Nuclear factor-kappaB is a critical mediator of Ste20-like proline-/alanine-rich kinase regulation in intestinal inflammation

Abstract

Inflammatory bowel disease (IBD) is thought to result from commensal flora, aberrant cellular stress, and genetic factors. Here we show that the expression of colonic Ste20-like proline-/alanine-rich kinase (SPAK) that lacks a PAPA box and an F-alpha helix loop is increased in patients with IBD. The same effects were observed in a mouse model of dextran sodium sulfate-induced colitis and in Caco2-BBE cells treated with the pro-inflammatory cytokine tumor necrosis factor (TNF)-alpha. The 5'-flanking region of the SPAK gene contains two transcriptional start sites, three transcription factor Sp1-binding sites, and one transcription factor nuclear factor (NF)-kappaB-binding site, but no TATA elements. The NF-kappaB-binding site was essential for stimulated SPAK promoter activity by TNF-alpha, whereas the Sp1-binding sites were important for basal promoter activity. siRNA-induced knockdown of NF-kappaB, but not of Sp1, reduced TNF-alpha-induced SPAK expression. Nuclear run-on and mRNA decay assays demonstrated that TNF-alpha directly increased SPAK mRNA transcription without affecting SPAK mRNA stability. Furthermore, up-regulation of NF-kappaB expression and demethylation of the CpG islands induced by TNF-alpha also played roles in the up-regulation of SPAK expression. In conclusion, our data indicate that during inflammatory conditions, TNF-alpha is a key regulator of SPAK expression. The development of compounds that can either modulate or disrupt the activity of SPAK-mediated pathways is therefore important for the control and attenuation of downstream pathological responses, particularly in IBD.

Figure 1

SPAK expression profile in colon tissue from patients with UC. A: Immunostaining of SPAK in normal human colon tissue and UC patient colon tissue from mucosal biopsies. SPAK expression (red); nuclear staining by DAPI (blue); SPAK is primarily expressed in epithelial cells. B: The expression of SPAK mRNA in normal human and UC patient colon tissues from mucosal biopsies were quantified by real-time PCR. **P < 0.01. C: Ten μg of protein from normal human colon and UC patient colon from mucosal biopsies were examined by Western blot with SPAK antibody, colon tissue from UC patients demonstrated a significantly higher level of SPAK expression (top) versus healthy colon, with GAPDH as the internal loading control.

Figure 2

SPAK expression profile in colon tissue from mice with experimental colitis. A: H&E-stained colon sections of mice treated with DSS at 0, 1, 3, 5, 9 (withdraw after 5 days treatment, 4 days to recover), and 14 (withdraw after 5 days treatment, 9 days to recover) days. B: Determination of MPO enzymatic activity in the colon as an index of neutrophil infiltration into the injured tissue. Results are expressed as MPO mU per μg protein and represent mean ± SEM of three determinations. *P < 0.05, **P < 0.01. C: Immunostaining of SPAK in same mice colon tissue as in H&E-stained sections. SPAK (red); nuclear staining by DAPI (blue). D: Real-time PCR analysis of SPAK mRNA expression in mucosa from colon tissue of DSS-treated mice. *P < 0.05, **P < 0.01. E: Western blot analysis of SPAK expression in mucosa from colon tissue of DSS-treated mice.

Figure 3

Real-time PCR (A) and Western blot (B) demonstrate dose responses of TNF-α on SPAK expression in Caco2-BBE cells. *P < 0.05, **P < 0.01. Real-time PCR (C) and Western blot (D) show the time course of TNF-α on SPAK expression in Caco2-BBE cells. *P < 0.05, **P < 0.01.

Figure 4

Characteristics of 5′ flanking region of human SPAK gene. A: Schematic presentation of characteristics of 5′ flanking region of SPAK gene. The red vertical line represents translational initiation site; two blue vertical lines represents the two TSSs; three green bars represent Sp1 binding sites; one gray bar represents NF-κB binding site; the horizontal line represent the 5′ flanking region of SPAK gene, of which the light blue line represents CpG island. The digits indicate the position of corresponding sites. B: Mapping of the TSS by primer extension analysis. Lane 1, Primer extension results with template; lane 2, Primer extension results without template. C: Mapping of the TSS by 5′-RACE. Lane M, 100-bp DNA molecular weight marker; lane 1, gel electrophoresis of PCR products; lane 2, gel electrophoresis of PCR products without template.

Figure 5

Characterization of SPAK promoter: A: Schematic representation of human SPAK promoter constructs. The full-length SPAK promoter (nt −1472 to +4); construct I (nt −1050 to +4); construct II (nt −398 to +4); construct III (nt −331 to +4); construct IV (nt −149 to +4); and construct V (nt −72 to +4). Numbers are given in relation to the translational start codon (+1) and indicate 5′-ends of the deletion constructs. The location of the identified positive regulatory region is indicated by a light blue box. Positions of the putative Sp1 (red) and NF-κB (yellow) sites are indicated by arrows. B: Promoter activities of the 5′ deleted constructs in untreated or TNF-α- and IL-1β-stimulated Caco2-BBE cells normalized to Renilla Luc activities driven by the phRL-CMV control vector. Activities are expressed as fold inductions over cells transfected with the empty pGL3-basic vector. Each value represents the mean ± SD of at least three independent sets of transfection experiments performed in triplicate. *P < 0.05, **P < 0.01. C: Schematic representation of mutated SPAK promoter constructs: the full-length SPAK promoter; I Sp1 binding site (−496); II Sp1 binding site (−303); III Sp1 binding site (−114); and NF-κB binding site (−354). The digits are given in relation to the translational start codon (+1). The location of the identified positive regulatory region is indicated by a light blue box. Positions of the putative Sp1 sites are indicated by arrows and NF-κB is indicated by a rectangle. The corresponding mutated transcription factor binding site is indicated by a black arrow or black rectangle. D: Effects of mutations of Sp1 or NF-κB binding sites on SPAK promoter activity. The various mutated constructs were transiently transfected into Caco2-BBE cells under the basal (gray bar) or TNF-α-stimulated promoter activities of the full-length wild-type construct were set to 100% (control). Values represent means ± SD of at least three independent sets of transfection experiments performed in triplicate. *P < 0.05, **P < 0.01.

Figure 6

EMSA of I Sp1 (−496) (A), II Sp1 (−303) (B), III Sp1 (−114) (C), NF-κB (−354) (D). Lane 1, biotin-labeled oligonucleotide alone; lane 2, biotin-labeled oligonucleotides incubated with 5 μg of Caco2-BBE nuclear extracts; lane 3, biotin-labeled oligonucleotides incubated with 5 μg of TNF-α-treated Caco2-BBE nuclear extracts; lane 4, biotin-labeled oligonucleotides incubated with 5 μg of Caco2-BBE nuclear extracts in the presence of anti-Sp1 (A–C) or NF-κB (p65) (D) antibodies; lane 5, biotin-labeled oligonucleotides incubated with 5 μg of Caco2-BBE nuclear extracts in the presence of nonspecific IgG; lane 6, biotin-labeled oligonucleotides incubated with 5 μg of Caco2-BBE nuclear extracts in the presence of a 50-fold excess of cold competitor oligonucleotide; lane 7, biotin-labeled binding site-mutated oligonucleotides incubated with 5 μg of Caco2-BBE nuclear extracts. ChIP assay: the antibodies indicated were incubated with cross-linked DNA isolated from Caco2-BBE cells treated with (+) or without (−) TNF-α (E) or IL-1β (F). Sp1 (I, II, and III) and NF-κB promoter elements in the immunoprecipitates were detected by PCR using the specific primers shown in Table 1. The bottom panel uses DNA input as template for internal control.

Figure 7

A: Western blots of Sp1 and NF-κB (p65) demonstrating DSS effect on Sp1 and NF-κB protein levels. Histone 1 acts as a control. *P < 0.05, **P < 0.01. B: Co-immunoprecipitation assay of TNF-α increasing phosphorylation of Sp1. C: Reduction of NF-κB not Sp1 expression reduced SPAK protein expression in unstimulated and in TNF-α-stimulated Caco2-BBE cells. Cells were harvested and subjected to Western blot analysis using Sp1, NF-κB (p65), and SPAK antibodies as described in the Materials and Methods. GAPDH acts as a loading control.

Figure 8

A: Nuclear run-on assay indicated the increase of SPAK mRNA transcription under the treatment of TNF-α, with the mRNA transcription of GAPDH as internal control. B: TNF-α does not change SPAK mRNA stability, the percentage of remaining SPAK mRNA is shown at the different time points. Filled circle represents the value of real-time PCR with the samples from Caco2-BBE cells without treatment, open circle represents the value of real-time PCR with the samples from Caco2-BBE treated with actinomycin D, solid triangle represents real-time PCR with the samples from Caco2-BBE treated with actinomycin D and TNF-α. C: Northern blot analysis of total RNA from Caco2-BBE cells. Lane 1, no treatment; lane 2, AcD; lane 3, TNF-α; and lane 4, AcD and TNF-α. The lower RNA electrophoresis shows equal loading of each condition. D: Methylation assay of CpG island in SPAK promoter. *P < 0.05 versus control.

Figure 9

Proposed model of activation and regulation of SPAK expression.