Loss of Mucosal p32/gC1qR/HABP1 Triggers Energy Deficiency and Impairs Goblet Cell Differentiation in Ulcerative Colitis

Abstract

Background & aims: Cell differentiation in the colonic crypt is driven by a metabolic switch from glycolysis to mitochondrial oxidation. Mitochondrial and goblet cell dysfunction have been attributed to the pathology of ulcerative colitis (UC). We hypothesized that p32/gC1qR/HABP1, which critically maintains oxidative phosphorylation, is involved in goblet cell differentiation and hence in the pathogenesis of UC.

Methods: Ex vivo, goblet cell differentiation in relation to p32 expression and mitochondrial function was studied in tissue biopsies from UC patients versus controls. Functional studies were performed in goblet cell-like HT29-MTX cells in vitro. Mitochondrial respiratory chain complex V-deficient, ATP8 mutant mice were utilized as a confirmatory model. Nutritional intervention studies were performed in C57BL/6 mice.

Results: In UC patients in remission, colonic goblet cell differentiation was significantly decreased compared to controls in a p32-dependent manner. Plasma/serum L-lactate and colonic pAMPK level were increased, pointing at high glycolytic activity and energy deficiency. Consistently, p32 silencing in mucus-secreting HT29-MTX cells abolished butyrate-induced differentiation and induced a shift towards glycolysis. In ATP8 mutant mice, colonic p32 expression correlated with loss of differentiated goblet cells, resulting in a thinner mucus layer. Conversely, feeding mice an isocaloric glucose-free, high-protein diet increased mucosal energy supply that promoted colonic p32 level, goblet cell differentiation and mucus production.

Conclusion: We here describe a new molecular mechanism linking mucosal energy deficiency in UC to impaired, p32-dependent goblet cell differentiation that may be therapeutically prevented by nutritional intervention.

Keywords: C1QBP; Inflammatory Bowel Disease; Mitochondrial Function; Mucus Barrier.

Graphical abstract

Figure 1

UC patients in remission not receiving azathioprine display reduced colonic p32 level. (A) A model for energy generation and goblet cell differentiation in the colonic crypt and (B) schematic subcellular localization of proteins of interest were generated by modifying images from Servier Medical Art. (C) Representative immunohistochemistry staining of p32 (clone EPR8871), Tomm22, and KLF4 in paraffin-embedded human colonic biopsies. Scale bar = 50 μM. (D) p32 mRNA expression was measured by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) in colonic biopsies from non-IBD and UC patients in remission. (E) p32 exon expression was analyzed by TaqMan assay. Non-IBD: n = 10; UC: n = 7 and 6 for exon 1 and exon 1–2, respectively, and n = 8–9 for all other exon junctions. (F) Binding of TaqMan probes to the p32 plasmid was analyzed by qRT-PCR. (G) Intestinal p32 transcript expression was correlated against age. (H) p32 mRNA level in colonic biopsies from UC patients in remission treated with or without mesalazine, prednisolone, or azathioprine was measured by qRT-PCR. (I) Exemplary measurement of patients receiving azathioprine or control treatment applying TaqMan probes for every exon-exon junction. UC w/o azathioprine n = 5 for exon 1–2 and n = 7–8 for all other exons; UC with azathioprine n = 3–4. (J) Representative immunohistochemistry staining and corresponding quantification of p32 (clone EPR8871) expression in the upper part of the colonic crypt in paraffin-embedded biopsies from non-IBD control subjects and UC patients in remission. Scale bar = 10 μM. (D, E) Unpaired t test with Welch’s correction; (G) Spearman’s rank correlation coefficient; (H–J) unpaired t test; results are shown as (D, J) mean ± 95% CI, (E, I) box-and-whisker plot min to max, or (H) mean ± SD. ∗P ≤ .05, ∗∗P ≤ .01.

Figure 2

UC patients in remission display mucosal energy deficiency and impaired goblet cell differentiation. (A) Comparison of energy generation cells of the transit amplifying and differentiated cell zone. (B) L-lactate level were measured in serum or plasma samples and (C) Western blot experiments were performed in colonic biopsies from non-IBD control subjects and UC patients in remission. (D) Expression of transcripts of interest was performed by qRT-PCR in colonic biopsies from non-IBD control subjects and UC patients in remission. (E) Colonic p32 mRNA expression was correlated against KLF4 mRNA expression in non-IBD control subjects and UC patients in remission. (B) Unpaired t test; (D) unpaired t test with Welch’s correction; (E) Spearman’s rank correlation coefficient; results are shown as (B) mean ± 95% CI or (D) median ± interquartile range. ∗P ≤ .05, ∗∗∗∗P ≤ .0001.

Figure 3

Goblet cell loss correlates with inflammasome activation and decrease of full-length p32 level in active UC. (A) Schematic visualization of p32 cleavage by active Caspase-1. (B) p32 mRNA expression in paired biopsies from noninflamed and inflamed intestinal tissue sections were quantified by qRT-PCR. (C) Representative immunohistochemistry staining and corresponding quantification of p32 protein expression (clone EPR8871, anti-p32 exon 5) in the upper part of the colonic crypt in paraffin-embedded biopsies from noninflamed and inflamed colonic tissue sections from UC patients. Scale bar = 100 μM. (D) KLF4 mRNA expression was quantified by qRT-PCR in noninflamed colonic biopsies from UC patients in remission and inflamed colonic biopsies from UC patients with active disease. (E) Representative immunohistochemistry staining against pro-caspase-1 and p32 exon 6 as well as PAS-Alcian staining in paraffin-embedded tissue biopsies from the descending colon or sigma; #1: non-IBD noninflamed; #2.1: UC noninflamed; #3: UC low-grade inflammation; #2.2: UC medium-grade inflammation; #4: UC high-grade inflammation. Representative images from 8 biopsies each categorized as non-IBD control subjects or UC noninflamed and 5 UC inflamed samples are displayed. Scale bar = 50 μM. Results are shown as (C) mean ± 95% CI or (D) median ± interquartile range. i., inflamed; n.i., noninflamed.

Figure 4

UC patients in remission display reduced PAS-Alcian staining intensity. Follow-up of representative immunohistochemistry staining against pro-caspase-1 and p32 exon 6 as well as PAS-Alcian staining in paraffin-embedded tissue biopsies from the descending colon or sigmoid colon; #5 and #6: non-IBD noninflamed; #7 and #8: UC noninflamed.

Figure 5

Goblet cell differentiation is dependent on p32 expression. (A) Transcripts of goblet cell differentiation factors, mucins, and p32 were measured in colorectal cancer cell lines by qRT-PCR. (B) Graphical setup of cell culture experiments. (C) Western blot experiments were performed from whole protein extracts with respective antibodies in cells stimulated with 1.25-mM butyrate and/or 1-μg/mL lipopolysaccharide (LPS) (p32 clone EPR8871). (D) Basal OCR and ECAR were measured by Seahorse assay. (E) Representative image of HT29-MTX cell growth characteristics. (F) Cell counts are presented as fold change for each individual experiment. (G) Muc5AC levels in the supernatant were measured by ELISA, were normalized to cell count, and are displayed as fold change for each individual experiment. (H) KLF4 and MUC5AC immunohistochemistry staining was performed in paraffin-embedded butyrate-stimulated or control HT29-MTX cells. Scale bar = 10 μM. (I) p32 mRNA expression in butyrate-treated or untreated HT29-MTX cells was measured by qRT-PCR. (J, K) For siRNA knockdown, HT29-MTX cells were stimulated with 50-nM p32 siRNA or respective control for 96 hours and butyrate for 72 hours. (J) Representative Western blot and quantification from whole protein extracts (p32 clone 60.11) and (K) L-lactate level from corresponding cell culture supernatants. (D) Paired t test; (F, G) unpaired t test; (J, K) uncorrected Fisher’s test; data are shown as mean ± SD with the exception of (A) mean ± SEM. ∗P ≤ .05, ∗∗P ≤ .01.

Figure 6

Mucin secretion is dependent on energy supplied by mitochondrial respiration. (A) Graphical setup of cell culture experiments. (B) HT29-MTX cells were grown for 3 or 8 days. Muc5AC was measured by direct ELISA in cell culture supernatants, was normalized to cell count, and is shown as fold change to 3 days grown HT29MTX. (C) Seahorse measurement of HT29-MTX cells before and after 2 μM oligomycin injection. Muc5AC level in the cell culture supernatant after 24-hour stimulation with (D) oligomycin or (F) DNP were measured by ELISA and normalized to each control. Intracellular Muc5AC level of (E) oligomycin or (G) DNP-stimulated HT29-MTX cells were measured by direct ELISA in native protein isolates. Intracellular Muc5AC level was normalized to corresponding protein concentrations. (B) Unpaired t test; (D, F) 1-way analysis of variance with Tukey’s post hoc test for multiple comparisons; results are shown as mean ± SD. ∗P ≤ .05, ∗∗P ≤ .01, ∗∗∗P ≤ .001, ∗∗∗∗P ≤ .0001.

Figure 7

Mitochondrial dysfunction in mice is accompanied by defective goblet cell differentiation. (A) Schematic overview of the mutation in subunit 8 of the ATP synthase in ATP8 mutant mice and published metabolic imbalance.^, (B) Representative colonoscopy image (n = 3 mice per group). (C) Kc mRNA expression was measured by qRT-PCR. (D) Fecal IgA was determined by ELISA. (E) Mucosa-attached bacteria (MAB) were extracted from colonic tissue by hypotonic cell lysis, stained with a commercial bacterial stain, quantified by fluorescence-activated cell sorting analysis, and normalized to tissue weight. (F, I, L) Expression of transcripts of interest was performed by qRT-PCR in colonic biopsies from B6 WT and ATP8 mutant mice. Data were normalized to β-actin and are displayed as relative values to B6 WT mice for each sampling round. (G) Representative immunohistochemistry staining and according quantification of p32 (clone EPR8871) expression in paraffin-embedded colonic biopsies of B6 WT and ATP8 mutant mice (n = 3 mice per group). Scale bar = 50 μM (20×), 10 μM (63×). (H) L-lactate levels were measured in serum samples from B6 WT and ATP8 mutant mice. (J) Colonic p32 mRNA expression was correlated against klf4 mRNA expression in B6 WT and ATP8 mutant mice. (K) Representative PAS-Alcian and Muc2 fluorescent staining with according quantification in Carnoy’s fixed colonic tissue samples from B6 WT and ATP8 mutant mice. Scale bar = 50 μM (PAS-Alcian), 100 μM (Muc2 IF). Arrow indicates inner mucus layer. (F, G, I) Unpaired t test; (J) Spearman’s rank correlation coefficient; (K) unpaired t test with Welch’s correction; results are shown as (C) median with interquartile range, (D, E, G, K) mean ± SD, or (F, I, L) mean ± 95% CI. ∗P ≤ .05, ∗∗P ≤ .01.

Figure 8

GFHP diet increased mucosal energy supply, induced colonic p32 protein expression, and promoted goblet cell differentiation. (A) Hypothesized metabolic switch upon GFHP dietary intervention in mice. Weekly (B) food consumption and (C) mice body weight was determined (n = 6 from 2 independent experiments). (D) Glucose and (E) L-lactate level were measured in serum samples from control and GFHP diet mice. Expression of transcripts was measured (F) via TaqMan probes for p32 exon 3–4 or (I, K) by SYBR qRT-PCR in colonic biopsies from control and GFHP diet mice. (G, J) Representative p32 (clone EPR8871) and KLF4-Alcian blue immunohistochemistry staining of paraformaldehyde-fixed colonic tissue samples as well as MUC2 fluorescent staining of Carnoy’s fixed tissue are presented with corresponding quantifications. Scale bar = 100 μM (10×), 50 μM (20×), 10 μM (63×). Arrow indicates inner mucus layer. (H) Western blot experiment of whole protein extracts from colonic samples from control and GFHP diet mice (p32 clone EPR8871). (G, J) (thickness of inner mucus layer) unpaired t test; (I, J) (KLF4 staining) unpaired t test with Welch’s correction; results are shown as mean ± SD. ∗P ≤ .05.