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Identity of Adenylyl Cyclase Isoform Determines the Rate of Cell Cycle Progression in NIH 3T3 Cells

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Identity of Adenylyl Cyclase Isoform Determines the Rate of Cell Cycle Progression in NIH 3T3 Cells Proc. Natl. Acad. Sci. USA Vol. 95, pp. 15084–15089, December 1998 Pharmacology Identity of adenylyl cyclase isoform determines the rate of cell cycle progression in NIH 3T3 cells MARTINE J. SMIT,DENNIS VERZIJL, AND RAVI IYENGAR* Department of Pharmacology, Mount Sinai School of Medicine, New York, NY 10029 Communicated by Lutz Birnbaumer, University of California, Los Angeles, CA, October 5, 1998 (received for review May 28, 1998) ABSTRACT Cell cycle progression is regulated by cAMP in protein kinase A (PKA) phosphorylation of Raf (16–19). Re- several cell types. Cellular cAMP levels depend on the activity of cently, cyclin D1 also has been found to be phosphorylated by different adenylyl cyclases (ACs), which have varied signal- PKA in vitro and has been described as a target of the cAMP receiving capabilities. The role of individual ACs in regulating inhibitory effects (20, 21). Hence, cAMP blocks transfer of signal proliferative responses was investigated. Native NIH 3T3 cells from the growth factor receptor and Ras to MAPK 1,2 and thus contain AC6, an isoform that is inhibited by a variety of signals. inhibits proliferation. Regulation of intracellular levels of cAMP Proliferation of exogenous AC6-expressing cells was the same as is controlled largely at the level of its synthesis by the ACs. Nine in control cells. In contrast, expression of AC2, an isoform ACs have been cloned and characterized (22, 23). They are stimulated by protein kinase C (PKC), resulted in inhibition of expressed in a tissue-selective manner and have different signal- cell cycle progression and increased doubling time. In AC2- receiving capabilities. ACs that are stimulated by Ca21 or protein expressing cells, platelet-derived growth factor (PDGF) elevated kinase C (PKC) are expressed mostly in neuronal and postmitotic cAMP levels in a PKC-dependent manner. PDGF stimulation of cells. On the other hand, many peripheral cells express AC a mitogen-activated protein kinases 1 and 2 (MAPK 1,2), DNA isoforms that are not stimulated by signals other than G s and are synthesis, and cyclin D1 expression was reduced in AC2- inhibited by a variety of signals. Because the different AC expressing cells as compared with control cells. Dominant neg- isoforms vary in their ability to receive and integrate signals, we ative protein kinase A relieved the AC2 inhibition of PDGF- were interested in determining whether the identity of AC induced DNA synthesis. Expression of AC2 also blocked H-ras- isoforms present in a cell affected cell cycle progression and induced transformation of NIH 3T3 cells. These observations proliferation. We were particularly interested in adenylyl cyclase indicate that, because AC2 is stimulated by PKC, it can be 2 (AC2) because it can be stimulated by PKC (24–26), and, hence, activated by PDGF concurrently with the stimulation of MAPK presumably by growth factors. We determined whether growth 1,2. The elevation in cAMP results in inhibition of signal flow factor stimulation of AC2, and the subsequent elevation of from the PDGF receptor to MAPK 1,2 and a significant reduc- cAMP, could modulate growth factor signaling through other tion in the proliferative response to PDGF. Thus, the molecular pathways and thus regulate proliferation. For this, we expressed identity and signal receiving capability of the AC isoforms in a AC2 in NIH 3T3 cells and studied the consequences of its cell could be important for proliferative homeostasis. expression. Homeostasis of proliferation is a complex process that is deter- MATERIALS AND METHODS mined by a balance between external signals triggering prolifer- Materials. Platelet-derived growth factor (PDGF), Ro-31– ation and intracellular components regulating this process. Many 8220, and Ro-20–1724 were purchased from Calbiochem, cyclin mitogens trigger proliferation by activation of Ras (1–4). Ras D1 antibody was from Santa Cruz Biotechnology, MAPK anti- activates Raf, initiating the protein kinase cascade to mitogen- bodies were from New England Biolabs, and fluorescein isothio- activated protein kinases 1 and 2 (MAPK 1,2) (extra cellular cyanate-conjugated anti-BrdU was from Becton Dickinson. signal-regulated kinases 1 and 2) ultimately triggering mitogen- DMEM and Lipofectamine were obtained from GIBCOyBRL, esis. The RasyMAPK 1,2 pathway has been shown to transduce and bovine calf serum was from HyClone. Phorbol 12-myristate signals from growth factors to the cell cycle machinery (5–8). Key 13-acetate (PMA), 4a phorbol, propidium iodide, and BrdU were 3 3 mediators of cell cycle progression through the G1 phase to the from Sigma, [ H]thymidine was from NEN, and [2,8- H]adenine S phase are the cyclins (D and E) that form complexes with was from ICN. specific cyclin-dependent kinases (9–11). Cyclin D1 appears to Cell Culture. NIH 3T3 cells were grown at 37°C, 5% CO2 in play a major role in cell cycle progression because inhibition of its DMEM containing 5% calf serum supplemented with L- expression resulted in growth arrest whereas overexpression glutamine (2 mM) and 50 unitsyml penicillin and 50 mgyl shortened the G1 phase (6). Inhibition of the MAPK 1,2 pathway streptomycin. NIH 3T3 cells were stably transfected with the blocks mitogen-induced expression of cyclin D1, and sustained vector pcDNA3 or pMam-neo (inducible promoter) containing activation of the MAPK 1,2 pathway is required for continued the AC2 or AC6 cDNA or vector without insert by using accumulation of cyclin D1, thus suggesting a role for MAPK 1,2 Lipofectamine. Clonal cell lines were obtained by selection with in cell cycle progression (5–8). G-418 (0.8 mgyml). Stable cell lines were maintained in DMEM In many cell types, intracellular cAMP affects proliferation. with 0.4 mgyml G-418. Expression of the insert was induced by Growth-stimulatory effects of cAMP have been reported in some dexamethasone (1 mM) and was monitored by measurement of cell types whereas growth inhibitory effects are observed in others accumulation of cAMP. Clonal cell lines were treated with a (12–15). Elevation of intracellular levels of cAMP, by G s- dexamethasone (1 mM) on alternative days for 2 weeks before mediated activation of adenylyl cyclase (AC), by addition of 8-Br-cAMP, or forskolin, suppresses mitogenic signaling through Abbreviations: AC, adenylyl cyclase; MAPK 1,2, mitogen-activated protein kinases 1 and 2; PDGF, platelet-derived growth factor; PKC, The publication costs of this article were defrayed in part by page charge protein kinase C; PKA, protein kinase A; PMA, phorbol 12-myristate 13-acetate. payment. This article must therefore be hereby marked ‘‘advertisement’’ in *To whom reprint requests should be addressed at: Department of accordance with 18 U.S.C. §1734 solely to indicate this fact. Pharmacology, Box 1215, Mount Sinai School of Medicine, One © 1998 by The National Academy of Sciences 0027-8424y98y9515084-6$2.00y0 Gustave L. Levy Place, New York, NY 10029. e-mail: iyengar@ PNAS is available online at www.pnas.org. msvax.mssm.edu. 15084 Downloaded by guest on October 1, 2021 Pharmacology: Smit et al. Proc. Natl. Acad. Sci. USA 95 (1998) 15085 performance of experiments (27). For the coexpression experi- (14 mg). Hygromycin-resistant clones were plated on soft agar to ments, AC2-expressing cells were cotransfected with the domi- assess colony formation. Cells (2 3 103) were suspended in 3 ml m nant negative subunit of PKA (pHL-REVABneo, 10 g) (AB) of medium (DMEMy5% calf serum) containing 0.3% agar. The (30) or wild-type PKA regulatory subunit (pHL-REVwtneo, 10 mixture was added over a layer of 0.5% agar in DMEM in a 6-well mg) (WT) and the hygromycin resistance gene (pRSV1.1, 1 mg). plate. Cells were fed weekly with 1 ml of DMEMy5% calf serum Hygromycin resistant clones were assessed for PDGF-induced and dexamethasone (1 mM). Three weeks later, plates were [3H]thymidine incorporation. stained with the vital stain 2-(-p-isodophenyl)-3-(-p-nitrophenyl)- Accumulation of cAMP. Cells were labeled with [3H]adenine 5-phenyltertazolium chloride hydrate for 2 days. Colonies larger (2 mCiyml) for 24 hr. Cells were preincubated for 30 min with the than 0.15 mm in diameter were scored. phosphodiesterase inhibitor Ro-20174 (0.3 mM), and accumula- tion of cAMP was measured for 30 min in the absence or presence RESULTS of the stimulus. Accumulation of cAMP was measured as de- Expression of Exogenous AC2 and AC6 in NIH 3T3 Cells: scribed (19) and was expressed as [3H]cAMPy([3H]ATP 1 Effect on Proliferation and Cell Cycle. To identify the ACs [3H]cAMP) (3103). expressed in NIH 3T3 cells, we performed reverse transcription– Reverse Transcription–PCR of AC Isoforms. Degenerate PCR analysis by using degenerate primers encompassing a con- primers (sense 59 KIKTIG, antisense 39 WG(NyK)TVN) encom- served domain within the ACs, with unique sizes for each of the passing a conserved domain within the ACs, with unique sizes for family members (28). Identification of the amplified products each of the family members (AC2, 291 bp; AC5y6 273 bp), were showed only a strong 273-bp band, a size corresponding to AC5 used to identify the ACs present in NIH 3T3 cells, as described or AC6, and no other bands corresponding to the other isoforms by Premont (28). Poly(A) mRNA was isolated and reverse (Fig. 1, lane a). Because AC9, like AC6, is thought to be expressed transcribed by using an oligo(dT) primer, and the first strand was ubiquitously (29), we checked with AC9 specific primers as to used for the PCR reaction. The amplified products were identi- whether this isoform was expressed in NIH 3T3 cells. However, fied on a 4% NuSieve gel (FMC). no detectable expression of AC9 isoform was found (data not Cell Growth Curve.
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