Colonic TRPV4 overexpression is related to constipation severity

Abstract

Background: Chronic constipation is prevalent and involves both colon sensitivity and various changes in intestinal bacteria, particularly mucosa-associated microflora. Here we examined regulatory mechanisms of TRPV4 expression by co-culturing colon epithelial cell lines with intestinal bacteria and their derivatives. We also investigated TRPV4 expression in colon epithelium from patients with constipation.

Methods: Colon epithelial cell lines were co-cultured with various enterobacteria (bacterial components and supernatant), folate, LPS, or short chain fatty acids. TRPV4 expression levels and promoter DNA methylation were assessed using pyrosequencing, and microarray network analysis. For human samples, correlation coefficients were calculated and multiple regression analyses were used to examine the association between clinical background, rectal TRPV4 expression level and mucosa-associated microbiota.

Results: Co-culture of CCD841 cells with P. acnes, C. perfringens, or S. aureus transiently decreased TRPV4 expression but did not induce methylation. Co-culture with clinical isolates and standard strains of K. oxytoca, E. faecalis, or E. coli increased TRPV4 expression in CCD841 cells, and TRPV4 and TNF-alpha expression were increased by E. coli culture supernatants but not bacterial components. Although folate, LPS, IL-6, TNF-alpha, or SCFAs alone did not alter TRPV4 expression, TRPV4 expression following exposure to E. coli culture supernatants was inhibited by butyrate or TNF-alphaR1 inhibitor and increased by p38 inhibitor. Microarray network analysis showed activation of TNF-alpha, cytokines, and NOD signaling. TRPV4 expression was higher in constipated patients from the terminal ileum to the colorectum, and multiple regression analyses showed that low stool frequency, frequency of defecation aids, and duration were associated with TRPV4 expression. Meanwhile, incomplete defecation, time required to defecate, and number of defecation failures per 24 h were associated with increased E. faecalis frequency.

Conclusions: Colon epithelium cells had increased TRPV4 expression upon co-culture with K. oxytoca, E. faecalis, or E. coli supernatants, as well as TNFα-stimulated TNFαR1 expression via a pathway other than p38. Butyrate treatment suppressed this increase. Epithelial TRPV4 expression was increased in constipated patients, suggesting that TRPV4 together with increased frequency of E. faecalis may be involved in the pathogenesis of various constipation symptoms.

Keywords: Chronic constipation; Colon; E. coli; E. faecalis; TRPV4.

Fig. 1

Effect of co-culturing CCD841 cells with bacterial suspensions on TRPV4 expression. A Decreased TRPV4 mRNA expression following 1 day co-culture of CCD841 cells with P. acnes, C. perfringens or S. aureus (clinical strain; black bars). Co-culture with K. oxytoca, E. faecalis or E. coli (white bars) for 7 days increased TRPV4 mRNA expression. Co-culture with the other types of bacteria (gray bars) did not affect TRPV4 expression. Asterisks indicate significant change in expression relative to untreated control cells. Error bars represent the mean ± S.E. of five to six trials. B For bacteria that decreased TRPV4 expression after one day of co-culture, suppression of TRPV4 expression returned after 3 days and 7 days of co-culture. C Increased TRPV4 expression in CCD841 cells co-cultured with K. oxytoca, E. faecalis or E. coli (standard strain) for 7 days

Fig. 2

Effect of co-culture with bacterial supernatants or components on cytokine expression in CCD184 cells. A Exposure of CCD841 cells to K. oxytoca supernatant (SN) for 1 day increased TRPV4 expression, but co-culture with K. oxytoca bacterial components (BC) for 1 day did not. Exposure to E. coli (O111) SN for 3 days increased TRPV4 expression, but E. coli BC for 3 days did not. Exposure to either E. faecalis SN or BC for 1 day increased TRPV4 expression. B Treatment with folic acid, LPS (1 ng ~ 20 µg/ml), or SCFA (butyrate, acetate, and propionate) at any concentration for 3 days had no effect on TRPV4 expression in CCD841 cells. C Human IL-6 or TNF-alpha at any concentration or any duration (6, 8 or 24 h) did not affect TRPV4 expression in CCD841. D Exposure to K. oxytoca or E. coli SN for 1 day increased TNF-alpha expression in CCD841 cells, but E. faecalis SN and BC for 1 day did not. * indicates significant difference from untreated control. Error bars represent the mean ± S.E. of five to six trials

Fig. 3

Bacterial supernatants affect gene expression in TNF-alpha signaling pathways. Exposure of CCD841 cells to E. coli (O111) supernatant (SN) increased relative amounts of TRPV4, TNF-alpha, TNFR1, TNFR2, IL-1, IL-6, NOD2, and MMP mRNA. # indicates significant difference (p < 0.05) between treated and untreated control cells. Error bars represent the mean ± S.E. of five to six trials

Fig. 4

Effect of E. coli. O111 supernatant on TRPV4 protein expression in CCD841 cells. Western blot of CCD841 cells co-cultured with E. coli (O111) supernatant (SN) for 3 days shows a significant increase in TRPV4 protein expression relative to untreated control cells (*p < 0.05). Error bars represent the mean ± S.E. of five to six trials. The rightmost lane of the blot shows the ladder

Fig. 5

Effect of TNF-alpha inhibitors on TRPV4 expression in CCD841 cells co-cultured with bacterial supernatants. Combination exposure of CCD841 cells to E. coli (O111) supernatant (SN) for 3 days. Butyrate or TNFaR1 inhibitor (TNFaR1I) inhibited TRPV4 expression induced by SN exposure, but the p38 inhibitor SB202190 at 10 μM (P38I) or 100 μM (P38*10) did not. # indicate significant differences from untreated control. Error bars indicate the mean ± S.E. of five to six trials

Fig. 6

TRPV4 expression in intestinal tissues from patients with chronic constipation is increased relative to healthy controls. Patients with chronic constipation (CC, n = 5) have increased TRPV4 expression in the terminal ileum (TI), cecum (Cec), sigmoid colon (Sig), and rectum (Rec) compared to healthy subjects (n = 9); the increase in the rectum was significant (#p < 0.05; n = 40). Error bars indicate the mean ± S.E