![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Previous Article | Next Article 
Molecular and Cellular Biology, October 2007, p. 6933-6947, Vol. 27, No. 19
0270-7306/07/$08.00+0 doi:10.1128/MCB.02385-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030,1 Department of Otorhinolaryngology, Yonsei University College of Medicine, Seoul 120-752, South Korea,2 Program in Cancer Biology, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas3
Received 20 December 2006/ Returned for modification 26 February 2007/ Accepted 9 July 2007
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
isozyme and transmits the activation signal to CREB via the Raf/MEK/extracellular signal-regulated kinase/p90 ribosomal S6 kinase (RSK) pathway. Activated RSK translocated from the cytoplasm to the nucleus, where it phosphorylates CREB. Activated CREB then binds to a cis-acting replication element motif on the promoter (at nucleotides [nt] –878 to –871) of the MUC5AC gene. The depletion of CREB using small interfering RNA abolished not only the RA-induced MUC5AC but also RA-induced MUC2 and MUC5B. Taken together, our findings demonstrate that CREB activation via this nonclassical RA signaling pathway may play an important role in regulating the expression of mucin genes and mediating the early biological effects of RA during normal mucous differentiation in NHTBE cells. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
RA, which is a metabolite of vitamin A, has an essential role in the development and maintenance of mucociliary differentiation of the epithelium in the conducting airways (18, 20, 21). When normal human tracheobronchial epithelial (NHTBE) cells are cultured in medium deficient in RAs, they undergo squamous differentiation; the addition of RAs restores mucous differentiation (31, 32) and induces mucin gene expression. We previously showed that RA receptor (RAR) played an important role in the induction of expression of the mucin genes MUC2, MUC5AC, and MUC5B and mucous cell differentiation of bronchial epithelial cells by RA (31). Unlike this genetic event, an early effect of RA on mucociliary differentiation of bronchial epithelial cells is the nongenomic activation of cyclic AMP (cAMP) response element (CRE)-binding protein (CREB), as we previously demonstrated that RA rapidly activated a CREB transcription factor without using its canonical RAR/retinoid X receptors (RXRs) (1).
The CREB family of transcription factors plays critical roles in controlling cell growth, survival, and cell cycle progression and determining the fate of many cell types (11, 26, 34, 35, 50). Studies have shown that CREB is important for the proliferation and differentiation of neuronal cells (9, 35, 59), vascular smooth muscle cells (28), adipocytes (49), thyrocytes (42), Sertoli cells (53), pancreatic beta cells (27), and hematopoietic cells (47). CREBs recognize the specific DNA sequence 5'-TGACGTCA-3', known as the CRE, in the transcription regulatory regions of many cAMP-regulated genes (8, 25, 38, 40, 61) with cell type specificity (5). Studies have shown that numerous kinases are involved in the activation of CREB in response to various extracellular stimuli (e.g., growth factors and stress signals) via diverse signaling pathways (28, 35, 55) including protein kinase A (PKA) (7, 15), protein kinase C (PKC) (64), p90 ribosomal S6 kinase 1/2 (RSK1/2) (63), mitogen- and stress-activated protein kinase 1 (10), mitogen-activated protein kinase (MAPK)-activated protein 2 kinase (61), Akt (12), calcium/calmodulin-dependent protein kinase II (58), and calcium/calmodulin-dependent protein kinase IV (37).
PKCs are a family of serine/threonine kinases that assume important physiological functions and are highly activated in certain malignancies (19). They can be categorized into three subfamilies, conventional, novel, and atypical, based on the structural domains that confer their coactivator dependency (41). A novel isoform, PKC
, has been shown to play an important role in mucin exocytosis stimulated by human neutrophil elastase (45) and mucin gene expression induced by phorbol 12-myristate 13-acetate (62). Conventional PKC (cPKC) isoforms were shown to be directly activated by RA (48). A recent crystallography study showed that RA may exert its effect by binding to the C2 domain of PKC, a structure domain that present only in conventional isoforms (43). However, as PKCs are differentially expressed in different cell types (19), it is not known which isoform of cPKC is involved in RA-induced CREB activation and whether such an activation has a role in mucin gene expression and the normal differentiation of lung epithelial cells. Here, we hypothesized that RA-induced CREB activation has a critical role in mucin gene (especially MUC5AC) regulation and triggers early events in the differentiation of the bronchial epithelia. To test our hypothesis, we performed the study described herein to further determine the role of molecules involved in the nonclassical signaling pathway of RA in the activation of CREB. In addition, we analyzed the transcriptional action of CREB on the promoter region of the MUC5AC gene in NHTBE cells. An exploration of transcriptional regulation demonstrated that the RA-induced up-regulation of the MUC5AC gene in NHTBE cells was mediated by the CRE motif on the promoter. RA transactivates the promoter function of the other secretory mucin genes, including MUC2 and MUC5B, which are also regulated by CREB. Together, these findings give new insight into the role of CREB in mucin gene expression in mucous cell differentiation.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, phospho-PKC (Ser-659 phosphorylated), PKCßII, RAR, RARß, RAR
, and RXR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). An antibody against ß-actin (clone AC-15) was purchased from Sigma-Aldrich (St. Louis, MO). Antibodies against CREB, phospho-CREB (Ser-133 phosphorylated), phospho-PKCßII (Thr-641 phosphorylated), and cPKCß were purchased from Upstate Biotechnology (Waltham, MA). MUC5AC antibody was purchased from NeoMarkers (Freemont, CA). All-trans RA, 9-cis RA, 13-cis RA, retinol, cycloheximide, and actinomycin D were purchased from Sigma-Aldrich. A pan-RAR antagonist (Ro 61-8431) and a pan-RXR antagonist (Ro 26-5405) were provided by Roche Bioscience (Palo Alto, CA). Other signal transduction inhibitors [H89, wortmannin, PP2, U0126, SB203580, Sp600125, staurosporine, Go6976, GF109203X, rottlerin, U73122, 2',5'-dideoxyadenosine, calphostin C, EGTA-ethyleneglycol-bis(ß-aminoethyl)-N,N,N',N'-tetraacetoxymethyl ester (EGTA-AM), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM), 12-O-tetradecanoyl-phorbol 13-acetate (TPA), and 8-bromo (8Br)-cAMP] were purchased from Calbiochem (San Diego, CA). All of the chemicals were dissolved in dimethyl sulfoxide (DMSO), and all other reagents were of analytical reagent grade or better. NHTBE cell culture. NHTBE cells were purchased from Clonetics (San Diego, CA). Second-passage NHTBE cells (1 x 105) were seeded onto a 24-mm Transwell plate (Corning, Acton, MA) and grown in serum-free growth factor-supplemented and hormone-supplemented culture medium as described previously (1, 18, 30, 32). After 7 days under immersed culture conditions, the cell culture was switched to an air-liquid interface system. For signal transduction experiments, NHTBE cells were incubated with bronchial epithelial cell basal medium for 24 h prior to RA treatment. To study the effect of chemical inhibitors on signal transduction pathways, cells were pretreated with each inhibitor 2 h prior to treatment with RA. To study the effect of RA on MUC5AC mRNA expression and secretion, NHTBE cells were grown in RA-deficient medium for 7 days and then further incubated with 1 µM RA for the indicated times or with various concentrations of RA for 24 h. All cells were grown at 37°C in a humidified atmosphere of 5% CO2.
QRT-PCR analysis.
Total RNA was extracted from NHTBE cells and the NCI-H292 (H292) lung cancer cell line using RNeasy Mini kits (QIAGEN, Valencia, CA). Each reverse transcription (RT) reaction was performed using 1 µg of total RNA that was reverse transcribed into cDNA using random hexamer primers (GeneAmp RNA PCR core kit; Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. PCR conditions were an initial reaction at 95°C for 5 min followed by 30 cycles at 95°C, 55°C, and 72°C for 30 s each. PCR analysis of ß-actin expression (Ambion, Austin, TX) was performed as an internal control. The primer sequences, which were described previously (24), were as follows: 5'-AGCCTACGTGCCAAAAAAGG-3' (PKC forward), 5'-TCTAGGTGTGGAGGCAAATGG-3' (PKC reverse), 5'-ACCATGGAATCTGGAGCCGAGAAC-3' (PKCßI forward), 5'-CTGTAGGAAGGCCTCCTTGAAAGA-3' (PKCßI reverse), 5'-ACCATGGAATCTGGAGCCGAGAAC-3' (PKCßII forward), 5'-CTGTAGGAAGGCCTCCTTGAAAGA-3' (PKCßII reverse), 5'-ACCATGGAATCTGGAGCCGAGAAC-3' (PKCß forward), 5'-CTGTAGGAAGGCCTCCTTGAAAGA-3' (PKCß reverse), 5'-AGCCTACGTGCCAAAAAAGG-3' (RARß forward), 5'-TCTAGGTGTGGAGGCAAATGG-3' (RARß reverse), 5'-ACCATGGAATCTGGAGCCGAGAAC-3' (CREB forward), and 5'-CTGTAGGAAGGCCTCCTTGAAAGA-3' (CREB reverse). PCR products were then separated in a 2% agarose DNA gel and stained with ethidium bromide. To investigate the expression level of MUC5AC, mRNA expression was determined using quantitative RT-PCR (QRT-PCR). MUC5AC primer sequences were as follows: 5'-TGTGGCGGGAAAGACAGC-3' (forward) and 5'-CCTTCCTATGGCTTAGCTTCAGC-3' (reverse). MUC5B primer sequences were as follows: 5'-CGATCCCAACAGTGCCTTCT-3' (forward) and 5'-CCTCGCTCCGCTCACAGT-3' (reverse). MUC2 primer sequences were as follows: 5'-GCTGGCTGGATTCTGGAAAA-3' (forward) and 5'-TGGCTCTGCAAGAGATGTTAGC-3' (reverse). Human GAPDH (glyceraldehyde-3-phosphate dehydrogenase) endogenous control reagents (Applied Biosystems) were used as normal controls. The PCR was performed in a 96-well format using the iCycler Real Time PCR detection system (Bio-Rad, Richmond, CA) by using SYBR green PCR core reagent (Applied Biosystems). Samples were analyzed in triplicate in a PCR program with an initial 3-min 95°C step followed by 40 cycles of 1 min at 95°C and 1 min at 65°C. After each analysis, a melting curve was performed to ensure that no primer dimers or secondary products were formed, and data were then analyzed using the accompanying iCycler version 3.1 software (Bio-Rad).
PKC activation assay and preparation of cell fractionation.
To investigate PKC activation in NHTBE cells, we measured the translocation of PKC and PKCßII from cytosolic to membrane fractions as described previously (14). Briefly, the cells were treated with 1 µM RA for the indicated time periods at 37°C and then washed twice with ice-cold phosphate-buffered saline (PBS), scraped, and lysed using Tris-sucrose buffer (10 mM Tris-HCl [pH 7.5], 0.25 M sucrose, 0.2 mM CaCl2, 1 mM EDTA) supplemented with protease inhibitor cocktail tablets (Roche Diagnostics, Indianapolis, IN). The cells were sonicated using a sonic dismembrator (Fisher Scientific, Pittsburgh, PA) three times for 30 s each with 5 min of incubation on ice between sonications. Nuclei and unbroken cells were removed by centrifugation at 1,000 x g for 10 min in a Beckman SW28 rotor (Beckman Coulter, Fullerton, CA) at 4°C. The resulting supernatant was further centrifuged at 100,000 x g for 60 min in a Beckman SW28 rotor at 4°C. The supernatant from this centrifugation (cytosolic fraction) was removed and saved. The pellet (membrane fraction) was resuspended in Tris-sucrose buffer containing 1% Triton X-100 and 10 mM 2-mercaptoethanol. The suspensions were then briefly sonicated and centrifuged (100,000 x g for 60 min at 4°C). Insoluble material was discarded, and the supernatant was collected as a Triton X-100-soluble membrane fraction after the last centrifugation. The protein concentrations of the cytosolic and nuclear fractions were determined according to the Bradford protocol (4).
Western blot analysis. Western blot analysis was performed as previously described (1). Briefly, whole-cell extracts were prepared using 2x sodium dodecyl sulfate (SDS) Laemmli lysis buffer. Equal amounts of total protein (20 µg) were resolved by 10% SDS-polyacrylamide gel electrophoresis. Proteins that were reactive with primary antibody were visualized with a horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence reagents (Amersham Bioscience, Arlington Heights, IL). Equal protein loading was confirmed by stripping the blots and reprobing them with an anti-ß-actin antibody.
RNA interference.
RNA interference was performed with NHTBE cells by using the siIMPORTER small interfering RNA (siRNA) transfection reagent (Upstate Biotechnology) as described previously (1). For target gene silencing, SMARTpool-sequenced siRNAs targeting human PKC (GenBank accession no. NM_002953), RSK1 (GenBank accession no. NM_002953), RSK2 (GenBank accession no. NM_004586), ERK2 (GenBank accession no. NM_002745), RAR (GenBank accession no. NM_000964), RARß (GenBank accession no. NM_000965), RAR (GenBank accession no. NM_000966), RXR (GenBank accession no. NM_002957), or CREB (GenBank accession no. NM_004379) and a nonspecific control pool (siRNA-negative control; Dharmacon RNA Technologies, Lafayette, CO) were diluted and stored according to the manufacturer's instructions. NHTBE cells at 60% or 70% confluence were transfected with a final concentration of 100 nM SMARTpool siRNA or the nonspecific control pool. Cells were analyzed 72 h after transfection.
Transient transfection and luciferase assays. Transfections of NHTBE cells were performed as described previously (1). Briefly, NHTBE cells (1 x 104 cells/well) were plated in 12-well plates using bronchial epithelial growth medium in the absence of RA. When the cells reached 60% to 70% confluence, transfections were performed using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA) with the CRE promoter-luciferase reporter plasmid (Stratagene, La Jolla, CA), the construct of deletion mutants of MUC5AC or point-mutated CRE sites of MUC5AC, and the ß-galactosidase (ß-gal) reporter construct (BD Biosciences Clontech, Palo Alto, CA) according to the manufacturer's instructions. Four hours after transfection, cells were treated with RA and cultured for another 48 h. The luciferase activity was measured using a Lumat LB 9507 luminometer (EG&G, Berthold, Germany). ß-gal activity was measured using a ß-gal enzyme assay system (Promega, Madison, WI) and used to normalize transfection efficiency.
For knockdown by siRNA, 100 nM siRNA of the indicated target gene or nonspecific siRNA was cotransfected with luciferase plasmid and ß-gal reporter constructs. The cells were treated with or without RA for another 24 h after 72 h of transfection, when target protein levels had been reduced by 70% to 80% as assessed by Western blot analysis. After transfection, whole-cell lysate was prepared for luciferase assay.
Immunofluorescence analysis. For immunofluorescence staining, NHTBE cells were grown on coverslips for 7 days in an RA-deficient medium. After treatment with RA, the cells were fixed in a methanol-acetone mixture (1:1, vol/vol), washed with PBS, and blocked with 5% preimmune serum for 30 min. The cells were then incubated with the indicated primary antibodies (1:100 dilution) for 2 h at room temperature. The coverslips were washed with PBS containing 0.1% Tween 20, incubated with an AlexaFluor 488-tagged secondary antibody (Molecular Probes, Eugene, OR) for 1 h at room temperature, and then counterstained for nuclei with 4',6-diamidino-2-phenylindole (DAPI) for 30 min. After being washed with PBS, slides were mounted using the SlowFade Antifade kit (Molecular Probes). The stained cells were visualized under a fluorescence microscope (Axioskop 40; Carl Zeiss, Thornwood, NY), and the images were captured at a magnification of x400 and stored using the AxioVision software program (Carl Zeiss) according to the instructions provided by the manufacturer.
Quantification of secreted MUC5AC. To analyze the production of MUC5AC, accumulating apical secretion fluid with or without RA was collected and analyzed by dot blot analysis (16). Briefly, diluted apical secretion fluid was applied to nitrocellulose membranes, which were incubated with human MUC5AC antibody, followed by a reaction with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG). The signal was detected by chemiluminescence using the ECL kit (Amersham, Little Chalfont, Buckinghamshire, United Kingdom). The blot intensities were analyzed densitometrically using Quantity One 4.6.1 software (Bio-Rad).
Preparation of MUC5AC promoter-luciferase constructs. Methods to measure MUC5AC promoter activity with luciferase as a reporter were reported previously (17). Fragments ranging in size from 3.7 kb (nt –3752 to +7) to 0.29 kb (nt –296 to +7), which were located immediately adjacent to the 5' transcriptional start site of human MUC5AC, were generated by digestion with exonuclease (Erase a Base system; Promega Corp., Madison, WI) of the 3.7-kb fragment of the MUC5AC promoter and cloned into the pGL3-Basic luciferase vector (Promega Corp.).
Site-directed mutations of the human MUC5AC promoter were made in the context of MUC5AC-LUC (positions –1366 to +7), using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and confirmed by sequencing. The oligomers used to introduce point mutations included the following: M1 (5'-CCATCAAGACTCTTGAACTGGCCC-3'), M2 (5'-CCATCAAGTGGTGTGAACTGGCCC-3'), and M3 (5'-CCATCAAGTGACTGACACTGGCCC-3') (mutations are shown in boldface type; underlined type indicates putative CRE sites) (56).
EMSA.
To assess the DNA-binding activity of CREB, we performed an electrophoretic mobility shift analysis (EMSA) as described previously (1), with necessary modifications. Briefly, nuclear proteins from RA-treated or untreated cells were prepared and stored at –80°C. For EMSA, oligonucleotides corresponding to the consensus CRE sequences (5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3' [boldface type indicates CRE {Santa Cruz Biotechnology}]), CRE-specific sequences in the MUC5AC promoter region at nt –878 to –871 (wild type [wt] [5'-CCATCAAGTGACTTGAACTGGCCC-3' {boldface type indicates putative CRE}]), and the CRE mutant sequence (M1 [5'-CCATCAAGACTCTTGAACTGGCCC-3'], M2 [5'-CCATCAAGTGGTGTGAACTGGCCC-3'], and M3 [5'-CCATCAAGTGACTGACACTGGCCC-3' [underlining indicates a CRE mutant]) were synthesized, annealed, and end labeled with [-32P]ATP using T4 polynucleotide kinase. Nuclear extract (10 µg) was incubated at room temperature for 30 min with the 32P-labeled CRE probe in binding buffer [20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM dithiothreitol, 250 mM NaCl, 50 mM Tris-HCl (pH 7.5), and 0.25 mg/ml poly(dI-dC)]. For supershift analysis, nuclear extracts were incubated with antibodies against phospho-CREB (pCREB) or CREB and wild-type CRE-oligonucleotide probe for 30 min at 37°C, and the complex was then analyzed by EMSA. Antibodies against preimmune serum were included as negative controls. The DNA-protein complexes were resolved by electrophoresis in 5% nondenaturing polyacrylamide gels in 0.5x TBE (1x TBE is 25 mM Tris base, 25 mM boric acid, and 0.5 mM EDTA). Gels were dried and autoradiographed at –80°C. For EMSA, all studies were repeated three times; consistent results were obtained.
ChIP assay. To determine transcription factor binding on the promoter region, we performed chromatin immunoprecipitation (ChIP) assays (34). NHTBE cells were treated with RA for 4 h, cross-linked with 1% formaldehyde for 10 min at 37°C, and then washed with cold PBS. The cell pellet was resuspended in 0.3 ml of lysis buffer (1% SDS, 100 mM NaCl, 50 mM Tris-HCl [pH 8.1], 5 mM EDTA), followed by sonication to an average DNA length of 500 to 1,000 bp, and then rotated at 4°C overnight. After interaction with protein A beads and incubation overnight at 65°C to reverse the cross-links, the DNA was dissolved in Tris-EDTA buffer and then analyzed by PCR. The antibodies anti-CREB, anti-phospho-CREB (Upstate Biotechnology), and normal rabbit IgG were added separately into the reaction solutions. Primers used for PCR were from MUC5AC promoter sequences 5'-TCACTGGGACCTTTCTGTGCT-3' (forward) and 5'-ACTGAAGTAGCGCCAGCCA-3' (reverse).
Data analysis. For all transfection assays and QRT-PCR analyses, values are given as means ± standard errors (SE) of triplicate assays in three independent experiments. Statistical analysis was performed with the Prism program (GraphPad Software, San Diego, CA) using one-way analysis of variance, followed by Dunnett's test for comparing experiment groups against a single control or by Bonferroni's test for paired comparison.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
is a predominant cPKC isozyme expressed in NHTBE cells.
We previously showed that RA induced CREB activation via PKC in NHTBE cells (1). A recent crystallography study indicates that RA activates PKC by binding to its C2 domain (43). Because this domain is present only in cPKC, to further determine which cPKC isotype mediates the RA activation of CREB, we measured the level of expression of cPKC isozymes in NHTBE cells using RT-PCR and Western blot analysis with isoform-specific primers and antibodies. H292 cells were included as a control for their known high expression level of PKC. As shown in Fig. 1, PKC was highly expressed in NHTBE and H292 cells. However, PKCßI and PKCßII were barely detectable in NHTBE cells although fairly detectable in H292 cells. PKC was not detected in either cell type (Fig. 1A). We further confirmed the predominant expression of PKC among cPKC isoforms in NHTBE cells using Western blot analysis with antibodies against PKC, PKC/ß/, and PKCßII (Fig. 1B). We used an antibody that recognizes all three types (/ß/) to assess the level of PKCßI because a PKCßI-specific antibody was not available. Immunoblotting of PKC was not performed because we did not detect mRNA of PKC in RT-PCR. This result is in agreement with data reported previously by Park et al. (45) showing that NHTBE cells express PKC, PKC, PKCµ, and PKC
but not PKCß, PKC, or PKC
. These results clearly showed that PKC is a major cPKC isozyme expressed in NHTBE cells.
|
and CREB, and activated PKC translocates from the cytoplasm to plasma and perinuclear membranes.
We next determined the effect of RA on the activation status of PKC. We analyzed the effect of RA on PKC phosphorylation at Ser-659, which indicates activation of PKC (3, 44), and PKC translocation using Western blot and immunofluorescence analysis. RA clearly induced phosphorylation of PKC and CREB in a time-dependent manner (Fig. 2A). We detected PKC phosphorylation as early as 5 min after treatment with RA; the level of phosphorylation reached a maximum at 120 min. CREB phosphorylation showed a similar pattern. The levels of expression of PKC and CREB remained unchanged. We also observed the occurrence of RA-induced phosphorylation of PKC and CREB in a dose-dependent manner (Fig. 2B). Both PKC and CREB were phosphorylated with treatment with as little as 10 nM RA for 30 min.
|
in bronchial epithelial cells, we isolated nuclear, cytoplasmic, and membrane fractions from the NHTBE cells treated with 1 µM RA for 15, 30, or 60 min. As shown in Fig. 2C, the majority of PKCs were located in the cytoplasm of RA-deficient NHTBE cells. However, upon treatment with RA, more than 80% of the PKCs were detected in the membrane and nuclear fractions within 15 min after treatment, demonstrating translocation of PKC by RA. As analyzed using Western blot analysis to detect phosphorylated PKC, PKC translocated to nuclear and membrane fractions in a phosphorylated form. In contrast, PKC residing in the cytoplasm was not phosphorylated. Treatment with RA did not cause the translocation of cytoplasmic PKCßII. These results are consistent with those of immunocytochemical analyses showing that most of the diffusely localized PKC in the cytoplasm when the cells were in an RA-deficient state was translocated to the plasma and perinuclear membranes within 15 min after treatment with RA (Fig. 2D). Consistent with our previous results, the majority of CREB was localized in nuclear fractions. Phosphorylation of CREB was maximal after 15 min of treatment with RA and continued for another 60 min with no change in the level of CREB expression in nuclear fractions (1).
Activation of PKC and CREB by RA is independent of RAR and RXR.
To determine whether RAR and RXR are involved in the activation of PKC by RA, we initially examined the effect of several retinoids, including 9-cis RA, 13-cis RA, and retinol, on the phosphorylation of PKC and CREB in NHTBE cells. We found that all these retinoids equipotently induced the phosphorylation of PKC and CREB (Fig. 3A). Furthermore, all-trans RA, 9-cis RA, 13-cis RA, and retinol induced the transactivation of the CRE-luciferase reporter four- to fivefold, which is similar to the capacity of TPA and 8Br-cAMP, which were used as positive controls for CRE activation (Fig. 3B). These findings clearly demonstrate that all-trans RA is not the only retinoid that activates PKC and CREB.
|
by RA, we next examined the effects of pan-RAR and pan-RXR antagonists on the activation of PKC. We incubated NHTBE cells with 10 µM Ro 61-8431 and Ro 26-5405 before treatment with RA. Pretreatment with either antagonist did not block RA-induced activation of PKC or CREB (Fig. 4A). As we previously reported (1), these antagonists did not affect the RA-induced CRE transactivation either.
|
by RA, we silenced the expression of RAR, RARß, RAR, and RXR using siRNA; we silenced only the type of RXR because it is known to be a predominant RXR isotype in eliciting the classical action of RA. At 72 h after transfection of siRNAs targeting RARs and RXR, we confirmed the maximal silencing of target gene expression (data not shown). Western blot analysis of RA-induced active PKC levels after transfection with pooled synthetic siRNAs for RARs and RXR revealed that the depletion of RARs and RXR in NHTBE cells did not affect RA-induced rapid phosphorylation of either PKC or CREB (Fig. 4B). Silencing of RARs, RXR, and a nonspecific control pool (negative control for siRNA) did not affect the activation status of PKC or CREB in the vehicle-treated control. We also examined the effect of RARs and RXR knockdown on CRE-luciferase activity and found no effect of such a knockdown on the RA-induced CRE transcriptional activity (data not shown). Taken together, these results confirmed that the RA-induced rapid activation of PKC and CREB does not require RAR or RXR function in NHTBE cells.
cPKC and ERK are critically involved in RA-induced activation of CREB.
To investigate the signaling pathway by which RA induces the activation of PKC and CREB, we treated NHTBE cells with various signaling inhibitors alone or in combination with RA and determined the activation status of PKC and CREB. We found that of the signal transduction inhibitors used, only staurosporine (general PKC inhibitor), Go6976 (cPKC inhibitor), and GF109203X (cPKC and novel PKC inhibitor) substantially blocked PKC and CREB activation induced by RA (Fig. 5A). Other inhibitors, including H89 (PKA inhibitor), wortmannin (phosphoinositide 3-kinase inhibitor), PP2 (Src tyrosine kinase inhibitor), SB203580 (p38 MAPK inhibitor), Sp600125 (c-Jun N-terminal kinase inhibitor), and rottlerin (novel PKC inhibitor), did not block RA activation of PKC or CREB. Also, U0126 (MEK1/2 inhibitor) did not inhibit the RA activation of PKC but did inhibit the activation of CREB. These results reconfirmed that cPKC isotypes and ERK1/2 are involved in the RA activation of CREB.
|
and CREB, we inhibited endogenous AC using the AC inhibitor 2',5'-dideoxyadenosine in NHTBE cells before treatment with RA. As shown in Fig. 5B, 2',5'-dideoxyadenosine did not inhibit the RA-induced activation of PKC or CREB. We also sought to determine whether phospholipase C (PLC) is involved in the RA activation of PKC and CREB by treating NHTBE cells with U73122 (an inhibitor of PLC) before treatment with RA. Treatment with 10 µM U73122 did not affect the activation of PKC or CREB (Fig. 5B).
To further determine whether diacylglycerol and Ca2+ are required for RA-induced PKC and CREB activation, we treated NHTBE cells with the indicated concentrations of calphostin C (diacylglycerol inhibitor), EGTA-AM, or BAPTA-AM (an intracellular Ca2+ chelator) before treatment with RA. As shown in Fig. 5B, treatment with calphostin C did not block RA-induced activation. However, treatment with inhibitors that specifically chelated intracellular calcium did not inhibit the phosphorylation of PKC but slightly blocked the phosphorylation of CREB (Fig. 5C).
RA activation of CREB is mediated by the PKC/Raf/MEK/ERK/RSK signaling pathway.
To determine whether RA-activated PKC uses the linear signal transduction of the Raf/MEK/ERK/RSK signaling pathway for CREB activation, we examined the effect of RA on the PKC/Raf/MEK/ERK/RSK/CREB signaling pathway after the knockdown of the expression of PKC with an siRNA targeting PKC. We verified the successful knockdown of PKC expression in NHTBE cells using Western blot and RT-PCR analyses (Fig. 6A). Treatment with RA resulted in the activation of the PKC/Raf/MEK/ERK/RSK/CREB signaling cascade in control transfections with a nonspecific siRNA. However, RA activation of this signaling pathway was completely blocked in PKC-silenced NHTBE cells (Fig. 6A). This result clearly demonstrated that PKC is indispensable for RA activation of the CREB signaling cascade.
|
(which is upstream of ERK) remained intact (Fig. 6B, left). The knockdown of RSK1/2 blocked the activation of CREB (downstream of RSK) only, without affecting upstream PKC or ERK (Fig. 6B, right). To determine whether the inhibition of this signaling cascade inhibits the transcriptional activity of CREB, we transfected NHTBE cells with a CRE-luciferase reporter vector alone or with small interfering PKC, siERK1/2, or small interfering CREB (siCREB). As shown in Fig. 6C, the knockdown of individual signaling mediators significantly blocked the RA-induced transcriptional activity of CREB. ERK and RSK are activated by RA, and RSK but not ERK is translocated to the nucleus. To determine which molecule enters the nucleus upon stimulation with RA, we examined the localization of activated RSK and ERK in NHTBE cells using Western blot analysis and immunocytofluorescence analysis. As shown in Fig. 7A, the majority of RSKs and ERKs were located in cytoplasmic fractions in the absence of RA. Treatment with RA (1 µM for 30 min) reduced the amount of RSK in the cytoplasm while increasing the amount of RSK in the nuclear fractions. We detected phosphorylated RSKs only in the nuclear fractions. However, we detected ERK only in the cytoplasmic fractions, and treatment with RA induced only the activation of ERK but not the translocation of it.
|
Mapping of RA-induced transactivation regions within the MUC5AC promoter. To determine whether RA induces the upregulation of MUC5AC, we treated the NHTBE cells with 1 µM RA for different periods of time or with different concentrations of RA for 24 h and measured MUC5AC gene expression and secretion. We found that RA increased both the MUC5AC mRNA level and protein secretion in a time- and dose-dependent manner (Fig. 8).
|
|
|
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
isoform of cPKC in primary bronchial epithelial cells and that RA-activated PKC transmitted the signal via the Raf/MEK/ERK/RSK pathway to induce the transcriptional activity of CREB. We further confirmed the critical role of CREB in the RA-induced expression of mucin genes, including MUC5AC, MUC5B, and MUC2.
Translocation of PKC from the cytoplasm to the membrane is a key event in classical PKC activation (2, 39). We showed that the majority of PKCs were rapidly phosphorylated and translocated from the cytoplasm to the plasma and perinuclear membranes after treatment with RA (Fig. 2D). A recent study (13) showed that the calcium-binding loop and ß-strands 3 and 4 in the C2 domain of PKC are essential for the specific translocation of PKC to the plasma membrane in response to Ca2+. Because the RA-binding site has been suggested to be the region of R239-V271 (48) that encompasses the third loop of the calcium-binding loop, RA may act like Ca2+ or enhance the effect of Ca2+ in coordinating the active structure of PKC. In fact, our study showed that the deprivation of cytosolic Ca2+ by a calcium chelator did not block the RA-induced activation of PKC but slightly reduced downstream CREB activation (Fig. 5C), indicating a cooperative effect between Ca2+ and RA, and that Ca2+ may be needed for the full activity of RA-activated PKC. Interestingly, unlike Ca2+-induced activation, which translocates PKC only to the inner leaflet of the plasma membrane (13), RA stimulation induced the translocation of PKC to both the plasma membrane and the perinuclear membrane. It has been suggested that nucleus-translocated PKC may play certain roles in transcriptional regulation (36). However, we did not observe a bypassing of the intermediate pathway in terms of RA-induced CREB activation. The role of nuclear PKC remains to be elucidated.
It has been shown that PKC isoforms are differentially expressed according to the cell types and the differentiational stages of the same cell linage (19). Other than cPKCs, a novel PKC has been reported to play an important role in mucin secretion in airway epithelial cells (45, 62). However, in our preliminary study, we did not detect any RA-induced phosphorylation of PKC in NHTBE cells (data not shown). Thereafter, we found that rottlerin, a novel PKC-selective inhibitor, was not able to inhibit RA-induced PKC phosphorylation and subsequent CREB activation (Fig. 5B). In contrast, the cPKC-selective inhibitors GF109203X and Go6976 completely abolished RA-induced PKC activation and its downstream signal transduction (Fig. 5A and Fig. 7B to D), indicating a cPKC-selective activation of RA. Moreover, treatment with calphostin C, a PKC inhibitor that competes with diacylglycerol for binding to the C1 domain of PKCs, did not block the aforementioned activation, which further supported the model that RA directly activates PKC by binding to its C2 domain (43).
It is well known that RA activity is mediated mainly through RAR/RXR. However, our results showed that the activation of PKC and CREB by RA does not require RAR/RXR. The functional nullification of RAR/RXR by treatment with pan-RAR and pan-RXR antagonists or by transfection of siRNAs targeting RARs and RXR did not block RA-induced activation of PKC or CREB (Fig. 4). Also, the treatment did not affect the transactivation activity of CREB in NHTBE cells. These results clearly demonstrated a nonclassical effect of RA on the activation of PKC and CREB. We sought to explore the role of other conventional pathways that are known to activate PKC or CREB and found that neither PLC (upstream activator of PKC) nor PKA (upstream activator of CREB) is required for the RA-induced activation of PKC and CREB (Fig. 5A).
Our previous study and other previous reports showed that the downstream signaling of PKC to CREB can be mediated through the Raf/MEK/ERK/RSK/CREB pathway (1, 29, 46, 52). Our results clearly showed that the RA activation of this signaling pathway was completely blocked in PKC-silenced NHTBE cells (Fig. 6A), which indicates that PKC is indispensable in this signaling cascade. Our previous studies and the current one also showed that the RA activation of CREB requires RSK (CREB kinase) and ERK (RSK kinase); the knockdown of either ERK or RSK abolished the RA-induced phosphorylation and transactivation activity of CREB but not the activation of PKC (Fig. 6B). In addition, our results clearly showed that RA induced the translocation of RSK but not ERK from the cytoplasm to the nucleus, implying that CREB remained in the nucleus and is activated by the translocated active RSK (Fig. 7). Our results confirmed that RA-activated PKC activates the signaling cascade of Raf/MEK/ERK/RSK/CREB during the mucociliary differentiation of primary bronchial epithelial cells.
Although we have previously shown that RA induces MUC5AC gene expression (32), it is not clear whether cis-regulatory and/or trans-acting factors control the expression of MUC5AC gene in response to RA. In this study, on the basis of the promoter reporter assay, we determined that the region from nt –1039 to nt –596 of the MUC5AC promoter is essential for the response to RA. We found that the CRE motif in the region at nt –878 of the MUC5AC promoter is critical for the RA-induced transcriptional activity of MUC5AC (Fig. 9). In addition, mutation of the CRE motif sequence abolished the responsiveness of the MUC5AC promoter reporter to RA. Further studies employing EMSA and ChIP analyses verified that RA induced the binding of activated CREB to this putative CRE motif of the MUC5AC promoter both in vitro and in vivo. The crucial role of CREB in the transactivation of MUC5AC was further demonstrated by the silencing of CREB with siRNA, which abolished the RA-induced transactivation of the MUC5AC promoter (Fig. 10A). The RA-induced promoter activity of MUC5AC was decreased as the concentration of transfected CREB siRNA increased, indicating a specific control of MUC5AC expression by CREB. However, CREB knockdown abolished not only RA-induced MUC5AC expression/protein secretion but also the expression of MUC2 and MUC5B. Using the TFsearch database, we found corresponding putative CRE sites in the MUC2 and MUC5B promoter regions (Fig. 12B). Taken together, these results strongly suggest that CREB is a potent transcriptional regulator of several mucin genes, including MUC2, MUC5AC, and MUC5B, in human primary airway epithelial cells.
|
/Raf/MEK/ERK/RSK signaling pathway has a priming effect on MUC5AC production and mucous differentiation in primary bronchial epithelial cells.
In summary, we demonstrated a nonclassical signaling pathway by which RA activates CREB. We showed that RA activates the PKC isozyme and that RA-activated PKC immediately translocates from the cytoplasm to the plasma and perinuclear membranes and transmits activation signals to CREB via the activation of a linear signaling cascade, Raf/MEK/ERK/RSK, without using conventional RAR and RXR in primary bronchial epithelial cells. Activated RSK was then translocated from the cytoplasm to the nucleus, where it activates CREB and where the activated CREB transactivates the MUC5AC gene. On the basis of our current and previous findings, we propose a new paradigm for RA-induced mucociliary differentiation of bronchial epithelial cells in which the nonclassical action (mediated through CREB) and the conventional action (mediated through RAR and RXR) of RA each assume important physiological roles and cooperatively facilitate the differentiation of bronchial epithelia.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Published ahead of print on 23 July 2007.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
2. Almholt, K., P. O. Arkhammar, O. Thastrup, and S. Tullin. 1999. Simultaneous visualization of the translocation of protein kinase C-green fluorescent protein hybrids and intracellular calcium concentrations. Biochem. J. 337:211-218.[CrossRef][Medline]
3. Bornancin, F., and P. J. Parker. 1997. Phosphorylation of protein kinase C-alpha on serine 657 controls the accumulation of active enzyme and contributes to its phosphatase-resistant state. J. Biol. Chem. 272:3544-3549.
4. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.[CrossRef][Medline]
5. Cha-Molstad, H., D. M. Keller, G. S. Yochum, S. Impey, and R. H. Goodman. 2004. Cell-type-specific binding of the transcription factor CREB to the cAMP-response element. Proc. Natl. Acad. Sci. USA 101:13572-13577.
6. Cho, K. N., J. Y. Choi, C. H. Kim, S. J. Baek, K. C. Chung, U. Y. Moon, K. S. Kim, W. J. Lee, J. S. Koo, and J. H. Yoon. 2005. Prostaglandin E2 induces MUC8 gene expression via a mechanism involving ERK MAPK/RSK1/cAMP response element binding protein activation in human airway epithelial cells. J. Biol. Chem. 280:6676-6681.
, P < 0.05; 