Introduction

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₁-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 ₁-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 ₁-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 ₁-adrenergic receptors induce c-fos expression.

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 ₁-adrenergic receptors induces intracellular Ca²⁺ mobilization in many cells (Guarino et al., 1996). In addition, stimulation of ₁-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 ₁ receptors activation of c-fos transcription, we used rat-1 fibroblast cell lines stably transfected with each of three ₁-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 ₁-adrenergic receptor-mediated c-fos induction.

	Experimental Procedures

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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. [³²P]ATP, [³²P]ATP, and DNA labeling system were from Amersham Co. (Arlington, IL).

Cell Culture and Transfection. Rat-1 fibroblasts stably transfected with human _1A, _1B, and _1D-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 ₁-adrenergic receptor-induced c-fos expression. For examination of c-fos expression 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 -adrenergic receptor in the cells) followed by stimulation with the ₁-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 with ³²P-labeled v-fos cDNA (pstI fragment) and reprobed with human -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 [³²P]ATP in kinase buffer at 30°C for 10 min. The ³²P-phosphorylated MBP was detected by electrophoresis on SDS-polyacrylamide gel electorphoresis followed by autoradiography.

	Results

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Ca²⁺ Responses Mediated by ₁-Adrenergic Receptor Subtypes Expressed in Rat-1 Cells. In cells expressing each of the three ₁-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 ₁-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 ₁-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 ₁-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).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   PE-induced Ca2+ responses in rat-1 cells stably expressing each of three 1-adrenergic receptor subtypes. Cells were cultured on a coverslip and loaded with 1.5 µM Fura-2/AM in HBSS containing 15 mM HEPES and 0.1% BSA at room temperature for 30 min. Ca2+ signaling in response to 10 µM PE was recorded using a spectrofluorometer (Hitachi F2000) and expressed as ratio (F340/F380) of fluorescence intensity at excitation of 340 nm and 380 nm. Left column, Ca2+ was measured in Ca2+-containing buffer (HBSS with 1.8 mM Ca2+), demonstrating PE-induced an initial transient Ca2+ increase and a sustained Ca2+ influx. Middle column, Ca2+ was measured in Ca2+-free buffer (Ca2+-deficient HBSS with 1 mM EGTA) with Ca2+ (2 mM) reintroduced after phenylephrine stimulation to dissociate the initial Ca2+ release phase from the sustained Ca2+ influx phase. Right column, cells were pretreated with thapsigargin (Tg, 2 µM for 15 min) to deplete Ca2+ stores in the endoplasmic reticulum to confirm further that the initial peak stimulated by 1-adrenergic receptors was due to release of internal Ca2+ stores. This experiment was replicated four to five times with similar results.

Induction of c-fos mRNA Expression by ₁-Adrenergic Receptor Subtypes in Rat-1 Cells. The ₁-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 _1D receptor activation was much less than for _1A and _1B 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 _1A and _1B receptor subtypes.

View larger version (69K): [in this window] [in a new window]   Fig. 2.   Time course and dose response of c-fos gene induction by phenylephrine in rat-1 fibroblasts stably expressing 1-adrenergic receptors subtypes. Rat-1 cells were made quiescent in serum-free DMEM overnight and then stimulated with 10 µM PE for different times (upper panel) or with various concentrations (0-100 µM) of phenylephrine for 30 min (lower panel) at 37°C. Total RNA was extracted from cells and 10 µg total RNA was fractionated on glyoxal gels, as described in Experimental Procedures. c-fos mRNA transcript was determined by Northern blotting using a v-fos cDNA probe.

Effects of Manipulating Ca²⁺ Signaling on c-fos mRNA Induction by _1A and _1B 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 _1A or _1B 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 ₁-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 _1A and _1B subtypes, respectively, as shown in Table 1.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Manipulation of 1-adrenergic receptor-mediated Ca2+ mobilization by intracellular and extracellular Ca2+ chelators. Fura-2-loaded cells were incubated with DMSO control (left column) or with the intracellular Ca2+ chelator BAPTA/AM (10 µM) for 30 min (middle column), or with the extracellular Ca2+ chelator EGTA (5 mM) for 2 min (right column) in HBSS at 37°C. Ca2+ mobilization in response to 10 µM PE after these pretreatments was measured in the same buffer. Control (DMSO) responses to PE and the Ca2+ ionophore ionomycin (Ion, 2 µM) in cells expressing 1A and 1B receptors are shown in left column. In BAPTA-preloaded cells (middle column), PE did not stimulate detectable Ca2+ responses for either 1 receptor subtype. Addition of supplemental Ca2+ (10 mM) after PE stimulation did not increase in [Ca2+]i until ionomycin was added, suggesting that pretreatment with BAPTA did not interfere with the Ca2+/Fura-2 signal nor with cell viability. EGTA pretreatment (right column) did not change the PE-induced initial Ca2+ increase phase, whereas addition of Ca2+ ionophore ionomycin (Ion, 2 µM) after PE stimulation showed only a slight Ca2+ influx, suggesting most extracellular Ca2+ has been removed by EGTA. Reintroduction of Ca2+ (6.8 mM) to the assay buffer to reach the same free Ca2+ concentration (1.8 mM) as that before EGTA treatment increased Ca2+ influx signals to control values. This experiment was replicated three to four times with similar results.

View larger version (62K): [in this window] [in a new window]   Fig. 4.   Intracellular Ca2+ chelator BAPTA inhibits c-fos induction mediated by 1A and 1B receptors. Quiescent cells were preloaded with 10 µM BAPTA/AM at 37°C for 30 min followed by stimulation with or without 10 µM PE for 30 min. mRNA of c-fos a (upper panel) and -actin (lower panel) was measured by Northern blot analysis with 10 µg total RNA per lane. This experiment was repeated five times with similar results.

View this table: [in this window] [in a new window]   TABLE 1 Quantitation of inhibitory effects of Ca2+ manipulation on 1-adrenergic receptor-mediated c-fos mRNA induction in stably transformed rat-1 cells c-fos mRNA expression in Northern blots, as shown in Figs. 4 and 5, was quantified with PhosphoImager analysis and normalized to -actin mRNA expression or 18s RNA. For each condition, a percentage of response using PE-induced c-fos mRNA expression in cells without pretreatment with BAPTA and EGTA as 100% response, was calculated. Data represents mean ± S.D. of five to six separate experiments.

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Extracellular Ca2+ chelator EGTA inhibits c-fos induction mediated by 1A and 1B subtypes. Quiescent cells in DMEM containing 1.8 mM Ca2+ and pretreated with 5 mM EGTA or EGTA plus 6.8 mM Ca2+ for 5 min followed by stimulation with or without 10 µM phenylephrine or 2 µM A23187 (as positive control) for 30 min. c-fos mRNA expression was determined by Northern blot analysis with 10 µg total RNA per lane. This experiment was repeated five times with similar results.

View larger version (59K): [in this window] [in a new window]   Fig. 6.   Brief stimulation of 1-adrenergic receptors does not maximally induce c-fos expression. Quiescent cells were stimulated with 10 µM PE or vehicle for 1 min and then medium was replaced with fresh medium containing 5 µM doxazosin (DOX) to terminate PE action. The cells were then incubated in the same medium for another 30 min. c-fos mRNA expression was analyzed by Northern blot, with 10 µg total RNA per lane. The 1-min stimulation partially induced c-fos expression, suggesting that initial Ca2+ transient increase (internal Ca2+ release) stimulated by 1-adrenergic receptors is not sufficient to fully induce c-fos gene expression and that a sustained Ca2+ increase (requiring extracellular Ca2+ influx) is required for further 1-adrenergic receptor-mediated c-fos induction. This experiment was replicated twice with similar results.

Role of CaM in Induction of c-fos by ₁-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 ₁-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 _1A and _1B 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-fos gene 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 ₁-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, and N-(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 ₁-adrenergic receptor-mediated Ca²⁺ responses in these cells (data no shown).

View larger version (33K): [in this window] [in a new window]   Fig. 7.   Effects of Ca2+/CaM on 1-adrenergic receptor-mediated c-fos induction. Quiescent rat-1 cells were pretreated with CaM antagonist calmidazolium (R24571, 10 µM) at 37°C for 1 h (A), or with CaM kinase inhibitor KN-62 (10 µM) for 30 min (B). Control cells were treated with vehicle DMSO. Pretreated cells were then stimulated with 10 µM PE at 37°C for another 30 min. Ten micrograms of total RNA per lane was used for Northern blot analysis of c-fos mRNA expression. This experiment was replicated three times with similar results.

Absence of Involvement of Ras/MAP Kinase, PKA, and PKC in c-fos Induction by ₁-Adrenergic Receptor Subtypes in Rat-1 Cells. As shown above, the intracellular Ca²⁺ chelator BAPTA completely inhibited ₁-adrenergic receptor-mediated increase in [Ca²⁺]_i but incompletely inhibited c-fos mRNA induction by either _1A- or _1B-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 ₁-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 _1A or _1B 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 ₁ subtypes. Although PMA alone slightly induced c-fos expression (Fig. 8), in repeated experiments (3-4 times), there was no difference among ₁ subtypes and empty vector-transfected rat-1 cells. However, we found previously that PMA-induced CREB phosphorylation is dependent on PKC and that ₁-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 ₁ receptors in rat-1 cells.

View larger version (39K): [in this window] [in a new window]   Fig. 8.   1-Adrenergic receptor-mediated c-fos induction is independent of PKC. Quiescent cells were pretreated with PKC inhibitor GF109203X (50 µM) for 30 min (A) or with 100 nM PMA for 24 h (B) followed by stimulation with 10 µM PE for 30 min. Neither inhibition nor down-regulation of PKC changed 1A and 1B induction of c-fos mRNA expression. This experiment was replicated twice with similar results.

View larger version (56K): [in this window] [in a new window]   Fig. 9.   MAP kinase activity in rat-1 cells stably expressing 1-adrenergic receptors after stimulation with PE. Quiescent cells were stimulated with or without 10 µM PE or 100 ng/ml EGF for 10 min. Cell lysis, immunoprecipitation with anti-ERK1, and kinase activity assay were done as described in Experimental Procedures. 32P-phosphorylated MBP was analyzed by running 15% SDS-polyacrylamide gel electrophoresis followed by autoradiography. Neither 1-adrenergic receptor subtypes activated MAP kinase in these cells; however, EGF powerfully activated MAP kinase in these cells, suggesting that MAP kinase pathway is not involved in c-fos induction mediated by 1-adrenergic receptors. This experiment was replicated four to five times with similar results.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The current study investigated the role of Ca²⁺ signaling pathways in the induction of c-fos gene expression mediated by ₁-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 ₁ receptors.

Previous studies have demonstrated the importance of Ca²⁺ influx in the induction of c-fos mRNA 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). ₁-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 ₁-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 ₁-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 ₁-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 ₁-adrenergic receptor agonists increase cAMP accumulation in the rat-1 cells stably expressing ₁-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 ₁-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 ₁-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-fos gene 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-fos expression. Also, two specific calcineurin inhibitors, FK506 and cyclosporine A, had no effect on c-fos expression induced by ₁-adrenergic receptors. These results suggest that the ₁-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 ₁-adrenergic receptors in rat-1 cells. We do not know the reason for the difference in these results.

In summary, ₁-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 ₁-adrenergic receptors-stimulated Ca²⁺ converges to activate regulatory elements in the c-fos gene promoter.