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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 204-213, 2002
© 2002 American Thoracic Society

Regulation of Airway Smooth Muscle Cyclin D1 Transcription by Protein Kinase C-{delta}

Kristen Page, Jing Li, Kevin C. Corbit, Kandelaria M. Rumilla, Jae-Won Soh, I. Bernard Weinstein, Chris Albanese, Richard G. Pestell, Marsha R. Rosner and Marc B. Hershenson

Department of Pediatrics and the Ben May Institute for Cancer Research, University of Chicago, Chicago, Illinois; Herbert Irving Comprehensive Cancer Center, College of Physicians and Surgeons, Columbia University, New York; and Department of Medicine and Developmental and Molecular Biology, Albert Einstein Cancer Center, Albert Einstein College of Medicine, New York, New York

Address correspondence to: Marc B. Hershenson, M.D., University of Chicago Children's Hospital, 5841 S. Maryland Avenue, MC 4064, Chicago, IL 60637-1470. E-mail: mhershen{at}midway.uchicago.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The precise mechanism by which protein kinase C-{delta} (PKC{delta}) inhibits cell cycle progression is not known. We investigated the regulation of cyclin D1 transcription by PKC{delta} in primary bovine airway smooth muscle cells. Overexpression of the active catalytic subunit of PKC{delta} attenuated platelet-derived growth factor (PDGF)-mediated transcription from the cyclin D1 promoter, whereas overexpression of a dominant-negative PKC{delta} increased promoter activity. A PKC{delta}-specific pseudosubstrate increased cyclin D1 protein abundance. To determine the transcriptional mechanism by which PKC{delta} negatively regulates cyclin D1 expression, we transiently transfected cells with cDNAs encoding cyclin D1 promoter 5' deletions and site mutations in the context of a -66 promoter fragment. We found that the -57 to -52 CRE/ATF2 site functions as a basal level and PDGF enhancer, whereas the -39 to -30 nuclear factor-{kappa}B site functions as a basal level suppressor. Further, PDGF and PKC{delta} responsiveness of the cyclin D1 promoter was maintained following 5' deletion to the Ets-containing -22 minimal promoter. Finally, using electrophoretic mobility gel shift and reporter assays, we determined that PKC{delta} inhibits CRE/ATF2 binding and transactivation, activates nuclear factor-{kappa}B binding and transactivation, and attenuates Ets transactivation. These data suggest that PKC{delta} attenuates cyclin D1 promoter activity via the regulation of three distinct cis-acting regulatory elements.

Abbreviations: activating transcription factor-2, ATF-2 • cAMP response element, CRE • CRE binding protein, CREB • cAMP-responsive element modulator, CREM • Dulbecco's Minimum Essential Medium, DMEM • extracellular signal regulated kinase, ERK • hemagglutinin, HA • mitogen-activated protein kinase, MAP kinase • myelin basic protein, MBP • nuclear factor-{kappa}B, NF-{kappa}B • phosphate buffered saline, PBS • platelet-derived growth factor, PDGF • phosphatidylinositol 3-kinase, PI 3 • PKC, protein kinase C • phenylmethylsulfonyl fluoride, PMSF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Increased airway smooth muscle mass has been demonstrated in patients with bronchopulmonary dysplasia (1, 2) and asthma (3, 4). These data highlight the need for a precise understanding of the events involved in airway smooth muscle mitogenesis. To that end, investigators have developed cell culture systems adopting tracheal and bronchial myocytes from different species. A growing body of literature suggests that common signal transduction pathways regulate airway smooth muscle cell cycle entry across species lines. The extracellular signal regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathways appear to constitute the major paths required for cell proliferation in both human (5–7) and bovine airway smooth muscle cells (8–10). However, little is known about the signaling pathways that limit airway smooth muscle cell proliferation.

Protein kinase C (PKC) is a complex family including three types of isoenzymes. The conventional isoforms ({alpha}, ß1, ß2, and {gamma}) are activated by calcium, phorbol esters, and phosphatidylserine, whereas the novel isoforms ({delta}, {varepsilon}, {eta}, {theta}, and µ) are calcium-insensitive and activated by phorbol esters and phosphatidylserine. The atypical isoforms ({zeta} and {tau}/{lambda}) are calcium- and phorbol ester–insensitive and activated by phosphatidylserine. PKC{alpha}, ß1, ß2, {delta}, {varepsilon}, and {zeta} are expressed in bovine tracheal myocytes (11). Recent studies have shown that different PKC isoforms may have distinct roles in the regulation of cell proliferation. In NIH3T3 cells, PKC{varepsilon} is a powerful growth stimulus, whereas PKC{alpha} and {delta} inhibit growth (12). PKC{delta} also inhibits cell cycle progression in A7r5 vascular smooth muscle cells (13), capillary endothelial cells (14), and rat colonic epithelial cells (15). In vascular smooth muscle cells, overexpression of PKC{delta} suppressed G1 cyclin expression (13), consistent with the hypothesis that cyclin D1 expression is under the transcriptional control of PKC{delta}. On the other hand, PKC{delta} induces S-phase arrest of capillary endothelial cells by increasing the level of p27Kip1, a cyclin-dependent kinase inhibitor (16).

Although PKC{delta} inhibits NIH 3T3 (12) and vascular smooth muscle (13) cell cycle traversal, it has been shown that platelet-derived growth factor (PDGF), a potent mitogen, activates PKC{delta} in these cell types (17, 18), suggesting that PDGF stimulation may elicit negative or counterregulatory responses which serve to limit excessive cell growth or prevent transformation. We therefore hypothesized that PKC{delta} negatively regulates airway smooth muscle cyclin D1 expression, and sought to determine the transcriptional mechanisms by which this occurs.

Previous studies have demonstrated a number of positive regulatory elements in the cyclin D1 promoter, including CREB/ATF2 (19–22), nuclear factor (NF)-{kappa}B (23–26), activator protein (AP)-1 (19), Sp1 (27), and Ets (28). The cyclin D1 E2F site is required for both Neu-induced transcription in MCF7 breast cancer cells (27) and E2F-1–mediated transcriptional repression in JEG-3 trophoblast cells (29). It is therefore conceivable that promoter element function may vary with cell type, perhaps due to differences in the transcription factor and co-activator environment.

In this report, we confirm that PKC{delta} negatively regulates PDGF-induced cyclin D1 expression. Furthermore, we found that, in airway smooth muscle, PKC{delta} may attenuate cyclin D1 promoter activity via the regulation of three distinct promoter elements, namely CRE/ATF2 and Ets enhancer sites and an NF-{kappa}B suppressor site.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Peroxidase-linked goat anti-rabbit IgG, protein G sepharose beads, myelin basic protein (MBP), o-nitrophenyl-ß-D-galactoside and trichloroacetic acid were from Sigma Chemical (St. Louis, MO). PDGF was obtained from Upstate Biotechnology (Lake Placid, NY). [{gamma}-32P]-ATP, [3H]thymidine, and an enhanced chemiluminescence kit were obtained from DuPont/NEN Research Products (Wilmington, DE). Antibodies against PKC{delta}, cyclin D1, CREB1, CREM1, ATF-2, p65 (Rel A), p50 (NF-{kappa}B1), RelB, and c-Rel were purchased from Santa Cruz (Santa Cruz, CA). HA6.11, a monoclonal antibody against hemagglutinin (HA) was purchased from Babco (Richmond, CA). A peroxidase-linked rat anti-mouse {kappa} light chain IgG was purchased from Zymed Laboratories (South San Francisco, CA). PKC{delta} myristolated pseudosubstrate (Myr-MNRRGAIKQAKI-OH) was synthesized by Research Genetics (Huntsville, AL). Bryostatin 1 was purchased from BioMol (Plymouth Meeting, PA). Luciferase assay buffer and transcription factor consensus oligonucleotides for CREB, NF-{kappa}B, and AP-2 were purchased from Promega (Madison, WI). An NF-{kappa}B reporter plasmid was purchased from Stratagene (La Jolla, CA).

Plasmid DNAs encoding the HA-tagged dominant-negative (pHANE-HA-PKC{delta}-KR) and constitutively active (pHANE-HA-PKC{delta}-CAT) forms of PKC{delta} were generated as described (30). pHANE is a derivative of pcDNA3. The construction of luciferase reporter plasmids encoding the full-length human cyclin D1 promoter (-1745CD1LUC), a series of 5' promoter deletions (-630 CD1LUC, -163 CD1LUC, -66 CD1LUC, -22CD1LUC), two site-directed mutants in the context of the -66 5' promoter fragment (-66 CREmut CD1LUC, -66 CREmut NF-{kappa}BLUC), and a CRE reporter plasmid (CRE-TK81-Luc) have been described previously (19, 28). cDNAs encoding a functionally intact I{kappa}B{alpha} mutant with a C-terminal deletion (pCMV4-I{kappa}B{alpha}-{Delta}C) and a nonphosphorylatable mutant with an N-terminal truncation (I-{kappa}B{alpha}-{Delta}N) were provided by D. Ballard (Vanderbilt University, Nashville, TN) (31). Dominant-negative Ets (Ets LacZ) (32) and an Ets reporter construct (pv13 Ets-LUC) (33) were gifts from Michael Ostrowski (Ohio State University, Columbus, OH).

Cell Culture
Primary bovine, rat, and human tracheal smooth muscle cells were isolated as described previously (34–36). Myocytes of passage number 5 or less were studied. Cells were maintained in Dulbecco's Minimum Essential Medium (DMEM) with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% nonessential amino acids. Twenty-four hours before each experiment, bovine tracheal myocytes were serum-starved in DMEM without serum.

Determination of Cyclin D1 Promoter Transcriptional Activity
Cells were transiently cotransfected with plasmids encoding the human cyclin D1 promoter subcloned into a luciferase reporter and cDNAs as described above or the appropriate empty vector (37). Concentration–response curves were generated for each expression vector to determine optimal concentration. Cells were serum-starved in DMEM and in some cases, were stimulated with PDGF (30 ng/ml for 16 h). After cell lysis, luciferase activity was measured at room temperature using a luminometer (Turner Designs, Sunnyvale, CA). Luciferase content was assessed by measuring the light emitted during the initial 30 s of the reaction and the values expressed in arbitrary light units. The background activity from cell extracts was typically less than 0.02 units. Cyclin D1 promoter transcriptional activation was normalized for transfection efficiency by cotransfecting cells with a cDNA encoding ß-galactosidase (pCMV-ßgal, 30 ng/plate). ß-Galactosidase activity was assessed by colorimetric assay using o-nitrophenyl-ß-D-galactoside as a substrate (38). Data were expressed as arbitrary light units/ß-galactosidase/h.

Immunoblot Analysis
Cells were cultured in six-well plates and serum starved for 24 h. Selected cells were pretreated with PKC{delta} pseudosubstrate for 1 h before PDGF treatment (30 ng/ml for 16 h). Cells were washed in phosphate-buffered saline (PBS; 150 mM NaCl, 0.1 M phosphate, pH 7.5) and extracted in a lysis buffer containing 50 mM Tris, pH 7.5, 40 mM ß-glycerophosphate, 100 mM NaCl, 2 mM EDTA, 50 mM NaF, 200 µM Na3VO4, 200 µM phenylmethylsulfonyl fluoride (PMSF), and 1% Triton X-100. Insoluble materials were removed by centrifugation (13,000 rpm for 10 min at 4°C). Lysates were centrifuged (13,000 rpm for 10 min at 4°C) and the supernatant transferred to fresh microcentrifuge tubes. Extracts (15 µg unless specifically mentioned) were resolved on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose by semidry transfer (Hoefer, San Francisco, CA). After incubation with antibody, signals were amplified and visualized using enhanced chemiluminescence.

PKC{delta} Activation Assay
Cells were serum starved for 24 h before treatment. Selected cells were pretreated with rottlerin (20 µM for 2 h) before treatment with bryostatin 1 (500 nM for 15 min). Cells were washed twice with PBS and incubated in a lysis buffer consisting of 50 mM Tris-HCl, pH 7.5, 1% NP-40, 150 mM NaCl, 10 mM NaF, 50 µg/ml aprotinin, 10 µg/ml leupeptin, 50 µg/ml pepstatin, 0.4 mM sodium pyrophosphate, 400 µM Na3VO4, and 500 µM PMSF (30 min at 4°C). Insoluble materials were removed by centrifugation (13,000 rpm for 10 min at 4°C). Cell lysates were then incubated overnight with 30 µl of protein-A sepharose beads precoupled for 2 h with the PKC{delta} antibody. Immunoprecipitates were washed three times with high salt buffer (0.5 M Tris-HCl, pH 7.4, 0.5M NaCl, and 1% NP-40), three times with lysis buffer (without protease inhibitors), and twice with kinase buffer containing 25 mM Hepes (pH 7.4), 20 mM MgCl2, 20 mM ß-phosphoglycerate, 2 mM dithiothreitol, 20 µM Na3VO4, and 20 mM p-nitrophenyl phosphate. Immune complexes were resuspended in a final volume of 30 µl kinase buffer and incubated (20 min at 30°C) with 5 µCi [{alpha}-32P]-ATP and 0.25 mg/ml MBP. Reactions were terminated by adding Laemmli buffer and boiling. Samples were resolved on a 10% sodium dodecyl sulfate gel and the proteins transferred to a nitrocellulose membrane by semidry transfer. After Ponceau staining, the membrane was exposed to film and substrate phosphorylation assessed by optical scanning (Jandel Scientific, San Rafael, CA).

Expression of the PKC{delta} Constructs
Cells were transiently cotransfected with cDNAs encoding HA-tagged PKC{delta}-CAT (active) or HA-tagged PKC{delta}-KR (dominant-negative). Three days later, cells were washed twice with PBS and incubated in a lysis buffer consisting of 50 mM Tris-HCl, pH 7.5, 1% Triton X-100, 40 mM ß-glycerophosphate, 100 mM NaCl, 50 mM NaF, 2 mM EDTA, 200 µM Na3VO4, and 0.2 mM phenylmethylsulfonyl fluoride (PMSF) (30 min at 4°C). Insoluble materials were removed by centrifugation (13,000 rpm for 10 min at 4°C). Cell lysates were incubated with protein G beads precoupled with the HA6.11 anti-hemagglutinin antibody. Immunoprecipitates were washed three times with lysis buffer before addition of Laemmli buffer and boiling. Samples were resolved on a 10% SDS-PAGE gel and the proteins transferred to a nitrocellulose membrane by semidry transfer. Western blot analysis using the HA6.11 anti-hemagglutinin antibody as the primary and goat anti-light chain as the secondary confirmed the presence of the hemagglutinin tag.

Preparation of Nuclear Extracts
Nuclear extracts were prepared by the method of Dignam and coworkers (39) with some modifications. Cultures were trypsinized, rinsed twice with PBS (0.1 M sodium phosphate, pH 7.5), and incubated on ice for 10 min with four volumes of buffer A, which consisted of 10 mM HEPES buffer (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM PMSF, and 0.5 mM DTT. After centrifugation (1,000 rpm for 5 min at 4°C), cells were resuspended in 1.5 original packed cell volume of buffer A. After centrifugation (10,000 x g for 20 min at 4°C), cells were suspended in 1.5 packed cell volume of extraction buffer C (20 mM HEPES, 25% glycerol, 1.5 mM MgCl2, 420 mM KCl, 0.2 mM EDTA, 0.5 mM PMSF, and 0.5 mM DTT) and rocked on a platform for 30 min at 4°C. After centrifugation (22,000 x g for 20 min at 4°C), supernatants were dialyzed for 1 h against three changes of 1 liter buffer D (20 mM HEPES, 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.5 mM PMSF, and 0.5 mM DTT). Following dialysis, nuclear extracts were clarified by centrifugation at 14,000 rpm for 20 min. Protease inhibitors (leupeptin, antipain, chymostatin, and pepstatin A, 5 µg/ml each) were added and aliquots stored at -80°C.

Electromobility Shift Assays
Electrophoretic mobility-shift assays were performed using nuclear extracts (4 µg) and binding buffer containing 5 mM Tris HCl (pH 7.5), 37.5 mM KCl, 0.5 mM EDTA, 2% Ficoll, 50 µg/ml poly (dI-dC), and 30–100,000 cpm of [{gamma}-32P]-labeled probe, and incubated on ice for 15 min. Nuclear extracts were added and the mixture was incubated at room temperature for 20 min. The protein–DNA complexes were analyzed by electrophoresis through a 5% polyacrylamide gel. The gels were dried and exposed to radiographic film. To detect supershift, specific antibodies were added to the complex for 20 min before running the samples on the gel.

Measurement of [3H]thymidine Incorporation
Cells were grown to near confluence in six-well plates and growth was arrested by incubation of cells in DMEM without serum for 24 h. Cells were pretreated with bryostatin 1 (500 nM for 1 h) before addition of PDGF (30 ng/ml) for 8 h. [3H]thymidine (4 µCi/ml) was added to each well, and 16 h later cells were washed twice with PBS. Enough 5% trichloroacetic acid was added to cover the bottom of the wells and cells were incubated for 30 min at 4°C. Following aspiration, the precipitate was washed in 1 ml 0.1 N NaOH for 30 min and the liquid transferred to a scintillation tube for counting.

Statistical Analysis
Data were expressed as mean ± SEM. When applicable, statistical significance was assessed by one-way ANOVA. Differences identified by ANOVA were pinpointed by the Student-Newman-Keuls' multiple range test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PKC{delta} Is Expressed in Airway Smooth Muscle
Whole cell lysates from bovine, human, and rat tracheal myocytes were probed with an antibody against the rat PKC{delta} sequence. PKC{delta} is endogenously expressed in airway smooth muscle cells of all three species (Figure 1A) . Further, anti-HA immunoblots of transfected cells demonstrated expression of the HA-tagged PKC{delta} proteins (Figure 1B).



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  		Figure 1. PKC{delta} expression in airway smooth muscle cells. (A) Whole cell lysates were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with an anti-PKC{delta} antibody. Bovine tracheal smooth muscle cells (50 µg protein), human airway smooth muscle cells (50 µg protein), and rat airway smooth muscle cells (10 µg protein) are shown. Rat brain lysate is shown as a control. (B) Cells were transiently cotransfected with plasmids encoding either constitutively active or dominant-negative PKC{delta}, or the vector control. Cell lysates were immunoprecipitated for the hemagglutinin tag. Cellular proteins were resolved by SDS-PAGE and probed for the hemagglutinin tag.

 
PKC{delta} Regulates Transcription from the Cyclin D1 Promoter
We investigated the regulation of cyclin D1 promoter activity by PKC{delta}. Cells were transiently transfected with a human full-length cyclin D1 promoter subcloned into a luciferase reporter and either a vector control or mutants of PKC{delta}. Overexpression of a dominant-negative PKC{delta} (pHANE-PKC{delta}-KR) increased basal and PDGF-mediated transcription from the cyclin D1 promoter (Figure 2A) . Furthermore, overexpression of a catalytically active PKC{delta} (pHANE-PKC{delta}-CAT) attenuated PDGF-mediated cyclin D1 promoter activity (Figure 2B). These data confirm that PKC{delta} attenuates G1 cyclin expression (13).



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  		Figure 2. Effect of PKC{delta} on bovine tracheal myocyte cyclin D1 transcription. (A) Cells were transiently cotransfected with cDNAs encoding the human full-length cyclin D1 promoter subcloned into a luciferase reporter gene and the dominant-negative mutant of PKC{delta} or a vector control. (B) Cells were transiently cotransfected with cDNAs encoding the cyclin D1 reporter, a constitutively active mutant of PKC{delta} or a vector control and ß-galactosidase. Selected cultures were treated with PDGF (30 ng/ml for 16 h). Data are calculated as luciferase/ß-galactosidase/h normalized to the control vector (n = 4–5). PKC{delta}-KR significantly increased both basal and PDGF-mediated activation of the cyclin D1 promoter, whereas PKC{delta}-CAT significantly decreased PDGF-mediated activation of the cyclin D1 promoter (*P < 0.05, ANOVA).

 
We asked whether inhibition of endogenous PKC{delta} would increase cyclin D1 protein abundance. To inhibit PKC{delta}, we employed a PKC{delta} pseudosubstate myristoylated peptide (Myr-MNRRGAIKQAKI-OH) derived from the sequence of the rat pseudosubstrate domain (40). The pseudosubstrate is thought to bind the autoinhibitory domain and maintain the enzyme in an inactive state. Inhibiting PKC{delta} with increasing doses of PKC{delta} pseudosubstrate increased cyclin D1 protein abundance in unstimulated and low dose PDGF-stimulated rat myocytes (Figure 3) . These data demonstrate that endogenous PKC{delta} is a negative regulator of cyclin D1 expression.



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  		Figure 3. Inhibition of PKC{delta} increases cyclin D1 protein abundance in rat airway smooth muscle cells. Selected cultures were pretreated with PKC{delta} pseudosubstrate (PKC{delta} PS; 10–100 µM for 1 h) before selected cultures being stimulated with low dose PDGF (3 ng/ml for 16 h). One culture was treated with high-dose PDGF (30 ng/ml for 16 h) as a control. Whole cell lysates were resolved by SDS-PAGE, transferred to nitrocellulose and probed with an anti-cyclin D1 antibody. This experiment was repeated twice with similar results.

 
Localization of the PKC{delta} Effect to a Minimal Region of the Cyclin D1 Promoter
To determine the transcriptional mechanism by which PKC{delta} negatively regulates cyclin D1 promoter activity, we transiently transfected cells with luciferase-tagged 5' promoter deletions of the full-length cyclin D1 promoter (-163 CD1LUC, -66 CD1LUC, or -22 CD1LUC) and either vector control or PKC{delta}-CAT. Luciferase activity was normalized to ß-galactosidase and compared with that of the full-length cyclin D1 promoter. Whereas deletion of promoter sequences 5' to -66 base pairs from the promoter start site reduced basal promoter activity by ~ 60%, deletion to -22 nearly abolished promoter activity (Figure 4A) , demonstrating the presence of at least one critical regulatory element between -66 and -22 base pairs 5' to the transcription start site. Also, with all 5' deletion constructs tested, including the minimal -22 CD1 promoter, PDGF responsiveness was maintained and overexpression of active PKC{delta} decreased basal and PDGF-induced promoter activity.



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  		Figure 4. Effect of PKC{delta} on cyclin D1 promoter deletion mutants. (A) Cells were transiently cotransfected with -1745CD1LUC, -163CD1LUC, -66CD1LUC, or -22CD1LUC and either empty vector or active PKC{delta}-CAT. Selected cultures were treated with PDGF (30 ng/ml for 16 h). Data are expressed as the fold increase in luciferase/ß-galactosidase/h relative to the control vector for the full length plasmid (n = 3–6). Note the broken axis for the -22CD1LUC plasmid data. (B) Cells were transiently cotransfected with -66CD1LUC, -66CRE mut CD1LUC, or -66NF-{kappa}B mut CD1LUC. Data are expressed as the fold increase in luciferase/ß-galactosidase/h relative to the control vector for -66CD1LUC (n = 3–5). PKC{delta}-CAT significantly decreased PDGF-mediated activation of the cyclin D1 promoter fragments (*P < 0.05, ANOVA).

 
The region of the cyclin D1 promoter -66 base pairs from the transcription start site contains at least three transcription factor binding sites; CRE/ATF2 (-57 to -52), NF-{kappa}B (-39 to -30) and Ets (-10 to -8). To determine the roles of CRE/ATF2 and NF-{kappa}B promoter sequences in the regulation of airway smooth muscle cyclin D1 transcription, we employed CRE/ATF2 and NF-{kappa}B mutants in the context of the -66CD1LUC 5' promoter construct. Mutation of the CRE/ATF2 binding site nearly abolished luciferase activity, suggesting that the CRE/ATF2 site is a basal level enhancer (Figure 4B). Deletion of the NF-{kappa}B site in the context of the -66 5' promoter deletion construct increased basal activity, suggesting a role as a transcriptional suppressor. PDGF and PKC{delta} responsiveness was maintained for each mutant, suggesting that neither element is required for PKC{delta}-mediated regulation. Together with data demonstrating maintenance of PKC{delta} responsiveness in the -22CD1 minimal promoter (above), these results suggest that the -10 to -8 Ets site is an important downstream target of PKC{delta}. On the other hand, given the dramatic reduction in promoter activity accompanying 5' deletion of the -66 reporter construct, as well as the apparent enhancer and suppressor roles for the CRE/ATF2 and NF-{kappa}B sites, respectively, we proposed a model in which PKC{delta} suppresses airway smooth muscle cyclin D1 promoter activity via the regulation of three distinct promoter elements: CRE/ATF2, NF-{kappa}B, and Ets.

PKC{delta} Inhibits DNA Binding and Transactivation at the Cyclin D1 Promoter CRE/ATF2 Site
To test whether PKC{delta} inhibited binding of nuclear proteins to the cyclin D1 CRE/ATF2 site, selected cultures were treated with bryostatin 1 before PDGF stimulation. Bryostatin 1 binds to and activates PKC but induces only a subset of the responses to phorbol esters. It is particularly potent for translocating PKC{delta} and, to a lesser extent, PKC{varepsilon} (41–43). Nuclear extracts were prepared and were incubated with an oligonucleotide encoding the consensus CRE/ATF2 binding site. Bryostatin 1 attenuated both basal and PDGF-induced nuclear protein binding to CRE/ATF2 (Figure 5A) . Supershift assays revealed that PDGF treatment induces binding of CREB1 and CREM1, but not ATF2, to the CRE/ATF2 transcription factor binding site. Activation of PKC{delta} by bryostatin 1 was confirmed by immunoprecipitation of endogenous PKC{delta} with a specific antibody, followed by in vitro phosphorylation assay using MBP as a substrate (Figure 5B). To examine CRE/ATF2 transactivation, we transiently transfected cells with a CRE/ATF2 reporter plasmid (CRE-TK81-LUC) along with either vector control or active PKC{delta}, and treated selected cultures with PDGF. Overexpression of active PKC{delta} attenuated PDGF-induced transactivation of the CRE reporter plasmid (Figure 5C). These data suggest that PKC{delta} inhibits nuclear protein binding and transactivation at the cyclin D1 promoter CRE/ATF2 enhancer region, an effect which would be expected to negatively regulate cyclin D1 expression.



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  		Figure 5. Effect of PKC{delta} on CRE binding and transactivation. (A) Cells were pretreated with bryostatin 1 (15 min) before PDGF stimulation (15 min). Nuclear extracts were obtained, incubated with a 32P end-labeled double-stranded CRE oligonucleotide, and resolved on a gel. Supershift analysis was performed by the addition of an antibody to sample complexes for 20 min before being resolved on the gel. DNA–protein complexes are shown with a filled arrowhead and supershift complexes are shown with the hollow arrowhead. (B) Cells were treated with bryostatin 1 (500 nM for 15 min), extracted, and lysates immunoprecipitated with a specific PKC{delta} antibody. Activation of PKC{delta} was determined by phosphorylation of MBP. (C) Cells were transiently cotransfected with the CRE-responsive element (CRE-TK81-LUC) and either empty vector or active PKC{delta}-CAT. Selected cultures were treated with PDGF (30 ng/ml for 16 h). Data are expressed as fold increase relative to the control vector (n = 4). PKC{delta}-CAT significantly decreased PDGF-mediated activation of the CRE-responsive element (*P < 0.05, ANOVA).

 
PKC{delta} Activates DNA Binding and Transactivation at the Cyclin D1 Promoter NF-{kappa}B Site
To test whether PKC{delta} activates NF-{kappa}B binding to DNA, nuclear extracts were incubated with an oligonucleotide encoding the consensus NF-{kappa}B binding site. Both PDGF and bryostatin 1 induced binding of nuclear proteins to the oligonucleotide, although bryostatin 1 appeared to have a greater effect (Figure 6A) . Supershift assays demonstrated that bryostatin 1 induces p65 RelA binding to the NF-{kappa}B oligonucleotide. To test whether PKC{delta} induces transactivation of the NF-{kappa}B site, we transiently transfected cells with a NF-{kappa}B reporter plasmid and either control vector or active PKC{delta}. Selected cells were stimulated with PDGF. Both PDGF and active PKC{delta} increased NF-{kappa}B transactivation (Figure 6B). These data suggest that PKC{delta} increases the DNA binding and transactivation of the cyclin D1 promoter NF-{kappa}B suppressor site.



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  		Figure 6. The effect of PKC{delta} on NF-{kappa}B binding and transactivation. (A) Cells were pretreated with bryostatin 1 (15 min) before PDGF stimulation (15 min). Nuclear extracts were incubated with a 32P end-labeled double-stranded NF-{kappa}B oligonucleotide. Complexes were resolved on a 5% acrylamide gel. In some cases, an antibody was added to the complex for 20 min before electrophoresis. DNA–protein complexes are shown with a filled arrowhead and supershift complexes are shown with the hollow arrowhead. (B) Cells were transiently cotransfected with an NF-{kappa}B–responsive element (NF-{kappa}B-TATA-LUC) and either empty vector or active PKC{delta}-CAT. Selected cultures were treated with PDGF (30 ng/ml for 16 h). Data are expressed as the fold increase relative to the control vector (n = 4). Both PDGF and PKC{delta}-CAT increased activation of the NF-{kappa}B–responsive element (*P < 0.05, ANOVA). (C) Cells were transiently cotransfected with the full-length or the –66CD1LUC promoter and either the functionally intact (I{kappa}B{alpha}-{Delta}C) or the unphosphorylatable mutant (I{kappa}B{alpha}-{Delta}N) and selected cultures were treated with PDGF (30 ng/ml for 16 h). Data are expressed as the fold increase relative to the control vector (n = 6). Overexpression of the unphosphorylatable I{kappa}B{alpha} (I{kappa}B{alpha}-{Delta}N) significantly increased PDGF-mediated activation of the cyclin D1 reporter constructs (*P < 0.05, ANOVA/Student-Newman-Keuls' multiple range test).

 
To confirm the suppressive effect of NF-{kappa}B activation on cyclin D1 expression, we examined the effects of NF-{kappa}B inhibition on transcription from the full-length and -66 cyclin D1 reporter genes. Cells were cotransfected with the reporter construct of interest and cDNAs encoding either a functionally intact I{kappa}B{alpha} mutant with a C-terminal deletion (I{kappa}B{alpha}-{Delta}C) or a N-terminal truncated unphosphorylatable form (I{kappa}B{alpha}-{Delta}N). Phosphorylation of I{kappa}B{alpha} at two N-terminal serine residues (Ser32 and Ser36) is necessary for I-{kappa}B{alpha} polyubiquitinylation and degradation, which in turn exposes a nuclear localization sequence on the NF-{kappa}B molecule. Expression of the unphosphorylatable mutant I{kappa}B{alpha} increased basal and PDGF-induced promoter activity in both the full-length and -66 cyclin D1 promoter constructs (Figure 6C), consistent with the notion that, in airway smooth muscle cells, the NF-{kappa}B site functions as a suppressor of cyclin D1 transcription.

Role of Ets in PKC{delta}-Mediated Regulation of the Cyclin D1 Promoter
Initial data (shown in Figure 4A) suggested that the Ets binding site may also be subject to PDGF and PKC{delta} regulation. To test this, cells were transfected with an Ets responsive reporter (33). Overexpression of active PKC{delta} inhibited PDGF-induced Ets activation (Figure 7A) . To further assess the role of Ets transactivation in cyclin D1 expression, we transfected cells with a cDNA encoding a mutant Ets DNA binding domain without transactivating sequences (Ets-LacZ) (32). Introduction of a dominant-negative Ets inhibited PDGF-induced responsiveness (Figure 7B). Together, these data are consistent with the notion that PKC{delta} attenuates cyclin D1 transcription by inhibition of the proximal Ets site.



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  		Figure 7. Effect of PDGF and PKC{delta} on Ets transactivation. (A) Cells were transiently cotransfected with an Ets-responsive element (Ets-LUC) and either empty vector or active PKC{delta}-CAT. Selected cultures were treated with PDGF (30 ng/ml for 16 h). Data are expressed as the fold increase relative to the control vector (n = 4). PKC{delta}-CAT significantly increased PDGF-mediated activation of the Ets-responsive element (*P < 0.05, ANOVA). (B) Cells were transiently cotransfected with a dominant-negative Ets element (Ets-LacZ) and either the full-length or the -66CD1LUC reporter plasmids. Selected cultures were treated with PDGF (30 ng/ml for 16 h). Data are expressed as the fold increase relative to the control vector (n = 4). Dominant-negative Ets significantly attenuated PDGF-mediated activation of the -66CD1 promoter (*P < 0.05, ANOVA).

 
PKC{delta} Attenuates PDGF-Induced DNA Synthesis in Bovine Tracheal Myocytes
We tested the effect of PKC{delta} activation on PDGF-induced DNA synthesis, as measured by [3H]thymidine incorporation. Activation of PKC{delta} by bryostatin 1 significantly decreased DNA synthesis (Figure 8) .



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  		Figure 8. PKC{delta} inhibits PDGF-induced DNA synthesis in bovine tracheal myocytes. Cells were grown to near-confluence in six-well plates and serum starved for 24 h. Cells were pretreated with bryostatin 1 (500 nM for 1 h) before addition of PDGF (30 ng/ml) for 8 h. [3H]thymidine was added to each well for 16 h. DNA was precipitated using trichloroacetic acid (5%), and the precipitate was washed in 0.1 N NaOH before counting. Data are expressed as the fold increase in [3H]thymidine incorporation relative to untreated cells (n = 4). Bryostatin 1 significantly attenuated PDGF-induced [3H]thymidine incorporation (*P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In vascular smooth muscle cells, overexpression of PKC{delta} suppressed G1 cyclin expression (13), consistent with the hypothesis that cyclin D1 expression is under the transcriptional control of PKC{delta}. In this report, we confirm these findings and offer potential mechanisms by which PKC{delta} negatively regulates cyclin D1 transcription. We found that in primary airway smooth muscle cells: (i) activation of PKC{delta} inhibits cyclin D1 transcription and protein expression, whereas inhibition increases expression; (ii) the cyclin D1 promoter CRE/ATF2 site functions a basal level and PDGF enhancer, whereas the NF-{kappa}B site functions as transcriptional suppressor; (iii) PDGF and PKC{delta} responsiveness of the cyclin D1 promoter is maintained following 5' deletion to the minimal promoter (-22 basepairs from the transcription start site); and (iv) PKC{delta} inhibits CRE/ATF2 binding and transactivation, activates NF-{kappa}B binding and transactivation, and attenuates Ets transactivation. Taken together, these data suggest that PKC{delta} attenuates cyclin D1 promoter activity via the regulation of three distinct cis-acting regulator elements.

Previous studies have demonstrated the importance of the CRE/ATF2, NF-{kappa}B, and Ets sites in cyclin D1 transcriptional regulation. In NIH3T3 cells, SV40 small t antigen-induced cyclin D1 transcription is mediated by the -57 CRE/ATF2 site (19). CRE/ATF2 functions as a basal level enhancer in chondrocytes (44). The CREB/ATF2 site is positively regulated by p21Ras activation in colon carcinoma cells (22) and by integrin-linked kinase in mammary epithelial cells (45). Finally, in the MCF-7 human mammary carcinoma cells, estrogen activates the cyclin D1 promoter via the CRE/ATF-2 site, inducing the binding of ATF-2 homodimers and ATF-2/c-Jun heterodimers (21). Consistent with this work, we found that the CRE/ATF2 site functions as a major basal level and PDGF response element in bovine tracheal myocytes. Unlike previous studies, however, mitogen-induced stimulation of the cyclin D1 promoter was associated with CREB1 and CREM1, but not ATF-2 binding. This discrepancy may reflect cell type–specific differences in the expression, availability, or function of various transcription factors. Nevertheless, given the critical role of the CRE/ATF2 site for cyclin D1 transcription in a variety of cell types, this site represents a likely candidate for PKC{delta} regulation. Accordingly, we found that PKC{delta} attenuates protein binding to and transactivation of the CRE/ATF2 site.

Previous studies have shown NF-{kappa}B to be an important positive regulator of cyclin D1 transcriptional control. NF-{kappa}B activation stimulates transcription from the cyclin D1 promoter in COS-7 cells and mouse embryo fibroblasts, whereas mutation of NF-{kappa}B–responsive elements attenuates serum-induced transcription (26). In NIH3T3 cells, Rac1-mediated cyclin D1 promoter activity requires the presence of the -39 to -33 NF-{kappa}B binding site (24). Supershift analysis revealed binding of both the p65 and p50 NF-{kappa}B subunits to the NF-{kappa}B site. In murine C2C12 myoblasts, inhibition of NF-{kappa}B activation reduced cyclin D1 expression while accelerating myogenic differentiation, as evidenced by their myotube phenotype and ability to express the late differentiation marker myosin heavy chain (25). However, we found in primary airway smooth muscle cells that deletion of the -39 to -32 NF-{kappa}B site and expression of a nonphosphorylatable I{kappa}B-{alpha} each increased, rather than decreased, transcription from the cyclin D1 promoter. Further, activation of PKC{delta}, a potent inhibitor of cyclin D1 expression, increased NF-{kappa}B binding and transactivation. Together, these data suggest that in airway smooth muscle, unlike other cell types previously reported, NF-{kappa}B functions as a transcriptional repressor. We have recently obtained preliminary data that the NF-{kappa}B site may also function as cis-acting transcriptional repressor in prostate cancer cells (R. Pestell, unpublished data). The mechanism by which NF-{kappa}B binding negatively regulates transcription from the cyclin D1 promoter in airway smooth muscle cells, as opposed to the positive regulation noted in previous studies, is unclear. As noted above, differences in cell type, which could dictate variation in the transcription factors or co-activators available for binding to the NF-{kappa}B site, could play a role. Supershift studies suggested binding of p65 RelA but not p50 NF-{kappa}B1 to the site, in contrast to results in NIH3T3 cells.

Although our results contrast with work in C2C12 myoblasts, it should be noted that unlike skeletal muscle cells, smooth muscle cells do not undergo differentiation into myotubes. Vascular and airway smooth muscle cells have been noted to switch, depending on culture conditions, between proliferative/synthetic and contractile phenotypes (46). However, we have found using bromodeoxyuridine incorporation that contractile smooth muscle cells, unlike skeletal muscle myotubes, do not lose the ability to proliferate (J. Solway and M. B. Hershenson, unpublished data). Further, our preliminary studies suggest that NF-{kappa}B does not regulate myosin heavy chain expression in bovine tracheal myocytes, as in C2C12 myoblasts (K. Page and M. B. Hershenson, unpublished data). Thus, specific differences in skeletal and smooth muscle biology may determine differences in NF-{kappa}B function.

The target DNA sequences of Ets proteins include a core motif with extensive variation at both the 5' and 3' sides of the invariant GGA core. In a variety of promoters lacking a TATA box, Ets binding sites have been localized close to the initiation site (47). Sequences analogous to the core motif required for Ets protein binding are located within the proximal cyclin D1 promoter at the transcription start site. In JEG-3 human trophoblast cells, epidermal growth factor- and ERK-induced transcription are dependent on this site (28). Similarly, we found that PDGF-induced stimulation is dependent on Ets binding in primary airway smooth muscle cells. However, we have extended these results by showing that the proximal promoter is negatively regulated by PKC{delta}.

The precise pathways by which PKC{delta} may regulate transcription factor binding to the cyclin D1 CRE/ATF2, NF-{kappa}B, and Ets sites remain unclear. Substrate proteins that appear to be selectively phosphorylated by PKC{delta} include the eukaryotic elongation factor (eEF)-1{alpha} (48), high-affinity IgE receptor (49), troponin I (50), and heat shock protein-25/27 (51). In rat aortic smooth muscle cells, overexpression of PKC{delta} but not PKCß1 increases p38 mitogen-activated protein (MAP) kinase activity (52), suggesting that p38 may be a proximal or distal downstream target of PKC{delta} phosphorylation. Like PKC{delta}, activation of p38 negatively regulates cyclin D1 promoter activity and protein expression in airway smooth muscle (53), consistent with the notion that inhibition of cyclin D1 transcription by PKC{delta} is mediated by p38. Little is known about the downstream transcription factor targets of PKC{delta}. It has been shown in NIH 3T3 cells that PKC{alpha} and PKC{varepsilon}, but not PKC{delta} or PKC{zeta}, mediate the serum response element (SRE) by activating the transactivation domain of Elk-1 (30). In COS cells, PKC{delta} has been shown to increase 12-O-tetradecanoylphorbol-13-acetate (TPA)-inducible genes via activation of AP-1 (54). In NIH3T3 cells, Ras activation of AP-1 is inhibited by a dominant-negative form of PKC{delta} (55). PKC{delta} and PKC{varepsilon} enhanced phosphorylation and transactivation of MEF2A, a skeletal muscle transcriptional regulatory protein, in COS and HeLa cells (56). Overexpression of PKC{delta} or PKC{varepsilon} enhanced E2F transactivation in rat fibroblast 3Y1 cells (57). Finally, it has recently been shown in human neutrophils that inhibition of PKC{delta} with rottlerin prevents I{kappa}B-{alpha} degradation and NF-{kappa}B activation (58), consistent with our finding that active PKC{delta} induces NF-{kappa}B DNA binding and transactivation in airway smooth muscle cells.

PDGF, a potent mitogen, has been noted to activate PKC{delta} in NIH3T3 (17), vascular smooth muscle (18), and airway smooth muscle cells (K. Page and M. Hershenson, unpublished data). Because PKC{delta} inhibits cyclin D1 expression in each of these cell types (12, 13), these data suggest that PDGF stimulation may elicit negative or counter-regulatory responses which serve to limit excessive cell growth or prevent transformation. Our finding that PKC{delta} regulates cyclin D1 transcription by PKC{delta} via three distinct mechanisms underlines the potential importance of this counterregulatory pathway. The observed increase in cyclin D1 expression following treatment of stimulated cells with either PKC{delta}-KR or pseudosubstrate peptide demonstrates that there is basal activation of PKC{delta} in bovine tracheal myocytes. These data are consistent with the notion that PKC{delta} may be an important negative regulator of cell cycle progression in these cells. Our data that bryostatin 1, a relatively selective activator of PKC{delta}, attenuates PDGF-induced DNA synthesis, is consistent with the notion that PKC{delta} negatively regulates airway smooth muscle cell proliferation.

Understanding the signaling pathways which regulate airway smooth muscle cyclin D1 expression may provide insight into the pathogenesis of asthma, which has been associated with an increase in airway smooth muscle cell number. Previous work in this area has focused on positive regulatory pathways of cyclin D1 expression, namely the ERK (37, 59, 60) and PI 3-kinase signaling pathways (10). In the present study, we demonstrate that PKC{delta} negatively regulates transcription from the cyclin D1 promoter via the regulation of multiple cis-acting regulatory sequences, including CRE/ATF2, NF-{kappa}B, and Ets. Because cyclin D1 expression is required for airway smooth muscle DNA synthesis (61), further studies examining the mechanisms that suppress cyclin D1 expression may lead to therapeutic interventions for asthma and other disease states in which abnormal cell proliferation is a feature.


   Acknowledgments
 
These studies were supported by National Institutes of Health grants CA26056 (J.W.S. and I.B.W.), CA75503, CA86071, CA86072 (R.G.P.), NS33858, GM61038 (M.R.R.), HL54685, HL63314 (M.B.H.), and HL56399 (M.B.H., R.G.P., and M.R.R.); the Susan G. Komen Breast Cancer Foundation, the Pfeiffer Foundation, the Breast Cancer Alliance (R.G.P.); the Cornelius Crane Trust (M.R.R.), and the Blowitz-Ridgeway Foundation (M.B.H.).

Received in original form December 27, 2001

Received in final form March 1, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hislop, A. A., and S. G. Haworth. 1989. Airway size and structure in the normal fetal and infant lung and the effect of premature delivery and artificial ventilation. Am. Rev. Respir. Dis. 140:1717–1726.[Medline]
  2. Sward-Comunelli, S. L., S. M. Mabry, W. E. Truog, and D. W. Thibeault. 1997. Airway muscle in preterm infants: changes during development. J. Pediatr. 130:570–576.[Medline]
  3. Carroll, N., J. Elliot, A. Morton, and A. James. 1993. The structure of large and small airways in nonfatal and fatal asthma. Am. Rev. Respir. Dis. 147:405–410.[Medline]
  4. Ebina, M., T. Takahashi, T. Chiba, and M. Motomiya. 1993. Cellular hypertrophy and hyperplasia of airway smooth muscles underlying bronchial asthma: a 3-D morphometric study. Am. Rev. Respir. Dis. 148:720–726.[Medline]
  5. Orsini, M. J., V. P. Krymskaya, A. J. Eszterhas, J. L. Benovic, R. A. Panettieri, and R. B. Penn. 1999. MAPK superfamily activation in human airway smooth muscle: mitogenesis requires prolonged p42/p44 activation. Am. J. Physiol. 277:L479–L488.[Abstract/Free Full Text]
  6. Krymskaya, V. P., R. Hoffman, A. Eszterhas, V. Ciocca, and P. A. Panettieri. 1997. TGF-1 modulates EGF-stimulated phosphatidylinositol 3-kinase activity in human airway smooth muscle cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 273:L1220–L1227.[Abstract/Free Full Text]
  7. Walker, T. R., S. M. Moore, M. F. Lawson, R. A. Panettieri, and E. R. Chilvers. 1998. Platelet-derived growth factor-BB and thrombin activate phosphoinositide 3-kinase and protein kinase B: role in mediating airway smooth muscle proliferation. Mol. Pharmacol. 54:1007–1015.[Abstract/Free Full Text]
  8. Karpova, A. Y., M. K. Abe, J. Li, P. T. Liu, J. M. Rhee, W. L. Kuo, and M. B. Hershenson. 1997. MEK1 is required for PDGF-induced ERK activation and DNA synthesis in tracheal myocytes. Am. J. Physiol. 272:L558–565.[Abstract/Free Full Text]
  9. Scott, P. H., C. M. Belham, J. al-Hafidh, E. R. Chilvers, A. J. Peacock, G. W. Gould, and R. Plevin. 1996. A regulatory role for cAMP in phosphatidylinositol 3-kinase/p70 ribosomal S6 kinase-mediated DNA synthesis in platelet-derived-growth-factor-stimulated bovine airway smooth-muscle cells. Biochem. J. 318:965–971.
  10. Page, K., J. Li, Y. Wang, S. Kartha, R. G. Pestell, and M. B. Hershenson. 2000. Regulation of cyclin D1 expression and DNA synthesis by phosphatidylinositol 3-kinase in airway smooth muscle cells. Am. J. Respir. Cell Mol. Biol. 23:436–443.[Abstract/Free Full Text]
  11. Webb, B. L. J., M. A. Lindsay, P. J. Barnes, and M. A. Giembycz. 1997. Protein kinase C isoforms in airway smooth muscle. Biochem. J. 324:167–175.
  12. Acs, P., Q. J. Wang, K. Bogi, A. M. Marquez, P. S. Lorenzo, T. Biro, Z. Szallasi, J. F. Muchinski, and P. M. Blumberg. 1997. Both the catalytic and regulatory domains of protein kinase C chimeras modulate the proliferative properties of NIH3T3 cells. J. Biol. Chem. 272:28793–28799.[Abstract/Free Full Text]
  13. Fukumoto, S., Y. Nishizawa, M. Hosoi, H. Koyama, K. Yamakawa, S. Ohno, and H. Morii. 1997. Protein kinase C inhibits the proliferation of vascular smooth muscle by suppressing G1 cyclin expression. J. Biol. Chem. 272:13816–13822.[Abstract/Free Full Text]
  14. Harrington, E. O., J. Loffler, P. R. Nelson, K. C. Kent, M. Simons, and J. A. Ware. 1997. Enhancement of migration by protein kinase C alpha and inhibition of proliferation and cell cycle progression by protein kinase C delta in capillary endothelial cells. J. Biol. Chem. 272:7390–7397.[Abstract/Free Full Text]
  15. Perletti, G. P., E. Marras, P. Concari, F. Piccinini, and A. H. Tashjian. 1999. PKC delta acts as a growth and tumor suppressor in rat colonic epithelial cells. Oncogene 18:1251–1256.[Medline]
  16. Ashton, A. W., G. Watanabe, C. Albanese, E. O. Harrington, J. A. Ware, and R. G. Pestell. 1999. Protein kinase C delta inhibition of S-phase transition in capillary endothelial cells involves the cyclin-dependent kinase inhibitor p27(Kip1). J. Biol. Chem. 274:20805–20811.[Abstract/Free Full Text]
  17. Li, W., J. C. Yu, P. Michieli, J. F. Beeler, N. Ellmore, M. A. Heidaran, and J. H. Pierce. 1994. Stimulation of the platelet-derived growth factor beta receptor signaling pathway activates protein kinase C-delta. Mol. Cell. Biol. 14:6727–6735.[Abstract/Free Full Text]
  18. Thommes, K. B., J. Hoppe, H. Vetter, and A. Sachinidis. 1996. The synergistic effect of PDGF-AA and IGF-1 on VSMC proliferation might be explained by the differential activation of their intracellular signaling pathways. Exp. Cell Res. 226:59–66.[Medline]
  19. Watanabe, G., A. Howe, R. J. Lee, C. Albanese, I. W. Shu, A. N. Karnezis, L. Zon, J. Kyriakis, K. Rundell, and R. G. Pestell. 1996. Induction of cyclin D1 by simian virus 40 small tumor antigen. Proc. Natl. Acad. Sci. USA 93:12861–12866.[Abstract/Free Full Text]
  20. Lee, R. J., C. Albanese, R. J. Stenger, G. Watanabe, G. Inghirami, G. K. Haines, M. Webster, W. J. Muller, J. S. Brugge, R. J. Davis, and R. G. Pestell. 1999. pp60(v-src) induction of cyclin D1 requires collaborative interactions between the extracellular signal-regulated kinase, p38, and Jun kinase pathways: a role for cAMP response element-binding protein and activating transcription factor-2 in pp60(v-src) signaling in breast cancer cells. J. Biol. Chem. 274:7341–7350.[Abstract/Free Full Text]
  21. Sabbah, M., D. Courilleau, J. Mester, and G. Redeuilh. 1999. Estrogen induction of the cyclin D1 promoter: involvement of a cAMP response-like element. Proc. Natl. Acad. Sci. USA 96:11217–11222.[Abstract/Free Full Text]
  22. Tetsu, O., and F. McCormick. 1999. ß-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature 398:422–426.[Medline]
  23. Herber, B., M. Truss, M. Beato, and R. Muller. 1994. Inducible regulatory elements in the human cyclin D1 promoter. Oncogene 9:2105–2107.[Medline]
  24. Joyce, D., B. Bouzahzah, M. Fu, C. Albanese, M. D'Amico, J. Steer, J. U. Klein, R. J. Lee, J. E. Segall, J. K. Westwick, C. J. Der, and R. G. Pestell. 1999. Integration of Rac-dependent regulation of cyclin D1 transcription through a nuclear factor-kappaB-dependent pathway. J. Biol. Chem. 274: 25245–25249.[Abstract/Free Full Text]
  25. Guttridge, D. C., C. Albanese, J. Y. Reuther, R. G. Pestell, and A. S. Baldwin. 1999. NF-{kappa}B controls cell growth and differentiation through transcriptional regulation of cyclin D1. Mol. Cell. Biol. 19:5785–5799.[Abstract/Free Full Text]
  26. Hinz, M., D. Krappmann, A. Eichten, A. Heder, C. Scheidereit, and M. Strauss. 1999. NF-{kappa}B function in growth control: regulation of cyclin D1 expression and G0/G1-to-S-phase transition. Mol. Cell. Biol. 19:2690–2698.[Abstract/Free Full Text]
  27. Lee, R. J., C. Albanese, M. Fu, M. D'Amico, B. Lin, G. Watanabe, G. K. Haines, P. M. Siegel, M. C. Hung, Y. Yarden, J. M. Horowitz, W. J. Muller, and R. G. Pestell. 2000. Cyclin D1 is required for transformation by activated Neu and is induced through an E2F-dependent signaling pathway. Mol. Cell. Biol. 20:672–683.[Abstract/Free Full Text]
  28. Albanese, C., J. Johnson, G. Watanabe, N. Eklund, D. Vu, A. Arnold, and R. G. Pestell. 1995. Transforming p21ras mutants and c-Ets-2 activate the cyclin D1 promoter through distinguishable regions. J. Biol. Chem. 270: 23589–23597.[Abstract/Free Full Text]
  29. Watanabe, G., C. Albanese, R. J. Lee, A. Reutens, G. Vairo, B. Henglein, and R. G. Pestell. 1998. Inhibition of cyclin D1 kinase activity is associated with E2F-mediated inhibition of cyclin D1 promoter activity through E2F and Sp1. Mol. Cell. Biol. 18:3212–3222.[Abstract/Free Full Text]
  30. Soh, J. W., E. H. Lee, R. Prywes, and I. B. Weinstein. 1999. Novel roles of specific isoforms of protein kinase C in activation of the c-fos serum response element. Mol. Cell. Biol. 19:1313–1324.[Abstract/Free Full Text]
  31. Brockman, J. A., D. C. Scherer, T. A. McKinsey, S. M. Hall, X. Qi, W. Y. Lee, and D. W. Ballard. 1995. Coupling of a signal response domain in I kappa B alpha to multiple pathways for NF-kappa B activation. Mol. Cell. Biol. 15:2809–2818.[Abstract]
  32. Langer, S. J., D. M. Bortner, M. F. Roussel, C. J. Sherr, and M. C. Ostrowski. 1992. Mitogenic signaling by colony-stimulating factor 1 and ras is suppressed by the ets-2 DNA-binding domain and restored by myc overexpression. Mol. Cell. Biol. 12:5355–5362.[Abstract/Free Full Text]
  33. Reddy, M. A., B.-S. Yang, X. Yue, C. J. K. Barnett, I. L. Ross, M. J. Sweet, D. A. Hume, and M. C. Ostrowski. 1994. Opposing actions of c-ets/PU.1 and c-myb protooncogenes products in regulating the macrophage-specific promoters of the human and mouse colony-stimulating factor-1 receptor (c-fms) genes. J. Exp. Med. 180:2309–2319.[Abstract/Free Full Text]
  34. Kelleher, M. D., M. K. Abe, T. S. Chao, M. Jain, J. M. Green, J. Solway, M. R. Rosner, and M. B. Hershenson. 1995. Role of MAP kinase activation in bovine tracheal smooth muscle mitogenesis. Am. J. Physiol. 268:L894–901.[Abstract/Free Full Text]
  35. Naureckas, E. T., M. B. Hershenson, M. K. Abe, M. D. Kelleher, C. Florio, S. I. Heisler, M. Absher, J. N. Evans, R. W. Samsel, and J. Solway. 1995. Bronchoalveolar lavage fluid from immature rats with hyperoxia-induced airway remodeling is mitogenic for airway smooth muscle. Am. J. Respir. Cell Mol. Biol. 12:268–274.[Abstract]
  36. Naureckas, E. T., I. M. Ndukwu, A. J. Halayko, C. Maxwell, M. B. Hershenson, and J. Solway. 1999. Bronchoalveolar lavage fluid from asthmatic subjects is mitogenic for human airway smooth muscle. Am. J. Respir. Crit. Care Med. 160:2062–2066.[Abstract/Free Full Text]
  37. Ramakrishnan, M., N. L. Musa, J. Li, P. Liu, R. G. Pestell, and M. B. Hershenson. 1998. Catalytic activation of extracellular signal-regulated kinases induces cyclin D1 expression in airway smooth muscle. Am. J. Respir. Cell Mol. Biol. 18:736–740.[Abstract/Free Full Text]
  38. Parmacek, M. S., A. J. Vora, T. Shen, E. Barr, F. Jung, and J. M. Leiden. 1992. Identification and characterization of a cardiac-specific transcriptional regulatory element in the slow cardiac tropinin C gene. Mol. Cell. Biol. 12:1967–1976.[Abstract/Free Full Text]
  39. Dignam, J. D., R. M. Lebovitz, and R. G. Roeder. 1983. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11:1475–1489.[Abstract/Free Full Text]
  40. Zoukhri, D., R. R. Hodges, C. Sergheraert, A. Toker, and D. A. Dartt. 1997. Lacrimal gland PKC isoforms are differentially involved in agonist-induced protein secretion. Am. J. Physiol. 272:C263–C269.[Abstract/Free Full Text]
  41. Lu, Z., A. Hornia, Y.-W. Jiang, Q. Zang, S. Ohno, and D. A. Foster. 1997. Tumor promotion by depleting cells of protein kinase C delta. Mol. Cell. Biol. 17:3418–3428.[Abstract]
  42. Szallasi, Z., C. B. Smith, G. R. Pettit, and P. M. Blumberg. 1994. Differential regulation of protein kinase C isozymes by bryostatin 1 and phorbol 12-myristate 13-acetate in NIH 3T3 fibroblasts. J. Biol. Chem. 269:2118–2124.[Abstract/Free Full Text]
  43. Wang, Q. J., D. Bhattacharyya, S. Garfield, K. Nacro, V. E. Marquez, and P. M. Blumberg. 1999. Differential localization of protein kinase C delta by phorbol esters and related compounds using a fusion protein with green fluorescent protein. J. Biol. Chem. 274:37233–37239.[Abstract/Free Full Text]
  44. Beier, F., R. J. Lee, A. C. Taylor, and R. G. Pestell. 1999. LuValle P. Identification of the cyclin D1 gene as a target of activating transcription factor 2 in chondrocytes. Proc. Natl. Acad. Sci. USA 96:1433–1438.[Abstract/Free Full Text]
  45. D'Amico, M., J. Hulit, D. F. Amanatullah, B. T. Zafonte, C. Albanese, B. Bouzahzah, M. Fu, L. H. Augenlicht, L. A. Donehower, K.-I. Takemaru, R. T. Moon, R. Davis, M. Lisanti, M. Shtutman, J. Zhurinski, A. Ben-Ze'ev, A. A. Troussard, S. Dedhar, and R. G. Pestell. 2000. The integrin-linked kinase regulates the cyclin D1 gene through glycogen synthase 3ß and cAMP-repsonse element binding protein-dependent pathways. J. Biol. Chem. 275:32649–32657.[Abstract/Free Full Text]
  46. Halayko, A. J., B. Camoretti-Mercado, S. M. Forsythe, J. E. Vieira, R. W. Mitchell, M. E. Wylam, M. B. Hershenson, and J. Solway. 1999. Divergent differentiation paths in airway smooth muscle culture: induction of functionally contractile myocytes. Am. J. Physiol. 276:L197–L206.
  47. Wasylyk, B., S. L. Hahn, and A. Giovane. 1993. The Ets family of transcription factors. Eur. J. Biochem. 211:7–18.[Medline]
  48. Kielbassa, K., H. J. Muller, H. E. Meyer, F. Marks, and M. Gschwendt. 1995. Protein kinase C delta-specific phosphorylation of the elongation factor eEF-alpha and an eEF-1 alpha peptide at threonine 431. J. Biol. Chem. 270:6156–6162.[Abstract/Free Full Text]
  49. Germano, P., J. Gomez, M. G. Kazanietz, P. M. Blumberg, and J. Rivera. 1994. Phosphorylation of the gamma chain of the high affinity receptor for immunoglobulin E by receptor-associated protein kinase C-delta. J. Biol. Chem. 269:23102–23107.[Abstract/Free Full Text]
  50. Jideama, N. M., T. A. Noland, R. L. Raynor, G. C. Blobe, D. Fabbro, M. G. Kazanietz, P. M. Blumberg, Y. A. Hannun, and J. Kuo. 1996. F, Phosphorylation specificities of protein kinase C isozymes for bovine cardiac troponin I and troponin T and sites within these proteins and regulation of myofilament properties. J. Biol. Chem. 271:23277–23283.[Abstract/Free Full Text]
  51. Maizels, E. T., C. A. Peters, M. Kline, R. E. Cutler, M. Shanmugam, and M. Hunzicker-Dunn. 1998. Heat-shock protein-25/27 phosphorylation by the delta isoform of protein kinase C. Biochem. J. 332:703–712.
  52. Igarashi, M., H. Wakasaki, N. Takahara, H. Ishii, Z.-Y. Jiang, T. Yamauchi, K. Kuboki, M. Meier, C. J. Rhodes, and G. L. King. 1999. Glucose or diabetes activates p38 mitogen-activated protein kinase via different pathways. J. Clin. Invest. 103:185–195.[Medline]
  53. Page, K., J. Li, and M. B. Hershenson. 2001. p38 mitogen-activated protein kinase negatively regulates cyclin D1 expression in airway smooth muscle cells. Am. J. Physiol. 280:L955–L964.
  54. Tseng, C. P., Y. J. Kim, R. Kumar, and A. K. Verma. 1994. Involvement of protein kinase C in the transcriptional regulation of 12-O-tetradecanoylphorbol-13-acetate-inducible genes modulated by AP-1 or non-AP-1 transacting factors. Carcinogenesis 15:707–711.[Abstract/Free Full Text]
  55. Hirai, S., Y. Izumi, K. Higa, K. Kaibuchi, K. Mizuno, S. Osada, K. Suzuki, and S. Ohno. 1994. Ras-dependent signal transduction is indispensable but not sufficient for the activation of AP1/Jun by PKC delta. EMBO J. 13: 2331–2340.[Medline]
  56. Ornatsky, O. I., D. M. Cox, P. Tangirala, J. J. Andreucci, Z. A. Quinn, J. L. Wrana, R. Prywes, Y. T. Yu, and J. C. McDermott. 1999. Post-translational control of the MEF2A transcriptional regulatory protein. Nucleic Acids Res. 27:2646–2654.[Abstract/Free Full Text]
  57. Nakaigawa, N., S. Hirai, K. Mizuno, T. Shuin, M. Hosaka, and S. Ohno. 1996. Differential effects of overexpression of PKC alpha and PKC delta/epsilon on cellular E2F activity in late G1 phase. Biochem. Biophys. Res. Commun. 222:95–100.[Medline]
  58. Vancurova, I., V. Miskolci, and D. Davidson. 2001. NF-kappaB activation in TNFalpha-stimulated neutrophils is mediated by protein kinase C delta: correlation to nuclear I-kappaB-alpha. J. Biol. Chem. 276:9746–9752.
  59. Fernandes, D., E. Guida, V. Koutsoubos, T. Harris, P. Vadiveloo, J. W. Wilson, and A. G. Stewart. 1999. Glucocorticoids inhibit proliferation, cyclin D1 expression, and retinoblastoma protein phosphorylation, but not activity of the extracellular-regulated kinases in human cultured airway smooth muscle. Am. J. Respir. Cell Mol. Biol. 21:77–88.[Abstract/Free Full Text]
  60. Lee, J. H., P. R. Johnson, M. Roth, N. H. Hunt, and J. L. Black. 2001. ERK activation and mitogenesis in human airway smooth muscle cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 280:L1019–L1029.[Abstract/Free Full Text]
  61. Xiong, W., R. G. Pestell, G. Watanabe, J. Li, M. R. Rosner, and M. B. Hershenson. 1997. Cyclin D1 is required for S phase traversal in bovine tracheal myocytes. Am J. Physiol. 272:L1205–L1210.[Abstract/Free Full Text]



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