Activated Protein Kinase Pathways in PC12 ... - Wiley Online Library

Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 1999 International Society for Neurochemistry

Differential Activation of Mitogen-Activated Protein Kinase Pathways in PC12 Cells by Closely Related ␣1-Adrenergic Receptor Subtypes Hongying Zhong and Kenneth P. Minneman Department of Pharmacology, Emory University Medical School, Atlanta, Georgia, U.S.A.

Abstract: Coupling of the three known ␣1-adrenergic receptor (␣1-AR) subtypes to mitogen-activated protein kinase (MAPK) pathways were studied in stably transfected PC12 cells. Subclones stably expressing ␣1A-, ␣1B-, and ␣1D-ARs under control of an inducible promoter, or at high and low receptor density, were isolated and characterized. Radioligand binding showed similar ranges of expression of each subtype. Norepinephrine (NE) increased inositol phosphate formation and intracellular Ca2⫹ level in these cells in a manner dependent on receptor density. However, ␣1A-ARs activated these second messenger responses more effectively than ␣1BARs, whereas ␣1D-ARs were least effective. NE stimulated activation of extracellular signal-regulated kinases (ERKs) in cells expressing all three ␣1-AR subtypes, although ␣1A- and ␣1B-ARs caused larger ERK activation than did ␣1D-ARs. Nerve growth factor (NGF) caused similar levels of ERK activation in all subclones. NE also activated p38 MAPK in ␣1A- and ␣1B- but not ␣1D-transfected cells and activated c-Jun NH2-terminal kinase (JNK) only in ␣1A-transfected cells. NE, but not NGF, strongly stimulated tyrosine phosphorylation of a 70-kDa protein only in ␣1A-transfected PC12 cells. NE caused neurite outgrowth only in ␣1A-expressing PC12 cells, but not in ␣1B- or ␣1D-transfected cells, whereas NGF caused neurite outgrowth in all cells. These studies show that ␣1A-ARs activate all three MAPK pathways, ␣1B-ARs activate ERKs and p38 but not JNKs, and ␣1D-ARs only activate ERKs. Only the ␣1A-AR-expressing cells differentiated in response to NE. The relationship of these responses to second messenger pathways activated by these subtypes is discussed. Key Words: Norepinephrine —PC12 cells—Mitogen-activated protein kinase — Adrenergic receptor. J. Neurochem. 72, 2388 –2396 (1999).

by growth factor receptors involves tyrosine phosphorylation and dimerization of the receptor, formation of Shc-Grb2-Sos complexes, and activation of Ras (Schlessinger and Ullrich, 1992; Lewis et al., 1998). However, the mechanisms by which GPCRs activate MAPK pathways are more complex and heterogeneous. In some cases, MAPK activation by GPCRs is mediated by second messengers such as cyclic AMP (Crespo et al., 1995), Ca2⫹ (Dikic et al., 1996), or protein kinase C (PKC) (Della Rocca et al., 1997). In other cases, MAPK activation appears to be mediated directly or indirectly through G␣ (Faure et al., 1994) or G␤␥ (Koch et al., 1994; van Biesen et al., 1995) or directly by heterotrimeric G proteins (Yamauchi et al., 1997). MAPKs are controlled by many stimuli in most cells and play critical roles in growth and differentiation. There are three major MAPK subfamilies, each with multiple isoforms: extracellular signal-regulated kinases (ERKs; also known as p42 and p44 MAPKs), c-Jun-NH2-terminal kinases (JNKs), and p38 MAPKs (Robinson and Cobb, 1997). Growth factor activation of ERKs contributes to growth and differentiation, with sustained activation of ERKs causing differentiation in PC12 cells (Marshall, 1995). JNKs and p38 MAPKs, on the other hand, are activated by stress stimuli such as heat, osmotic shock, or UV irradiation. Although JNKs and p38 MAPKs may play a role in preventing apoptosis in cells (Xia et al., 1995), their Received November 3, 1998; revised manuscript received January 21, 1999; accepted January 22, 1999. Address correspondence and reprint requests to Dr. H. Zhong at Department of Pharmacology, Emory University Medical School, Atlanta, GA 30322, U.S.A. Abbreviations used: AR, adrenergic receptor; BE 2254, [2-␤-(4hydroxyphenyl)ethylaminomethyl]tetralone; [Ca2⫹]i, intracellular Ca2⫹ concentration; DMEM, Dulbecco’s modified Eagle’s medium; ERK, extracellular signal-regulated protein (also known as p42 and p44 mitogen-activated protein kinases); GPCR, G protein-coupled receptor; 125 IBE, 125I-[2-␤-(4-hydroxyphenyl)ethylaminomethyl]tetralone; InsP, inositol phosphate; IPTG, isopropylthiogalactose; JNK, c-Jun NH2terminal kinase; MAPK, mitogen-activated protein kinase; NE, norepinephrine; NGF, nerve growth factor; PBS, phosphate-buffered saline; PKC, protein kinase C.

Mitogen-activated protein kinase (MAPK) pathways are activated by G protein-coupled receptors (GPCRs) as well as by growth factor receptors with intrinsic tyrosine kinase activity. Receptors that signal through Gs (Faure et al., 1994; Wan and Huang, 1998), Gi (van Biesen et al., 1995), and Gq (Hawes et al., 1995) have all been shown to activate these pathways. Activation of MAPK 2388

␣1-AR SUBTYPES AND PC12 MAPK PATHWAYS role in cell growth is not yet clear. JNK activation has been found to rescue cells from apoptosis (Sakata et al., 1995) but has also been associated with hepatic regeneration and T-cell activation (Hsueh and Lai, 1995; Westwick et al., 1995). Activation of p38 inhibits cyclin D1 transcription and therefore cell cycle progression (Lavoie et al., 1996), suggesting a negative role in cell proliferation. The balance between these pathways may determine cell fate (Xia et al., 1995). Adrenergic receptors (ARs) are involved in control of growth in several cell types. ␣1-ARs are Gq-coupled receptors, which regulate growth of myocardial (Zechner et al., 1997) and aortic smooth muscle (Nishio et al., 1996; Xin et al., 1997) cells, possibly through activation of MAPK pathways. The ␣1-AR subfamily includes three separate gene products (␣1A, ␣1B, and ␣1D) with different pharmacological properties (Hieble et al., 1995). We have shown previously that in PC12 cells stably transfected with ␣1A-ARs, norepinephrine (NE) activates all three MAPK pathways and causes differentiation similar to that caused by nerve growth factor (NGF) (Williams et al., 1998). These MAPK responses do not depend on either mobilization of intracellular Ca2⫹ or PKC activation (A. Berts et al., manuscript submitted for publication). Although all three ␣1-AR subtypes increase intracellular Ca2⫹ levels and activate PKC, they do so with different coupling efficiencies (␣1A ⬎ ␣1B ⬎ ␣1D) (Theroux et al., 1996). We have now examined whether these subtypes show similar differences in activating the various MAPK pathways in transfected PC12 cells. MATERIALS AND METHODS Materials PC12 cells were obtained from Cindy Miranti and Michael Greenberg (Harvard Medical School). The human ␣1A-AR cDNA (Hirasawa et al., 1993) was from Gozoh Tsujimoto (National Children’s Hospital, Tokyo, Japan), the human ␣1B-AR cDNA (Ramarao et al., 1992) was from Dianne Perez (Cleveland Clinic), and the human ␣1D-AR cDNA was cloned in our laboratory (Esbenshade et al., 1995). Other materials were obtained from the following sources: Lac-Switch vector system from Stratagene (La Jolla, CA, U.S.A.); fura-2 acetoxymethyl ester from Calbiochem (La Jolla); (⫺)-NE bitartrate, yohimbine, Dulbecco’s modified Eagle’s medium (DMEM), penicillin, and streptomycin from Sigma Chemical Co. (St. Louis, MO, U.S.A.); prazosin from Pfizer (Groton, CT, U.S.A.); carrier-free Na[125I] from Amersham (Arlington Heights, IL, U.S.A.); [3H]inositol (20 – 40 Ci/mmol) from American Radiolabelled Chemicals (St. Louis); phentolamine mesylate from Ciba-Geigy (Summit, NJ, U.S.A.); BE 2254 {[2-␤-(4-hydroxyphenyl)ethylaminomethyl]tetralone} from Beiersdorf AG (Hamburg, Germany); (⫹)niguldipine and BMY 7378 from Research Biochemicals International (Natick, MA, U.S.A.); phosphospecific MAPK (Thr202/Tyr204) antibody, phosphospecific JNK (Thr183/Tyr185) antibody, phosphospecific p38 MAPK (Thr180/Tyr182) antibody, MAPK antibody, JNK antibody, and p38 MAPK antibody from New England Biolabs (Beverly, MA, U.S.A.); phos-

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photyrosine antibody (P-Y 99) from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.); horseradish peroxidase-conjugated anti-mouse IgG and ECL reagent from Amersham; and horseradish peroxidase-conjugated anti-rabbit IgG from Bio-Rad (Hercules, CA, U.S.A.).

Cell culture Transfected PC12 cells were propagated in 75-cm2 flasks at 37°C in a humidified 5% CO2 incubator in DMEM containing 4.5 g/L glucose, 1.4% glutamine, 20 mM HEPES, 100 mg/L streptomycin, 105 units/L penicillin, 10% donor horse serum, and 5% fetal bovine serum. The cells were detached by gentle trituration and subcultured at a ratio of 1:3 on reaching confluency. Receptor expression was induced by treatment with 1 mM isopropylthiogalactose (IPTG) at least 24 h before each experiment. For measurements of MAPK, JNK, and p38 phosphorylation, 35-mm-diameter dishes were seeded at a density of 600,000 cells/2 ml. For studies involving Ca2⫹ level measurements, 100-mm-diameter dishes were seeded at a density of 6 ⫻ 106 cells/10 ml. Cells were grown to confluency before use.

Transfection PC12 cells were cotransfected with the Lac-Switch repressor (p3⬘SS) and the pRSVICAT operator vectors containing the human ␣1A-AR, ␣1B-AR, or ␣1D-AR cDNAs by Lipofectamine (GibcoBRL). Cells were propagated for several weeks in the presence of 250 ␮g/ml hygromycin and 500 ␮g/ml geneticin, and subclones were screened by radioligand binding for low constitutive and high inducible receptor expression.

Radioligand binding Confluent 100-mm-diameter plates were washed with phosphate-buffered saline (PBS; 20 mM sodium phosphate plus 154 mM NaCl, pH 7.6) and harvested by scraping. Cells were collected by centrifugation and homogenized with a Polytron. Cell membranes were collected by centrifugation at 30,000 g for 10 min and resuspended by homogenization. Receptor density was determined by saturation analysis of the ␣1-specific antagonist radioligand 125I-BE 2254 (125IBE; 20 – 800 pM) (Theroux et al., 1996). For analysis of competition by selective drugs, 50 pM radioligand was used. Nonspecific binding was defined as binding in the presence of 10 ␮M phentolamine.

Inositol phosphate (InsP) formation Accumulation of 3H-InsPs was determined in 35-mm-diameter dishes. Cells were treated with or without 1 mM IPTG for 48 h and labeled with myo-[3H]inositol (2 ␮Ci per plate) for 1–2 days in regular cell culture medium. Production of total 3 H-InsPs in the presence of 10 mM LiCl was determined as previously described (Esbenshade et al., 1993).

Immunoblotting Confluent cells were serum-starved for 2 h before use, and drug treatments were carried out for 15 min at 37°C. After stimulation, monolayers were lysed with Laemmli sample buffer. Cell lysates were centrifuged, and proteins (20 ␮g per lane) were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose. Phosphorylation of ERKs, JNKs, and p38 was detected by protein immunoblotting using a 1:1,000 dilution of rabbit polyclonal dual phosphospecific antibodies with horseradish peroxideconjugated goat anti-rabbit IgG as a secondary antibody (Williams et al., 1998). Protein tyrosine phosphorylation was detected by immunoblotting using a 1:2,000 dilution of mouse polyclonal phosphotyrosine-specific antibody with horseradish

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H. ZHONG AND K. P. MINNEMAN FIG. 1. Saturation binding of ␣1A-, ␣1B-, and ␣1DARs in subclones of PC12 cells. Cells were transfected with human ␣1A-, ␣1B-, and ␣1D-AR cDNA in the Lac-Switch vector system, selected, and propagated as described in Materials and Methods. Receptor density was determined in ␣1A subclone 3 and ␣1B subclone 8 treated without (control) or with (induced) 1 mM IPTG for 48 h by saturation analysis of specific 125IBE binding. Two ␣1D subclones (low and high) that constitutively expressed these receptors at low and high densities, respectively, were also used, because inducible expression in the appropriate range was not obtained. Data are mean ⫾ SEM (bars) values of three experiments performed in duplicate.

peroxidase-conjugated goat anti-mouse IgG as a secondary antibody. Quantification was performed by densitometry after development of membranes with ECL reagent and exposure to Hyperfilm (Amersham).

Immunoprecipitation Confluent cells were serum-starved for 2 h before further treatment. Cells were washed twice with ice-cold PBS containing 1 mM sodium orthovanadate and lysed on ice with RIPA lysis buffer (1% Nonidet P-40, 25 mM HEPES, 50 mM NaCl, 50 mM NaF, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, 10 ␮g/ml aprotinin, and 10 ␮g/ml leupeptin). Cell lysate was centrifuged at 3,000 g for 15 min at 4°C. The supernatant containing 1 mg of protein was incubated with 10 ␮g of anti-phosphotyrosine (P-Y 99; Santa Cruz) antibody at 4°C for 2 h followed by addition of 20 ␮l of protein A-agarose. After overnight incubation at 4°C, the sample was centrifuged, and the immunoprecipitates were washed three times with lysis buffer. After boiling in 30 ␮l of 2⫻ sodium dodecyl sulfate-sample buffer, 15 ␮l of supernatant was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Protein bands were detected by probing sequentially with primary antibody (anti-phosphotyrosine, 1:1,000), horseradish peroxidase-conjugated secondary antibody (1:5,000), and ECL reagent.

Intracellular Ca2ⴙ concentration [Ca2ⴙ]i determinations [Ca2⫹]i transients were determined using fura-2 as described previously (Esbenshade et al., 1993). Confluent 100-mm-diameter plates were washed with balanced salt solution (130 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.5 mM CaCl2, 20 mM HEPES, 10 mM glucose, and 0.1% bovine serum albumin), and cells were detached by mild trypsinization (0.25% trypsin). Cells were rinsed three or four times with balanced salt solution and stored on ice. One milliliter of cell suspension (1 ⫻ 106 cells/ml) was incubated with 1 ␮M fura-2 acetoxymethyl ester for 10 min at 37°C, rinsed 10 min with balanced salt solution, and diluted to 3 ml before the experiment. The cell suspension was transferred to a cuvette and placed in a Perkin-Elmer (Beaconsfield, Buckingshamshire, U.K.) model LS 50B luminescence spectrofluorometer with a thermostatted (37°C) stirred cell holder. The excitation wavelengths were 340 and 380 nm, and the emission wavelength was 510 nm. Calibration of the fluorescence signals for calculation of [Ca2⫹]i was performed by equilibrating intracellular and extracellular Ca2⫹ with 30 ␮M digitonin (Rmax), followed by addition of 300 mM EGTA and 1 M Tris (pH 9.0) (Rmin), and using a KD of 225 nM for fura-2 (Grynkiewicz et al., 1985).


RESULTS Expression of ␣1-AR subtypes in PC12 cells Because PC12 cells do not natively express any detectable levels of AR subtypes (Williams et al., 1998), these cell lines provide a useful system to study MAPK responses to ␣1A-, ␣1B-, and ␣1D-ARs. Human ␣1A-, ␣1B-, and ␣1D-AR cDNAs in the operator vector of the Lac-Switch inducible expression system were cotransfected with the lac repressor vector into PC12 cells. Subclones were screened for low constitutive and highly inducible receptor expression by radioligand binding, and subclones with expression levels similar to those found commonly in vivo were isolated and propagated. We were unable to obtain subclones with inducible expression of ␣1D-ARs in this range; thus, subclones that constitutively express ␣1D-ARs at low and high densities were chosen for the study. As shown in Fig. 1, 1 mM IPTG caused a six- to ninefold increase in ␣1A- and ␣1B-AR expression in the subclones studied (␣1A subclone 3 and ␣1B subclone 8), without significant changes in their affinity for the radioligand 125IBE (Table 1). In the two ␣1D subclones (high and low), there was a 6.5-fold difference in expression levels of low- and highexpressing clones, comparable to those of the uninduced and induced ␣1A and ␣1B subclones. These subclones were used in all further experiments, except where noted. Pharmacological characteristics of ␣1A-, ␣1B-, and ␣1D-ARs in PC12 cells Displacement of specific radioligand binding to the expressed receptors by subtype-selective antagonists was TABLE 1. Bmax and KD values for PC12 subclones stably expressing human ␣1A-, ␣1B-, and ␣1D-AR subtypes Bmax (fmol/mg)

␣1A subclone 3 ␣1B subclone 8 ␣1D subclones

KD (fmol)

Control

Induced

Control

Induced

47 ⫾ 9.9 28 ⫾ 8.6 106 ⫾ 8.8a

268 ⫾ 10 241 ⫾ 28.6 698 ⫾ 49.1

66 ⫾ 42.2 63 ⫾ 59.7 50 ⫾ 2.8a

32 ⫾ 4.3 97 ⫾ 30.3 76 ⫾ 14.6

Data are mean ⫾ SEM values (n ⫽ 3). These values are from subclones constitutively expressing these receptors at low (control) or high (induced) levels, respectively (see text). a

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cellular Ca2⫹ responses to NE in the PC12 subclones. Figure 4 shows that NE increased [Ca2⫹]i in all cell lines and that the increase was dependent on receptor density. Consistent with results in other cell lines (Theroux et al., 1996), ␣1A-ARs had the largest effect, and ␣1D-ARs had the smallest effect, as shown by fold stimulation over basal values, given in Table 2.

FIG. 2. Displacement of specific 125IBE binding by selective antagonists in membranes from ␣1A-, ␣1B-, or ␣1D-AR-expressing PC12 cells. The ␣1A subclone 3 and ␣1B subclone 8 were induced with 1 mM IPTG for 48 h before membranes were isolated for experiments. ␣1D high, which expressed a higher receptor density, was used for these experiments. Data are mean ⫾ SEM (bars) values of two experiments, each performed in triplicate.

used to confirm their pharmacological properties. Figure 2 shows that the ␣1-selective antagonist prazosin inhibited specific 125IBE binding with similar high potency for all three subtypes (top left), whereas the ␣2-selective antagonist yohimbine showed the expected low potency at all three subtypes (top right). The ␣1A-selective antagonist (⫹)-niguldipine was 100-fold more potent in competing for the ␣1A-AR than the other two subtypes (bottom left), whereas the ␣1D-selective antagonist BMY 7378 was 100-fold more potent at the ␣1D-AR than either of the other two subtypes (bottom right), showing that the expressed receptors show the expected pharmacological properties. InsP formation The functional coupling of the receptors was assessed by examining NE-stimulated InsP formation in ␣1A-, ␣1B-, and ␣1D-expressing PC12 subclones. Figure 3 shows that NE stimulated InsP formation in all subclones tested. In ␣1A and ␣1B subclones, induction of receptor expression with 1 mM IPTG increased the response to NE as expected. In ␣1D subclones, higher receptor density caused a greater InsP response to NE, although an increase in basal InsP formation was apparent compared with ␣1A and ␣1B subclones. Although all three subtypes stimulated InsP formation in PC12 cells, differences in coupling efficiency (␣1A ⬎ ␣1B ⬎ ␣1D) were observed similar to those observed previously in transfected HEK 293 and SK-N-MC neuroblastoma cells (Theroux et al., 1996). Increases in [Ca2ⴙ]i Activation of ␣1-ARs increases [Ca2⫹]i through a phospholipase C-mediated release of inositol 1,4,5trisphosphate (Hieble et al., 1995). We measured intra-

Activation of ERKs, JNKs, and p38 MAPK by ␣1-AR subtypes We next compared the ability of each subtype to activate the three parallel MAPK pathways. Figure 5 (upper panel) shows that NE caused inducible increases in ERK activation in response to induction of both ␣1Aand ␣1B-AR expression. However, activation of ␣1AARs had a larger effect than activation of ␣1B-ARs. In the low-expressing ␣1D subclone, NE caused no detectable ERK activation (data not shown). However, in the high-expressing ␣1D subclone, NE caused a weak ERK activation (Table 3). NGF activated ERKs to similar extents in all three subclones (Table 3), suggesting that differences in NE response are not due to differences among subclones. Blotting with total ERK antibody shows that all three subclones express similar levels of ERK (Fig. 5, lower panel). NE also strongly activated JNKs in ␣1A-AR PC12 cells where receptor density had been increased by induction with IPTG (Fig. 6, upper panel). However, NE caused no significant activation of JNKs in either ␣1B- or ␣1D-AR PC12 cells, even at high receptor densities (Table 3). All subclones showed similar levels of total JNK expression (Fig. 6, lower panel). NE stimulation of ␣1A-ARs also strongly activated p38 MAPK, whereas activation of ␣1B-ARs had a smaller effect, and activation of ␣1D-ARs caused no detectable activation of p38 MAPK (Fig. 7, upper panel, and Table 3). All three subclones have a similar level of p38 expression (Fig. 7, lower panel). These results were verified in at least one additional PC12 subclone expressing each subtype (␣1A subclone 28, ␣1B subclone 4, ␣1D subclone 3, and ␣1D subclone E) at densities similar to those in the subclones shown in Table 3. This confirms that these differences are due to ␣1A-AR subtype rather than subclone.

FIG. 3. InsP formation in ␣1A subclone 3, ␣1B subclone 8, and ␣1D low and high PC12 subclones. The ␣1A subclone 3 and ␣1B subclone 8 cells were treated without (Ctl) or with (Ind) 1 mM IPTG for 48 h before stimulation without (basal) or with (NE) 100 ␮M NE, and 3H-InsP formation was measured as described in Materials and Methods. Data are mean ⫾ SEM (bars) values of two experiments performed in triplicate, expressed as percent conversion of [3H]inositol into the phosphate form.


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H. ZHONG AND K. P. MINNEMAN FIG. 4. Increases in [Ca2⫹]i in ␣1A subclone 3, ␣1B subclone 8, and ␣1D high PC12 cells. The ␣1A subclone 3 and ␣1B subclone 8 cells were treated without (Ctl) or with (Ind) 1 mM IPTG for 48 h before Ca2⫹ level measurements. The ␣1D high clone was used for measurement of the Ca2⫹ response. Where indicated, 100 ␮M NE and 10 ␮M phentolamine (Phent) were added. Shown is one representative experiment from each cell line. Fold increase over basal [Ca2⫹]i from three experiments is shown in Table 2.

In conclusion, the three ␣1-AR subtypes showed clear differences in their abilities to activate the different MAPK pathways. ␣1A-ARs were the most efficient in activating all three arms of MAPK pathways, ␣1B-ARs activated ERKs and weakly activated p38, whereas ␣1DARs weakly activated ERKs but showed little or no activation of either JNKs or p38 MAPK. Protein tyrosine phosphorylation We next examined the pattern of protein tyrosine phosphorylation stimulated by NE in these cell lines, comparing this pattern with that caused by NGF. Figure 8 (left panel) shows that NE and NGF caused different patterns of protein tyrosine phosphorylation. More interesting is that a strong tyrosine phosphorylated band of ⬃70 kDa appeared in response to NE in ␣1A-AR PC12 cells, which was not observed in response to NGF. This is further confirmed by immunoprecipitation studies with an anti-phosphotyrosine antibody (Fig. 8, right panel). However, ␣1B- and ␣1D-AR PC12 cells did not show this band following NE stimulation.

cells. However, exposure to NGF caused differentiation of the cells within 24 – 48 h to a similar extent as that observed in ␣1A subclone 3 cells (data not shown). Therefore, the differential effect of NE on differentiation of cells expressing the three ␣1-AR subtypes correlates with the effects on MAPK activation, with the ␣1A-AR subtype being the most effective. DISCUSSION Although control of growth and differentiation has traditionally been associated with peptides acting through tyrosine kinase receptors, we now know that many neurotransmitters and hormones acting through GPCRs are also important in these processes. Various Gq/11-coupled receptors, including ␣1-ARs, have been found to activate ERKs in many cell types, and ␣1-ARs appear to be particularly important in control of cell growth by catecholamines (LaMorte et al., 1994; Hawes et al., 1995; Sugden and Clerk, 1997). We have reported previously that NE causes ERK, JNK, and p38 MAPK

Differential effect of NE on differentiation in ␣1A-, ␣1B-, and ␣1D-transfected PC12 cells Exposure of ␣1A subclone 3 PC12 cells to either NE or NGF caused differentiation of the cells within 24 – 48 h after exposure (Fig. 9, top). The extent of NE-induced differentiation of ␣1A subclone 3 PC12 cells was dependent on the level of receptor expression. Cells expressing high levels of ␣1A-ARs (following induction with IPTG) displayed NE-induced differentiation similar to that observed with NGF alone, whereas cells expressing lower levels of ␣1A-ARs (not induced with IPTG) showed NE-induced differentiation only slightly higher than that of untreated cells (data not shown). Exposure of ␣1B subclone 8 (Fig. 9, middle) or ␣1D (Fig. 9, bottom) cells to NE failed to cause any detectable differentiation of the 2⫹

TABLE 2. NE-stimulated increases in [Ca ]i in PC12 cells that express each of the three ␣1-AR subtypes Fold over basal

␣1A subclone 3 ␣1B subclone 8 ␣1D high

5.05 ⫾ 0.77 2.64 ⫾ 0.10 1.65 ⫾ 0.13

Responses are expressed as the maximal fold stimulation over basal (see Fig. 4). Data are mean ⫾ SEM values of at least four experiments.


FIG. 5. Upper panel: Activation of ERKs by ␣1-AR subtypes. The ␣1A subclone 3 and ␣1B subclone 8 cells were induced without (Ctl) or with (Ind) 1 mM IPTG for 48 h before experiments. The ␣1D high cells were not treated with IPTG. Cells were serumstarved for 2 h before adding vehicle (C), 100 ␮M NE, or 100 ng/ml NGF for 15 min. Ten micrograms of protein was used for western blotting with phosphospecific ERK1/2 antibody as described in Materials and Methods. Lower panel: Total level of ERK expression in the three subclones studied. Ten micrograms of protein was used for western blotting with ERK1/2 antibody as described in Materials and Methods. Shown here is a representative experiment, with averaged results from multiple experiments shown in Table 3.

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FIG. 7. Upper panel: Activation of p38 MAPK by ␣1-AR subtypes. The ␣1A subclone 3 and ␣1B subclone 8 cells were induced (Ind) with 1 mM IPTG for 48 h before experiments. The ␣1D high cells were not treated with IPTG. Cells were serum-starved for 2 h before adding vehicle (C), 100 ␮M NE, or 100 ng/ml NGF for 15 min. Ten micrograms of protein was used for western blotting with dual phosphospecific p38 MAPK antibody as described in Materials and Methods. Lower panel: Total level of p38 expression in the three subclones studied. Ten micrograms of protein was used for western blotting with p38 antibody as described in Materials and Methods. Shown here is a representative experiment, with averaged results from multiple experiments shown in Table 3.

FIG. 6. Upper panel: Activation of JNKs by ␣1-AR subtypes. The ␣1A subclone 3 and ␣1B subclone 8 cells were induced without (Ctl) or with (Ind) 1 mM IPTG for 48 h before experiments. The ␣1D high cells were not treated with IPTG. Cells were serumstarved for 2 h before adding vehicle (C), 100 ␮M NE, or 100 ng/ml NGF for 15 min. Ten micrograms of protein was used for western blotting with dual phosphospecific JNK antibody as described in Materials and Methods. Lower panel: Total level of JNK expression in the three subclones studied. Ten micrograms of protein was used for western blotting with JNK antibody as described in Materials and Methods. Shown here is a representative experiment, with averaged results from multiple experiments shown in Table 3.

major MAPK pathways (ERKs, JNKs, and p38). Induction with 1 mM IPTG caused a six- to ninefold increase in ␣1A- and ␣1B-AR density, respectively, without significantly changing their KD values for the nonselective radioligand 125IBE. We were unable to obtain subclones with inducible expression of ␣1D-ARs at similar expression levels; however, we did isolate different subclones constitutively expressing ␣1D-ARs at low and high densities. Increased receptor expression was associated with corresponding increases in NE-stimulated InsP formation and [Ca2⫹]i for all three subtypes but with different efficiencies. The subtypes activated both second messenger responses in PC12 cells with an efficiency order of ␣1A ⬎ ␣1B ⬎ ␣1D, consistent with previous work in other cell lines (Theroux et al., 1996). ␣1A-ARs had the highest efficiency in activating these responses, whereas ␣1DARs showed the lowest efficiency. At lower densities (⬍200 fmol/mg), we failed to observe significant increases in InsP formation or [Ca2⫹]i in response to ␣1D-AR activation. At higher expression levels (500 – 600 fmol/mg), ␣1D-ARs did activate both responses but to a much lower degree than ␣1A- or ␣1B-ARs at similar expression levels. This agrees with previous studies in other cell lines and supports the conclusion that the ␣1A is the ␣1-AR subtype with the highest efficiency in activating these second messenger responses.

activation through ␣1A-ARs expressed in PC12 cells (Williams et al., 1998). We used this system to determine whether closely related ␣1-AR subtypes activate similar mitogenic responses. Several ARs have been reported to be able to activate MAPK pathways; however, there has been no systematic comparison of the abilities of closely related subtypes to activate these pathways. It is clear that the three known ␣1-AR subtypes all activate phospholipase C through Gq/11, increasing InsP formation and [Ca2⫹]i. However, these subtypes have different efficiencies in activating these responses, as expression of ␣1A-, ␣1B-, or ␣1D-ARs over similar density ranges resulted in different maximal responses (␣1A ⬎ ␣1B ⬎ ␣1D) in both HEK 293 and SK-N-MC cell lines (Theroux et al., 1996). The relative abilities of these subtypes to activate mitogenic pathways have not previously been examined. In this study, we heterologously expressed ␣1A-, ␣1B-, and ␣1D-ARs in PC12 cells using an inducible expression system and studied the ability of NE to activate the three

TABLE 3. Activation of ERKs, JNKs, and p38 MAPK by NE and NGF in PC12 cells expressing three ␣1-AR subtypes ERKs

␣1A subclone 3 ␣1B subclone 8 ␣1D high

JNKs

p38 MAPK

NE

NGF

NE

NGF

NE

NGF

4.45 ⫾ 1.32 4.29 ⫾ 1.19 2.58 ⫾ 0.90

5.13 ⫾ 1.56 7.10 ⫾ 2.51 4.10 ⫾ 1.02

4.14 ⫾ 1.14 1.33 ⫾ 0.08 1.24 ⫾ 0.13

1.73 ⫾ 0.18 2.13 ⫾ 0.40 1.65 ⫾ 0.53

4.84 ⫾ 0.83 2.70 ⫾ 0.15 1.17 ⫾ 0.21

1.99 ⫾ 0.16 18.8 ⫾ 9.24 1.78 ⫾ 0.78

Responses were quantified by densitometry and expressed as fold stimulation over basal. Data are mean ⫾ SEM values from three to eight experiments.


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FIG. 8. Protein tyrosine phosphorylation stimulated by NE and NGF in PC12 cells expressing ␣1A-, ␣1B-, and ␣1D-AR subtypes. The ␣1A subclone 3 and ␣1B subclone 8 cells were induced with 1 mM IPTG for 48 h, whereas ␣1D high cells were not treated with IPTG. Cells were then treated with 100 ␮M NE or 100 ng/ml NGF for 15 min. Ctl, control. Left panel: Ten micrograms of protein was loaded into each lane, and gels were probed with phosphotyrosine-specific antibody as described in Materials and Methods. Right panel: One microgram of total protein from ␣1A subclone 3 cell lysate was immunoprecipitated (IP) with an antiphosphotyrosine antibody (P-Tyr). The precipitated proteins were eluted and probed (IB) with the same antibody after electrophoresis and electrotransfer as described in Materials and Methods.

Similar, although not identical, differences were observed among the three ␣1-AR subtypes with regard to ERK activation. Stimulation of ␣1A- and ␣1B-ARs caused similar levels of ERK activation, whereas ␣1D-AR activation resulted in a smaller ERK activation. This contrasts with a previous study by Xin et al. (1997), where ␣1D-ARs specifically activated ERKs in smooth muscle cells. However, these prior experiments were complicated by coexpression of multiple subtypes in smooth muscle cells and the difficulties in pharmacologically separating responses to coexisting subtypes. In our experiments where subtypes were expressed in isolation, ␣1D-ARs caused only a low level of ERK activation, even at expression levels higher than that of ␣1A- or ␣1B-ARs. This discrepancy could also be due to the different cell types used in these studies, because activation of ERKs by GPCRs is known to be highly dependent on cell phenotype (Robinson and Cobb, 1997). Activation of ␣1-ARs has been shown to activate JNKs in several cell types (Nishio et al., 1996; Spector et al., 1997). In aortic smooth muscle cells, Nishio et al. (1996) showed that the ␣1-agonist phenylephrine caused pertussis toxin-sensitive activation of JNKs but not of ERKs and that this JNK activation was required for ␣1-AR-mediated arachidonic acid release. However, this study did not address the question of which ␣1-AR subtype mediated these responses, and responses may have been due to a combination of subtypes. In PC12 cells, we found that neither ␣1B- nor ␣1D-AR activation caused activation of the JNK pathways, despite robust activation of the traditional second messenger responses, particularly by the ␣1B subtype. This suggests that either JNK activation is a specific consequence of ␣1A-AR J. Neurochem., Vol. 72, No. 6, 1999

activation or that this selectivity is peculiar to the PC12 cell phenotype. Studies by Ramirez et al. (1997) showed that ␣1-AR stimulation caused transient activation of ERKs but a sustained activation of JNK activity in cardiac myocytes. This sustained increase in JNK activity was associated with in vitro and in vivo hypertrophy. It is not clear which subtype of ␣1-AR was involved in mediating these responses, although ␣1A-ARs have been primarily implicated in the hypertrophic actions of catecholamines on neonatal rat ventricular myocytes (Knowlton et al., 1993). However, all three ␣1-AR subtypes are known to be expressed in cardiac myocytes (Rokosh et al., 1996),

FIG. 9. Effects of NE on differentiation of the ␣1A subclone 3 (top), ␣1B subclone 8 (middle), and ␣1D high (bottom) PC12 cells. Cells were plated on collagen-coated plates and treated with 1 mM IPTG for 48 h to induce receptor expression. Cells were then treated for 48 h with 50 ␮M NE, adding fresh NE every 24 h. A representative field of cells is shown in each case. Original magnification, ⫻100.

␣1-AR SUBTYPES AND PC12 MAPK PATHWAYS and the role of specific subtypes in catecholamine responses is still controversial. Activation of p38 MAPK by ␣1-ARs has been reported by Spector et al. (1997), who also showed that decreases in ␣1-AR density were associated with corresponding decreases in ␣1-AR-stimulated p38 MAPK activation. We found that ␣1-AR subtypes caused activation of p38 MAPK in PC12 cells with efficacies similar to those observed for activation of InsP and Ca2⫹ second messenger responses (␣1A ⬎ ␣1B ⬎ ␣1D), although the ␣1D subtype did not actually cause a measurable activation of this pathway. This finding suggests that p38 MAPK activation is more likely to be downstream of the classical second messenger responses known to be activated by ␣1-ARs than JNK or ERK activation. Previous studies from our laboratory have shown that addition of NE to ␣1A-AR-expressing PC12 cells caused cell differentiation to a similar extent as treatment with NGF (Williams et al., 1998). Results from the current study confirmed this effect of NE in cells expressing ␣1A-ARs. However, there was no measurable effect of NE on differentiation of ␣1B- or ␣1D-AR expressing cells. Because treatment with NGF was able to cause differentiation in all subclones, the differential effects of NE are likely to be due to subtype-specific differences in stimulating MAPK pathways. This is the first study to compare directly the ability and effectiveness of three ␣1-AR subtypes in activating MAPK pathways. Expression of all three subtypes in the same cell phenotype at similar receptor densities allowed us to compare directly differences among these subtypes in activating mitogenic pathways and their relationship to second messenger responses. Previous studies from our laboratory have shown that ␣1A-AR-stimulated ERK and JNK activations are not dependent on mobilization of intracellular Ca2⫹ or PKC activation (A. Berts et al., manuscript submitted for publication). This is consistent with the present study, because all three subtypes can activate InsP formation and increase [Ca2⫹]i, but they show different patterns of MAPK activation. This supports the hypothesis that mechanisms upstream of InsP formation and [Ca2⫹]i must participate in MAPK activation. Comparison of protein tyrosine phosphorylation stimulated by the three ␣1-AR subtypes supports this hypothesis. The unique tyrosine phosphorylation of a 70-kDa protein only by ␣1A-ARs suggests that this subtype may activate different signaling pathways from the other two ␣1-AR subtypes. The fact that this protein is not phosphorylated in response to NGF suggests that it may play some unique role in the signaling by the G proteincoupled ␣1A-AR. However, whether this tyrosine phosphorylation is involved in any of the mitogenic responses to ␣1A-AR activation remains to be determined. In conclusion, our results demonstrate that there are significant differences in the mitogenic pathways activated by ␣1A-, ␣1B-, and ␣1D-ARs in PC12 cells. The ␣1A subtype is most effective in activating all three MAPK pathways, whereas ␣1B-ARs show a similar activation of

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ERKs and a smaller activation of p38 MAPK but do not activate JNKs. ␣1D-ARs cause a small activation of ERKs but no significant activation of activity of either JNKs or p38 MAPK. This is the first report demonstrating that closely related subtypes show markedly different patterns of mitogenic activation, suggesting that ␣1-AR subtypes may play different roles in controlling growth, differentiation, and cell fate. Acknowledgment: This work was supported by grant NS 32706 from the National Institutes of Health. We thank Deborah Lee for excellent technical assistance.

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