent study of glioblastoma cells, PMA-induced activation of ERK1/2 also appeared to involve both EGF receptor-dependent and -independent pathways (1).
Gi-independent activation of ERKs by -adrenergic receptor-stimulated cAMP production is thought to be mediated by PKA and/or the cAMP-regulated guanine nucleotide exchange factor EPAC (exchange protein directly activated by cAMP), which in turn cause sequential activation of the downstream intermediates Rap1 and B-Raf (18, 19, 30). In HSY cells stimulation of ERK1/2 by the cAMP-elevating agents IBMX and cholera toxin was unaffected by pretreatment of cells with H-89 (Fig. 5). Thus we found no evidence in HSY cells for PKA involvement in ERK activation by cAMP, although as noted above experiments using H-89 may be difficult to interpret, given the complex actions of PKA on ERK activation in any given cell type. We have detected immunoreactive EPAC and B-Raf in HSY cells (data not shown), yet it remains to be determined whether isoproterenol stimulation of ERKs in these cells might occur via an EPAC-Rap1-B-Raf signaling cascade.
Src tyrosine kinases are thought to act as early signaling intermediates in both G-mediated EGF receptor transactivation and PKA-dependent ERK activation induced by GPCR stimulation (18, 19, 30). In our studies of HSY cells, isoproterenol-responsive phosphorylation of the EGF receptor was attenuated by the Src inhibitor PP2, whereas Src inhibition by either PP2 or transfection of a DnSrc blocked the ERK response to
-adrenergic agonist (Fig. 6). These findings suggest that Src is required for EGF receptor transactivation and possibly additional signaling events essential to ERK activation by isoproterenol. However, the precise target(s) for Src regulation in Gi- and/or cAMP-mediated pathways of isoproterenol action remains to be identified.
Although -adrenergic receptors are linked to the ERK cascade in multiple cell types, functional responses to the ERK-mediated actions of isoproterenol may be cell type specific. Of particular note in this regard, the growth-promoting effects of isoproterenol appear to be specific for the salivary gland and a few other tissues (i.e., pancreas and heart) (3, 27). The specificity of target cell responsiveness to
-adrenergic receptor signaling through the ERK pathway may reside, at least in part, in the downstream effectors generated in any given cell type. The CD44 proteins are a family of transmembrane glycoproteins thought to regulate cell proliferation, survival, and differentiation through transduction of signals originating in the extracellular matrix (24). CD44 is present in normal human salivary tissue and has been proposed to play a role in adult salivary gland regeneration and/or regrowth (8, 24). Moreover, in some tissues, alterative splicing of the CD44 gene is regulated by activation of the ERK pathway (32). In the current study, isoproterenol treatment of HSY cells caused a profound increase in surface CD44 expression, which was completely blocked by the ERK pathway inhibitor PD-98059 (Fig. 7). Thus CD44 could function as a downstream target mediating salivary cell growth responsive to
-adrenergic receptor-induced activation of the ERK pathway. EGF and IBMX were each found to increase CD44 expression, suggesting the involvement of both EGF receptor and cAMP signaling pathways in isoproterenol-induced regulation of CD44. Although treatment of cells over 24 h with the EGF receptor inhibitor AG-1478 completely blocked isoproterenol-induced CD44 expression, whether prolonged exposure to AG-1478 might have adverse effects on both pathways implicated in isoproterenol signaling requires further elucidation.
In summary, we propose that activation of -adrenergic receptors in HSY cells induces the mitogenic signaling pathways depicted schematically in Fig. 8. Distinct pathways of ERK activation mediated by Gi-dependent transactivation of EGF receptors or cAMP signaling processes have been individually demonstrated in several cell types (18, 19). To our knowledge, however, the coexistence of both pathways operating simultaneously in a single cell line has not been described previously. In HSY cells, both pathways appear to be Gs dependent because isoproterenol-induced phosphorylation of ERKs was abolished under conditions (i.e., prolonged incubation with cholera toxin) found to downregulate G
s in other systems (5, 21, 29). Hence, in Fig. 8, the two signaling pathways are shown to diverge at a step involving either G
s activation of adenylyl cyclase or switching of
-adrenergic receptor coupling from G
s to G
i (18). Although the process of G protein switching is generally thought to require PKA-mediated phosphorylation of the
-adrenergic receptor, we found no evidence to indicate that isoproterenol-responsive ERK activation in HSY cells is PKA dependent; nonetheless, a possible role for PKA in Gi- and/or cAMP-mediated signaling pathways has not been fully excluded. Finally, the identification of CD44 as a likely effector downstream of ERK1/2 is also summarized in Fig. 8. In a related study (34), we recently reported that EGF receptor-mediated stimulation of ERKs in HSY cells increases expression of
-adrenergic receptors coupled to activation of adenylyl cyclase. Additional investigations are required to clarify the mechanisms by which cross-talk between
-adrenergic receptor- and receptor tyrosine kinase-linked pathways elicits mitogenic signals promoting tissue-specific growth of salivary gland cells.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* C.-K. Yeh and P. M. Ghosh contributed equally to this work.
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