Am J Physiol Cell Physiol AJP citation statistics
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 293: C1093-C1102, 2007. First published July 11, 2007; doi:10.1152/ajpcell.00225.2007
0363-6143/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/3/C1093    most recent
00225.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kanematsu, A.
Right arrow Articles by Adam, R. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kanematsu, A.
Right arrow Articles by Adam, R. M.

MUSCLE CELL BIOLOGY AND CELL MOTILITY

GATA-6 mediates human bladder smooth muscle differentiation: involvement of a novel enhancer element in regulating {alpha}-smooth muscle actin gene expression

Akihiro Kanematsu, Aruna Ramachandran, and Rosalyn M. Adam

Urological Diseases Research Center, Children's Hospital Boston, and Department of Surgery, Harvard Medical School, Boston, Massachusetts

Submitted 31 May 2007 ; accepted in final form 8 July 2007


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hollow organs exposed to pathological stimuli undergo phenotypic modulation characterized by altered expression of smooth muscle contractile proteins and loss of normal function. The molecular mechanisms that regulate smooth muscle differentiation, especially in organs other than the vasculature, are poorly understood. In this study, we describe a role for the GATA-6 transcription factor in regulation of human bladder smooth muscle differentiation. Knockdown of endogenous GATA-6 in primary human bladder smooth muscle cells (pBSMC) led to decreased mRNA levels of the differentiation markers {alpha}-smooth muscle actin ({alpha}-SMA), calponin, and smooth muscle myosin heavy chain. Similar effects were obtained following downregulation of GATA-6 by forskolin-induced elevation of intracellular cAMP levels. Forskolin treatment of pBSMC abolished recruitment of GATA-6 to the {alpha}-SMA promoter in vivo and reduced activity of human {alpha}-SMA promoter-directed gene expression by >60%. This inhibitory effect was rescued by enforced expression of wild-type GATA-6 but not by a zinc-finger-deleted mutant, GATA-6-{Delta}ZF, which lacks DNA-binding ability. In silico analysis of a region of the human {alpha}-SMA promoter, described previously as a transcriptional enhancer, identified a putative GATA-binding site at position –919/–913. Point mutation of this site in SMA-Luc abrogated GATA-6-induced activation of promoter activity. Together, these results provide the first evidence for a functional role for GATA-6 in regulation of bladder smooth muscle differentiation. In addition, these findings demonstrate that GATA-6 regulates human {alpha}-SMA expression via a novel regulatory cis element in the {alpha}-SMA promoter-enhancer.

hollow organs; contractile proteins; dedifferentiation; adenosine 3',5'-cyclic monophosphate

THE HOLLOW ORGANS of the urinary tract, vasculature, and airways become phenotypically modified in response to pathological insults or other metabolic perturbations (5, 38, 48). Differentiation marker proteins, including {alpha}-smooth muscle actin ({alpha}-SMA), calponin, and smooth muscle myosin heavy chain (SM-MHC) have been used to objectively monitor pathological changes associated with smooth muscle growth and remodeling (16, 27, 38). Although phenotypic responses to stress are often initially adaptive, allowing for compensatory reactions to overload or injury, excessive deviation from the normal state can impair muscle contractility and compliance and lead to organ dysfunction.

The smooth muscle cell (SMC) phenotype is regulated, in part, by changes in the transcriptional program (4, 38). The GATA transcription factors are a highly conserved family of zinc finger proteins that mediate tissue-specific gene expression. They can be broadly classified into transcriptional regulators of the hematopoietic lineage (GATA-1, -2, -3) or of the mesodermal and/or endodermal lineages (GATA-4, -5, -6) (reviewed in Ref. 31). Of the latter group, all three GATAs have been implicated in regulation of cardiac muscle phenotype, both during development and in response to pathological stimuli (29, 31, 32, 52). Both GATA-5 and GATA-6 are expressed in SMC (33, 34). Although the functional significance of GATA-5 expression in smooth muscle has not been addressed, GATA-6 has been implicated in regulation of SMC contractile protein expression in vascular SMC, suggesting that the protein is an important contributor toward maintenance of the differentiated state (10, 43, 49, 50, 53). However, inhibition of GATA-6 by expression of a dominant negative engrailed GATA-6 fusion protein or conditional inactivation of GATA-6 expression in SMC by tissue-specific targeting of Cre recombinase led to little or no change in expression of genes encoding contractile proteins in vascular SMC, arguing against a role for GATA-6 in smooth muscle differentiation (25, 26). Thus the specific mechanisms whereby GATA-6 might participate in regulation of the differentiated smooth muscle phenotype and the extent to which GATA-6 activity is tissue or organ specific remain poorly understood.

In this study, we demonstrate that GATA-6 mediates smooth muscle differentiation in primary culture SMC derived from the human urinary tract. We identify GATA-6 as a direct transcriptional regulator of {alpha}-SMA expression in this cell type.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. After receiving institutional review board approval and parental consent, we obtained human bladder tissue from patients undergoing ureteral reimplantation for vesicoureteral reflux but with no bladder abnormality otherwise. Tissue was processed for isolation of cells or immunohistochemical analysis as described previously (5). Briefly, primary culture human bladder smooth muscle cells (pBSMC) were isolated by explant from fragments of bladder muscle tissue and propagated as described previously (5). Cells were maintained in low-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Invitrogen, Carlsbad, CA) at 37°C in a humidified atmosphere of 95% air-5% CO2. All experiments were performed on cells between passages 2 and 5. Based on immunocytochemical staining, the majority of cells stained positively for {alpha}-SMA and calponin (data not shown). Rat pBSMC were isolated from neonatal rat bladders by enzymatic dispersion as described previously (39) and maintained in medium 199–10% FBS. Human embryonic kidney (HEK)-293 and 293T cells were maintained in DMEM-10% FBS.

Reagents and antibodies. Forskolin, isoproterenol, carbachol, N6, 2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP), and 8'-bromoadenosine-3',5'-cyclic monophosphate (8-BrcAMP) were purchased from Sigma Chemical (St. Louis, MO). Antibodies used were anti-GATA-6 (H-92 and N-18), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), both from Santa Cruz Biotechnology (Santa Cruz, CA), mouse monoclonal anti-GATA-6 (R&D Systems, Minneapolis, MN), anti-{alpha}-SMA, anti-calponin (Sigma), anti-hemagglutinin (HA) (Covance Research Products, Denver, PA), and Cy3-conjugated goat anti-rabbit and anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). The Expand long-template PCR reaction system and FuGENE6 transfection reagent were obtained from Roche Applied Science (Indianapolis, IN). Platinum blue PCR Supermix was obtained from Invitrogen, and SYBR green PCR Master Mix was obtained from Applied Biosystems (Foster City, CA). The RNeasy RNA isolation kit was obtained from Qiagen (Valencia, CA), and the iScript cDNA synthesis reagent was obtained from Bio-Rad Laboratories (Hercules, CA). The Micro BCA protein assay and SuperSignal chemiluminescence reagents were obtained from Pierce Chemical (Rockford, IL).

Treatments and cell extractions. pBSMC were seeded at a density of 2 x 105 cells/well in six-well plates. At ~80% confluence, cells were serum-deprived in DMEM containing 0.5% FBS for 48 h and then subjected to treatment with the indicated agents. Under these conditions, cells were confluent at the time of treatment. For mRNA analysis, total RNA was extracted using either Trizol reagent or RNeasy columns and reverse-transcribed using the iScript cDNA synthesis kit. PCR was performed as described previously (1) using gene-specific primers (Table 1) and either Platinum blue Supermix or SYBR green PCR Master Mix for qualitative or semiquantitative real-time analyses, respectively. Whole cell lysates were retrieved in 20 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, and 1% Triton X-100 supplemented with protease and phosphatase inhibitors. Nuclear extracts were obtained according to the method of Dignam as described previously (39). Immunoblot analysis was performed as described previously (45).


View this table:
[in this window]
[in a new window]

  		Table 1. Primers for PCR and EMSA

 
Construction of GATA-6 expression vectors. Sequences corresponding to the short and long isoforms of human GATA-6 (6) were amplified from pBSMC cDNA using the Expand long-template PCR reaction system supplemented with 5% dimethylsulfoxide and ligated to the BglII site of pCMV-HA vector (Clontech Laboratories, Mountain View, CA). A mutant form of GATA-6 lacking the entire NH2-terminal zinc finger domain and part of the COOH-terminal zinc finger domain ({Delta}ZF-GATA-6) was a kind gift from Dr. Kenneth Walsh, Boston University School of Medicine (40). The {Delta}ZF-GATA-6 coding region was subcloned into pCMV-HA for expression analyses.

Generation of BSMC with stable knockdown of GATA-6. To suppress expression of endogenous GATA-6 in human pBSMC, double-stranded 21-base length oligonucleotides targeting the 3'-untranslated region of the human GATA-6 transcript were designed and subcloned into the lentiviral vector pLKO.1-puro (46), a generous gift from Dr. William C. Hahn, Dana-Farber Cancer Institute. The sequence of the GATA-6-targeting oligonucleotide was 5'-GCGCTGACAGAACGTGATTCT-3'. A control vector containing sequence targeting green fluorescent protein (GFP: 5'-GGCTACGTCCAGGAGCGCACC-3') was also generated, as described elsewhere (46). An additional control vector comprising a scrambled sequence (42) was purchased from Addgene (Cambridge, MA). Several candidate sequences targeting GATA-6 were screened by transient analyses in 293T cells. Based on the efficacy of reduction in expression of endogenous GATA-6 in 293T cells, sequences were selected for subsequent packaging into viral particles. To generate viral supernatants encoding GATA-6-specific or control short hairpin RNA (shRNA), 293T cells were cotransfected with the desired lentiviral construct and the packaging and envelope plasmids pCMV {Delta}R8.91 and pCMV-VSV-G. Viral supernatants were retrieved 72 h posttransfection, passed through a 0.45-µm filter, and immediately used for infection. Human pBSMC were infected in the viral supernatant for 4 h and then maintained in medium with 0.5 µg/ml puromycin for ~2 wk until no more uninfected control cells survived. Puromycin-resistant cells were passaged for assays. Three independent sets of stable cell lines were generated for the assays described.

Promoter-reporter assays. The human disabled-2 (Dab-2) promoter-reporter construct (pGL3-Dab2) was a kind gift from Dr. Jer-Tsong Hsieh, University of Texas Southwestern Medical Center (56). The human {alpha}-SMA promoter-reporter (pCI-{alpha}-SMA-Luc) and appropriate negative and positive control constructs (pCI and pCI-Luc) were a generous gift from Dr. Michael Keogh, Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School (24). Site-directed mutagenesis of pCI-{alpha}-SMA-Luc was performed by overlap PCR as described previously (39). Promoter constructs were transfected along with GATA-6 expression vectors or pEGFP-N1 (Clontech) as control into HEK-293 cells or rat pBSMC as follows. HEK-293 cells were transfected in DMEM-10% FBS. pBSMC were transfected under serum-free conditions for 1 h as described previously (24). Cells were treated with forskolin in M199–10% FBS 24 h later. Lysates were harvested 48 h posttransfection, and luciferase activity was measured using the luciferase assay reagent (Promega, Madison, WI).

Chromatin immunoprecipitation. Formaldehyde-cross-linked chromatin was obtained from pBSMC and sheared as described previously (14) before immunoprecipitation. Briefly, sheared chromatin was precleared with preimmune mouse serum and protein G beads (22) and then immunoprecipitated with 5 µg of mouse anti-GATA-6 antibody (R&D Systems) or mouse IgG1 (Sigma). The enrichment of GATA-binding sequences in eluted DNA was quantified by real-time PCR and expressed as a percentage of input DNA (14, 20). Primers used for PCR are listed in Table 1.

Immunofluorescence analysis. Formalin-fixed paraffin-embedded bladder tissue was sectioned as described previously. For antigen retrieval, the sections were heated in 0.01 M citrate buffer for 20 min. GATA-6 in intact human cells and bladder tissue was detected by immunofluorescence using rabbit anti-GATA-6 polyclonal primary antibody (1:500) and Cy3-conjugated anti-rabbit IgG (1:500) as secondary antibody (8). Nuclei were counterstained with 4',6-diamidino-2-phenylindole, and specimens were visualized using an Axioplan2 fluorescence microscope (Carl Zeiss MicroImaging, Thornwood, NY).

Electromobility shift analysis. Electromobility shift analyses (EMSAs) for GATA-6 were carried out with 3–5 µg of nuclear extract and 0.5 ng of 32P-labeled oligonucleotide probe in a 20-µl reaction volume containing 25 mM Tris·HCl, pH 8, 50 mM KCl, 5 mM MgCl2, 0.5 mM EDTA, 8% glycerol, 2 µg of BSA, and 0.5 µg of poly(dI-dC). The reaction was incubated for 20 min at room temperature, and the DNA-protein complexes were resolved on 5% polyacrylamide gels in 0.5x Tris-borate-EDTA (TBE) buffer at 4°C. The gels were dried, and the complexes were visualized by autoradiography using a Typhoon Trio variable mode phosphorimager (GE Healthcare Biosciences, Piscataway, NJ). For competition experiments, a 50-fold molar excess of the cold competitor oligonucleotide was added simultaneously with the probe. For supershift experiments, a GATA-6 antibody raised against the NH2 terminus (N-18, sc-7245X; Santa Cruz Biotechnology) was preincubated on ice for an hour with nuclear extract followed by addition of the other components for 20 min at room temperature. The following oligonucleotides were used in EMSAs: GATA wt, containing consensus GATA-6 binding sites (5'-CAC TTG ATA ACA GAA AGT GAT AAC TCT-3'); GATA mut, containing mutations (underlined) in the consensus GATA sites (5'-CAC TTC TTA ACA GAA AGT CTT AAC TCT-3'); G1 wt, corresponding to the GATA-6 binding motif present in the {alpha}-SMA enhancer (5'-GGG ATT ATG GAG ATT AGA ATT CGA GAC-3'); and G1 mut, containing a point mutation (underlined) in the {alpha}-SMA GATA-6 motif (5'-GGG ATT ATG GAG GTT AGA ATT CGA GAC-3').

Statistical analysis. Where appropriate, comparisons between experimental groups were performed using Student's t-test. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
GATA-6 mRNA was shown previously to be expressed in the urogenital ridge and bladder of the developing mouse (33). However, its expression in tissues of the human urinary tract has not been described. Initially, we performed qualitative RT-PCR to determine which members of the mesodermal cohort of GATA factors (GATA-4, -5, -6) were expressed in pBSMC. As shown in Fig. 1A, pBSMC express GATA-5 and GATA-6 mRNA, whereas GATA-4 mRNA levels were negligible, consistent with the reported expression pattern of GATA factors in the developing urinary tract (33, 34). Immunoblot analysis indicated that pBSMC express two molecular forms of GATA-6 (Fig. 1B), consistent with previous descriptions of two GATA-6 isoforms arising from the use of alternative promoters and/or initiation codons (6, 7). Based on the migration pattern relative to ectopically expressed GATA-6, endogenous GATA-6 expressed in human pBSMC predominantly comprises the short isoform. Despite detection of GATA-5 mRNA in pBSMC, we were unable to detect GATA-5 protein in these cells (data not shown). Immunofluorescence imaging demonstrated that GATA-6 localized primarily to nuclei in cultured cells and tissues (Fig. 1C).


Figure 1
View larger version (37K):
[in this window]
[in a new window]

  		Fig. 1. Expression profile of endogenous GATA-6 in human primary human bladder smooth muscle cells (pBSMC). A: cDNA from human pBSMC was amplified with primers specific for GATA-4, GATA-5, or GATA-6 (see Table 1). cDNA from Caco-2 cells served as a positive control for GATA-4 and GATA-6, whereas cDNA from HT29 cells treated with sodium butyrate was a positive control for GATA-5. A size marker in base pairs is shown at left. B: 20 µg of nuclear fractions from confluent human pBSMC expressing vector only (–) or hemagglutinin (HA)-tagged GATA-6 expression construct (+) were resolved by 10% SDS-PAGE and immunoblotted with antibodies to GATA-6 or the HA epitope tag. In pBSMC expressing vector alone, endogenous GATA-6 comigrated with ectopically expressed GATA-6. Lane 1 is an overexposure of lane 2. GATA-6-L, long isoform; GATA-6-S, short isoform; MW, molecular weight. C: immunofluorescence analysis of endogenous GATA-6 in human pBSMC (top) and human bladder muscle tissue (bottom). Following antigen retrieval, human bladder tissue was stained with anti-GATA-6 polyclonal antibody (1:1,000) and Cy3-conjugated secondary antibody before being mounted in VectaShield medium containing 4',6-diamidino-2-phenylindole (DAPI) to counterstain nuclei (iiand v). Original magnifications: x40 for SMC, x20 for muscle tissue. Scale bars: 25 µm for SMC, 50 µm for muscle tissue.

 
Recent reports asserted that a dominant inhibitor of GATA-6 activity, or genetic ablation of the protein, had minimal effect on expression of {alpha}-SMA, SM-MHC, and calponin in vascular SMC (25, 26). To assess whether this was the case in the SMC of the bladder, we employed RNA interference to knock down GATA-6 protein levels and evaluated GATA-6 target gene expression using real-time RT-PCR. Suppression of GATA-6 expression led to a reduction in {alpha}-SMA, calponin, and SM-MHC mRNA levels (Fig. 2A), suggesting that GATA-6 plays an important role as a transcriptional regulator of contractile protein expression. Parallel changes in protein expression for {alpha}-SMA and calponin (not shown) were also observed (Fig. 2B). GATA-6 knockdown also reduced levels of the known GATA-6 target Dab2 (35). In contrast, cyclin D1 mRNA levels were increased in SMC lacking GATA-6 expression, suggesting that effects on gene expression from GATA-6 knockdown were more extensive and were not restricted to genes specific to the muscle lineage.


Figure 2
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 2. GATA-6 knockdown decreases contractile protein gene expression in human BSMC. A: total RNA from stable populations of human pBSMC infected with viral supernatants encoding short hairpin RNA (shRNA) to GATA-6 or scrambled sequence was reverse-transcribed, and cDNAs were amplified by semiquantitative real-time PCR using gene-specific primers as described in text. Data represent the degree of change in gene expression in cells harboring GATA-6 knockdown relative to cells expressing scrambled shRNA, after normalization to GAPDH. RNAi, RNA interference. B: following antibiotic selection, stable pBSMC populations expressing GATA-6 or control shRNAs were harvested for immunoblot analysis of GATA-6 and {alpha}-SMA protein levels. An additional control comprising cells infected with supernatant encoding green fluorescent protein (GFP) shRNA was also included in this analysis. Scram, scrambled sequence.

 
As an independent test of the effect of GATA-6 downregulation on the SMC gene expression pattern, we exploited the observation that elevation of intracellular cAMP levels can decrease GATA-6 protein levels (21, 30, 36). Initially, we determined the effect of increasing cAMP on GATA-6 expression in SMC. Elevation of intracellular cAMP levels by exposure of cells to the direct adenylate cyclase activator forskolin (Fig. 3A, ii) or to the cAMP analog 8-BrcAMP (Fig. 3A, iii) led to a time- and dose-dependent reduction in GATA-6 protein levels. A similar effect was observed with DBcAMP (not shown). The forskolin-induced loss of GATA-6 expression did not result from apoptotic effects on cells, since flow cytometry analysis revealed no change in the extent of the sub-G1 peak following forskolin treatment (data not shown).


Figure 3
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 3. Elevation of intracellular cAMP levels downregulates GATA-6 expression in human BSMC. A: schematic summarizing the effect of cAMP manipulation on GATA-6 protein levels (i). Confluent and quiescent pBSMC were treated with the indicated doses of forskolin (Fsk; ii) for 8 or 24 h or 8'-bromoadenosine-3',5'-cyclic monophosphate (8-BrcAMP; iii) for 24 h. Whole cell lysates were resolved by SDS-PAGE and blotted with antibodies to GATA-6 or GAPDH. B: relative expression of beta-adrenergic and muscarinic (M2 and M3) acetylcholine receptors in pBSMC was determined by real-time RT-PCR (i). Data are presented as ratios to the housekeeping gene, GAPDH. pBSMC were exposed to increasing doses of isoproterenol (Iso), a beta-adrenergic agonist (ii). Untreated cells (C) or Fsk (100 µM)-treated cells were included as negative and positive controls, respectively. pBSMC were exposed to 10–6 M Iso for 8 h in the absence or presence of 10–4 M carbachol (CCh), a muscarinic agonist (iii). In each case, GATA-6 and GAPDH expression were assessed as described in A. C: pBSMC were treated with 50 µM Fsk for 24 h. Total RNA from control and Fsk-treated cells was reverse-transcribed, and cDNAs were amplified by semiquantitative real-time PCR using gene-specific primers as described in text. Data represent the degree of change in gene expression in treated vs. untreated cells following normalization to GAPDH (i). Lysates of control and Fsk-treated cells were resolved by SDS-PAGE and blotted with antibodies to GATA-6, {alpha}-SMA, and GAPDH (ii). Dab2, human disabled-2; SM-MHC, smooth muscle myosin heavy chain.

 
Alterations in intracellular cAMP levels are known to lie downstream of both adrenergic and cholinergic receptor activation (2, 15, 51). To determine whether activation of these receptors could affect GATA-6 levels in pBSMC, we first performed real-time RT-PCR to assess relative receptor expression. As shown in Fig. 3B,i, the predominant species observed in pBSMC in culture and in human detrusor tissue (data not shown) were the beta2-adrenergic and M2 muscarinic cholinergic receptors. Treatment of pBSMC with isoproterenol, a beta2-adrenergic receptor agonist and inducer of cAMP synthesis, downregulated GATA-6 levels (Fig. 3B, ii). This reduction could be reversed in the presence of carbachol, a muscarinic receptor agonist and inhibitor of cAMP elevation (Fig. 3B, iii, compare lane 4 with lane 2). Forskolin treatment of SMC also led to reduced expression of {alpha}-SMA, calponin, and Dab2 mRNA (Fig. 3C), similar to the profile observed following GATA-6 knockdown by RNA interference (RNAi) (see Fig. 2A). Downregulation of {alpha}-SMA protein was also evident in forskolin-treated SMC. Together, these observations suggest that GATA-6-mediated regulation of contractile protein expression in bladder SMC is sensitive to intracellular cAMP levels.

To determine the effect of cAMP on transcriptional regulation of {alpha}-SMA by GATA-6, we employed a human {alpha}-SMA promoter-luciferase reporter construct. The fragment comprises 1 kb upstream of the transcription start site and incorporates the proximal core promoter and a distal enhancer element (24). Initially, we evaluated GATA-6-mediated stimulation of the {alpha}-SMA promoter in the highly transfectable HEK-293 cell line and observed greater than fourfold induction of promoter activity relative to control (Fig. 4A). Based on our observations that elevation of intracellular cAMP decreased GATA-6 levels (Fig. 3), we tested whether forskolin caused a concomitant reduction in {alpha}-SMA promoter activation in pBSMC. As shown in Fig. 4B, forskolin treatment led to a >60% decrease in reporter activity, whereas a constitutively active luciferase construct under control of the cytomegalovirus (CMV) promoter was unaffected by forskolin treatment. Notably, the forskolin-induced decrease in {alpha}-SMA promoter activity could be rescued almost completely by ectopic expression of wild-type GATA-6, but not by a zinc finger-deleted mutant, GATA-6{Delta}ZF, that lacks DNA-binding ability (Fig. 4C, i), suggesting that direct binding of GATA-6 to the promoter is required for its regulatory activity. The inability of GATA-6{Delta}ZF to rescue {alpha}-SMA promoter activity was not a result of variation in protein levels, since the deletion mutant was expressed at levels comparable to those of the wild-type protein (Fig. 4C, ii). Together, these findings suggest that GATA-6 directly regulates the {alpha}-SMA promoter in a cAMP-sensitive manner.


Figure 4
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 4. GATA-6 stimulates the {alpha}-SMA promoter in a cAMP-sensitive manner. A: HEK-293 cells were cotransfected with pCI-{alpha}-SMA-luciferase reporter construct (SMA-Luc) and GATA-6 or enhanced GFP (EGFP) expression plasmids. Luciferase activity was determined 48 h later as described in text. RLU, relative light units. B: rat pBSMC were transfected with SMA-Luc and treated without or with 50 µM Fsk for 24 h. The reporterless construct pCI and the cytomegalovirus promoter-driven pCI-Luc (CMV-Luc) were included as negative and positive controls, respectively. Luciferase activity was determined as described in text. C: rat pBSMC were cotransfected with SMA-Luc and wild-type (Wt, GATA-6) or mutant (Mut, {Delta}ZF-GATA-6) expression constructs. Cells were exposed to 100 µM Fsk for 24 h, and luciferase activity was determined as described in text (i). Expression of wild-type and mutant GATA-6 proteins in cell extracts was confirmed by immunoblot analysis using anti-GATA-6 antibody (ii).

 
Our observation that {alpha}-SMA mRNA levels decreased following downregulation of GATA-6 protein levels, as well as the ability of GATA-6 to activate {alpha}-SMA reporter activity, suggested that GATA-6 was recruited to the {alpha}-SMA promoter in vivo. The {alpha}-SMA gene is known to be regulated at the transcriptional level through the binding of serum-response factor (SRF) and one or more coregulatory factors to CArG boxes located within the core promoter region (10, 37, 53, 55). In addition, Keogh et al. (24) identified a region within the human {alpha}-SMA promoter located at –999/–890 relative to the transcriptional start site that behaved as an enhancer in SMC. To determine whether this region might be targeted by GATA-6, we analyzed the –999/–890 fragment in silico using MatInspector (9) and identified a putative GATA-binding site at position –919/–913 (AGATTA). To determine whether GATA-6 was recruited to the human {alpha}-SMA promoter in vivo, we performed chromatin immunoprecipitation (ChIP) and amplified the eluted DNA with primers flanking either the core or enhancer GATA sites. GATA-6 bound to both the {alpha}-SMA core promoter and enhancer regions (Fig. 5A, top). In addition, elevation of SMC cAMP levels by forskolin treatment abolished GATA-6 binding to both sites (Fig. 5A, bottom) and was accompanied by a reduction in {alpha}-SMA transcription as measured by real-time RT-PCR (Fig. 5B).


Figure 5
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 5. GATA-6 is recruited to the {alpha}-SMA promoter in vivo. A: confluent and quiescent human pBSMC were treated without (control, top) or with 50 µM Fsk (bottom) for 24 h and harvested for chromatin immunoprecipitation analysis with anti-GATA-6 or isotype control mouse IgG as described in text. In each case, eluted DNA was amplified by real-time PCR with primers for upstream negative control (5' negative), upstream enhancer, core promoter region, and downstream negative control (intron 2; 3' negative) as listed in Table 1. Data are presented as the percentage of input. The cycle time (Ct) value of each input sample was within one cycle. B: total RNA isolated from pBSMC treated without or with 50 µM Fsk for 8 h was reverse-transcribed and amplified with {alpha}-SMA-specific primers. Data are presented as ratios to control following normalization to GAPDH.

 
The identification of a putative GATA binding site (AGATTA) in the enhancer region of the {alpha}-SMA promoter suggested a role for this motif in GATA-6 recruitment. To address this question, we performed EMSA using nuclear extracts of pBSMC and oligonucleotides incorporating the GATA site. As shown in Fig. 6A, i, we observed robust complex formation with labeled probe corresponding to the GATA binding site at –919/–913. The complex was competed out with the corresponding unlabeled {alpha}-SMA GATA or consensus GATA oligonucleotides, but not with mutant versions of these sequences (Fig. 6A, i). In addition, we observed immunodepletion of the complex in the presence of GATA-6 antibody but not control IgG (Fig. 6A, ii), consistent with the presence of GATA-6 in the complex.


Figure 6
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 6. GATA-6 binds to the {alpha}-SMA promoter-enhancer to regulate activity. A: nuclear extracts from human pBSMC were incubated with 32P-labeled oligonucleotide corresponding to the promoter-enhancer GATA-binding site in {alpha}-SMA, and DNA-GATA-6 complexes were analyzed by EMSA as described. Competition assays were performed with either WT or mutant (Mut) unlabeled GATA-site containing oligos or WT or mutant consensus (Cons) GATA oligos (i). Arrow indicates the specific GATA-6-DNA complex. Nuclear extracts were incubated with IgG control or GATA-6 antibody for 20 min before addition of 32P-labeled probe (ii). FP, free probe; NE, nuclear extract. B: pBSMC were cotransfected with WT or GATA-binding site-mutated (Mut) versions of SMA-Luc and either vector alone (pcDNA), GATA-6-WT, or GATA-6-{Delta}ZF. Luciferase activity was determined after 48 h, as described.

 
Having confirmed GATA-6 binding to the GATA site-containing enhancer region by ChIP and EMSA analyses, we next investigated the functional consequence of mutating this site on {alpha}-SMA promoter activity. We engineered a point mutation in the enhancer GATA motif (AGATTA->AGGTTA) in the SMA-Luc construct and compared basal and GATA-6-activated transcription from the wild-type and mutant {alpha}-SMA promoters. As shown in Fig. 6B, point mutation of the GATA site did not affect basal transcription but abrogated GATA-6-induced promoter activation. Together, these findings implicate GATA-6 as a regulator of {alpha}-SMA expression that acts through a specific site in the enhancer region.


   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we have demonstrated that GATA-6 mediates differentiation of primary human bladder SMC and regulates {alpha}-SMA expression via a novel cis element in the {alpha}-SMA promoter-enhancer. The evidence supporting these conclusions is as follows: 1) downregulation of GATA-6 protein levels in primary bladder SMC by RNA interference led to a reduction in mRNAs encoding multiple smooth muscle differentiation marker proteins; 2) reduction of GATA-6 levels by elevation of intracellular cAMP levels suppressed {alpha}-SMA promoter activation, mRNA expression, and protein production; 3) GATA-6 was recruited to the human {alpha}-SMA promoter in vivo; 4) cAMP-mediated inhibition of {alpha}-SMA promoter activity could be rescued by enforced expression of wild-type GATA-6 but not by a DNA-binding incompetent GATA-6 mutant; and 5) point mutation of the GATA-binding motif in the {alpha}-SMA enhancer abolished GATA-6 binding as shown by EMSA and abrogated GATA-6-induced promoter activation. Although GATA-6 mRNA has been reported in bladder SMC in the developing mouse (33, 34), the functional role of this factor in the bladder has not been explored, and its role as a mediator of smooth muscle differentiation in general has been in dispute (25, 26). Our demonstration that loss of endogenous GATA-6 expression by genetic or pharmacological means resulted in downregulation of {alpha}-SMA and other contractile proteins strongly implicates GATA-6 in maintenance of the differentiated, contractile phenotype in bladder smooth muscle.

The specific molecular mechanisms that govern smooth muscle differentiation in hollow organs other than the vasculature are almost completely unknown. Recent findings from Li et al. (28) provided evidence for SRF as a key regulator of bladder smooth muscle differentiation during murine development. In that study, SRF expression was noted in the developing bladder mesenchyme and smooth muscle before the appearance of {alpha}-SMA. These observations are consistent with findings from vascular smooth muscle, where SRF regulates the expression of several smooth muscle-specific genes via CArG boxes located within their core promoter regions (55). Since SRF is expressed ubiquitously, specificity of smooth muscle marker expression is achieved through the interaction of SRF with one or more binding partners, including myocardin (55), GATA-6 (37), and the cysteine-rich LIM-only proteins CRP1 and CRP2 (10). Consistent with these findings, we observed recruitment of GATA-6 to the core promoter region of the human {alpha}-SMA promoter in pBSMC as shown by ChIP. Interestingly, elevation of intracellular cAMP has been reported to inhibit SRF activity (12, 19) in addition to reducing GATA-6 protein levels (21, 30). Thus it is possible that the observed inhibition of {alpha}-SMA promoter activity in pBSMC treated with forskolin may result from negative regulation of both GATA-6 and SRF. In support of this possibility, deletion analysis of the {alpha}-SMA promoter indicated that although loss of the distal GATA binding site prevented rescue of forskolin-induced repression, its absence did not preclude inhibition of {alpha}-SMA promoter activity by forskolin (data not shown). This latter effect is likely mediated, at least in part, through inhibition of SRF (12, 19). Despite the potential involvement of SRF, however, the demonstration that ectopic expression of GATA-6 could recover {alpha}-SMA promoter activity almost completely strongly implicates GATA-6 as a dominant regulator of {alpha}-SMA expression in bladder smooth muscle.

In addition to its interaction with the core promoter, we also observed binding of GATA-6 in a region ~1 kb upstream of the {alpha}-SMA transcriptional start site (TSS). This region has been implicated previously in {alpha}-SMA transcriptional regulation, with the demonstration that the region from –999/–890 functioned as a transcriptional enhancer in SMC (24). However, the mechanism underlying its function is unknown. With the exception of the current report, the promoter constructs used in other studies of human {alpha}-SMA gene regulation terminated proximal to –999/–890 (3, 11, 41) and so were studied out of context and, as this study shows, in the absence of an endogenous regulatory feature that elicits activity from a regulator of smooth muscle differentiation. We have shown that point mutation of a putative GATA binding motif within this region prevented GATA-6-mediated activation of the promoter, demonstrating that GATA-6 binding to this site is required for {alpha}-SMA expression in SMC. Thus our study is the first to identify a specific transcription factor that is recruited to the {alpha}-SMA enhancer and that regulates its function. In addition to its obvious relevance to regulation of smooth muscle phenotype, this observation may also have implications for cells undergoing epithelial-mesenchymal transition (EMT). The EMT is implicated in several physiological and pathological conditions, including development, fibrosis, and tumor cell metastasis (44, 47) and is characterized by increased {alpha}-SMA expression. It is tempting to speculate that factors recruited to the enhancer region of the {alpha}-SMA promoter may elicit tissue-specific and context-specific regulation of {alpha}-SMA expression.

Another important implication of our findings is that stimuli that alter intracellular cAMP levels, such as effectors of the autonomic nervous system, are likely to be important regulators of GATA-6-mediated expression of {alpha}-SMA and possibly other contractile proteins in hollow organs. Intracellular cAMP levels in SMC are controlled, at least in part, by the balance between neurotransmitters such as beta-adrenergic agonists that activate adenylate cyclase and muscarinic cholinergic agonists that inhibit the enzyme (2). Based on our observations, under conditions where cholinergic stimuli predominate and cAMP levels are attenuated, expression of GATA-6 and its target genes/proteins is elevated. Conversely, beta-adrenergic stimulation of SMC, which upregulates cAMP, decreases GATA-6 and its targets. These observations are consistent with a model where GATA-6 acts as a switch to regulate {alpha}-SMA expression downstream of autonomic inputs. Consistent with this hypothesis, conditions characterized by excessive contraction of smooth muscle, such as certain types of urinary tract dysfunction, can be alleviated by administration of agents that restore the appropriate balance between beta-adrenergic and muscarinic cholinergic signaling in bladder SMC and normalize smooth muscle contractility (17, 18, 23).

The complexity underlying specification of SMC phenotype is only beginning to be appreciated. Although SMC are classified broadly into those of vascular or visceral origin, even SMC from the same tissue type display striking differences in gene expression profiles. For example, expression profiling of SMC isolated from different locations within the vasculature revealed numerous differentially expressed genes (13). One such gene, PRISM, was found to regulate SMC fate by transcriptional repression of differentiation-inducing genes, including GATA-6, and activation of proliferative genes (13). GATA-6 itself is not expressed in SMC from all smooth muscle-containing tissues, showing robust expression in SMC of the vasculature and bladder wall but no expression in gut or uterine smooth muscle (33). Although its functional significance remains to be determined, this restricted expression pattern is consistent with a tissue-specific role for GATA-6 and may also reflect differential expression of factors that regulate the expression of GATA-6 itself.

In summary, we have demonstrated that GATA-6 is an important regulator of human bladder smooth muscle differentiation. We have also identified a new mode of regulation of {alpha}-SMA expression in primary human bladder SMC, via GATA-6 binding to a novel cis-acting element in the human {alpha}-SMA promoter-enhancer. Together, these observations provide a point of departure for future studies aimed at understanding the GATA-6-dependent signaling web in the bladder.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R21 DK-66412 (to R. M. Adam), the Edwin Beer Fellowship from the New York Academy of Medicine (to R. M. Adam), the Robert Leet and Clara Guthrie Patterson Trust (to R. M. Adam), and the Children's Urological Foundation. This work was also supported in part by grants from the American Urological Association (AUA) Foundation Research Scholar Program and Children's Hospital Boston (to A. Kanematsu) and the AUA Foundation Research Scholar Program and Boehringer Ingelheim (to A. Ramachandran).


   ACKNOWLEDGMENTS
 
We thank Drs. Michael Keogh, Jer-Tsong Hsieh, Jiang Zhou, Kenneth Walsh, and William C. Hahn for reagents; Drs. Mohini Lutchman, Carlos R. Estrada, and Samuel H. Eaton as well as Michelle Mulone for technical assistance; and Drs. Stephen D. Krasinski, Nishit K. Mukhopadhyay, Sean Li, and Michael R. Freeman for helpful discussions.

Present address of A. Kanematsu: Dept. of Urology, Kyoto University, 54 Shogoin Kawaracho, Sakyo, Kyoto, Japan.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. M. Adam, Urological Diseases Research Center, John F. Enders Research Laboratories, Rm. 1077, 300 Longwood Ave., Boston, MA 02115 (e-mail: rosalyn.adam{at}childrens.harvard.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Adam RM, Eaton SH, Estrada C, Nimgaonkar A, Shih SC, Smith LE, Kohane IS, Bagli D, Freeman MR. Mechanical stretch is a highly selective regulator of gene expression in human bladder smooth muscle cells. Physiol Genomics 20: 36–44, 2004.[Abstract/Free Full Text]

2. Andersson KE, Arner A. Urinary bladder contraction and relaxation: physiology and pathophysiology. Physiol Rev 84: 935–986, 2004.[Abstract/Free Full Text]

3. Andrawis NS, Ruley EH, Abernethy DR. Angiotensin II regulates human vascular smooth muscle alpha-actin gene expression. Biochem Biophys Res Commun 196: 962–968, 1993.[CrossRef][Web of Science][Medline]

4. Black BL, Olson EN. Transcriptional control of muscle development by myocyte enhancer factor-2 (MEF2) proteins. Annu Rev Cell Dev Biol 14: 167–196, 1998.[CrossRef][Web of Science][Medline]

5. Borer JG, Park JM, Atala A, Nguyen HT, Adam RM, Retik AB, Freeman MR. Heparin-binding EGF-like growth factor expression increases selectively in bladder smooth muscle in response to lower urinary tract obstruction. Lab Invest 79: 1335–1345, 1999.[Web of Science][Medline]

6. Brewer A, Gove C, Davies A, McNulty C, Barrow D, Koutsourakis M, Farzaneh F, Pizzey J, Bomford A, Patient R. The human and mouse GATA-6 genes utilize two promoters and two initiation codons. J Biol Chem 274: 38004–38016, 1999.[Abstract/Free Full Text]

7. Brewer A, Nemer G, Gove C, Rawlins F, Nemer M, Patient R, Pizzey J. Widespread expression of an extended peptide sequence of GATA-6 during murine embryogenesis and non-equivalence of RNA and protein expression domains. Gene Expr Patterns 2: 123–131, 2002.[CrossRef][Medline]

8. Capo-chichi CD, Roland IH, Vanderveer L, Bao R, Yamagata T, Hirai H, Cohen C, Hamilton TC, Godwin AK, Xu XX. Anomalous expression of epithelial differentiation-determining GATA factors in ovarian tumorigenesis. Cancer Res 63: 4967–4977, 2003.[Abstract/Free Full Text]

9. Cartharius K, Frech K, Grote K, Klocke B, Haltmeier M, Klingenhoff A, Frisch M, Bayerlein M, Werner T. MatInspector and beyond: promoter analysis based on transcription factor binding sites. Bioinformatics 21: 2933–2942, 2005.[Abstract/Free Full Text]

10. Chang DF, Belaguli NS, Iyer D, Roberts WB, Wu SP, Dong XR, Marx JG, Moore MS, Beckerle MC, Majesky MW, Schwartz RJ. Cysteine-rich LIM-only proteins CRP1 and CRP2 are potent smooth muscle differentiation cofactors. Dev Cell 4: 107–118, 2003.[CrossRef][Web of Science][Medline]

11. Comer KA, Dennis PA, Armstrong L, Catino JJ, Kastan MB, Kumar CC. Human smooth muscle alpha-actin gene is a transcriptional target of the p53 tumor suppressor protein. Oncogene 16: 1299–1308, 1998.[CrossRef][Web of Science][Medline]

12. Davis A, Hogarth K, Fernandes D, Solway J, Niu J, Kolenko V, Browning D, Miano JM, Orlov SN, Dulin NO. Functional significance of protein kinase A activation by endothelin-1 and ATP: negative regulation of SRF-dependent gene expression by PKA. Cell Signal 15: 597–604, 2003.[CrossRef][Web of Science][Medline]

13. Davis CA, Haberland M, Arnold MA, Sutherland LB, McDonald OG, Richardson JA, Childs G, Harris S, Owens GK, Olson EN. PRISM/PRDM6, a transcriptional repressor that promotes the proliferative gene program in smooth muscle cells. Mol Cell Biol 26: 2626–2636, 2006.[Abstract/Free Full Text]

14. Edelstein LC, Pan A, Collins T. Chromatin modification and the endothelial-specific activation of the E-selectin gene. J Biol Chem 280: 11192–11202, 2005.[Abstract/Free Full Text]

15. Ehlert FJ. Contractile role of M2 and M3 muscarinic receptors in gastrointestinal, airway and urinary bladder smooth muscle. Life Sci 74: 355–366, 2003.[CrossRef][Web of Science][Medline]

16. Halayko AJ, Solway J. Molecular mechanisms of phenotypic plasticity in smooth muscle cells. J Appl Physiol 90: 358–368, 2001.[Abstract/Free Full Text]

17. Hegde SS. Muscarinic receptors in the bladder: from basic research to therapeutics. Br J Pharmacol 147, Suppl 2: S80-S87, 2006.[CrossRef][Web of Science][Medline]

18. Hegde SS, Eglen RM. Muscarinic receptor subtypes modulating smooth muscle contractility in the urinary bladder. Life Sci 64: 419–428, 1999.[CrossRef][Web of Science][Medline]

19. Hogarth DK, Sandbo N, Taurin S, Kolenko V, Miano JM, Dulin NO. Dual role of PKA in phenotypic modulation of vascular smooth muscle cells by extracellular ATP. Am J Physiol Cell Physiol 287: C449–C456, 2004.[Abstract/Free Full Text]

20. Horak CE, Mahajan MC, Luscombe NM, Gerstein M, Weissman SM, Snyder M. GATA-1 binding sites mapped in the beta-globin locus by using mammalian chIp-chip analysis. Proc Natl Acad Sci USA 99: 2924–2929, 2002.[Abstract/Free Full Text]

21. Ishida A, Iijima R, Kobayashi A, Maeda M. Characterization of cAMP-dependent proteolysis of GATA-6. Biochem Biophys Res Commun 332: 976–981, 2005.[CrossRef][Web of Science][Medline]

22. Johnson KD, Bresnick EH. Dissecting long-range transcriptional mechanisms by chromatin immunoprecipitation. Methods 26: 27–36, 2002.[CrossRef][Web of Science][Medline]

23. Kaefer M, Pabby A, Kelly M, Darbey M, Bauer SB. Improved bladder function after prophylactic treatment of the high risk neurogenic bladder in newborns with myelomeningocele. J Urol 162: 1068–1071, 1999.[CrossRef][Web of Science][Medline]

24. Keogh MC, Chen D, Schmitt JF, Dennehy U, Kakkar VV, Lemoine NR. Design of a muscle cell-specific expression vector utilising human vascular smooth muscle {alpha}-actin regulatory elements. Gene Ther 6: 616–628, 1999.[CrossRef][Web of Science][Medline]

25. Lepore JJ, Cappola TP, Mericko PA, Morrisey EE, Parmacek MS. GATA-6 regulates genes promoting synthetic functions in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 25: 309–314, 2005.[Abstract/Free Full Text]

26. Lepore JJ, Mericko PA, Cheng L, Lu MM, Morrisey EE, Parmacek MS. GATA-6 regulates semaphorin 3C and is required in cardiac neural crest for cardiovascular morphogenesis. J Clin Invest 116: 929–939, 2006.[CrossRef][Web of Science][Medline]

27. Levin RM, Haugaard N, O'Connor L, Buttyan R, Das A, Dixon JS, Gosling JA. Obstructive response of human bladder to BPH vs. rabbit bladder response to partial outlet obstruction: a direct comparison. Neurourol Urodyn 19: 609–629, 2000.[CrossRef][Web of Science][Medline]

28. Li J, Shiroyanagi Y, Lin G, Haqq C, Lin CS, Lue TF, Willingham E, Baskin LS. Serum response factor, its cofactors, and epithelial-mesenchymal signaling in urinary bladder smooth muscle formation. Differentiation 74: 30–39, 2006.[CrossRef][Web of Science][Medline]

29. Liang Q, De Windt LJ, Witt SA, Kimball TR, Markham BE, Molkentin JD. The transcription factors GATA4 and GATA6 regulate cardiomyocyte hypertrophy in vitro and in vivo. J Biol Chem 276: 30245–30253, 2001.[Abstract/Free Full Text]

30. Maeda M, Ishida A, Ni L, Kobayashi A. Isolation of CHO-K1 clones defective in cAMP-dependent proteolysis, as determined by the stability of exogenously expressed GATA-6. Biochem Biophys Res Commun 329: 140–146, 2005.[CrossRef][Web of Science][Medline]

31. Molkentin JD. The zinc finger-containing transcription factors GATA-4, -5, and -6. Ubiquitously expressed regulators of tissue-specific gene expression. J Biol Chem 275: 38949–38952, 2000.[Free Full Text]

32. Morimoto T, Hasegawa K, Kaburagi S, Kakita T, Masutani H, Kitsis RN, Matsumori A, Sasayama S. GATA-5 is involved in leukemia inhibitory factor-responsive transcription of the beta-myosin heavy chain gene in cardiac myocytes. J Biol Chem 274: 12811–12818, 1999.[Abstract/Free Full Text]

33. Morrisey EE, Ip HS, Lu MM, Parmacek MS. GATA-6: a zinc finger transcription factor that is expressed in multiple cell lineages derived from lateral mesoderm. Dev Biol 177: 309–322, 1996.[CrossRef][Web of Science][Medline]

34. Morrisey EE, Ip HS, Tang Z, Lu MM, Parmacek MS. GATA-5: a transcriptional activator expressed in a novel temporally and spatially-restricted pattern during embryonic development. Dev Biol 183: 21–36, 1997.[CrossRef][Web of Science][Medline]

35. Morrisey EE, Musco S, Chen MY, Lu MM, Leiden JM, Parmacek MS. The gene encoding the mitogen-responsive phosphoprotein Dab2 is differentially regulated by GATA-6 and GATA-4 in the visceral endoderm. J Biol Chem 275: 19949–19954, 2000.[Abstract/Free Full Text]

36. Nakagawa R, Sato R, Futai M, Yokosawa H, Maeda M. Gastric GATA-6 DNA-binding protein: proteolysis induced by cAMP. FEBS Lett 408: 301–305, 1997.[CrossRef][Web of Science][Medline]

37. Nishida W, Nakamura M, Mori S, Takahashi M, Ohkawa Y, Tadokoro S, Yoshida K, Hiwada K, Hayashi K, Sobue K. A triad of serum response factor and the GATA and NK families governs the transcription of smooth and cardiac muscle genes. J Biol Chem 277: 7308–7317, 2002.[Abstract/Free Full Text]

38. Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 84: 767–801, 2004.[Abstract/Free Full Text]

39. Park JM, Adam RM, Peters CA, Guthrie PD, Sun Z, Klagsbrun M, Freeman MR. AP-1 mediates stretch-induced expression of HB-EGF in bladder smooth muscle cells. Am J Physiol Cell Physiol 277: C294–C301, 1999.[Abstract/Free Full Text]

40. Perlman H, Suzuki E, Simonson M, Smith RC, Walsh K. GATA-6 induces p21(Cip1) expression and G1 cell cycle arrest. J Biol Chem 273: 13713–13718, 1998.[Abstract/Free Full Text]

41. Reddy S, Ozgur K, Lu M, Chang W, Mohan SR, Kumar CC, Ruley HE. Structure of the human smooth muscle {alpha}-actin gene. Analysis of a cDNA and 5' upstream region. J Biol Chem 265: 1683–1687, 1990.[Abstract/Free Full Text]

42. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307: 1098–1101, 2005.[Abstract/Free Full Text]

43. Shirvani S, Xiang F, Koibuchi N, Chin MT. CHF1/Hey2 suppresses SM-MHC promoter activity through an interaction with GATA-6. Biochem Biophys Res Commun 339: 151–156, 2006.[CrossRef][Web of Science][Medline]

44. Shook D, Keller R. Mechanisms, mechanics and function of epithelial-mesenchymal transitions in early development. Mech Dev 120: 1351–1383, 2003.[CrossRef][Web of Science][Medline]

45. Stehr M, Estrada CR, Khoury J, Danciu TE, Sullivan MP, Peters CA, Solomon KR, Freeman MR, Adam RM. Caveolae are negative regulators of transforming growth factor-beta1 signaling in ureteral smooth muscle cells. J Urol 172: 2451–2455, 2004.[CrossRef][Web of Science][Medline]

46. Stewart SA, Dykxhoorn DM, Palliser D, Mizuno H, Yu EY, An DS, Sabatini DM, Chen IS, Hahn WC, Sharp PA, Weinberg RA, Novina CD. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA 9: 493–501, 2003.[Abstract/Free Full Text]

47. Thiery JP. Epithelial-mesenchymal transitions in development and pathologies. Curr Opin Cell Biol 15: 740–746, 2003.[CrossRef][Web of Science][Medline]

48. Tschumperlin DJ, Drazen JM. Chronic effects of mechanical force on airways. Annu Rev Physiol 68: 563–583, 2006.[CrossRef][Web of Science][Medline]

49. Wada H, Hasegawa K, Morimoto T, Kakita T, Yanazume T, Abe M, Sasayama S. Calcineurin-GATA-6 pathway is involved in smooth muscle-specific transcription. J Cell Biol 156: 983–991, 2002.[Abstract/Free Full Text]

50. Wada H, Hasegawa K, Morimoto T, Kakita T, Yanazume T, Sasayama S. A p300 protein as a coactivator of GATA-6 in the transcription of the smooth muscle-myosin heavy chain gene. J Biol Chem 275: 25330–25335, 2000.[Abstract/Free Full Text]

51. Werstiuk ES, Lee RM. Vascular beta-adrenoceptor function in hypertension and in ageing. Can J Physiol Pharmacol 78: 433–452, 2000.[CrossRef][Web of Science][Medline]

52. Xin M, Davis CA, Molkentin JD, Lien CL, Duncan SA, Richardson JA, Olson EN. A threshold of GATA4 and GATA6 expression is required for cardiovascular development. Proc Natl Acad Sci USA 103: 11189–11194, 2006.[Abstract/Free Full Text]

53. Yin F, Herring BP. GATA-6 can act as a positive or negative regulator of smooth muscle-specific gene expression. J Biol Chem 280: 4745–4752, 2005.[Abstract/Free Full Text]

54. Yoshida T, Owens GK. Molecular determinants of vascular smooth muscle cell diversity. Circ Res 96: 280–291, 2005.[Abstract/Free Full Text]

55. Yoshida T, Sinha S, Dandre F, Wamhoff BR, Hoofnagle MH, Kremer BE, Wang DZ, Olson EN, Owens GK. Myocardin is a key regulator of CArG-dependent transcription of multiple smooth muscle marker genes. Circ Res 92: 856–864, 2003.[Abstract/Free Full Text]

56. Zhou J, Hernandez G, Tu SW, Scholes J, Chen H, Tseng CP, Hsieh JT. Synergistic induction of DOC-2/DAB2 gene expression in transitional cell carcinoma in the presence of GATA6 and histone deacetylase inhibitor. Cancer Res 65: 6089–6096, 2005.[Abstract/Free Full Text]





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/3/C1093    most recent
00225.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kanematsu, A.
Right arrow Articles by Adam, R. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kanematsu, A.
Right arrow Articles by Adam, R. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.