The role of Sp1 in IL-1beta and H. pylori-mediated regulation of H,K-ATPase gene transcription

Abstract

Helicobacter pylori infection of the gastric body induces transient hypochlorhydria and contributes to mucosal progression toward gastric carcinoma. Acid secretion is mediated by parietal cell H,K-ATPase, in which the catalytic alpha-subunit (HKalpha) promoter activity in transfected gastric epithelial [gastric adenocarcinoma (AGS)] cells is repressed by H. pylori through NF-kappaB p50 homodimer binding to the promoter. IL-1beta, an acid secretory inhibitor whose mucosal level is increased by H. pylori, upregulates HKalpha promoter activity in AGS cells. Because IL-1beta also activates NF-kappaB signaling, we investigated disparate HKalpha regulation by H. pylori and IL-1beta, testing the hypothesis that IL-1beta-induced HKalpha promoter activation is mediated by the transcription factor Sp1. DNase I footprinting revealed Sp1 binding to the HKalpha promoter at -56 to -39 bp. IL-1beta stimulated the activity of three HKalpha promoter constructs containing NF-kappaB and Sp1 sites transfected into AGS cells and also stimulated a construct containing only an Sp1 site. This stimulation was abrogated by mutating the HKalpha promoter Sp1 binding site. Gelshift assays showed that IL-1beta increased Sp1 but not p50 binding to cognate HKalpha probes and that Sp1 also interacts with an HKalpha NF-kappaB site when bound to its cognate HKalpha cis-response element. H. pylori did not augment Sp1 binding to an HKalpha Sp1 probe, and small interfering RNA-mediated knockdown of Sp1 expression abrogated IL-1beta-induced HKalpha promoter stimulation. We conclude that IL-1beta upregulates HKalpha gene transcription by inducing Sp1 binding to HKalpha Sp1 and NF-kappaB sites and that the H. pylori perturbation of HKalpha gene expression is independent of Sp1-mediated basal HKalpha transcription.

Fig. 1.

A: schematic depiction of 7 progressively truncated human catalytic α-subunit of H,K-ATPase (HKα) 5′-flanking sequence deletion constructs. Numbering represents base pair position with respect to transcription initiation site (TIS) shown as bent arrows. HKα2179 shows 5′-terminus of homology domain I at −322 bp. NF-κB₁, NF-κB₂, and Sp1 represent the 2 NF-κB and 1 Sp1 binding regions in HKα promoter. B: HKα promoter activity is increased by IL-1β and decreased by Helicobacter pylori. Human gastric adenocarcinoma (AGS) cells were independently transfected with HKα deletion-Luc constructs followed by incubation with H. pylori [25 multiplicity of infection (MOI); 6 h] or IL-1β (10 ng/ml; 24 h). HKα promoter activity was expressed in terms of relative luciferase activity. White bar, control AGS cells; light gray bar, IL-1β-treated AGS cells; dark gray bar, H. pylori-treated AGS cells. Data are shown as means ± SD of 3 separate experiments. For HKα206, HKα177, HKα165, and HKα102 deletion constructs, IL-1β-induced promoter activity differed significantly from control (P < 0.001). For shorter HKα58 and HKα37 deletion constructs, IL-1β-induced and control promoter activity did not differ significantly. For HKα2179, HKα206, HKα177, and HKα165 deletion constructs, H. pylori-induced promoter activity differed significantly from control (P < 0.001). For HKα102, HKα58, and HKα37 deletion constructs, IL-1β-induced and basal promoter activity did not differ significantly.

Fig. 2.

IL-1β activates both Sp1 and NF-κB in AGS cells. AGS cells were treated with IL-1β (10 ng/ml) for 4 h, and cellular content of ERK1/2, Sp1, and NF-κB p50 subunits was assayed by immunoblotting using antibodies recognizing phosphorylated (p) and nonphophosphorylated forms of ERK1/2, Sp1, and NF-κB p50 subunit. Left: content of p-ERK1/2, Sp1, and NF-κB p50 in untreated, IL-1β-treated, and PD-98059 + IL-1β-treated AGS cells. Right: cellular content of total ERK1/2, Sp1, and NF-κB p50 subunits, acting as normalization control for left. Representative data are from 3 independent sets of experiments. Con, control.

Fig. 3.

Sp1 binding region is located upstream of TATA box region in HKα promoter. Representative DNA sequencing gel electropherogram showing DNase I footprinting of the HKα 5′-flanking sequence with recombinant human Sp1 protein is shown. Protected and hypersensitive regions are identified by base pair numbering with respect to transcription initiation site. DNase I digestion products in the absence of recombinant Sp1 are shown in lanes 1 and 3, and digestion products in the presence of recombinant Sp1 are shown in lanes 2 and 4. Lanes G, A, T, and C represent the sequencing lanes.

Fig. 4.

IL-1β but not H. pylori increases binding of Sp1 to the HKα promoter. AGS cells were treated with IL-1β (10 ng/ml; A–C) or with H. pylori (25 MOI) for 4 h (D). Interactions of AGS cell nuclear extract proteins with ³²P-labeled HKα-specific Sp1 oligonucleotide probes were assessed by EMSA and supershift analysis with Sp1 antibody (Ab) or nonimmune rabbit IgG. A: EMSA and supershift analysis with ³²P-labeled HKα Sp1 probe or HKαΔSp1 probe (6 nucleotide mutation in the core Sp1 binding region), IL-1β-treated AGS cells, and Sp1-specific antibody or nonimmune rabbit IgG. B: densitometric quantitation of DNA-protein complexes from lanes 1, 2, 7, and 8 in A. Data are shown as means ± SD of 3 independent experiments. C: EMSA with ³²P-labeled HKα Sp1probe (lanes 1 and 2) and with 200× molar excess of cold (nonradioactive) HKα Sp1 probe (lanes 3 and 4). D: EMSA and supershift analysis with HKα Sp1 probe and H. pylori-treated AGS cells and Sp1-specific antibody. Gels are representative of 3 replicates for A and 2 replicates for experiments shown in C and D. Long and short arrows indicate DNA-protein complexes; arrowheads indicate supershifted complexes.

Fig. 5.

IL-1β modulates Sp1 binding to NF-κB binding region in HKα promoter. A: interactions of AGS cell nuclear extract (NE) with and without IL-1β treatment (10 ng/ml) with ³²P-labeled HKα-specific NF-κB₁ oligonucleotide probe (T18; Ref. 25) by EMSA and supershift analysis with NF-κB p50 antibody (lanes 3 and 4) or Sp1 antibody (lanes 5 and 6). B: interactions of AGS nuclear extracts (lanes 1-4) or recombinant (r) human Sp1 protein (lanes 5 and 6) with an equimolar mixture of ³²P-labeled NF-κB₁ T18 probe and cold (nonradioactive) HKα Sp1 probe. Long and short arrows indicate DNA-protein complexes; arrowheads indicate supershifted complexes. Note the supershifts induced by addition of Sp1 antibody in lanes 3 and 4 (change in long arrow band to arrowheads). The gel is a representative EMSA gel from 2 independent experiments.

Fig. 6.

IL-1β-induced increase in HKα promoter activity is regulated by Sp1 and not NF-κB. AGS cells were transiently transfected with either wild-type HKα206 or double-mutated HKα206 (HKα NF-κB₁) or Sp1 core-mutated HKα206 (HKαΔSp1) and incubated with (gray bars) or without (white bars) IL-1β (10 ng/ml) for 24 h. HKα206 promoter activity was measured as normalized relative light units. Data are shown as means ± SD of 3 independent experiments. ***P < 0.001.

Fig. 7.

Sp1 downregulation abrogates IL-1β-induced increase in HKα promoter activity. A: AGS cells were transfected with Sp1-specific small interfering RNA (siRNA) for 48 h, and the cellular content of Sp1 in presence and absence of siRNA was measured by real-time PCR using β₂-microglobin as the internal control. Control, mock-transfected AGS cells; siRNA pool, AGS cells transfected with a mixture of 4 different siRNAs; siRNA 1, siRNA 2, and siRNA 3, AGS cells transfected with individual siRNAs; nontargeting (nt) siRNA, AGS cells transfected with nt siRNA as a negative control. Control and nt siRNA-transfected cells were harvested at the same time point as siRNA-treated cells. Data are shown as means ± SD of 3 independent experiments (***P < 0.001 compared with control). B: AGS cells were transfected with Sp1-specific siRNA 3, and the cellular content of Sp1 protein was measured after 72 h by immunoblotting using β-actin as the internal control. Control, mock-transfected AGS cells; siRNA, AGS cells transfected with siRNA 3 for 72 h. C: AGS cells were transfected with Sp1-specific siRNA 3 for 48 h, transfected with HKα206 for 24 h, and then treated with IL-1β for 24 h. HKα206 promoter activity was expressed as normalized relative light units (RLU) of luciferase activity. Data are shown as means ± SD of 3 independent experiments. ***P < 0.001.

Fig. 8.

Schematic diagram of proposed interaction of Sp1 transcription factor with Sp1 and NF-κB binding sites on the proximal HKα promoter. The proximal 5′-flanking sequence of the HKα promoter is represented by the black hairpin; gray boxes represent transcription factor binding sites, and white circles represent Sp1 protein and putative cofactor(s) required for Sp1-mediated coupling of the Sp1 and NF-κB binding sites.