Abstract
α1-Adrenergic receptors mediate mitogenic responses and increase intracellular free Ca2+([Ca2+]i) in vascular smooth muscle cells. Induction of c-fos is a critical early event in cell growth; expression of this gene is regulated by a number of signaling pathways including Ca2+. We wondered whether Ca2+ signaling plays a critical role in the induction of c-fos gene by α1-adrenergic receptors. Using stably transfected rat-1 fibroblasts, we confirmed that PE induced c-fos mRNA expression in a time- and dose-dependent manner, and also increased [Ca2+]i (measured with Fura-2 AM). These responses were blocked by the α1-adrenergic receptor antagonist doxazosin. Both intracellular Ca2+ chelation (using BAPTA/AM) and extracellular Ca2+ depletion (using EGTA) significantly inhibited PE-induced c-fosexpression by α1A and α1B receptors. Brief (1-min) stimulation of α1A and α1Breceptors with PE did not maximally induce c-fosexpression, suggesting that a sustained increase in [Ca2+]i due to Ca2+ influx is required. The calmodulin (CaM) antagonists, R24571, W7, and trifluoperazine, but not the CaM-dependent protein kinases inhibitor KN-62, significantly inhibited c-fos induction by α1A and α1B receptors. Neither inhibition of protein kinase C nor inhibition of adenylyl cyclase modified c-fos induction by PE. These results suggest that α1-adrenergic receptor-induced c-fosexpression in rat-1 cells is dependent on a Ca2+/CaM-associated pathway.
α1-Adrenergic receptors mediate a variety of the important physiological effects of catecholamines, such as vascular smooth muscle contraction, glycogenolysis, and myocardial inotropic responses. In addition to these well-known functions, increasing evidence indicates that these receptors may mediate growth responses in vascular smooth muscle and myocardial cells. For example, stimulation of α1-adrenergic receptors induces cell proliferation, DNA synthesis (Blaes and Boissel, 1983; Bell and Madri, 1989; Nakaki et al., 1990), and protein synthesis (Chen et al., 1995;Xin et al., 1997) in vascular smooth muscle cells in culture. Growth-related proto-oncogenes are activated early during the development of smooth muscle cell hypertrophy (Naftilan et al., 1989;Neyses and Vetter, 1992). In rat aorta, activation of α1-adrenergic receptors markedly induces expression of the proto-oncogene c-fos and other growth-stimulating genes including platelet-derived growth factor (Majesky et al., 1990; Okazaki et al., 1994). The product of c-fos gene, c-FOS protein, forms heterodimers with c-JUN via leucine zipper domains that binds to the activator protein-1 consensus site (TGACTCA) and functions as a transcription factor to regulate cell proliferation and differentiation (Angel and Karin, 1991). However, little is known about signaling mechanisms by which α1-adrenergic receptors induce c-fosexpression.
The c-fos gene promoter contains multiple enhancer elements located upstream of the transcription start site that regulate c-fos transcription in response to a variety of extracellular stimuli (Roche and Prentki, 1994; Rosen et al., 1995;Karin, 1995). Two major inducible elements located in the c-fos gene promoter region are a cAMP response element (CRE) or Ca2+ response element, and a serum response element (SRE). These specific sequence regions can be stimulated by phosphorylated transcription factors, such as cAMP response element binding protein (CREB) and serum response factor, respectively, leading to activation of c-fos gene transcription. Several signal transduction pathways are involved in c-fos gene induction, including protein kinase A (PKA), protein kinase C (PKC), Ras/mitogen-activated protein (MAP) kinase, and Ca2+/calmodulin (CaM)-dependent kinases (Rosen et al., 1995). Intracellular Ca2+ signaling is important in activating enhancer elements of the c-fos gene (Roche and Prentki, 1994; Rosen et al., 1995; Finkbeiner and Greenberg, 1996).
Activation of α1-adrenergic receptors induces intracellular Ca2+ mobilization in many cells (Guarino et al., 1996). In addition, stimulation of α1-adrenergic receptors may activate MAP kinase (Thorburn and Thorburn, 1994; Bogoyevitch et al., 1996; Hu et al., 1996; Xin et al., 1997), protein kinase C (Puceat et al., 1994), and increase protein tyrosine phosphorylation (Meucci et al., 1995) in many cells. Also, these receptors may stimulate cAMP production (Perez et al., 1993), leading to activation of PKA in some cells. Each of these pathways could potentially regulate transcription of the c-fos gene (Rosen et al., 1995). To investigate mechanisms of α1 receptors activation of c-fostranscription, we used rat-1 fibroblast cell lines stably transfected with each of three α1-adrenergic receptor subtypes as a model system. The results indicate that intracellular Ca2+, rather than MAP kinase and cAMP signaling pathways, play an important role in α1-adrenergic receptor-mediated c-fos induction.
Experimental Procedures
Materials.
1,2-bis-(o-Aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA/AM), calmidazolium chloride (R24571), and 1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyll-tyrosyl]-4-phenylpiperazine (KN-62) were purchased from Calbiochem (San Diego, CA). Bisindolylmaleimide I (GF109203X) was obtained from LC Laboratory (Woburn, MA). Fura-2/AM was from Molecular Probes, Inc. (Eugene, OR). Phenylephrine (PE), EGTA, phorbol 12-myristate 13-acetate (PMA), and Hank’s balanced salt solution (HBSS) were from Sigma (St. Louis, MO). G418, lipofectamine reagent, and tissue culture chemicals were supplied by GIBCO-BRL (Grand Island, NY). Anti-extracellular stimulus response kinase (ERK) 1 antibody was from Santa Cruz Biotechnology. [α32P]ATP, [γ32P]ATP, and DNA labeling system were from Amersham Co. (Arlington, IL).
Cell Culture and Transfection.
Rat-1 fibroblasts stably transfected with human α1A, α1B, and α1D-adrenergic receptors were obtained as gifts from Dr. G Johnson of Pfizer Laboratory (Kenny et al., 1996) and maintained in DMEM containing 5% FBS and 400 μg/ml G418. Some cells were transiently transfected with plasmid construct of PKA inhibitory peptide [PKI; a kind gift from Dr. J. Avruch (Grove et al., 1989)] using lipofectamine to determine the role of PKA in α1-adrenergic receptor-induced c-fos expression. For examination of c-fosexpression and MAP kinase activity, the cells were made quiescent in serum-free medium overnight and then pretreated with tested agents including 1 μM timolol (to block possible β-adrenergic receptor in the cells) followed by stimulation with the α1-adrenergic receptor selective agonist PE.
[Ca2+]i Measurement.
The rat-1 cells were plated on coverslips to form a monolayer and loaded with 1.5 μM Fura-2/AM in HBSS containing 0.1% BSA. Cytoplasmic-free Ca2+([Ca2+]i) was determined at excitation of 340 nm and 380 nm and at an emission of 510 nm using a spectrofluorometer (Hitachi F-2000) (Chen and Giri, 1997). Cell Ca2+ responses are expressed as the ratio (F340/F380) of fluorescence intensity at excitation of 340 and 380 nm.
Northern Blot Analysis.
Total RNA of cells was extracted with the single-step method of acid guanidinium thiocyanate-phenol-chloroform (Chomczynski and Sacchi, 1987), denatured with glyoxal, fractionated by electrophoresis on 1% agarose gel, and transferred to Nytran membranes. The blot was hybridized with32P-labeled v-fos cDNA (pstI fragment) and reprobed with human β-actin cDNA in ExpressHyb Hybridization solution (Clontech, Palo Alto, CA) following the manufacturer’s instructions.
MAP Kinase Activity Assay.
The MAP kinase activity was assayed following the method described previously (Hu et al., 1996). Briefly, cells were lysed in lysis buffer (1% Triton X-100, 25 mM HEPES, pH 7.5, 50 mM NaCl, 50 mM NaF, 5 mM EDTA, 10 nM okadaic acid, 0.1mM sodium orthovanadate, 1 mM PMSF, and 10 μg/ml aprotinin and leupeptin) after exposure to tested agents. MAP kinase was precipitated from the cell lysate by incubation with anti-ERK1 antibody (2 μg/mg protein) on ice for 2 h. The immunocomplex was then collected with protein-A/G agarose beads followed by washing four times with lysis buffer and once with kinase buffer (25 mM HEPES, pH 7.4, 8 mM MaCl2, 1 mM EGTA, 1 mM DTT, and 40 μM ATP) and incubated with 5 μg of myelin basic protein (MBP, as substrate for MAP kinase) and 1 μCi of [γ32P]ATP in kinase buffer at 30°C for 10 min. The32P-phosphorylated MBP was detected by electrophoresis on SDS-polyacrylamide gel electorphoresis followed by autoradiography.
Results
Ca2+ Responses Mediated by α1-Adrenergic Receptor Subtypes Expressed in Rat-1 Cells.
In cells expressing each of the three α1-adrenergic receptor subtypes, PE stimulated an initial rapid transient increase in intracellular Ca2+ concentration ([Ca2+]i); this response was followed by a sustained increase in [Ca2+]i (Fig.1 left column). The rapid initial phase was preserved in Ca2+-free buffer, whereas the subsequent sustained phase required the presence of extracellular Ca2+ (Fig. 1, middle column). Pretreatment of the cells with thapsigargin (2 μM), which depletes internal Ca2+ stores (Thastrup et al., 1990), completely inhibited the PE-induced initial transient increase in [Ca2+]i in Ca2+ free assay buffer. As expected, thapsigargin had no effect on the sustained increase in [Ca2+]i after reintroduction of Ca2+ to the buffer (Fig. 1, right column). The sustained Ca2+ increase was not sensitive to blockers of voltage-dependent Ca2+ L-channels (nifedipine and verapamil; data not shown). These results indicate that each of the three subtypes of α1-adrenergic receptor trigger Ca2+ release from internal Ca2+ stores (rapid initial [Ca2+]i increase) and Ca2+ influx from extracellular Ca2+ (sustained [Ca2+]i increase). The Ca2+ responses activated by PE stimulation were completely blocked by pretreatment of the cells with the α1-adrenergic receptor antagonist doxazosin and there were no Ca2+ responses in cells transfected with an empty vector DNA (data not shown). The Ca2+ response data from the rat-1 cells stably expressing α1-adrenergic receptors is consistent with most previous studies in vascular smooth muscle cells (Lepretre et al., 1994), a neuronal cell line (Esbenshade et al., 1993), and transfected COS and Chinese hamster ovary (CHO) cell lines (Horie et al., 1994; Awaji et al., 1996).
Induction of c-fos mRNA Expression by α1-Adrenergic Receptor Subtypes in Rat-1 Cells.
The α1-adrenergic receptor-selective agonist PE stimulated induction of c-fos mRNA expression in a time- and dose-dependent manner (Fig. 2). The induction of c-fos mRNA by α1Dreceptor activation was much less than for α1Aand α1B receptors. This may be related, at least in part, to different levels of expression of these receptors in transfected rat-1 cells, as indicated by ligand binding experiments (Kenny et al., 1996) or by a lower efficacy of these receptors (Taguchi et al., 1998). Subsequent experiments were conducted in cells expressing α1A and α1Breceptor subtypes.
Effects of Manipulating Ca2+ Signaling on c-fos mRNA Induction by α1A and α1B Receptors.
Using BAPTA/AM, an intracellular Ca2+ chelator (Tsien, 1980), preliminary experiments confirmed that PE did not induce an increase in [Ca2+]i in the BAPTA-loaded cells expressing α1A or α1B receptors (Fig.3, middle column). When supplemental free Ca2+ (10 mM) was added to the assay buffer, the Ca2+/Fura-2 fluorescence signal was not detected until the Ca2+ ionophore ionomycin (2 μM) was added, at which point the Ca2+ signal gradually increased to the same values found in control cells (without preloaded BAPTA). These results suggest that intracellular BAPTA not only completely blocked increases in [Ca2+]i induced by α1-adrenergic receptors but also caused no interference with Ca2+ measurements and did not damage cell viability (cells still normally restricted Ca2+ entry in the absence of the Ca2+ ionophore). Preloading of cells with BAPTA attenuated the induction of c-fos mRNA in response to PE. BAPTA preloading itself had no effect on basal expression of c-fos mRNA in the cells (Fig.4). BAPTA inhibited c-fos mRNA expression by 80 ± 9% (n = 6) and 70 ± 12% (n = 6) for α1A and α1B subtypes, respectively, as shown in Table1.
The next experiments investigated the relative importance of the source of Ca2+ in the induction of c-fos gene transcription by α1-adrenergic receptors. To determine a possible role of extracellular Ca2+influx in the induction of c-fos mRNA, we removed extracellular Ca2+ from culture medium by addition of the Ca2+ chelator EGTA (5 mM). EGTA had no effects on PE-induced Ca2+ release from internal Ca2+ stores, but the extracellular Ca2+ influx increased by ionomycin was decreased by more than 90% in the presence of EGTA (Fig. 3, right column). As shown in Table 1, removal of extracellular Ca2+significantly decreased PE-induced c-fos mRNA expression by 61 ± 12% (n = 5) and 46 ± 11% (n = 4) for α1A and α1B, respectively, with p < .01 (t test) as compared with EGTA-untreated cells. When the free Ca2+ concentration in the culture medium was restored by addition of supplemental Ca2+, the inhibitory effect of EGTA was completely reversed for both receptor subtypes (Figs. 5 and Table 1), suggesting that EGTA was not having nonspecific effects on the cells. These results suggest that sustained Ca2+ influx is important for c-fos mRNA induction.
We next determined the potential importance of the initial transient rise in [Ca2+]i occurring immediately after stimulation of α1 receptors on induction of c-fos mRNA expression. Cells were stimulated with PE for 1 min; at that point, the medium was replaced with fresh medium containing the α1-adrenergic receptor antagonist doxazosin (5 μM) to terminate further PE stimulation. After a total of 30 min, c-fos mRNA expression in the cells was determined. Under these conditions, the induction of c-fos mRNA was about 30 to 40% of the response found after cells were stimulated for 30 min with PE (Fig.6). The rinsing procedure with doxazosin- or dimethyl sulfoxide (DMSO)-containing medium had no effects on basal level of c-fos mRNA expression. The results suggest that brief stimulation with PE, which triggers Ca2+release from internal Ca2+ stores, only partially induces c-fos expression. These experiments are in good agreement with the results indicating the important role of extracellular Ca2+ in inducing c-fosexpression (Fig. 5).
Role of CaM in Induction of c-fos by α1-Adrenergic Receptor Subtypes.
Increases in [Ca2+]i are known to regulate a variety of intracellular enzymes through association with CaM (Vogel, 1994; Braun and Schulman, 1995). To determine whether activation of CaM was involved downstream of α1-adrenergic receptor-stimulated Ca2+ responses to induce c-fos gene transcription, cells were incubated with the CaM antagonist calmidazolium (R24571). As shown at Fig.7, preincubation of the cells with 10 μM R24571 for 1 h significantly decreased PE-induced c-fos mRNA expression by both α1Aand α1B receptors; indeed, the extent of inhibition was similar to that caused by the intracellular Ca2+ chelator BAPTA. Because R24571 itself slightly stimulated c-fos expression (Fig. 7), this action could hypothetically function as an autorepressor of c-fosgene transcription in response to other stimuli including PE (Ofir et al., 1990). To rule out this possibility, we preincubated cells with the protein synthesis inhibitor cycloheximide (3 μM for 5 min) (Zinck et al., 1995) before addition of R24571 to the culture medium. The inhibition of protein synthesis by cycloheximide did not prevent the inhibitory effect of R24571 on α1-adrenergic receptor-mediated c-fos induction, suggesting that the effects of R24571 were not due to FOS-mediated inhibition of the c-fos gene. Two other structurally distinct CaM antagonists, Trifluoperazine dimale, andN-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7), similarly inhibited c-fos induction; neither of these antagonists stimulated basal c-fos mRNA expression (data not shown). None of these CaM antagonists modified α1-adrenergic receptor-mediated Ca2+ responses in these cells (data no shown).
Ca2+/CaM-dependent protein kinases (CaM kinases), particularly types II and IV, have been implicated in induction of c-fos gene transcription through phosphorylating transcription factors (Miranti et al., 1995; Wang and Simonson, 1996). KN-62 is a selective inhibitor for CaM kinases (Mochizuki et al., 1993;Enslen and Soderling, 1994). Cells were preincubated with 10 μM KN-62 for 30 min before stimulation with PE (10 μM). This inhibitor did not antagonize induction of c-fos mRNA expression (Fig. 7B). We next asked whether the CaM-dependent protein phosphatase calcineurin is a candidate for Ca2+/CaM modulation of c-fos induction by α1-adrenergic receptors. Pretreatment of cells with FK506 or cyclosporine A (0.2 μM, 30 min for both inhibitors), specific and potent inhibitors of the calcineurin (Liu et al., 1991), had no significant effects on PE-induced c-fos induction in the rat-1 transformants expressing both α1A and α1B receptors (data not shown).
Absence of Involvement of Ras/MAP Kinase, PKA, and PKC in c-fos Induction by α1-Adrenergic Receptor Subtypes in Rat-1 Cells.
As shown above, the intracellular Ca2+ chelator BAPTA completely inhibited α1-adrenergic receptor-mediated increase in [Ca2+]i but incompletely inhibited c-fos mRNA induction by either α1A- or α1B-subtype receptors (Fig. 6). To determine the potential role of alternative signaling pathways in inducing c-fos mRNA expression, we examined whether activation of Ras/MAP kinase, PKC, or PKA were involved in c-fos induction in rat-1 cells expressing α1-adrenergic receptors. Cells were pretreated with the PKC inhibitor, GF109203X (10 μM 30 min), which inhibits all PKC isozymes (Martiny-Baron et al., 1993), or with prolonged pretreatment with PMA (100 nM, 24 h) to deplete PKC before PE stimulation. To determine the potential involvement of PKA, we treated the cells with the adenylyl cyclase inhibitor didexyadenosine (10 μM, 30 min) or transfected them with the cDNA for the PKA inhibitory peptide PKI (Grove et al., 1989). None of these approaches inhibited c-fos induction by α1A or α1B receptors in the rat-1 cells (Fig. 8). Neither PMA (100 nM, 30 min) nor increasing cellular cAMP (by stimulation with forskolin or adding dibutyryl-cAMP) had much capacity to induce c-fos expression in the cells (data not shown). There was also no difference in PMA-induced c-fos expression between cells expressing the various α1 subtypes. Although PMA alone slightly induced c-fos expression (Fig.8), in repeated experiments (3–4 times), there was no difference among α1 subtypes and empty vector-transfected rat-1 cells. However, we found previously that PMA-induced CREB phosphorylation is dependent on PKC and that α1-adrenergic receptor-mediated CREB phosphorylation involves the cAMP signaling pathway in the rat-1 cells (Lin et al., 1998). This suggests that the approaches used for manipulation of PKC and cAMP were efficient in this study. These findings suggest that activation of neither PKC nor PKA pathways is involved in c-fos induction by α1receptors in rat-1 cells.
We examined whether α1-adrenergic receptors stimulated MAP kinase activity in rat-1 cells. MAP kinase was immunoprecipitated from cell lysates of PE (10 μM, 10 min)-stimulated rat-1 cells with anti-ERK1/ERK2 antibodies. Activity of the kinase in the immunocomplex was measured based on32P-phosphorylation of substrate MBP. As shown at Fig. 9, PE did not stimulate activity of MAP kinase for either α1A or α1B receptors in the rat-1 cells. We have previously found that α1 receptors activate MAP kinase in cultured vascular smooth muscle cells (Hu et al., 1996). Epidermal growth factor (EGF), as positive control, increased MAP kinase activity about 10-fold in the rat-1 cells; however, under these conditions EGF very weakly induced c-fos mRNA expression in the same cells (data not shown). Together, these results suggest that the MAP kinase pathway is not involved in the c-fosinduction in rat-1 cells.
Discussion
The current study investigated the role of Ca2+ signaling pathways in the induction of c-fos gene expression mediated by α1-adrenergic receptors in rat-1 fibroblasts. We demonstrated that induction of the c-fos gene expression by these receptors is importantly dependent on an increase in intracellular free Ca2+ rather than activation of Ras/MAP kinase, protein kinase C or cAMP signaling pathways. Ca2+ activation of CaM-associated signaling contributes significantly to induction of c-fos mRNA. However, the well-characterized Ca2+/CaMs, such as CaM kinase II and IV and Ca2+/CaM-dependent protein phosphatase calcineurin, are not likely involved in the activation of expression of the c-fos gene by α1 receptors.
Previous studies have demonstrated the importance of Ca2+ influx in the induction of c-fosmRNA expression, for example, via voltage-sensitive Ca2+ channels in PC12 cells (Thompson et al., 1995) and via voltage-insensitive Ca2+ channels in mesangial cells (Wang and Simonson, 1996). α1-Adrenergic receptors induce initial, rapid transient increases in [Ca2+]i (due to Ca2+ release from inositol triphosphate-sensitive stores) and a sustained slow increase in [Ca2+]i (due to extracellular Ca2+ influx) in the rat-1 cells. However, the strong transient Ca2+ increase in the initial phase (less than 1 min) was not enough to fully stimulate c-fos induction, suggesting that sustained increases in Ca2+ are required for maximal induction of c-fos transcription by α1-adrenergic receptors. On the other hand, a 1-min transient increase in [Ca2+]i induced by a Ca2+ ionophore was sufficient for full induction of c-fos expression in promyelocytic HL-60 cells (Werlen et al., 1993). Also, a brief activation of muscarinic receptors resulted in a maximal increase in c-fos transcription induced by intracellular Ca2+ increase, although activation of PKC was required for this response (Trejo and Brown, 1991). These differences in response to brief changes in Ca2+concentrations require further explanation, and are likely dependent on cell- or receptor-specific factors.
Ca2+ can activate multiple signaling pathways that ultimately converge on activation of c-fos gene transcription (Roche and Prentki, 1994; Rosen et al., 1995; Karin, 1995). Two major inducible enhancer elements, namely the CRE or Ca2+ response element, and the SRE, are activated by Ca2+-dependent pathways (Rosen et al., 1995). Multiple signaling pathways are associated with phosphorylation and activation of CRE- and SRE-binding transcription factors. CREB is activated by phosphorylation on serine-133 by a number of kinases that may be directly or indirectly activated by increased intracellular Ca2+; for example, by CaM kinases (Sheng et al., 1991; Sun et al., 1994), cAMP-dependent PKA (Sheng et al., 1991;Hagiwara et al., 1993), the Ras/MAP kinase pathway (Segal and Greenberg, 1996), and Ca2+-dependent PKC (Xie and Rothstein, 1995). Our data suggest that in the rat-1 cells Ca2+ mediates c-fos induction by α1-adrenergic receptors without requiring activation of PKA, Ras/MAP kinase, or PKC. This conclusion is further supported by evidence that direct activation of these pathways using forskolin (for cAMP/PKA), EGF (for Ras/MAP kinase), or PMA (for PKC) had little or no effect on c-fos induction in the rat-1 cells transfected with or without α1-adrenergic receptors.
Elevated concentrations of cAMP lead to the induction of c-fos gene expression through activating PKA, which then translocates to the nucleus and catalyzes the phosphorylation of CREB at serine-133 (Gonzalez and Montminy, 1989; Hagiwara et al., 1993). Although α1-adrenergic receptor agonists increase cAMP accumulation in the rat-1 cells stably expressing α1-adrenergic receptors (Lin et al., 1998), as in other cells (Graham et al., 1996; Guarino et al., 1996), treatment of the rat-1 cells with either adenylyl cyclase activator forskolin or a cAMP analog did not effectively stimulate c-fos mRNA expression. Transfection of cells with PKI (Grove et al., 1989) did not attenuate induction of c-fos by α1-adrenergic receptors. These results indicate that cAMP is unlikely involved in activation of c-fos gene promoter in the rat-1 cells. However, we have found that stimulation of α1-adrenergic receptors in these cells induces CREB phosphorylation at serine-133 through a cAMP-dependent pathway (Lin et al., 1998). Although serine-133 phosphorylation frequently activates gene transcription through CRE regulation (Ginty et al., 1994), taken together our results suggest that serine-133 phosphorylation of CREB is insufficient to induce the c-fosgene in these cells.
Increased intracellular Ca2+ frequently regulates cellular responses via association with CaM. The Ca2+/CaM complex binds to and modulates the activities of multiple enzymes, including CaM-pendent protein kinases (CaM kinases) (Vogel, 1994; Braun and Schulman, 1995) and Ca2+-dependent protein phosphatases such as calcineurin (Fruman et al., 1992; Enslen and Soderling, 1994; Chen et al., 1996; Schaefer et al., 1996). Activation of CaM kinases II and IV by Ca2+/CaM may induce Ca2+-mediated CREB phosphorylation (Sheng et al., 1991; Enslen et al., 1994; Enslen and Soderling, 1994; Sun et al., 1994), which then activates a CRE enhancer in the c-fos gene promoter. Calcineurin has been also implicated in the regulation of Ca2+-induced immediate early gene expression (Enslen and Soderling, 1994; Schaefer et al., 1996). In the current study, inactivation of CaM with the CaM antagonist R24571 (Fig. 7), trifluoperazine dimale, and W7 (data not shown) significantly inhibited PE-induced c-fos induction in the rat-1 cells. However, pretreatment of cells with KN-62, a specific inhibitor of CaM kinases II, IV, and V (Tokumitsu et al., 1990; Mochizuki et al., 1993; Enslen and Soderling, 1994), did not block PE-induced c-fosexpression. Also, two specific calcineurin inhibitors, FK506 and cyclosporine A, had no effect on c-fos expression induced by α1-adrenergic receptors. These results suggest that the α1-adrenergic receptor-induced Ca2+-dependent c-fos expression depends on CaM but does not involve these specific CaM-associated proteins.
A recent study found that prolonged pretreatment of transfected rat-1 cells with PMA inhibited c-fos expression induced by norepinephrine, and suggested that PKC may play a key role (Garcia-Sainz et al., 1998). In our study, neither prolonged pretreatment with PMA nor the PKC inhibitor GF109203X inhibited c-fos expression mediated by α1-adrenergic receptors in rat-1 cells. We do not know the reason for the difference in these results.
In summary, α1-adrenergic receptor-induced c-fos gene transcription is critically dependent on increased intracellular Ca2+ and is mediated by CaM. In rat-1 cells, c-fos induction is independent of PKA, PKC, and the Ras/MAP kinase pathway, and appears independent of well-known Ca2+/CaM-associated protein kinases and protein phosphatases. Further study will determine possible signaling mechanisms by which α1-adrenergic receptors-stimulated Ca2+ converges to activate regulatory elements in the c-fos gene promoter.
Acknowledgments
We thank Dr. G. Johnson of the Pfizer Laboratory for allowing us to use rat-1 cells stably expressing α1-adrenergic receptor subtypes, and Dr. J. Avruch for the PKI expression plasmid. Dr. Paul De Koninck made helpful suggestions. Xiaoyou Shi provided excellent technical assistance.
Footnotes
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Send reprint requests to: Brian B. Hoffman, M.D., Veterans Affairs Medical Center, Geriatrics Research, Education and Clinical Center 182B, 3801 Miranda Ave., Palo Alto, CA 94304. E-mail:bhoffman{at}leland.stanford.edu
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↵1 This study was supported in part by a grant (HL41315) from National Institutes of Health and the Research Service of the VA.
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↵2 Recipient, National Research Service Award (Institutional), and Fellowship for Careers in Clinical Pharmacology from the Pharmaceutical Research and Manufacturers of America (PhRMA) Foundation.
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↵3 Current address: University of Texas Health Science Center at San Antonio, Department of Pharmacology, San Antonio, TX 78284.
- Abbreviations:
- CaM
- calmodulin
- CaM kinase
- Ca2+/CaM-dependent kinases
- PKA
- protein kinase A
- PKC
- protein kinase C
- MAP kinase
- mitogen-activated protein kinase
- ERK
- extracellular stimulus response kinase
- CREB
- cAMP response element binding protein
- CRE
- cAMP response element
- SRE
- serum response element
- R24571
- calmidazolium chloride
- PMA
- phorbol 12-myristate 13-acetate
- HBSS
- Hanks’ balanced saline solution
- W7
- N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide
- DMEM
- Dulbecco’s modified Eagle’s medium
- BAPTA/AM
- 1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra (acetoxymethyl) ester
- MBP
- myelin basic protein
- PKI
- protein kinase A inhibitory peptide
- Received August 31, 1998.
- Accepted January 29, 1999.
- The American Society for Pharmacology and Experimental Therapeutics