Pectin Modulates Calcium Absorption in Polarized Caco-2 Cells via a Pathway Distinct from Vitamin D Stimulation

Abstract

Pectin, a type of soluble fiber, promotes morphological changes in the small intestinal villi. Although its physiological significance is unknown, we hypothesized that changes in villus morphology enhance the efficiency of nutrient absorption in the small intestine and investigated the effect of pectin derived from persimmon on calcium absorption using polarized Caco-2 cells. In polarized Caco-2 cells, pectin altered the mRNA expression levels of substances involved in calcium absorption and the regulation of intracellular calcium concentration and significantly reduced calcium absorption. Although this was comparable to the results of absorption and permeability associated with the addition of active vitamin D, the simultaneous action of pectin and active vitamin D did not show any additive effects. Furthermore, as active vitamin D significantly increases the activity of intestinal alkaline phosphatase (ALP), which is known to be involved in the regulation of intestinal absorption of calcium and lipids, we also investigated the effect of pectin on intestinal ALP activity. As a result, it was found that, unlike the effect of active vitamin D, pectin significantly reduced intestinal ALP activity. These results suggest that pectin stimulates polarized Caco-2 cells through a mechanism distinct from the regulation of calcium absorption by vitamin D, modulating total calcium absorption from the elongated villi through morphological changes in the small intestine by suppressing it at the cellular level.

Abbreviations

1,25(OH)₂D₃, 1,25-dihydroxyvitamin D₃; AIS, alcohol-insoluble solid ; ALP, alkaline phosphatase; Ca_v1.3, voltage-dependent L-type calcium subunit alpha-1D; DEAE, diethylaminoethyl; EDTA, ethylenediaminetetraacetic acid; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LPS, lipopolysaccharide; PMCA, plasma membrane Ca²⁺ ATPase; RT-PCR, reverse transcription polymerase chain reaction; TBS, Tris-buffered saline; TRPV, transient receptor potential channels, vanilloid subtype; VDR, vitamin D receptor.

INTRODUCTION

Ingestion of pectin, a type of soluble dietary fiber, promotes the formation of small intestinal crypts, resulting in morphological changes in the villi of the small intestine.^1)2)3)4) Several studies have reported the underlying mechanisms of the effects, and it has been shown that in vitro, pectin stimulate intestinal epithelial-like cells to secrete growth factors, which in turn stimulate the proliferation of intestinal crypt-like cells.⁵⁾⁶⁾ However, the physiological significance of pectin in the morphological changes of the small intestinal crypts remains unclear. Pectin is generally thought to exert its effect as a prebiotic; for example, pectin has been reported to modulate inflammatory responses by acting on immune cells.^7)8)9)10) However, we considered other possibilities in addition to its importance as a prebiotic.

The epithelium of the small intestine is the most important site for the absorption of nutrients and functional ingredients from food. Both intracellular and intercellular pathways absorb nutrients and transport them to blood vessels. In the intracellular pathway, substances are transported through carriers or channels in the cell membrane. Epithelial cells contain many transporters involved in the absorption of sugars, amino acids, and lipids. For example, calcium crosses the brush border membrane via epithelial Ca²⁺ channels, such as transient receptor potential channels, vanilloid subtype (TRPV) 5, TRPV6, and voltage-dependent L-type calcium subunit alpha-1D (Ca_v1.3). It reaches the basolateral membrane by binding a protein with high affinity to Ca²⁺, such as Calbindin-D_9k.¹¹⁾ In contrast, the intercellular pathway is regulated by adhesion molecules between epithelial cells.¹²⁾¹³⁾ Previous animal studies, mainly those on rats, have reported that dietary fiber and indigestible oligosaccharides promote the absorption of minerals, such as calcium and magnesium.^14)15)16)17) This is reported to be caused by short-chain fatty acids produced by intestinal bacteria feeding on dietary fiber, mainly in the colon.¹⁸⁾ In the small intestine, raffinose and di-fructose anhydrate III in the diet have been suggested to enhance mineral absorption by widening the tight junctions between cells.¹⁹⁾²⁰⁾

Active vitamin D, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), regulates calcium absorption in the intestine and has been reported to upregulate molecules involved in calcium transport in the intracellular pathway.^21)22)23) The active form of vitamin D has been reported to enhance calcium absorption in some animals.^24)25)26) Vitamin D is involved in the regulation of alkaline phosphatase (ALP) activity, and when vitamin D intake is restricted, ALP activity in the small intestine of rats is significantly reduced.²⁷⁾ Furthermore, in unpolarized and polarized Caco-2 cells, the addition of 1,25(OH)₂D₃ has been reported to increase ALP activity.²⁸⁾²⁹⁾

In this study, to clarify the effects of pectin on nutrient absorption and morphological changes in the small intestine, the effects of pectin on calcium absorption were investigated using polarized Caco-2 cells of the small intestinal epithelial-like cell line. In particular, calcium absorption and intestinal ALP activity were compared with the effects of active vitamin D, which has been reported to be effective.

MATERIALS AND METHODS

Purification of pectin from persimmon. Persimmons (Diospyros kaki L. cv. Fuyu) were kindly provided by the Gifu Agricultural Technology Center (Gifu, Japan). The lyophilized persimmons were crushed, a 10-fold amount of water was added, and the mixture was autoclaved at 121 °C for 30 min. The water-soluble fraction was precipitated with 80 % ethanol, and the alcohol-insoluble solid (AIS) was recovered. AIS was applied to a diethylaminoethyl (DEAE)-cellulose (HCO₃⁻ form) column (7.2 × 30 cm) and eluted with water, 0.2 M, 0.3 M, 0.35 M, 0.4 M NaHCO₃, and 0.1 M NaOH at a flow rate of 2.0 mL/min. Each fraction was quantified using the carbazole sulfate method,³⁰⁾ dialyzed against water after adding ethylenediaminetetraacetic acid (EDTA) solution (final concentration 5 mM), and then lyophilized.

Cell culture. Caco-2 cells used in this study were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured as described by Nishida et al.⁵⁾⁶⁾ The polarization of Caco-2 cells was confirmed by measuring transepithelial electrical resistance every 4 days using the Millicell electrical resistance system (Merck KGaA, Darmstadt, Germany). Twenty days after seeding, the polarized cells were used as small intestinal epithelial-like cells. After the cells were polarized, the medium in the outer wells was replaced with a normal medium (Dulbecco Modified Eagle Medium with 10 % fetal bovine serum (FBS)). The medium in the insert wells was replaced with normal medium containing 0.1 mg/mL pectin and cultured for 1 h to provide a short-term stimulation to polarized Caco-2 cells. After culturing for 1 h, the medium was replaced with a medium containing 0.2 mg/mL CaCl₂, and cultured for 3, 6, 12, or 24 h. In addition, when contacting with pectin for a long time, the medium in the insert wells was replaced with normal medium containing 0.1 mg/mL pectin, 100 nM 1,25(OH)₂D₃, or both, followed by 72 h of incubation. The concentrations of pectin and vitamin D added were set to concentrations that sufficiently affect Caco-2 cells (pectin, 0.1 mg/mL; 1,25(OH)₂D₃, 100 nM), following Nishida et al.⁶⁾ and Noda et al.²⁹⁾ Then, 1,25(OH)₂D₃ was dissolved in ethanol and adjusted to a final concentration of 0.1 % in the medium. The medium added to the insert wells contained 0.2 mg/mL CaCl₂.

Measurement of calcium permeation and absorption. Calcium quantification was performed using a Calcium Colorimetric Assay Kit (BioVision, Milpitas, CA, USA), according to the manufacturer's protocol. The medium of the insert or outer well was used as the sample. The calcium absorption rate was calculated by formula (1) from the amount of calcium initially added to the insert (A) and the amount of calcium in the insert medium after incubation (B). The calcium permeation rate was calculated by formula (2) from the increase in calcium in the outer medium before and after the incubation.

  

$$\mathit{Absorption}\mathit{rate}(\text{\%})=(A-B)/A\times 100$$(1)

  

$$\mathit{Permeation}\mathit{rate}(\text{\%})=(C-D)/D\times 100$$(2)

Here, C was the amount of calcium in the outer medium after incubation, and D was the amount of calcium in the normal medium.

Evaluation of the mRNA expression involved in permeation and absorption of calcium by real-time reverse transcription polymerase chain reaction (RT-PCR). The RNAiso Plus RNA extraction reagent (Takara Bio Inc., Shiga, Japan) was used to extract total RNA from polarized Caco-2 cells. Approximately 1 μg of the total RNA obtained was treated with RNase-free DNase I (Worthington Industries, Columbus, OH, USA) and reverse-transcribed using oligo(dT)20 primers (TOYOBO Co., Ltd., Osaka, Japan) and ReverTra Ace (TOYOBO). The primer sequences used were as follows: Ca_v1.3 (114 bp), 5′-TGATCCAAGTGGAGCAGTCA-3′ (F), 5′-GTGTGAAAGTCCGGTAGGAGA-3′ (R); TRPV6 (235 bp), 5′-TTCCTGCGGGTGGAAGACAGGCA-3′ (F), 5′- ACGCAGGTCTCTCCTCAGGGTCCC-3′ (R); vitamin D receptor (VDR, 157 bp), 5′- TGACCCTGGAGACTTTGACC-3′ (F), 5′- GTTGAAGGGGCAGGTGAATA-3′ (R); Calbindin-D_9k (237 bp), 5′- ATGAGTACTAAAAAGTCTCCT-3′ (F), 5′- CTGGGATATCTTTTTTACTAA-3′ (R); plasma membrane Ca²⁺ ATPase 1 (PMCA1, 281 bp), 5′- AAACACAGATTCGAGTGGTGAATG-3′ (F), 5′- GGGATGAAGAGGTAGCAGACTTGT-3′ (R); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 177 bp), 5′-ATGACATCAAGAAGGTGGTG-3′ (F), 5′-CATACCAGGAAATGAGCTTG-3′ (R). The effect of pectin on the mRNA expression of Ca_v1.3 in Caco-2 cells was investigated by incubating with 0.1 mg/mL pectin in a six-well plate for 1 h and replacing the medium with an FBS-free medium (0 % FBS). Subsequently, it was incubated for several hours. The effects of pectin or 1,25(OH)₂D₃ on the mRNA expression of TRPV6, VDR, Calbindin-D_9k, and PMCA1 were examined by incubating a six-well plate with 0.1 mg/mL pectin or 100 nM 1,25(OH)₂D₃ for 72 h. Relative mRNA expression levels were analyzed by real-time RT-PCR with a Thermal Cycler Dice^® Real-Time system (Takara Bio) using THUNDERBIRD SYBR qPCR Mix (TOYOBO). Real-time RT-PCR was performed by heating at 95 °C for 60 s, followed by 45 cycles of denaturation at 95 °C for 15 s, annealing for 30 s, and extension at 72 °C for 60 s. The annealing temperatures were 62, 70, 61, 55, 57 °C, and 60 °C for Ca_v1.3, TRPV6, VDR, Calbindin-D_9k, PMCA1, and GAPDH, respectively

ALP activity assay. After washing the cells with Tris-buffered saline (TBS), intracellular proteins were extracted as follows: The cells were suspended in TBS containing 1 % Triton X-100 and 1 % protease inhibitor and then left on ice for 5 min. The cells were homogenized using an ultrasonic homogenizer VP-55 (TAITEC Corporation, Saitama, Japan) and centrifuged at 1,000 × G for 10 min at 4 °C. The supernatant obtained was used as the enzyme extract. ALP activity was measured at 37 °C in 100 mM carbonate-bicarbonate buffer (pH 10.0) containing 5 mM MgCl₂ and 10 mM p-nitrophenyl phosphate as the substrate. Aliquots of 20 μL enzyme extract and 200 μL substrate solution were mixed in 96-well plates and incubated at 37 °C for 15 min. p-Nitrophenol was used as the standard solution, and the absorbance was measured at 405 nm. The enzyme activity of intestinal ALP was measured using 20 mM L-phenylalanine as the inhibitor. Enzyme activity was measured as the rate of hydrolysis of p-nitrophenyl phosphate and was indicated in units (U = μmol p-nitrophenol formed/min). ALP-specific activity (mU/μg protein) was calculated based on ALP activity and protein content. The BCA Protein Assay kit (Pierce, Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to measure the protein concentration.

Statistical analysis. All results are represented as the mean ± S.E. Unpaired two-tailed Student's t-test was used to compare the mean values of the two groups. Other data were analyzed using Tukey's test after a one-way analysis of variance. Differences between means were considered statistically significant at P < 0.05.

RESULTS

Effects of pectin stimulation on the absorption and permeation of calcium in human intestinal Caco-2 cells.

The effect of pectin on the amount of calcium absorbed and permeated was calculated from the increased or reduced amount of calcium added. After 1 h of stimulation with pectin and 6 h of incubation with a CaCl₂-containing medium, a significant decrease in calcium absorption was confirmed compared to the control group that was not administered pectin (Fig. 1A). At the same incubation time, calcium absorption in the control group was the highest. Calcium permeability was significantly increased in the group administered pectin compared with that in the control group when incubated for 3 h after 1 h of pectin stimulation (Fig. 1B). The effects of calcium absorption on gene expression were also examined under the same conditions. Ca_v1.3 is an L-shaped channel located in the apical membrane and is involved in Ca²⁺ absorption in the intracellular pathway in the intestine.¹¹⁾ Therefore, using polarized Caco-2 cells, the effect of pectin treatment on the mRNA expression of Ca_v1.3 involved in calcium transport was investigated. The expression of Ca_v1.3 mRNA was upregulated after 1 h of pectin stimulation followed by 12 h of incubation compared with that in the control group that was not administered pectin (Fig. 1C), suggesting that stimulation with pectin affected the expression of Ca_v1.3 in epithelial cells.

Fig. 1. Effect of stimulation with pectin for 1 h on the permeation and absorption rate of calcium.

　(A, B) Caco-2 cells were stimulated with 0.1 mg/mL pectin for 1 h and then cultured in a medium containing 0.2 mg/mL CaCl₂ for 3, 6, 12, and 24 h. The absorption rate (A) and permeation rate of calcium (B) were calculated by measuring the amounts of calcium in the insert or outer medium. (C) Caco-2 cells were stimulated with 0.1 mg/mL pectin for 1 h and then cultured in fresh pectin-free 0 % FBS medium for 3, 6, 12, and 24 h. The transcript level of Ca_v1.3 was measured by real-time RT-PCR in the two groups and then corrected and compared relative to the expression level of GAPDH. The values are shown as means ± S.E. of three independent experiments. Comparisons between each control group and the pectin-treated group was performed using the unpaired two-tailed Student's t-test. ^*P < 0.05, ^**P < 0.01.

Effect of prolonged pectin contact on calcium absorption and permeation.

Since stimulation with pectin affected calcium absorption and permeation in polarized Caco-2 cells, the effect of continuous contact with pectin on the amount of calcium absorbed and permeated was examined. In this study, active vitamin D was used as a positive control because it has been reported to enhance calcium absorption.³¹⁾³²⁾ Pectin and 1,25(OH)₂D₃ were added to the insert wells on the apical side and incubated for 72 h in the same way as that for ALP activity change monitoring. The calcium absorption rate was calculated from the decrease in the amount of calcium in the medium of the insert (Fig. 2A). Calcium permeability was calculated based on the increase in the amount of calcium in the outer medium after incubation (Fig. 2B). In the pectin-supplemented group, the calcium absorption rate was significantly lower than that in the control group, similar to that in the 1,25(OH)₂D₃-supplemented group. Furthermore, compared with the groups with pectin and 1,25(OH)₂D₃ added simultaneously, there were no significant differences among the three groups, and there was no additive effect. In contrast, no significant change in calcium permeability was observed in any group.

Fig. 2. Effects of prolonged contact with pectin on the permeation and absorption rate of calcium.

　Caco-2 cells on permeable membranes were incubated for 72 h in a medium containing 0.2 mg/mL CaCl₂ with or without pectin and 1,25(OH)₂D₃. The absorption rate (A) and permeation rate (B) of calcium were calculated by measuring the amount of calcium in the insert or outer medium. Values are shown as the mean ± S.E. of three independent experiments. Statistical analyses were performed using the Tukey's test. Values not sharing a common letter (a or b) are significantly different (P < 0.05).

Effects of pectin on ALP activity.

Intestinal ALP has been reported to be closely related to dietary factors, such as regulating the intestinal absorption of calcium, phosphorus, and lipids.³³⁾³⁴⁾ Intestinal ALP is an ALP isozyme localized in the small intestine, and its activity increases by the action of active vitamin D.²⁹⁾ Therefore, since the effects of pectin on calcium absorption and permeation in polarized Caco-2 cells were similar to those of active vitamin D, we compared whether the activity of intestinal ALP was similarly increased. Both intracellular (Fig. 3A) and medium (Fig. 3B) ALP activity in Caco-2 cells supplemented with pectin was significantly lower than that in control cells without pectin. In contrast, when 1,25(OH)₂D₃ was added to the cells, ALP activity significantly increased compared to that in control. Cells treated with pectin and 1,25(OH)₂D₃ showed a significant difference in ALP activity compared to those treated with 1,25(OH)₂D₃ alone. The apparent activity was comparable to that of the control.

Fig. 3. Effects of pectin and 1,25(OH)₂D₃ on ALP activity.

　Caco-2 cells on permeable membranes were incubated for 72 h with or without pectin or 1,25(OH)₂D₃. ALP activities of Caco-2 cells (A) or insert medium (B) were assayed, and the protein level compensated for intracellular activity. As the medium in the insert contained 10 % FBS and had a high protein content, it was not corrected by protein content. Values are shown as the mean ± S.E. of three independent experiments. Statistical analyses were performed using Tukey's test.

Effects of pectin and active vitamin D on regulators involved in calcium absorption in the small intestine.

To compare changes in mRNA expression levels associated with calcium absorption in polarized Caco-2 cells 72 h after the addition of pectin or 1,25(OH)₂D₃, we investigated changes in the mRNA expression levels of vitamin D action-dependent molecules (TRPV6, VDR, Calbindin-D_9k, and PMCA1) among the transport molecules involved in calcium absorption efficiency in the small intestine. The results showed that the expression of TRPV6, a calcium channel present in the epithelial cell membrane, was significantly downregulated in the 1,25(OH)₂D₃ group than that in the control and pectin groups (Fig. 4A). In addition, the expression of VDR, which is a nuclear receptor for vitamin D, was significantly upregulated in the pectin group than that in the control group; however, there was no difference compared to the 1,25(OH)₂D₃-supplemented group (Fig. 4B). Furthermore, the effect of pectin on the mRNA expression of the calcium-binding protein Calbindin-D_9k was significantly different between the 1,25(OH)₂D₃ group and the pectin group, although there was no difference in mRNA expression levels compared with those in the control group (Fig. 4C). In contrast, the expression of PMCA1 in the basement membrane of epithelial cells was not significantly different among the groups (Fig. 4D).

Fig. 4. Effects of pectin and 1,25(OH)₂D₃ on the expression levels of calcium absorption-related mRNA.

　Caco-2 cells on permeable membranes were incubated for 72 h with or without pectin or 1,25(OH)₂D₃. The transcription levels of TRPV6 (A), VDR (B), Calbindin-D_9k (C), and PMCA1 (D) were determined by real-time RT-PCR and corrected and compared relative to the expression level of GAPDH. Values are shown as the mean ± S.E. of three independent experiments. Statistical analyses were performed using Tukey's test.

DISCUSSION

This study investigated whether pectin-induced morphological changes in the small intestinal villi improve nutrient absorption. Several pathways are involved in transporting substances in the intestinal epithelium, the site of nutrient absorption. In the intracellular pathway, substances are transported via transporters and channels on the cell membrane.¹²⁾ As vegetables and fruits rich in pectin also contain abundant minerals, the effects of pectin on the permeation and absorption of calcium were investigated. Calcium is absorbed mainly in the small intestine and transported through intracellular and intercellular pathways.³⁵⁾ In this study, in polarized Caco-2 cells, calcium absorption showed a maximum at 6 h of incubation. This was significantly different from the group stimulated with pectin for 1 h. In the pectin group, the calcium absorption rate varied only by 1-1.5 % over time, suggesting that pectin regulates calcium absorption in polarized Caco-2 cells at a constant level. In addition, permeability showed a significant increase in the pectin-supplemented group after 3 h of incubation.

In the pectin group, the amount of calcium in the outer medium, which indicates permeability, increased more significantly than the decrease in calcium in the insert medium, which indicates absorption. This suggest that, in addition to the calcium absorption from the apical side, intracellular calcium was significantly released outside the cell membrane. It has been suggested that pectin stimulation affects the localization and transport of calcium in intracellular calcium stores, resulting in a faster release of calcium to the basolateral side. The upregulated Ca_v1.3 mRNA expression observed after 12 h of incubation after pectin stimulation might be due to a transient increase in Ca_v1.3 mRNA expression caused by the difference in calcium concentration inside and outside the cells.

Polarized Caco-2 cells stimulated by pectin contact for 1 h showed temporary effects on calcium absorption and permeability. It was expected that a long contact time with pectin would also continuously alter calcium absorption and permeability; however, the group that reacted with pectin for 72 h showed a decrease in absorption and no significant difference in permeability compared to the control group. This effect was similar to that of active vitamin D. Ingestion of a calcium-rich diet reduces the need for an active calcium absorption mechanism because adequate calcium absorption is achieved by passive calcium transport through the intercellular space.³¹⁾ Therefore, in this study, calcium may have been transported passively, primarily through intercellular transport pathways or voltage-dependent channels on the plasma membrane. It has been reported that indigestible saccharides, in vitro, are involved in the passage of tight junctions and promote calcium absorption in the small intestinal epithelium.¹⁹⁾²⁰⁾ However, the addition of pectin decreased the calcium absorption. It has been suggested that the function of pectin in improving and enhancing intestinal barrier,³⁶⁾³⁷⁾ affects the intercellularity of polarized Caco-2 cells, resulting in decreased calcium absorption. Furthermore, low-methoxy (LM) pectin forms cross-linked structures in the presence of calcium. The LM pectin, persimmon-derived pectin, was used in this study. Therefore, it is possible that not only the effect of pectin on the intestinal barrier function, but also the properties of LM pectin in the presence of calcium ions affected the calcium absorption rate.

Intestinal ALP has been reported to be closely related to dietary factors, such as regulating the intestinal absorption of calcium, phosphorus, and lipids.³³⁾³⁴⁾ In this study, in polarized Caco-2 cells, calcium absorption and permeability were similar after prolonged contact with pectin or active vitamin D. In contrast, ALP activity was reduced by pectin and increased by active vitamin D, and the addition of both pectin and vitamin D had the same effect as that of the control. The fact that there was a difference in ALP activity despite the lack of difference in calcium absorption and permeation of pectin and active vitamin D supports the hypothesis that ALP does not act directly as a calcium transport protein.³²⁾³⁸⁾ Guar gum, a water-soluble polysaccharide, has previously been reported to reduce small intestinal ALP activity.³⁹⁾⁴⁰⁾ ALP activity is increased by inflammation caused by lipopolysaccharide (LPS),⁴¹⁾ and LPS is involved in the control of inflammation in the intestinal tract by the dephosphorylation of ALP.⁴²⁾⁴³⁾ Pectin has been reported to protect against LPS-induced inflammation and its anti-inflammatory mechanism.^{9)44)45)46)47)} Pectin, which has an anti-inflammatory effect, can reduce ALP activity, which is increased by inflammation, and may contribute to the cellular homeostasis of ALP activity. In addition, the reduction in intestinal ALP activity was consistent with the inhibitory effect of pectin on lipid absorption,⁴⁸⁾⁴⁹⁾ which is one of the physiological functions of pectin.

Many reports have shown that the presence of active vitamin D increases calcium absorption,²⁶⁾⁴⁶⁾ but this was not the case. The action of active vitamin D involves regulating calcium absorption in the small intestine via VDR.²⁶⁾⁵⁰⁾ Calcium flows into the cells through the calcium channels Ca_v1.3 and TRPVs localized in the epithelial cell membrane. Furthermore, when calcium flows into the cell, it is captured by the calcium-binding protein calbindin-D and diffuses into the cell. Calbindin-D_9k has a buffering effect on the movement of calcium to the basement membrane and regulates the intracellular Ca²⁺ concentration. In this study, when we compared the expression of a series of molecules involved in calcium absorption and permeation, TRPV6 showed a significant difference between the control and 1,25(OH)₂D₃ groups. Although TRPV6 is regulated through the 1,25(OH)₂D₃-VDR signal,⁵¹⁾ VDR expression was not different from that in the control group, regardless of the change in TRPV6. This result was consistent with reports that active vitamin D does not affect or reduce the expression of VDR in unpolarized and polarized Caco-2 cells.^29)45)52) In this study, the expression of highly calcium-selective TRPV6 was downregulated in polarized Caco-2 cells regardless of the addition of active vitamin D because sufficient calcium was added to the cells.

Ca²⁺ efflux from intestinal cells is mediated by two proteins, PMCA1 and the Na⁺/Ca²⁺ exchanger, which regulate intracellular Ca²⁺ concentration. In this study, calcium permeability to the basement membrane was not affected by the addition of pectin or 1,25(OH)₂D₃. It did not differ significantly from the control group, regardless of the change in calcium absorption rate. Furthermore, it did not affect the expression of PMCA1, which is involved in the calcium transport from the basement membrane to the extracellular space. In addition, the expression of VDR was significantly upregulated in the pectin group compared to that in the control group, and there was a significant difference in the expression of TRPV6 and Calbindin-D_9k between pectin and 1,25(OH)₂D₃. Therefore, it was suggested that polarized Caco-2 cells are stimulated by pectin or active vitamin D to affect the molecules involved in calcium absorption and permeation, efficiently permeating the absorbed calcium extracellularly.

Calbindin-D_9k is involved in regulating intracellular calcium absorption rates and concentrations. Calbindin-D_9k knockout mice, which are involved in the regulation of intracellular calcium absorption rate and concentration, have been reported not to reduce calcium absorption;⁵³⁾ therefore, it can be said that there are multiple alternative molecules for capturing intracellular calcium. Thus, pectin and active vitamin D have different receptors in intracellular calcium transport, suggesting that the cellular response to each stimulus occurs via different signal transduction pathways.

In conclusion, pectin significantly altered the mRNA expression of calcium transporters and affected calcium absorption and permeability. In particular, the activity of ALP in the small intestine was significantly reduced by pectin, which was opposite to that of active vitamin D. However, calcium absorption and permeability in polarized Caco-2 cells were similar to those induced by active vitamin D and were closely related to intestinal calcium absorption and regulation of serum calcium concentration. In other words, pectin stimulates polarized Caco-2 cells and regulates calcium absorption and permeability through a mechanism different from vitamin D-induced calcium absorption, suggesting that calcium is efficiently excreted outside cells on the blood vessel side. The decrease in ALP activity, a phosphatase, may be due to an increase in phosphorylated materials. In a model system of the small intestinal epithelial environment, it has been reported that pectin stimulation alters the glycan structures on the surface of epithelial-like cells and that Wnt proteins released into the medium by the stimulation are indirectly involved in the growth of crypt-like cells.⁵⁾⁶⁾ The decrease of ALP activity by the addition of pectin may affect Wnt signaling and the pathways involved in calcium absorption, and the increased release of cell growth factors and its activation may be involved in the morphological changes in the small intestine. Morphological changes in the small intestinal villi induced by pectin ingestion have been shown to promote an increase in the number of proliferating cells in the crypts and increase the area of nutrient absorption through villus elongation. Therefore, we speculate that the phenomenon observed in this study might be due to a mechanism regulating the overall absorption rate by decreasing the absorptive capacity of individual epithelial cells.

CONFLICTS OF INTEREST

The authors declare no conflicts of interest associated with this manuscript.

ACKNOWLEDGMENTS

We would like to thank Dr. Yasunori Chiba of the National Institute of Advanced Industrial Science and Technology (AIST), Japan, for his valuable suggestions. We would also like to thank the consortium members for supporting the industry projects in Gifu, including Ichimaru Pharcos Co., Ltd., Gifu Prefectural Research Institute for Food Science, Gifu Prefectural Agricultural Technology Center, and Gifu Economic and Industrial Promotion Center. This work was supported by the Strategic Foundational Technology Improvement Support Operation of the Ministry of Economy, Trade, and Industry (No. 17941167). We would like to thank Editage (www.editage.com) for English language editing.

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