Vitamin D increases tight-junction conductance and paracellular Ca2+ transport in Caco-2 cell cultures

Mary V. Chirayath, Leszek Gajdzik, Wolfgang Hulla, Jürg Graf, Heide S. Cross, and Meinrad Peterlik

Department of General and Experimental Pathology, University of Vienna, A-1090 Vienna, Austria

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

We investigated the effects of 1alpha ,25-dihydroxyvitamin D3 [1,25(OH)2D3] on paracellular intestinal Ca2+ absorption by determination of transepithelial electric resistance (TEER), as a measure of tight-junction ion permeability and bidirectional transepithelial 45Ca2+ fluxes in confluent Caco-2 cell cultures. The rise of TEER to steady-state levels of ~2,000 Omega  · cm2 was significantly attenuated by 1,25(OH)2D3 (by up to 50%) in a dose-dependent fashion between 10-11 and 10-8 M. Synthetic analogs of 1,25(OH)2D3, namely, 1alpha ,25-dihydroxy-16-ene,23-yne-vitamin D3 and 1alpha ,25-dihydroxy-26,27-hexafluoro-16-ene,23-yne-vitamin D3, exhibited similar biopotency, whereas their genomically inactive 1-deoxy congeners were only marginally effective. Enhancement of transepithelial conductance of Caco-2 cell monolayers by vitamin D was accompanied by a significant increase in bidirectional transepithelial 45Ca2+ fluxes. Although 1,25(OH)2D3 also induced cellular 45Ca2+ uptake from the apical aspect of Caco-2 cell layers and upregulated the expression of calbindin-9kDa mRNA, no significant contribution of the Ca2+-adenosinetriphosphatase-mediated transcellular pathway to transepithelial Ca2+ transport could be detected. Therefore stimulation of Ca2+ fluxes across confluent Caco-2 cells very likely results from a genomic effect of vitamin D sterols on assembly and permeability of tight-junctional complexes.

intestinal calcium absorption; 1alpha ,25-dihydroxyvitamin D3; synthetic vitamin D compounds; vitamin D receptor; genomic action; ionic conductance; cellular calcium uptake; calbindin-9kDa; calcium-adenosinetriphosphatase

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

IT IS STILL a matter of debate as to whether Caco-2 cells display vitamin D-dependent Ca2+ transport. Giuliano and Wood (21) have reported a stimulatory effect of the steroid hormone on Ca2+ transport in the mucosal-to-serosal direction but did not address the question of whether the vitamin D-dependent Ca2+ binding protein, or calbindin, which is the sole mediator of vitamin D-dependent transcellular Ca2+ transport (for review, see Ref. 8) is involved in this process. Furthermore, Bindels et al. (4) presented evidence that vitamin D also increases Ca2+ transport to the same extent in the serosal-to-mucosal direction, so that its effect on net transepithelial Ca2+ transport was zero. These conflicting views could be reconciled if one assumes that vitamin D-dependent Ca2+ transport across Caco-2 cell monolayers as measured by both groups proceeds mainly on a paracellular route. In this respect, we and others (12, 16) have previously shown that 1alpha ,25-dihydroxyvitamin D3 [1,25(OH)2D3] in fact is able to increase paracellular ion permeability of the intestinal epithelium.

It therefore appeared worthwile to reexamine the effect of vitamin D on intercellular ion permeability in Caco-2 cells to dissect the transcellular from the paracellular route of transepithelial transport.

    EXPERIMENTAL METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Caco-2 cells. The Caco-2 cell clone AQ, which was used in the present study, was originated in our laboratory by subcloning of an established clone, Caco-2/15 (cf. Ref. 3) after passage 100 by dilution plating. The population doubling time of the Caco-2/AQ clone during the logarithmic growth phase was estimated as 24 h (vs. 36 h of Caco-2/15 clone). The activity of the differentiation marker, alkaline phosphatase, increased during 20 days of confluent growth from an average of 20 to 60 mU/mg cellular protein in Caco-2/AQ cells, whereas the respective values for the parent clone Caco-2/15 were 25 and 190 mU/mg protein.

Cell culture. Caco-2/AQ cells (between passages 20 and 50) were grown either in 24-well Falcon culture plates (Becton-Dickinson, Bedford, MA) or on filters with 0.4-µm pore size (Falcon cell culture inserts), as appropriate, in Dulbecco's modified Eagle's medium [supplemented with 10% fetal calf serum, 4.0 mM glutamine, 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 50 U/ml penicillin, and 50 µg/ml streptomycin] at 37°C in a 5% CO2-95% air atmosphere.

Vitamin D compounds were dissolved in ethanol and added to cultures so that the final solvent concentration in the medium did not exceed 0.01%.

Measurement of transepithelial electrical resistance. Transepithelial electrical resistance (TEER) of the Caco-2 monolayers was measured by a high-precision technique as described previously in detail (30). Current pulses of 55 µA, 0.5-s duration, were passed across the monolayers with Ag-AgCl electrodes from an ESCOM 486 SX computer equipped with a high-performance Labcard (PCL-818). Resulting voltages were recorded with the aid of a differential amplifier with a high input resistance. Data were corrected for well area (given in Omega  · cm2).

Transport studies. If not indicated otherwise, the medium used for determination of transepithelial transport of 45Ca2+, 86Rb+, [14C]mannitol, or [14C]inulin, respectively, contained (in mM) 134 NaCl, 4.1 KCl, 2.1 CaCl2, 1.0 KH2PO4, 1.0 MgSO4, and 12.3 HEPES. pH was adjusted to 7.4 with 1.0 N NaOH. Specific activity of radiotracers was 0.5 µCi/ml.

Transepithelial transport across confluent Caco-2 cells was evaluated as described by Giuliano and Wood (21). Briefly, at time 0, transport buffer containing the radiolabeled solute at an appropriate concentration, i.e., 2.1 mM 45Ca2+, 0.1 mM 86Rb+, 1.0 mM [14C]mannitol, or 36 µM [14C]inulin, respectively, was filled into the filter well (1.0 ml) or the outside compartment (3.0 ml) of the filter unit as appropriate. In each case, the concentration of the solute under investigation in the contralateral compartment was zero. The filter plates were shaken horizontally at a frequency of 50 oscillations/min at room temperature for 60 min. Linearity of transport rates was monitored by determination of radioactivity in 20-µl aliquots drawn from the contralateral solutions at 15-min intervals.

Uptake studies. Caco-2 cells grown in 24-well plates were allowed to equilibrate with room temperature for 30 min before experimentation. After aspiration of the culture medium, 0.5 ml of a "low-sodium" mannitol buffer containing 45Ca2+ (0.5 µCi/ml) was added into each well. The buffer composition was (in mM) 198 mannitol, 25 KCl, 1.2 NaH2PO4, 25 NaHCO3, 1.2 MgSO4, 0.25 CaCl2, and 20 glucose (24). The uptake experiment was carried out for 10 min under horizontal shaking (50 oscillations/min) at room temperature. For termination of uptake, the transport medium was sucked off, and the cells were washed three times with 1.0 ml of ice-cold phosphate-buffered saline (pH 7.4). Cells were then suspended in 1.0 ml of 1.0 N NaOH and allowed to solubilize by overnight standing at 4°C.

Calbindin-9kDa mRNA isolation and Northern blotting. Total RNA was prepared from cells grown on filters in six-well plates until day 15 according to Ref. 11; 20 µg were used for Northern blotting (as described in Ref. 2). There was no obvious need for the use of semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) as performed by Fleet and Wood (19), since specific message was sufficient for conventional Northern analysis of calbindin-9kDa (CaBP-9kDa) mRNA expression. Probes for human (h) CaBP-9kDa were generated by RT-PCR. The following primers were selected from hCaBP-9kDa cDNA: Cal9.1.1 (1. for coding), <UNL>CAACCAGACA</UNL> <UNL>CCAGAATGAG</UNL>; Cal9.2.1 (1. for coding), <UNL>GTCTCCTGAG</UNL> <UNL>GAACTGAAGA</UNL>; Cal9.3.0 (0. for reverse), <UNL>GACCGACACT</UNL> <UNL>CCAAGTCAAT</UNL>; Cal9.4.0 (0. for reverse), <UNL>TAACTGAACC</UNL> <UNL>TCACAGCCAG</UNL>. The primers' specificity was determined by searching data bases with the respective sequences (FASTA), and primers with 100% homology to only hCaBP-9kDa were accepted. Products of ~380 and 290 bp with primers Cal9.1. and Cal9.4. ("outside") and Cal9.2. and Cal9.3. ("inside"), respectively, were obtained after 30 cycles. The longer PCR product was then reamplified with the inside primers. The expected length together with a characteristic restriction fragment pattern definitely identified the product as hCaBP-9kDa (RT-PCR from human brain cDNA yielded no product and thus served as a negative control). The PCR product was cloned into a pCRII vector (Invitrogen) and used as probe for the expression of hCaBP-9kDa mRNA thereafter.

Data presentation and statistical analysis. In each experimental series, at least three separate experiments were performed. Data are presented as means ± SE. Differences were considered statistically significant at the 5% confidence level with P values calculated by Student's unpaired t-test.

Materials. 25-Hydroxyvitamin D3 [25(OH)D3] was purchased from Sigma (Deisenhofen, Germany). 1alpha ,25-Dihydroxyvitamin D3 was generously supplied by Hoffmann-LaRoche (Basel, Switzerland). Synthetic vitamin D analogs, 25-hydroxy-16-ene,23-yne-vitamin D3 [25(OH)-16ene,23yne-D3], 25-hydroxy-26,27-hexafluoro-16-ene,23-yne-vitamin D3 [25(OH)-26,27-F6-16ene,23yne-D3], 1alpha ,25-dihydroxy-16-ene,23-yne-vitamin D3 [1,25(OH)2-16ene,23yne-D3], and 1alpha ,25-dihydroxy-26,27-hexafluoro-16-ene,23-yne-vitamin D3 [1,25(OH)2-26,27-F6-16ene,23yne-D3], were a generous gift from Dr. Milan R. Uskokovic' (Roche, Nutley, NJ). D-[1-14C]mannitol (sp act 2.1 GBq/mmol), 45CaCl2, and 86RbCl were purchased from New England Nuclear (Vienna, Austria) and [carboxyl-14C]inulin (sp act 2.05 mCi/g) from ARC (St. Louis, MO).

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Development of paracellullar permeability in confluent Caco-2 cells. Mucosal-to-serosal transepithelial transport of extracellular markers, namely, [14C]inulin (~5,000 mol wt) and [14C]mannitol (182.2 mol wt), was determined in parallel to 45Ca2+ transfer across confluent Caco-2 cell layers. As shown in Fig. 1, exposure of Caco-2 cells to 10-8 M 1,25(OH)2D3 for 2 wk past confluence had a distinct effect on the extent to which extracellular markers of different molecular weight could penetrate the Caco-2 cell layer. Although exposure to the steroid hormone had no effect whatsoever on transfer of the high molecular weight compound inulin, a small but significant vitamin D-related increment of transport of the considerably smaller molecule mannitol could be observed. Expectedly, 1,25(OH)2D3 elicited an approximately threefold rise in transepithelial transport of 45Ca2+ (Fig. 1).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1.   Time course of [14C]inulin (star , black-lozenge ), [14C]mannitol (triangle , black-triangle), and 45Ca2+ (square , black-square) transfer across confluent Caco-2 cells and effect of 1alpha ,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Cells were grown until day 14 past confluence. Solid symbols, presence of 10-8 M 1,25(OH)2D3 in culture medium from day 1 after confluence on; open symbols, untreated controls. Concentrations in transport buffer (apical compartment only): inulin, 36 µM; mannitol, 1.0 mM; Ca2+, 2.1 mM. Data from a typical experiment are shown as means from 6 determinations ± SE (vertical bars).

This observation suggested to us that 1,25(OH)2D3 could attenuate the closure of intercellular junctions during postconfluent differentiation to such an extent that the passage of bulky extracellular markers such as inulin would be severely restricted, whereas smaller molecules and ions such as mannitol, or even more importantly Ca2+, could still traverse the cell layer on a paracellular route.

To substantiate this notion, we measured TEER of confluent Caco-2 cell layers as a highly sensitive parameter of paracellular permeability simultaneously with mucosal-to-serosal transepithelial transport of [14C]mannitol and 45Ca2+ at different time points after confluence.

Figure 2A shows that 1,25(OH)2D3 markedly retards the development of TEER of Caco-2 cell layers during postconfluent growth. A significant effect of the sterol becomes visible at day 4. Figure 2B shows that the rate of [14C]mannitol is inversely related to the development of TEER (32). Consequently, treatment with 1,25(OH)2D3 made Caco-2 cells to a small but significant extent (P < 0.05) more permeable to [14C]mannitol (Fig. 2B). A similar relation to TEER was observed in the case of transepithelial 45Ca2+ transport (Fig. 2C). The ~50% reduction of TEER observed in 1,25(OH)2D3-treated Caco-2 cultures between days 10 and 15 past confluence (Fig. 2A) was accompanied by an about threefold increase in the amount of mucosal-to-serosal Ca2+ transfer (Fig. 2C).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2.   Development of transepithelial electric resistance (TEER) and transepithelial transport in postconfluent Caco-2 cells: effect of 1,25(OH)2D3. black-square, 10-8 M 1,25(OH)2D3 in culture medium from day of confluence on; square , untreated controls. Data from a typical experiment are shown as means from 6 determinations ± SE (vertical bars). A: TEER. B: mucosal-to-serosal [14C]mannitol transport. C: mucosal-to-serosal 45Ca2+ transport.

A dose-response study was carried out at two different time points after Caco-2 cells reached confluence (Fig. 3). On day 4, a significant reduction of TEER and a concomitant rise in Ca2+ transport was observed only in cells exposed to the highest 1,25(OH)2D3 concentration tested, i.e., 10-8 M, whereas, on day 12, linear dose responses were observed at sterol concentrations between 10-10 and 10-8 M. 


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3.   Dose-response relations of 1,25(OH)2D3 effects on TEER (A) and mucosal-to-serosal 45Ca2+ transport (B) in confluent Caco-2 cells. bullet , Day 4 past confluence; black-square, day 12 past confluence. Data are means from 6 determinations and are expressed as percentage of untreated control.

A plot of transport rate vs. conductance (i.e., 1/resistance) generated from single measurement values at various time points after confluence (Fig. 4), yielded identical curves for both 1,25(OH)2D3-treated and untreated Caco-2 cell layers. The absence of any vitamin D-related transport increment at a given conductance value strongly indicates that 1,25(OH)2D3 had no visible influence on apical-to-basolateral Ca2+ transfer other than on the paracellular route.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 4.   Transepithelial conductance and apical-to-basolateral (A) and basolateral-to-apical 45Ca2+ transport (B) in confluent Caco-2 cells. open circle , No addition to culture; bullet , 1,25(OH)2D3 (10-8 M) in culture medium from day of confluence on. TEER and Ca2+ transport were measured at various time points after confluence (cf. legend to Fig. 2). Each data point represents a single measurement. A and B each contain cumulative data from 3 separate experiments.

If transepithelial Ca2+ transport as measured is solely determined by the capacity of the paracellular route, then Ca2+ transfer both in the apical-to-basolateral and in the opposite direction should occur to an equal extent. In some experiments, we measured also the influence of 1,25(OH)2D3 on basolateral-to-apical transepithelial Ca2+ transport. At any time point, transport rates across either 1,25(OH)2D3-treated or untreated Caco-2 cell layers were equal to respective flux rates in the opposite direction (cf. also Table 1). Again, an identical relationship between Ca2+ transport rates and conductance as measure of paracellular permeability was observed in both hormone-treated and control groups (Fig. 4).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Short-term effect of 1,25(OH)2D3 on TEER and bidirectional 45Ca2+ fluxes across confluent Caco-2 cell layers

Effect of 1,25(OH)2D3 analogs on TEER of confluent Caco-2 cells. Caco-2 cells respond not only to 1,25(OH)2D3 but also to a number of its synthetic analogs by changes in growth and morphological appearance, provided that those compounds bear a 1alpha -hydroxy group (5), which is a prerequisite for binding to the vitamin D receptor (VDR) and hence for genomic activity. We therefore tested the effect of 1,25(OH)2D3 and of two potent antimitogenic compounds, 1,25(OH)2-16ene,23yne-D3 and 1,25(OH)2-26,27-F6-16ene,23yne-D3, as well as of the respective 1alpha -deoxy compounds, [25(OH)D3, 25(OH)-16ene,23yne-D3], and 25(OH)-26,27-F6-16ene,23yne-D3, on TEER and transepithelial Ca2+ transport across Caco-2 cell layers. Figure 5 depicts the effect of the vitamin D compounds at 10-8 M on the development of TEER of postconfluent Caco-2 cell layers. Although 25(OH)D3, with the exception of day 5, had no significant effect, 25(OH)-16ene,23yne-D3 and 25(OH)-26,27-F6-16ene,23yne-D3 from day 8 or 5 on, respectively, significantly reduced TEER by an average of <= 10%. In contrast, 1,25(OH)2D3 and both synthetic 1alpha -hydroxylated analogs reduced TEER to <= 50% of control levels at any time point. It should be noted that the decrement in TEER induced by the synthetic 1alpha -hydroxyvitamin D compounds tended to be even higher than that induced by 1,25(OH)2D3.


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of vitamin D compounds on TEER of confluent Caco-2 cells. Vitamin D sterols were present in culture medium at 10-8 M from confluence on. Data are means ± SE (n = 6) from a typical experiment. If not labeled NS (not significant), treatment groups were statistically different from control group at least at P < 0.05. 25(OH)D3, 25-hydroxyvitamin D3; 25(OH)-16ene,23yne-D3, 25-hydroxy-16-ene,23-yne-vitamin D3; 25(OH)-26,27-F6-16ene,23yne-D3, 25-hydroxy-26,27-hexafluoro-16-ene,23-yne-vitamin D3; 1,25(OH)2-16ene,23yne-D3, 1alpha ,25-dihydroxy-16-ene,23-yne-vitamin D3; 1,25(OH)2-26,27-F6-16ene,23yne-D3, 1alpha ,25-dihydroxy-26,27-hexafluoro-16-ene,23-yne-vitamin D3.

Time course of 1,25(OH)2D3 effects on TEER and transepithelial Ca2+ transport. The increase in TEER during growth of confluent Caco-2 cells in all likelihood reflects the development of tight junctions (cf. Ref. 28). It was therefore of interest to know whether vitamin D would affect not only the assembly of intercellular junctions during confluent cell growth but also influence their barrier function in a more advanced state of development. Thus, in another series of experiments, Caco-2 cells were allowed to grow for 12 days past confluence before treatment with 1,25(OH)2D3 was begun. Table 1 shows that, after 48-72 h, a highly significant reduction of TEER with a concomitant rise in transepithelial Ca2+ transport could be observed. It should be noted that Ca2+ transport in the mucosal-to-serosal as well as in the opposite direction was influenced by the hormone to the same extent (Table 1).

Effect of 1,25(OH)2D3 on cellular 45Ca2+ uptake and CaBP-9kDa mRNA expression. To evaluate a possible contribution of the transcellular route to vitamin D-related transepithelial Ca2+ transport as measured, we determined the effect of 1,25(OH)2D3 and analogs on Ca2+ uptake by Caco-2 cells at different growth stages (Fig. 6). Basal cellular Ca2+ uptake in vitamin D-free control cultures conspicuously increased during transition from the log growth phase into the confluent state. During this time period, the 1alpha -hydroxylated vitamin D compounds under investigation were most effective in raising cellular 45Ca2+ accumulation, whereas the 1alpha -deoxy compound, 25(OH)-16ene,23yne-D3, had no effect at all.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of vitamin D compounds on cellular uptake of 45Ca2+ by Caco-2 cells at different growth stages. Vitamin D sterols were present in culture medium at 10-8 M from confluence on. open circle , No treatment; star , 25(OH)-16ene,23yne-D3; black-square, 1,25(OH)2D3; black-triangle, 1,25(OH)2-16ene,23yne-D3; bullet , 1,25(OH)2-26,27-F6-16ene,23yne-D3. Data are means ± SE (n = 6) from a typical experiment.

In another experiment, the effect of 1,25(OH)2D3 on cellular Ca2+ accumulation was studied in confluent filter-grown Caco-2 cells, which were selectively exposed to the radiotracer 45Ca2+ either on their apical (i.e., mucosal) or basolateral (i.e., serosal) aspect, respectively. As expected, 1,25(OH)2D3, when added on day 12 past confluence at 10-8 M, after 72 h raised cellular 45Ca2+ accumulation due to uptake from the mucosal compartment from 6.8 ± 0.04 to 15.2 ± 0.3 nmol · h-1 · well-1 (n = 6, P < 0.01) but had no significant effect on uptake from the contralateral compartment, which was raised from 18.0 ± 1.8 to only 21.4 ± 0.3 nmol · h-1 · well-1 (n = 6, P > 0.05).

In the same experiment, we determined CaBP-9kDa mRNA levels by Northern blot analysis (Fig. 7). Caco-2 cells were able to express CaBP-9kDa message even in the absence of 1,25(OH)2D3. As expected (cf. Refs. 18, 19), a two- to threefold rise in mRNA levels was induced by 10-9 to 10-8 M 1,25(OH)2D3 within 48-72 h.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 7.   Northern blot analysis of CaBP-9kDa mRNA in confluent Caco-2 cells treated with 1,25(OH)2D3. Data are expressed in arbitrary densitometric units normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA. Open bars, untreated control; solid bars, 1,25(OH)2D3-treated groups. A: time course of hormone effect at 10-8 M; B: dose-response relationship. Duration of hormone treatment: 72 h.

Ca2+-adenosinetriphosphatase inhibition and transepithelial Ca2+ transport. Because the last step of mucosal-to-serosal transcellular Ca2+ transport involves extrusion of Ca2+ across the basolateral aspect of the cell by the Ca2+-adenosinetriphosphatase (ATPase), we sought to evaluate the contribution of active Ca2+ pumping to net transepithelial transport by blocking the activity with a potent inhibitor, calmidazolium. The data collated in Table 2 show that, apart from the fact that pretreatment with the inhibitor had no influence on TEER in either controls or 1,25(OH)2D3-treated Caco-2 cells, a block of the Ca2+ pump did not change the extent of basal mucosal-to-serosal transepithelial Ca2+ transfer as measured but, even more important, by no means reduced its 1,25(OH)2D3-related increment.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Ca2+-ATPase inhibition and transepithelial 45Ca2+ transport in Caco-2 cells

1,25(OH)2D3 and transepithelial 86Rb+ transport. Further proof for the assumption that 1,25(OH)2D3 can modulate ion transport on the paracellular route was obtained when we measured transepithelial 86Rb+ transport across confluent Caco-2 cell layers (Table 3). 86Rb+ is widely used as a substitute for K+ for assessment of Na+-K+-ATPase activity in whole cell preparations. It should be noted that, under the experimental conditions employed, transepithelial 86Rb+ transport was completely insensitive to ouabain treatment (Table 3). Because this excludes any contribution from the Na+-K+-ATPase-mediated transcellular pathway, 86Rb+ transport as measured mainly reflects ion flux on a paracellular route. The data collated in Table 3 therefore strongly suggest that stimulation of 86Rb+ transport across confluent Caco-2 cell layers in the serosal-to-mucosal direction by the steroid hormone occurs in parallel with reduction of TEER during postconfluent cell growth.

                              
View this table:
[in this window]
[in a new window]
 
Table 3.   Effect of 1,25(OH)2D3 on paracellular 86Rb+ flux and TEER in confluent Caco-2 cells

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Caco-2 cells, though originally derived from a human colon adenocarcinoma, are still able to undergo spontaneous differentiation into enterocyte-like cells. Thereby Caco-2 cells form confluent monolayers consisting of well-polarized cells with tight junctions and a typical apical brush border (28).

It is generally accepted that transepithelial electric conductance across Caco-2 cell layers is mainly determined by the ionic permeability of the intercellular junctions which develop during postconfluent cell growth (28). The fact that the paracellular route is the predominant pathway of transepithelial ion flux can be inferred from the observations that ~80% of total electrical resistance of Caco-2 cells is located in the mucosal membrane and that, in addition, its Na+ conductance is very limited (22). Changes in TEER can also not be explained by activation of Na+-D-glucose cotransport, because the Caco-2 cell clone used in the present study is devoid of any Na+-dependent D-glucose transport activity (10, 22), and in addition, no D-glucose was present in the incubation medium. Thus the decline in TEER largely reflects an effect on tight junction-mediated paracellular ion permeability (27). The present study documents that 1,25(OH)2D3 as well as its synthetic D-ring and side-chain-modified analogs substantially reduce TEER of confluent Caco-2 cells. This must be considered as clear evidence for the ability of genomically active vitamin D compounds to increase bidirectional paracellular flux of all ion species including Ca2+. In this respect, it is interesting to note that Favus et al. (16) had observed that 1,25(OH)2D3 caused a significant increase of tissue conductance and, most notably, also of bidirectional mannitol fluxes in the duodenum and descending colon of rats, whereas Cross et al. (12) reported on stimulation of paracellular ion transport, i.e., Na+, K+, and Rb+, in organ-cultured embryonic chick small intestine.

Caco-2 cells express VDR mRNA and protein during the log growth phase as well as after confluence (20, 23). This is apparently the basis for the action on paracellular ion permeability of vitamin D compounds, since all 1alpha -deoxyvitamin D compounds under investigation were either completely ineffective in reducing TEER, namely, 25(OH)-D3, or, like the two synthetic compounds, 25(OH)-16ene,23yne-D3 and 25(OH)-26,27-F6-16ene,23yne-D3, showed only marginal activity compared with their 1alpha -hydroxylated congeners (cf. Fig. 5). Because the 1alpha -hydroxy group mediates high-affinity binding to the VDR (cf. Ref. 6), it is reasonable to assume that the observed effects on TEER of 1,25(OH)2D3 and its two synthetic side-chain- and D-ring-modified analogs result from a genomic rather than from a nongenomic action. The latter possibility seems unlikely also for a number of other reasons. First, typical nongenomic effects of 1,25(OH)2D3 (for review see, e.g., Ref. 7) involve interactions with plasma membrane activities and are observed within seconds or minutes, whereas reduction of TEER requires at least 48-h exposure to the hormone (cf. Table 1). Second, a rapid membrane action of 1,25(OH)2D3 cannot be easily reconciled with the observation that the sensitivity of Caco-2 cells varies with ongoing differentiation between days 4 and 12 past confluence (cf. Fig. 3). It has been shown, however, that the expression of genomic effects of 1,25(OH)2D3, particularly in enterocytes, can depend to a large extent on the degree of their differentiation (13). Third, 25(OH)-16ene,23yne-D3 was shown to be most potent in eliciting nongenomic effects such as activation of voltage-gated Ca2+ channels in rat osteosarcoma cells (15), whereas the same analog was only weakly effective in attenuating TEER (cf. Fig. 5). Fourth, it is conceivable that the observed small effects of synthetic 1-deoxyvitamin D compounds on TEER of Caco-2 cells reflect their small genomic potency due to the ability to bind weakly to the VDR (6, 15) or, fifth, result from conversion into genomically active 1-hydroxy compounds. Although substantial 25-hydroxyvitamin D3-1-hydroxylase activity has been observed only in serum-free cultures of Caco-2 cells (14), it is conceivable that, even under the culture conditions employed in the present study, a small fraction of the 25-hydroxy compounds tested is converted to respective 1alpha -hydroxy derivatives, which could then be responsible for the observed effects on TEER (Fig. 5).

Both assembly and barrier properties of tight junctions depend on the formation of a bipartite functional complex with adjacent adherens junctions as well as on an appropriate organization of the latter with the actin cytoskeleton (1, 26). Fialka et al. (17) showed that estrogen-related upregulation of the c-Jun oncoprotein diminishes TEER in mammary epithelial cells and, at the same time, disrupts the polarized expression of the tight junction-associated protein zonin-1 as well as of the constituents of adherens junctions, E-cadherin and beta -catenin. Because the c-jun protooncogene is also a well-known target for signaling from the VDR (9, 25), we suggest that upregulation of c-Jun expression could also explain the observed effects of genomically active vitamin D compounds on tight-junctional permeability of Caco-2 cells. In fact, we have obtained evidence from Western blot analysis that treatment with 10-8 M 1,25(OH)2D3 for 5 days leads to reduced expression of E-cadherin in Caco-2 cells (unpublished results).

A strong argument for the notion that vitamin D stimulates transepithelial Ca2+ transport by an increase in junctional ion permeability rather than by stimulation of transcellular calbindin-mediated transport, as suggested by Fleet et al. (18, 19), can be derived from the following observations: 1) an identical relationship between TEER or conductance, respectively, and Ca2+ transport exists in untreated and vitamin D-treated Caco-2 cell cultures, and hence no conductance-independent vitamin D-related increment exists; 2) vitamin D has an identical effect on apical-to-basolateral as well as on basolateral-to-apical Ca2+ fluxes, which would not be the case if there were a major contribution from vectorial transcellular calbindin-mediated transport that proceeds exclusively in the apical-to-basolateral direction; and 3) the effect of vitamin D is not specific for Ca2+ transport but is visible also on bidirectional Rb+ fluxes, which are certainly not calbindin mediated.

As far as the existence of a major transcellular Ca2+ path in confluent Caco-2 cells is concerned, we were able to confirm the observation of Surendran et al. (29) that blocking Ca2+ extrusion across the basolateral membrane by Ca2+-ATPase inhibition does not alter the extent of transepithelial Ca2+ transport. Because this is valid also for 1,25(OH)2D3-treated Caco-2 cells (cf. Table 2), this observation must be considered as additional support for the assumption that vitamin D affects Ca2+ transport mainly through its effect on tight-junctional ion permeability.

In probing the vitamin D sensitivity of the consecutive steps of apical-to-basolateral transcellular Ca2+ transport, we could show that cellular Ca2+ uptake from the apical aspect of confluent Caco-2 cell layers involves a genomic action of vitamin D sterols. Furthermore, consistent with the results of Fleet et al. (18, 19), 1,25(OH)2D3 upregulates CaBP-9kDa mRNA levels (Fig. 7). However, it must be borne in mind that as long as direct measurement of human CaBP-9kDa protein in Caco-2 cells is not available, it remains questionable whether the vitamin D actions on mucosal Ca2+ influx and CaBP-9kDa mRNA are of a magnitude to efficiently raise the rate of transcellular transport of Ca2+.

Another explanation for the difference in the interpretation of our results and those of Fleet et al. (18, 19) lies in the fact that these authors did not observe any effect of vitamin D sterols on transepithelial transfer of phenol red, which they used as a marker for paracellular permeability. However, the relatively high molecular weight and negative charge of this compound may have compromised its use to detect changes in paracellular permeability of ions with a much smaller atomic radius, such as Ca2+. Because of the lack of any substantial contribution of the transcellular route to transepithelial Ca2+ transport, the Caco-2 system could serve as an excellent model for the study of vitamin D effects on intestinal Ca2+ absorption via the paracellular route (for review, see Ref. 31).

    ACKNOWLEDGEMENTS

The authors thank Teresa Manhardt for skillful technical assistance.

    FOOTNOTES

These investigations were supported by Grant P-10133-MED from the Austrian Science Foundation.

M. V. Chirayath was on leave of absence from Dept. of Physiology, PSG Medical College, Coimbatore, South India, and was the recipient of a fellowship of the International Academy of Pathology, Austrian Section.

Address for reprint requests: M. Peterlik, Dept. of General and Experimental Pathology, Waehringer Guertel 18-20, A-1090 Vienna, Austria.

Received 12 December 1996; accepted in final form 20 October 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Anderson, J. M., and C. M. Van Itallie. Tight junctions and the molecular basis for regulation of paracellular permeability. Am. J. Physiol. 269 (Gastrointest. Liver Physiol. 32): G467-G475, 1995[Abstract/Free Full Text].

2.   Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. Current Protocols in Molecular Biology. New York: Wiley, 1994, vol. 1.

3.   Beaulieu, J.-F., and A. Quaroni. Clonal analysis of sucrase-isomaltase expression in the human colon adenocarcinoma Caco-2 cells. Biochem. J. 280: 599-608, 1991[Medline].

4.   Bindels, R. J. M., J. A. H. Timmermans, A. Hartog, and C. H. van Os. Transcellular calcium transport across cultured intestinal and renal cells. In: Extra- and Intracellular Calcium and Phosphate Regulation: From Basic Research to Clinical Medicine, edited by F. Bronner, and M. Peterlik. Boca Raton, FL: CRC, 1992, p. 9-20.

5.   Bischof, M. G., K. Redlich, C. Schiller, M. V. Chirayath, M. Uskokovic, M. Peterlik, and H. S. Cross. Growth inhibitory effects on human colon adenocarcinoma-derived Caco-2 cells and calcemic potential of 1alpha ,25-dihydroxyvitamin D3 analogs: structure-function relationships. J. Pharmacol. Exp. Ther. 275: 1254-1260, 1995[Abstract].

6.   Bishop, J. E., E. D. Collins, W. H. Okamura, and A. W. Norman. Profile of ligand specificity of the vitamin D binding protein for 1alpha ,25-dihydroxyvitamin D3 and its analogs. J. Bone Miner. Res. 9: 1277-1288, 1994[Medline].

7.   Boland, A. R., and I. Nemere. Rapid actions of vitamin D compounds. J. Cell. Biochem. 49: 32-36, 1992[Medline].

8.   Bronner, F. Intestinal calcium transport: the cellular pathway. Miner. Electrolyte Metab. 16: 94-100, 1990[Medline].

9.   Candeliere, G. A., J. Prud'homme, and R. St.-Arnaud. Differential stimulation of Fos and Jun family members by calcitriol in osteoblastic cells. Mol. Endocrinol. 5: 1780-1788, 1991[Abstract].

10.   Chirayath, M. V., W. Hulla, W.-M. Tong, H. S. Cross, and M. Peterlik. Differential expression of genomic vitamin D effects in Caco-2 cells. Calcif. Tissue Int. 56: 469, 1995.

11.   Chomczinski, P., and N. Sacchi. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987[Medline].

12.   Cross, H. S., R. A. Corradino, and M. Peterlik. Calcitriol-dependent, paracellular sodium transport in the embryonic chick intestine. Mol. Cell. Endocrinol. 53: 53-58, 1987[Medline].

13.   Cross, H. S., and M. Peterlik. Differentiation-dependent expression of calcitriol actions on absorptive processes in cultured chick intestine: modulation by triiodothyronine. Acta Endocrinol. 124: 679-684, 1991[Medline].

14.   Cross, H. S., M. Peterlik, G. S. Reddy, and I. Schuster. Vitamin D metabolism in human colon adenocarcinoma-derived Caco-2 cells: expression of 25-hydroxyvitamin D3-1alpha -hydroxylase activity and regulation of side-chain metabolism. J. Steroid Biochem. Mol. Biol. 62: 21-28, 1997[Medline].

15.   Farach-Carson, M., I. Sergeev, and A. W. Norman. Nongenomic actions of 1,25-dihydroxyvitamin D3 in rat osteosarcoma cells: structure-function studies using ligand analogs. Endocrinology 129: 1876-1884, 1991[Abstract].

16.   Favus, M. J., E. Angeid-Backman, M. D. Breyer, and F. L. Coe. Effects of trifluoperazine, ouabain, and ethacrynic acid on intestinal calcium transport. Am. J. Physiol. 244 (Gastrointest. Liver Physiol. 7): G111-G115, 1983[Abstract/Free Full Text].

17.   Fialka, I., H. Schwarz, E. Reichmann, M. Oft, M. Busslinger, and H. Beug. The estrogen-dependent c-JunER protein causes a reversible loss of mammary epithelial cell polarity involving a destabilization of adherens junctions. J. Cell Biol. 132: 1115-1132, 1986[Abstract].

18.   Fleet, J. C., J. Bradley, G. S. Reddy, R. Ray, and R. J. Wood. 1alpha ,25-(OH)2-vitamin D3 analogs with minimal in vivo calcemic activity can stimulate significant transepithelial calcium transport and mRNA expression in vitro. Arch. Biochem. Biophys. 329: 228-234, 1996[Medline].

19.   Fleet, J. C., and R. J. Wood. Identification of calbindin D9k mRNA and its regulation by 1,25-dihydroxyvitamin D3 in Caco-2 cells. Arch. Biochem. Biophys. 308: 171-174, 1994[Medline].

20.   Giuliano, A. R., T. R. Franceschi, and R. J. Wood. Characterization of the vitamin D receptor from the Caco-2 human colon carcinoma cell line: effect of cellular differentiation. Arch. Biochem. Biophys. 285: 261-269, 1991[Medline].

21.   Giuliano, A. R., and R. J. Wood. Vitamin D-regulated calcium transport in Caco-2 cells: a unique in vitro model. Am. J. Physiol. 260 (Gastrointest. Liver Physiol. 23): G207-G212, 1991[Abstract/Free Full Text].

22.   Grasset, E., M. Pinto, E. Dussaulx, A. Zweibaum, and J.-F. Desjeux. Epithelial properties of human colonic carcinoma cell line Caco-2: electrical parameters. Am. J. Physiol. 247 (Cell Physiol. 16): C260-C267, 1984[Abstract].

23.   Hulla, W., E. Kállay, W. Krugluger, M. Peterlik, and H. S. Cross. Growth control of human colon adenocarcinoma-derived Caco-2 cells by vitamin D compounds and extracellular calcium in vitro: relation to c-myc oncogene and vitamin D receptor expression. Int. J. Cancer 62: 711-716, 1995[Medline].

24.   Hurwitz, S., H. C. Harrison, and H. E. Harrison. Effect of vitamin D3 on the in vitro transport of calcium by the chick intestine. J. Nutr. 91: 319-323, 1967[Medline].

25.   Lasky, S. R., K. Iwata, A. G. Rosmarin, D. G. Caprio, and A. L. Maizel. Differential regulation of JunD by dihydroxycholecalciferol in human chronic myelogenous leukemia cells. J. Biol. Chem. 270: 19676-19679, 1995[Abstract/Free Full Text].

26.   Madara, J. L., and J. R. Pappenheimer. Structural basis for physiological regulation of paracellular pathways in intestinal epithelia. J. Membr. Biol. 100: 149-164, 1987[Medline].

27.   McRoberts, J. A., R. Aranda, N. Riley, and H. Kang. Insulin regulates the paracellular permeability of cultured intestinal epithelial cell monolayers. J. Clin. Invest. 85: 1127-1134, 1990[Medline].

28.   Pinto, M., S. Robine-Leon, M. D. Appay, M. Kedinger, N. Triadou, E. Dussaulx, B. La Croix, P. Simon-Assman, K. Haffen, J. Fogh, and A. Zweibaum. Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture. Biol. Cell 47: 323-330, 1983.

29.   Surendran, N., L. D. Nguyen, A. R. Giuliano, and J. Blanchard. Mechanisms of acylcarnitine-mediated enhancement of calcium transport in the Caco-2 cell monolayer model. J. Pharm. Sci. 84: 269-274, 1995[Medline].

30.   Tschugguel, W., Z. Zhegu, L. Gajdzik, M. Maier, B. R. Binder, and J. Graf. High precision measurement of electrical resistance across endothelial cell monolayers. Pflügers Arch. 430: 145-147, 1995[Medline].

31.   Wasserman, R. H., and C. S. Fullmer. Vitamin D and intestinal calcium transport: facts, speculations and hypotheses. J. Nutr. 125: 1971S-1979S, 1995[Medline].

32.   Zucco, F., I. DeAngelis, O. Vincentini, L. Rossi, C. Steinkuhler, and A. Stammati. Potential use of the human intestinal cell line Caco-2 in toxicologic investigation. In Vitro Toxicol. 7: 107-112, 1994.


AJP Gastroint Liver Physiol 274(2):G389-G396
0193-1857/98 $5.00 Copyright © 1998 the American Physiological Society