Volume 129, Issue 5, Pages 1518-1532 (November 2005) Negative Transcriptional Regulation of Human Colonic Smooth Muscle Cav1.2 Channels by p50 and p65 Subunits of Nuclear Factor-κB  Xuan–Zheng Shi, Konrad Pazdrak, Nehad Saada, Bosong Dai, Philip Palade, Sushil K. Sarna  Gastroenterology  Volume 129, Issue 5, Pages 1518-1532 (November 2005) DOI: 10.1053/j.gastro.2005.07.058 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 (A) Quantitative PCR to detect transcripts containing exons 1a or 1b showed an abundance of those containing exon 1b and very few of those containing exon 1a. (B) The α1C mRNA in HCCSMCs decreased time dependently on treatment with 20 ng/mL TNF-α (n = 3 or 4). *P < .05. Gastroenterology 2005 129, 1518-1532DOI: (10.1053/j.gastro.2005.07.058) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Nuclear run-on assay showed that the transcription rate of α1C mRNA decreased, that of the housekeeping β-actin was not affected, and that of inducible interleukin 8 was increased in response to TNF-α treatment of HCCMCs for 24 hours with respect to that in cells treated with medium alone. The nuclear run-on assays on β-actin and interleukin 8 were used as controls. The bar graph shows quantitative data for α1C mRNA (n = 3). Gastroenterology 2005 129, 1518-1532DOI: (10.1053/j.gastro.2005.07.058) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 (A) Western blot analysis of α1C expression in response to TNF-α indicated a time-dependent decrease in its abundance (n = 4). (B) The increase of intracellular calcium (340:380 ratio) in response to BAY K 8644, a calcium channel opener, was blunted in cells treated with TNF-α for 24 hours when compared with those treated with control medium only. The cells were loaded with fura 2-AM. kD, kilodaltons. Gastroenterology 2005 129, 1518-1532DOI: (10.1053/j.gastro.2005.07.058) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 The effect of TNF-α on hα1C1b promoter activity in HCCSMCs. The pGL2-hα1C1b luciferase activity was normalized with SEAP activity (4 separate experiments; *P < .05 vs pGL2- hα1C1b control). Gastroenterology 2005 129, 1518-1532DOI: (10.1053/j.gastro.2005.07.058) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 (A) TNF-α treatment of cells transiently activated IKKα and IKKβ and degraded IκBα. (B) TNF-α–induced suppression of α1C was partially reversed in the cells transfected with dominant negative (d.n.) mutant IKKβ (top) or treated with NF-κB inhibitor MG-132 (25 μmol/L). Similar results were obtained from 3 separate experiments. Ctr., control. Gastroenterology 2005 129, 1518-1532DOI: (10.1053/j.gastro.2005.07.058) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 Representative NF-κB nuclear binding in tissues taken from 3 normal subjects (N), 2 ulcerative colitis (UC) patients, and 1 Crohn’s colitis (CC) patient. NF-κB/DNA binding was significantly greater in patients when compared with that in normal subjects. Similar data were obtained in 1 additional UC patient. Gastroenterology 2005 129, 1518-1532DOI: (10.1053/j.gastro.2005.07.058) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 (A) TNF-α–induced suppression of α1C expression in HCCSMCs is reversed by pretreatment of the cells with antisense oligonucleotides to p65 (A) or p50 (B). Cells were transfected with the sense and antisense oligonucleotides (10 μmol/L) before their treatment with control medium or TNF-α for 24 hours. The efficiency of antisense/sense treatments was confirmed by detection of nuclear abundance of p65 or p50 (lower panel). Similar results were obtained in 4 separate experiments. Ctr, control. Gastroenterology 2005 129, 1518-1532DOI: (10.1053/j.gastro.2005.07.058) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 8 (A and B) Overexpression of p65 or p50 suppressed the constitutive expression of α1C in HCCSMCs (C). Cells were transfected with 0.5 μg of pCMV-p65 or pCMV-p50 for 24 hours before their 24-hour treatment with control medium or TNF-α. Results are representative of 3 separate experiments. Gastroenterology 2005 129, 1518-1532DOI: (10.1053/j.gastro.2005.07.058) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 9 (A) Agarose–oligonucleotide pulldown showed time-dependent nuclear translocations of p65 (left) and p50 (right). (B) Nuclear binding of p65 and p50 to the hα1C1b NF-κB response elements RE1, RE2, and RE3 in response to TNF-α 20 ng/mL. The p65 (upper) and p50 (lower) bound to the wild-type, but not the mutant, probes of RE1 (left), RE2 (middle), and RE3 (right). The wild-type, but not the mutant, sequences competed for specific binding of p65 and p50 to RE1, RE2, and RE3. The sequences of the wild-type and mutant RE1, RE2, and RE3 probes are shown. Wt, wild type; Mut or Mt, mutant. Gastroenterology 2005 129, 1518-1532DOI: (10.1053/j.gastro.2005.07.058) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 10 Luciferase activity of full-length and 4 deletions of the hα1C1b promoter in the absence and presence of TNF-α. All luciferase activities were expressed as a percentage of the activity shown by the wild-type promoter–reporter construct. The vector alone, pGL2 basic, served as a negative control. *P < .05 vs full-length control. The results were obtained from 5 separate experiments. Wt, wild type. Gastroenterology 2005 129, 1518-1532DOI: (10.1053/j.gastro.2005.07.058) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 11 HCCSMCs were transiently transfected with the wild-type promoter construct and the constructs containing mutations of the first 3 nucleotides of RE1, RE2, or both RE1 and RE2 for 24 hours before incubation with control medium or TNF-α 20 ng/mL for another 24 hours. The luciferase activity was expressed as a percentage of wild-type control. The results are obtained from 3 separate experiments. *P < .05 vs the control in each group. mut, mutant. Gastroenterology 2005 129, 1518-1532DOI: (10.1053/j.gastro.2005.07.058) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 12 The p65 and p50 sense and antisense nucleotides were transfected in freshly obtained human colon circular muscle strips for 24 hours. The contractile response to ACh was suppressed by 24-hour TNF-α treatment of muscle strips transfected with sense oligonucleotides (A and B, bottom). This effect was similar to that seen in muscle strips treated with control medium (data not shown). The antisense treatments of both NF-κB subunits reversed the TNF-α–induced suppression of colonic circular muscle contractility in response to ACh. Antisense, but not sense, oligonucleotides to p65 (A) or p50 (B) suppressed the expression levels of p65 and p50 and blocked TNF-α–induced down-regulation of α1C in muscle homogenates. The sense and antisense treatments by themselves had little effect on the contractile response to ACh when compared with medium-treated muscle strips (data not shown for medium-treated muscle strips). Results were obtained from 4 separate experiments. •, sense oligo control; ○, sense oligonucleotide + TNF-α treatment; ▴, antisense oligonucleotide control; ▵, antisense oligonucleotide + TNF-α treatment. *P < .05 with respect to muscle strips treated with sense oligonucleotides only. AUC, area under the curve. Ctr, control; wt., weight. Gastroenterology 2005 129, 1518-1532DOI: (10.1053/j.gastro.2005.07.058) Copyright © 2005 American Gastroenterological Association Terms and Conditions