Vitamin D-inducible calcium transport and gene expression in three Caco-2 cell lines

Abstract

The parental cell line (P) of Caco-2 cells and two clones, BBe and TC7, were studied at 11 days postconfluence to test the facilitated diffusion model of vitamin D-mediated intestinal calcium absorption (CaTx). Nuclear vitamin D receptor (nVDR) and calbindin D 9k (CaBP) were measured by Western blot; 1,25-hydroxyvitamin D 3 24-hydroxylase (CYP24), CaBP, plasma membrane Ca-ATPase (PMCA), and Ca transport channel (CaT1) mRNA levels were examined by RT-PCR; and net apical-to-basolateral CaTx was examined after treating cells with vehicle or 10 nM calcitriol for 8 (mRNA levels) or 48 h (protein, CaBP mRNA, CaTx). nVDR level was lowest in BBe (38% P value) and directly related to CYP24 induction (TC7 ϭ P, which were 1.56 times greater than BBe). nVDR was inversely related to the vitamin D-induced levels of CaT1 mRNA, CaBP mRNA, PMCA mRNA, and net CaTx, with the highest induction seen in BBe. Basal CaBP mRNA (86 times greater than P) and protein levels were highest in TC7 cells and were not associated with higher net CaTx, suggesting CaBP may not be rate limiting for CaTx in these cells.intestine; calcium absorption; calbindin D 9k; calcium transport channel; plasma membrane Ca-adenosinetriphosphatase INTESTINAL CALCIUM ABSORPTION and its regulation by vitamin D status have been studied for over 60 years (26). The active metabolite that controls this process is 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] (27). To date, two models have been proposed to explain this process: the facilitated diffusion model (2) and the vesicular model (23). In the facilitated diffusion model, summarized in Fig. 1, the basal rate of calcium uptake and calcium extrusion from the absorptive epithelial cell is proposed to be sufficient to accommodate the elevated rate of transport seen after vitamin D stimulation. In contrast, mathematical modeling shows that intracellular diffusion is the rate-limiting, as well as the most vitamin D responsive, step. Calbindin D 9k has been proposed as the protein that binds calcium and facilitates its diffusion between the apical and basolateral surfaces of the cell (8). The level of calbindin D 9k in the intestine is closely correlated with the efficiency of calcium absorption (2), and thus this protein plays a central role in the facilitated diffusion model. In the vesicular model, the cell uses lysosomes to sequester calcium and facilitate its movement to the basolateral membrane for extrusion (24). While calbindin D 28k (the form found in avian intestine) has been associated with lysosomes (25), the role of calbindin in this model is less clear. In the past, we have tested the facilitated diffusion model using cultures of the colonic carcinoma cell line Caco-2 (9, 11, 12, 32).Caco-2 cells are a human colon adenocarcinoma cell line that can spontaneously differentiate in culture and acquire a small intestinal phenotype, e.g., they become polarized columnar epithelial cells, they form tight junctions and domes, and they express several markers that are unique to differentiated small intestinal epithelium...

The epithelial calcium channels TRPV5 and TRPV6: regulation and implications for disease

“…The reduced renal TRPV5 expression and accompanied hypocalcemia were completely restored after 1,25(OH) 2 D 3 supplementation ). Similar results were obtained using different animal models and cell lines, including VDR and 1α-OHase knockout mice and the human intestinal cell line Caco-2, confirming the vitamin D-dependent regulation of TRPV5 and TRPV6 (Fleet et al 2002;Hoenderop et al 2002a;van Abel et al 2003;van Cromphaut et al 2001;Weber et al 2001;Wood et al 2001). Together these findings indicate that the expression of TRPV5 and TRPV6 is indeed controlled by 1,25(OH) 2 D 3 .…”

“…VDR presence was demonstrated in prostate epithelial cells and vitamin D analogues were shown to inhibit the growth of primary cultures of human prostate tissue and cancer cell lines (Miller et al 1992;Peehl et al 1994;Zhao et al 2000). As discussed previously, the positive regulation of TRPV6 by 1,25(OH) 2 D 3 and several analogues has been clearly established (Fleet et al 2002;Weber et al 2001;Wood et al 2001). Apoptosis is induced by either depleting the cell or by overloading the cell with Ca 2+ (Haverstick et al 2000;Nicotera and Orrenius 1998;Rabinovitch et al 2001).…”

Nucleo‐cytoplasmic cycling of the vitamin D receptor in the enterocyte‐like cell line, Caco‐2

“…In contrast we did not observe an increase in endogenous VDR protein abundance in parental Caco-2 cells. In addition, Figure 1B shows that ligand does not significantly alter the proportion of endogenous VDR in the nuclear extracts (1.2±0.2 after vitamin D treatment vs. 1.0±0.16 arbitrary units for control, P=0.4299, n=9) or in the whole cell extracts (P=0.9768, n=9) from Caco-2 cells, even though we have previously shown that this dose can activate CYP24 and TRPV6 mRNA accumulation in Caco-2 cells [Fleet et al, 2002]. …”

“…NIH-PA Author Manuscript NIH-PA Author Manuscript of critical proteins: calbindin D 9k (CaBPD9k), the transient receptor vanelloid family member 6 (TRPV6), and the plasma membrane calcium ATPase 1b (PMCA1b) [Fleet et al, 2002;Song et al, 2003b;Fleet, 2006].…”

“…The total protein concentration in the samples was determined using BioRad protein assay (Hercules, CA). The samples containing 20 µg of total protein were analyzed for VDR and general transcription factor II B(TFIIB) protein levels as described previously [Fleet et al, 2002]. The VDR protein level was normalized to TFIIB protein content in each sample to correct for protein loading.…”