GATA-6 and NF-κB activate CPI-17 gene transcription and regulate Ca2+ sensitization of smooth muscle contraction

Abstract

Protein kinase C (PKC)-potentiated inhibitory protein of 17 kDa (CPI-17) inhibits myosin light chain phosphatase, altering the levels of myosin light chain phosphorylation and Ca(2+) sensitivity in smooth muscle. In this study, we characterized the CPI-17 promoter and identified binding sites for GATA-6 and nuclear factor kappa B (NF-κB). GATA-6 and NF-κB upregulated CPI-17 expression in cultured human and mouse bladder smooth muscle (BSM) cells in an additive manner. CPI-17 expression was decreased upon GATA-6 silencing in cultured BSM cells and in BSM from NF-κB knockout (KO) mice. Moreover, force maintenance by BSM strips from KO mice was decreased compared with the force maintenance of BSM strips from wild-type mice. GATA-6 and NF-κB overexpression was associated with CPI-17 overexpression in BSM from men with benign prostatic hyperplasia (BPH)-induced bladder hypertrophy and in a mouse model of bladder outlet obstruction. Thus, aberrant expression of NF-κB and GATA-6 deregulates CPI-17 expression and the contractile function of smooth muscle. Our data provide insight into how GATA-6 and NF-κB mediate CPI-17 transcription, PKC-mediated signaling, and BSM remodeling associated with lower urinary tract symptoms in patients with BPH.

Fig 1

GATA-6 and NF-κB are recruited to the promoter. (A) Mapping the promoter and regulatory regions of the CPI-17 gene. 5′ upstream nucleotide sequences (0.85 kb or 1.333 kb) of the CPI-17 gene were fused to the luciferase reporter (PGL4.1 basic vector) and tested in transient expression assays in murine primary BSM cells. The firefly luciferase activities of the constructs were normalized to Renilla luciferase activity. Values are means ± standard deviations (SD) of the results of four independent transfection assays. ***, P < 0.001. (B) Schematic representation of the murine CPI-17 promoter. Structural details and putative transcription factor binding sites are shown. The horizontal arrow denotes the direction of transcription. (C) DNA affinity column chromatography. Protein fractions eluted from the DNA affinity column (bp −850 to −1333 region of the CPI-17 promoter) with various KCl concentrations were assayed by TF-ELISA. Binding of NF-κB p50 and GATA-6 to their consensus oligonucleotides was expressed as absorbance units at 450 nm (O.D., optical density). Data are representative of at least three independent triplicate experiments. (D) Oligonucleotide (Oligo) competition assay. Increasing amounts of WT or mutant NF-κB and GATA-6 binding consensus DNA sequence were added to the ELISA plate. (E) Immunoblot analysis of protein fractions bound to CPI-17 promoter. Purified fractions from the DNA affinity column (bp −850 to −1333 region of CPI-17 promoter) were analyzed by immunoblotting. GATA-6, NF-κB p50, and NF-κB c-Rel were detected in the fraction eluted with 1 M KCl. (F) ChIP analysis. Chromatin samples prepared from murine BSM were immunoprecipitated with anti-p50, -c-Rel, and -GATA-6 antibodies or preimmune rabbit IgG as a negative control. Precipitated fragments were PCR amplified using primers specific for κB and GATA motifs of mouse CPI-17 promoter. Quantified band intensities from the agarose gel are presented as percentages of input chromatin. Values are averages of six separate experiments using BSM from six mice (n = 6; **, P < 0.01).

Fig 2

GATA-6 and NF-κB bind to CPI-17 promoter. Gel mobility shift assay was performed using 10 fmol 5′-end biotin-labeled double-stranded oligonucleotide probe from CPI-17 promoter (GATA motifs −1220 to −1270 and κB motif −920 to −950) and 15 μg nuclear extracts (NE) from murine BSM. The reaction mixture was preincubated with the indicated antibodies (ab) or rabbit preimmune (PI) IgG for 20 min on ice before the addition of the labeled DNA probe. Competitor (Compet) containing 100-fold excess of unlabeled self-oligonucleotide was added to the binding reaction mixture 15 min prior to the addition of labeled probe. The migration positions of the free probe, the specific GATA-6, NF-κB p50, and c-Rel DNA complexes, and the supershifted complexes (arrows) are indicated. The boldface letters in the probe sequences indicate the putative GATA-6 and NF-κB binding nucleotides.

Fig 3

Transcriptional activation of CPI-17 promoter by GATA-6 and NF-κB in primary BSM cells. (A) Murine BSM cells were cotransfected with murine WT or mutant CPI-17 promoter luciferase constructs and GATA-6, NF-κB p50, and c-Rel cDNA. The NF-κB binding site (κB motif) mutation in the 1.33-kb CPI-17 promoter is designated Mut1. The GATA-6 binding site (multiple GATA motifs) mutation is designated Mut2. The double mutant with both Mut1 and Mut2 mutations is designated Mut3. Mutant nucleotides are shown in boldface font. Luciferase activity was measured in each sample after 72 h and is presented relative to that of Renilla luciferase. Data are presented as means ± SD from five independent experiments. ≠≠, P < 0.01 compared with results for 0.85-kb CPI-17 promoter; **, P < 0.01 compared with results for 1.33-kb CPI-17 promoter. (B) Representative immunoblot showing the expression levels of ectopically expressed GATA-6, NF-κB p50, and c-Rel compared with their endogenous levels. (C and D) ChIP analysis of CPI-17 promoter in WT and c-Rel KO murine BSM with GATA-6 antibody (n = 5) (C) and of CPI-17 promoter in scrambled (Control) and GATA-6 shRNA knockdown BSM cells with NF-κB p50 and c-Rel antibodies (D). GATA motifs (C) and κB motif (D) in the murine CPI-17 promoter were amplified by PCR. Preimmune serum (IgG) was used as a negative control in both ChIP experiments. Data are presented as the means ± SD of five experiments. (E) Coimmunoprecipitation of GATA-6, NF-κB p50, and c-Rel in WT and c-Rel KO mouse BSM. Immunoprecipitates from WT and c-Rel KO murine BSM nuclear extracts by GATA-6 antibody were subjected to immunoblot analysis with GATA-6, NF-κB p50, and c-Rel antibodies. An immunoblot representative of five different experiments is presented (n = 5).

Fig 4

Silencing of GATA-6 downregulates CPI-17 gene expression. (A and B) RT-PCR (A) and immunoblot analysis (B) of GATA-6 and total and phosphorylated CPI-17 in murine BSM cells transduced with scrambled (control) and GATA-6 shRNA. Representative blots from three different experiments are presented. (C) ChIP analysis of the CPI-17 promoter using GATA-6 antibody in murine BSM cells transfected with scrambled or GATA-6 shRNA. The GATA-6 binding motif in the murine CPI-17 promoter was amplified by PCR. Preimmune serum (IgG) was used as a negative control. (D) Relative luciferase activities in murine BSM cells transfected with scrambled and GATA-6 siRNA. Reporter firefly luciferase activities were normalized to Renilla luciferase activity. Data are presented as means ± SD of three independent experiments. **, P < 0.01 compared with the results for scrambled shRNA. (E and F) Immunoblot analysis of the expression levels of endogenous GATA-6 and exogenously transfected GATA-6 in scrambled shRNA and GATA-6 shRNA knockdown cells. Representative blots from three different experiments are presented.

Fig 5

GATA-6 and NF-κB upregulate endogenous CPI-17 expression in primary BSM cells. (A to C) Murine BSM cells were transduced with adenovirus encoding GATA-6, LacZ, or NF-κB p50 and c-Rel cDNA individually and in combination for 72 h. RNA and protein were extracted and subjected to RT-PCR (A) and immunoblot analysis (B and C) for total and phospho-Thr³⁸-CPI-17 (pCPI-17), GATA-6, NF-κB p50, c-Rel, and GAPDH. (D to F) Data from the experiments for which representative results are shown in panels A to C are presented as means ± SD from four independent experiments. **, P < 0.01 compared with control.

Fig 6

NF-κB silencing downregulates CPI-17 gene expression. RT-PCR (A and B) and immunoblot (C and D) analyses of CPI-17 mRNA and total and phospho-CPI-17 protein in WT, NF-κB p50 KO, and NF-κB c-Rel KO mouse BSM tissues. Data are presented as the means ± SD of five experiments. **, P < 0.01 compared with WT. (E) Immunofluorescence analysis of CPI-17 in WT and c-Rel KO mouse bladders. BSM tissue was stained with anti-CPI-17 and anti-SM22 polyclonal antibody (1:100) followed by Cy3- and FITC-conjugated secondary antibodies. Scale bars = 20 μm. Representative confocal images are presented.

Fig 7

NF-κB silencing upregulates RhoA and ROCKβ expression in murine BSM. (A and C) Immunoblot analyses of RhoA (A) and ROCKβ (C) expression in WT, p50 KO, and c-Rel KO mouse BSM tissue. (B, D) Data are presented as means ± SD of five experiments. **, P < 0.01 compared with the results for WT mice. (E) Expression of total MYPT, phospho-Thr⁶⁹⁶-MYPT1, and phospho-Thr⁸⁵⁰-MYPT1 in WT, p50 KO, and c-Rel KO BSM tissues. An immunoblot representative of five different experiments is presented.

Fig 8

Effects of NF-κB silencing on PKC- and RhoA-mediated BSM contraction. (A and B) Representative force profiles for WT and NF-κB c-Rel KO mouse BSM strips in response to PDBu and KCl. Isolated BSM strips were stretched intermittently while being stimulated with electrical field stimulation (80 V, 32 Hz for 1 ms) to determine optical length (Lo). Individual BSM strips were stimulated with 1.0 μM PDBu, a PKC activator (A), and force was recorded at the peak of contraction. Similarly, the strips were stimulated with 125 mM KCl (B) to determine the maximal contractile response. (C and D) Means of five separate experiments using BSM strips from five WT and five NF-κB c-Rel KO mice stimulated with PDBu (C) (n = 5, P < 0.05) or KCl (D) (n = 5, P < 0.05). (E and F) Representative profiles of 2-D gel electrophoresis showing MLC₂₀ phosphorylation. Protein extracts were prepared from muscle strips freeze-clamped at the peak of maximal force in response to PDBu. Arrows indicate spots for phosphorylated (p) and unphosphorylated (u) MLC₂₀ in Coomassie blue-stained 2-D gels. (G) Quantification of the results from five experiments (n = 5) determined by densitometry.

Fig 9

Carbachol-induced BSM contraction in NF-κB c-Rel KO mice. (A and B) Representative force profiles of WT and NF-κB c-Rel KO mouse BSM strips in response to carbachol as a function of time (in minutes). Isolated BSM strips were stretched intermittently while being stimulated with electrical field stimulation (80 V, 32 Hz for 1 ms) to determine optical length (Lo). Individual strips were stimulated with 10 μM carbachol, and force was recorded at the peak of contraction. (C) Quantification of force maintenance in NF-κB c-Rel KO and WT BSM in response to carbachol stimulation. The graph shows the means ± standard errors of the mean of five separate experiments (n = 5, P < 0.05). (D) Representative immunoblot showing CPI-17 phosphorylation in response to carbachol stimulation in WT and c-Rel KO BSM. Protein extract prepared from muscle strips freeze-clamped at the peak of maximal force in response to carbachol was analyzed by immunoblotting for total and phospho-CPI-17 expression levels. GAPDH was used as a loading control. (E) Immunoblot analysis of PKCα, PKCβ, and PKCδ expression in WT and c-Rel KO mouse BSM. A representative immunoblot from five separate experiments is presented. (F) Effects of carbachol and PDBu on BSM contraction in permeabilized WT and c-Rel KO tissue. Alpha-toxin-permeabilized BSM strips were contracted with low Ca²⁺ (pCa 7.5) alone, with low Ca²⁺ (pCa 7.5) plus carbachol plus GTP, or with low Ca²⁺ (pCa 7.5) plus PDBu. Contractions were normalized to the maximal response elicited by pCa 4.5. Data are presented as the means ± SD of five experiments. n = 5; **, P < 0.05 compared with low Ca²⁺ alone; pCa 7.5.

Fig 10

Overexpression of CPI-17 in murine model of PBOO. (A and B) RT-PCR (A) and immunoblot (B) analysis of CPI-17 mRNA and protein expression in sham-operated and PBOO mouse BSM tissues. GAPDH served as a control. (C and D) Quantification of RT-PCR (A) and immunoblot (B) data presented as means ± SD (n = 10 each for sham-operated and PBOO mouse BSM). **, P < 0.01 compared with the results for sham-operated control mice. (E) Murine bladder sections prepared from sham-operated and PBOO mice were stained with anti-CPI-17 (Cy3, red) and anti-SM22 (FITC, green) antibodies. Negative-control sections were prepared using antibody preabsorbed with the antigen used to raise the antibody (data not shown). Scale bars = 20 μm.

Fig 11

Overexpression of NF-κB p50 and c-Rel in murine model of PBOO. Nuclear protein (A and B) and RNA (C and D) were prepared from sham-operated and PBOO mouse BSM tissues and analyzed for NF-κB p50, c-Rel, GAPDH, and p97 expression. GAPDH and p97 served as loading controls. (E and F) Data are presented as means ± SD (n = 10 each for sham-operated and PBOO mice). **, P < 0.01 compared with sham-operated control mice.

Fig 12

In situ localization of NF-κB p50 in murine model of PBOO. Bladder sections prepared from sham-operated and PBOO mice were stained with anti-NF-κB p50 (Cy3, red) and anti-SM22 (FITC, green). Scale bars = 15 μm.

Fig 13

PBOO increased NF-κB and GATA-6 DNA binding activity. (A to C) Chromatin samples prepared from sham-operated and PBOO mouse BSM were immunoprecipitated with anti-GATA-6, NF-κB p50, and c-Rel antibodies or preimmune rabbit serum. Precipitated fragments were PCR amplified using primers specific for GATA and κB motifs on the mouse CPI-17 promoter or primers specific for CPI-17 exon-1 as a negative control. (D and E) Quantification of band intensities from agarose gels is presented as percentages of input chromatin (average of six sham-operated and six PBOO mouse samples). Data are presented as means ± SD. **, P < 0.01 compared with the results for sham-operated control mice.

Fig 14

Overexpression of NF-κB and GATA-6 in BPH-induced bladder outlet obstruction correlates with CPI-17 gene expression. (A to E) RNA and protein samples prepared from BSM of control and BPH patients were subjected to RT-PCR and immunoblot analysis for NF-κB p50, c-Rel, CPI-17, and phospho-CPI-17. GAPDH and p97 were used as controls. Data are presented as means ± SD. **, P < 0.01 compared with the results for control human BSM. (F) Chromatin samples from BSM of control and BPH patients were immunoprecipitated with antibodies specific for GATA-6, NF-κB p50, and c-Rel or preimmune rabbit IgG. Precipitated fragments were PCR amplified using primers specific for GATA and κB motifs in the human CPI-17 promoter. Rabbit IgG was used as a negative control. Quantification of band intensities from agarose gels is presented as percentage of input chromatin. Data are presented as means ± SD. **, P < 0.01 compared with the results for control human BSM.