Expression of Cav1.3 calcium channel in the human and mouse colon: posttranscriptional inhibition by IFNγ

Abstract

It has been hypothesized that apically expressed L-type Ca²⁺ channel Ca_v1.3 (encoded by CACNA1D gene) contributes toward an alternative TRPV6-independent route of intestinal epithelial Ca²⁺ absorption, especially during digestion when high luminal concentration of Ca²⁺ and other nutrients limit TRPV6 contribution. We and others have implicated altered expression and activity of key mediators of intestinal and renal Ca²⁺ (re)absorption as contributors to negative systemic Ca²⁺ balance and bone loss in intestinal inflammation. Here, we investigated the effects of experimental colitis and related inflammatory mediators on colonic Ca_v1.3 expression. We confirmed Ca_v1.3 expression within the segments of the mouse and human gastrointestinal tract. Consistent with available microarray data (GEO database) from inflammatory bowel disease (IBD) patients, mouse colonic expression of Ca_v1.3 was significantly reduced in trinitrobenzene sulfonic acid (TNBS) colitis. In vitro, IFNγ most potently reduced Ca_v1.3 expression. We reproduced these findings in vivo with wild-type and Stat1^-/- mice injected with IFNγ. The observed effect in Stat1^-/- suggested a noncanonical transcriptional repression or a posttranscriptional mechanism. In support of the latter, we observed no effect on the cloned Ca_v1.3 gene promoter activity and accelerated Ca_v1.3 mRNA decay rate in IFNγ-treated HCT116 cells. While the relative contribution of Ca_v1.3 to intestinal Ca²⁺ absorption and its value as a therapeutic target remain to be established, we postulate that Ca_v1.3 downregulation in IBD may contribute to the negative systemic Ca²⁺ balance, to increased bone resorption, and to reduced bone mineral density in IBD patients.

Keywords: calcium absorption; calcium channel; inflammation; interferon; intestine.

Fig. 1.

Expression of Ca_v1.3 in the murine and human gastrointestinal tract. qPCR detection of Ca_v1.3 mRNA expression in different segments of the mouse (normalized to TBP) (A) and human (normalized to GAPDH) (B) gastrointestinal tract. In both species, renal expression was used as a calibrator. C and D: immunofluorescent detection of Ca_v1.3 in the mouse colon. Red, Ca_v1.3; blue, nuclei (DAPI). E: Ca_v1.3 protein expression in the human colon (image credit: Human Protein Atlas (34); cropped image from http://www.proteinatlas.org/ENSG00000157388-CACNA1D/tissue/colon).

Fig. 2.

Mucosal expression of Ca_v1.3 mRNA in experimental colitis. A: representative hematoxylin and eosin staining of colonic mucosa from control and TNBS-treated mice. B: qPCR detection of Ca_v1.3 mRNA expression in the colonic mucosa of control and TNBS-treated mice. N = 3 for control, N = 5 for TNBS. *P = 0.02, Student's t-test.

Fig. 3.

Mucosal expression of Ca_v1.3 protein in experimental colitis. Immunofluorescent detection of Ca_v1.3 in the mouse colon. Red, Ca_v1.3; blue, nuclei (DAPI).

Fig. 4.

Effects of proinflammatory cytokines on Ca_v1.3 mRNA and protein expression in vitro and in vivo. A: qPCR analysis of Ca_v1.3 mRNA expression in HCT116 colonic adenocarcinoma cells treated for 24 h with IFNγ (100 U/ml), IL1β (2 ng/ml), and TNFα (10 ng/ml) individually or in combination (cytomix). Data were analyzed with 1-way ANOVA followed by Fisher PLSD post hoc test. *Differences between respective treatment and control (Ctrl; P < 0.05). B: colonic expression of Ca_v1.3 mRNA and protein in WT or Stat1^−/− mice injected with PBS or IFNγ (50,000 units/mouse ip daily for 3 days). qPCR data normalized to PBS-injected controls for each genotype. *Differences between respective treatment and control (P < 0.05). Western blotting summary with a representative image of Ca_v1.3 and β-actin expression; P value of Student's t-test indicated.

Fig. 5.

Posttranscriptional mechanism of reduced Ca_v1.3 mRNA expression in HCT116 cells treated with IFNγ. A: HCT116 cells were treated with ActD to inhibit de novo transcription and treated with 100 U/ml of IFNγ for 24 h. *Differences between IFNγ and control (P < 0.05). B: 4 deletion constructs of the cloned murine Ca_v1.3 gene promoter in Firefly luciferase (Luc) reporter vector pGL-basic (Promega), or promoterless vector, were cotransfected with Renilla luciferase vector pRL-null into HCT116 cells and treated with control medium or with 100 U/ml of IFNγ for 24 h. All tested promoter constructs were functional, but no significant effects of IFNγ were detected.

Fig. 6.

IFNγ accelerates Ca_v1.3 mRNA decay in colonic epithelial cells. HCT116 cells were treated with ActD to inhibit de novo gene transcription in control or IFNγ-supplemented medium (100 U/ml). Cells were harvested at 2, 4, 8, 12, and 24 h into treatment and expression of Ca_v1.3 mRNA was analyzed by qPCR. qPCR data were normalized to GAPDH transcript, with time “zero” as the calibrator. A t-test at individual time points was used for statistical analysis. *Differences between IFNγ and control (P < 0.05).