Effects of Calcitonin on Porcine Intestinal Epithelial Cells Proliferation, Phosphorus Absorption, and NaPi-IIb Expression
Effects of Calcitonin on Porcine Intestinal Epithelial Cells Proliferation, Phosphorus Absorption, and NaPi-IIb Expression
Xiaopeng Tang1, Wen-qin Su1 and Re-jun Fang1,2,*
1College of Animal Science and Technology, Hunan Agricultural University, Changsha 410128, China
2Hunan Co-Innovation Center of Animal Production Safety, Changsha, 410128, China
To investigate the effects of calcitonin (CT) on cell proliferation, NaPi-IIb expression, and the absorption of phosphorus in porcine intestinal epithelial cells (IPEC-1). The experiment consisted of 1 control treatment and 5 levels of CT treatments (1×10-12 to 1×10-8 mol/L), and each treatment has 6 replications. Cell proliferation was determined by the MTT method. Cell morphology and phosphorus concentration in the cell supernatant were measured after 24 h of CT treatment. The NaPi-IIb protein expression was determined by Western Blot, and the NaPi-IIb mRNA expression was determined by RT-PCR. The results showed that, compared with the control group, different levels of CT had no effect on cell proliferation, but it inhibited (P < 0.05) the absorption of phosphorus at CT concentration of 1×10-11, 1×10-10 mol/L and 1×10-9 mol/L. There was no effect of CT on NaPi-IIb mRNA and NaPi-IIb protein expression. The study suggested that CT can inhibit the absorption of phosphorus in porcine intestinal epithelial cells without affects NaPi-IIb expression.
Received 09 July 2017
Revised 12 May 2018
Accepted 05 June 2018
Available online 21 August 2019
XT and WS designed and performed the experiment. XT wrote the experiment. RF provided the technical and financial support and revised the paper.
Phosphorus absorption, Calcitonin, NaPi-IIb, Porcine intestinal epithelial cell.
* Corresponding author: email@example.com
0030-9923/2019/0006-2167 $ 9.00/0
Copyright 2019 Zoological Society of Pakistan
Inorganic phosphate (Pi) plays a major role in growth, development, bone formation and cellular metabolism. Pi absorption from small intestine and reabsorption from rental play key roles in phosphate homeostasis (). Phosphorus is mainly absorbed by simple diffusion and active absorption. Many researchers have demonstrated that the active absorption of Pi is mediated by sodium-dependent transport family protein NaPi-IIb (; ; ; ; ), which accounts for 70% to 90% of the body phosphorus active absorption (; ). NaPi-IIb expression in vivo is regulated by many physiological factors, such as estrogen (; ), epidermal growth factor (EGF) (), glucocorticoid (), vitamin D3 and its intermediate 1,25(OH)2D3 (), insulin-like growth factors (IGF) (), and protein kinase including Serum and Glucocorticoid inducible Kinase (SGK) (). Thus, investigating the regulatory factors of NaPi-IIb deeply is critically important for improving intestinal phosphorus utilization, decreasing manure phosphorus excretion and reducing environmental pollution. Calcitonin (CT) is an important regulator hormone of calcium and phosphorus in the body, and can reduce the absorption of phosphorus (). Early studies about CT were focused on the influence of osteoclasts and calcium metabolism (; ). Studies on Pi uptake and NaPi-IIb expression mediated by CT in the small intestine are still lacking. Therefore, the objective of this study was to investigate the effects of CT on IPEC-1 growth, NaPi-IIb expression, and absorption of phosphorus, to further elucidate the mechanism of Pi absorption in vitro.
Materials and Methods
Porcine intestinal epithelial cells (IPEC-1) were kindly provided by Institute of Subtropical Agriculture, Chinese Academy of Science (Changsha, China). IPEC-1 cells were cultured in DMEM/F12 medium (Invitrogen, Grand Island, NY, USA) containing 10% FBS (HyClone, South Logan, Utah, USA), 1% antibiotics (Penicillin-Streptomycin) (Santa cruz, CA, USA), and grown in a humidified incubator at 37°C with 5% CO2 and 95% air.
Determination of cell proliferation
To determine the effects of CT on IPEC-1 proliferation, cells were seeded in 96-well cell culture plates with approximately 1 × 104 cells per well, and cultured in DMEM-F12 containing 0 mol/L, 1 × 10-12 mol/L, 1 × 10-11 mol/L, 1 × 10-10 mol/L, 1 × 10-9 mol/L or 1 × 10-8 mol/L of CT (GDBIO, Qingdao, China). After a 24 h of culture, the cells were observed under inverted microscope (XDS-1A, Shanghai Precision Instrument Co., Ltd., Shanghai, China). The cell proliferation was determined by methyl-thiazolyl-tetrazalium (MTT) method (). At each time point (24, 48, 72, 96h) 20 μL of MTT (Amresco, Solon, OH, USA) was added to each well and further cultured in 37°C, 5% CO2 atmosphere for 4 h. Removed the supernatant, rinsed the plates twice with PBS buffer (Solarbio, Beijing, China), added 150 μL dimethylsulfoxide (DMSO) (Amresco, Solon, OH, USA) in each well, shaken the plates for 15 min at room temperature to dissolve the blue-violet crystals (Formazan), then measured the optical density (OD) at a wavelength of 490 nm by enzyme-linked immune detector (Bio-Rad, Hercules, CA, USA).
Phosphorus absorption analysis
To determine the effects of CT on phosphorus absorption, cells were seeded in 6-well cell culture plates with approximately 1 × 105 cells per well, and cultured in DMEM-F12 containing 0 mol/L, 1 × 10-12 mol/L, 1 × 10-11 mol/L, 1 × 10-10 mol/L, 1 × 10-9 mol/L or 1 × 10-8 mol/L of CT. After a 24 h of culture, the supernatant was collected in 0.5 mL centrifuge tube for phosphate analysis according to the guidelines of biochemical reagents (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The absorbed phosphorus of cells equals to phosphorus in the medium minus the phosphorus in supernatant.
Real-time PCR (RT-PCR) analysis of NaPi-IIb expression
To determine the effects of CT on NaPi-IIb expression, cells were seeded in 6-well cell culture plates with approximately 1 × 105 cells per well, and cultured in DMEM-F12 containing 0 mol/L, 1 × 10-12 mol/L, 1 × 10-11 mol/L, 1 × 10-10 mol/L, 1 × 10-9 mol/L or 1 × 10-8 mol/L of CT. After a 24 h of incubation, total cell RNA was extracted and purified according to the guidelines of biochemical kit (Tiandz Inc., CAT #: 71201-50, Beijing, China). Total RNA was quantified at an OD of 260 nm, and its purity was assessed by determining the OD260/OD280 ratio. Meanwhile, RNA integrity in each sample was determined using 1 % denatured agarose gel electrophoresis. Total RNA was reverse transcribed using a PrimeScript® RT reagent kit with gDNA Eraser (Takara, Dalian, China) according to the manufacturer’s instruction. cDNA was synthesized and stored at -20°C.
The primers used to detect NaPi-IIb were designed from the reported porcine NaPi-IIb sequence (), and the primers used to detect β-actin were designed from the reported porcine (Susscrofa) β-actin cDNA sequence (U07786) with software Primer 5.0. All primers were synthesized by Shanghai Biological Engineering Company (Shanghai, China). The forward primer of NaPi-IIb: 5’-CAGCCCGAAACAAGAGTATGAT-3’; the reverse primer of NaPi-IIb: 5’-TACAAACCATCCGTCCAACAG-3’; the forward primer of β-actin: 5’-CTTCCTGGGCATGGAGTCCT-3’; The reverse primer of reverse: 5’-CGTGTTGGCGTAGAGGTCCTT-3. The RT-PCR was performed using the SYBR® Premix Ex TaqTM (Takara, Dalian, China) on an Applied Biosystems 7500 Fast Real-Time PCR System (Foster City, CA, USA). The total volume of PCR reaction system was 25 μL. Briefly, 12.5 μL SYBR® Premix Ex TaqTM, 4 μLcDNA, 1 μL (10 mmol/L) forward/ reverse primers and 8.5 μL dH2O. All PCRs were performed in triplicate on a 96-well RT-PCR plate under the following conditions: 95°C for 30s followed by 39 cycles of 95°C for 5s, 58°C for 30s and 72°C for 60s.
Western blotting analysis of NaPi-IIb protein
To determine the effects of CT on NaPi-IIb expression, cells were seeded in 6-well cell culture plate with approximately 1 × 105 cells per plate, and cultured in DMEM-F12 containing 0 mol/L, 1 × 10-12 mol/L, 1 × 10-11 mol/L, 1 × 10-10 mol/L, 1 × 10-9 mol/L or 1 × 10-8 mol/L of CT. After a 24 h of incubation, cells were obtained for analysis of protein and Western blotting. Cells in 6-well culture plates were gently washed for 3 times with pre-cooled PBS. Cells were collected and lysed in RIPA Lysis Buffer (containing 1% PMSF, Beyotime, Haimen, China). Cellular debris were removed through centrifugation at 12,000 g at 4°C for 10 min, and the total protein concentration was detected using the Bradford Protein Assay Kit according to the manufacturer’s instructions (Beyotime, Haimen, China). The extracted protein sample was denatuned at 100°C for 5 min in a loading buffer containing 5% β- mercaptoethanol and stored at -20°C.
Protein samples were separated by 10% SDS-PAGE, and then electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes with transferred proteins were blocked for 1 h at room temperature in blocking buffer containing 5 % Skim-milk in TBST buffer (1× Tris-buffered saline including 0.1 % Tween 20). Membranes were incubated overnight at 4°C with following primary antibodies: rabbit anti-human SLC34A2 antibody (1:200, Santa Cruz Biotechnology, CA, USA), or α-Tubulin (1:1,000, Santa Cruz Biotechnology, CA, USA). After washed 3 times with TBST, the membranes were incubated for 1 h at room temperature with goat anti- rabbit IgG secondary antibody (Anti-Rabbit IgG (H+L) Antibody, 1:1,000, Proteintech Rosemont, IL, USA). Membranes were washed 3 times with 1 × TBST and colored with a chemiluminescence reagent (ECL-plus, Beyotime, Haimen, China), and the chemifluorescence was detected using the Image Quant LAS 4000 mini system (GE Healthcare Bio-sciences AB, Inc., Sweden), and quantified using a gel-imaging system with Image Quant TL software (GE Healthcare life Science, Inc., USA). Abundances of all proteins of interest were normalized to those for α-Tubulin.
Data were analyzed by One-way ANOVA analysis using the SAS software version 9.2 (SAS Institute Inc., NC, USA). All treatments were subjected to linear and quadratic regression analysis of hosphorus absorption to CT concentration. All values were expressed as mean±standard deviation (SD). Differences among treatment mean were determined using Duncan’s multiple comparison test, P < 0.05 was considered significant.
Effect of CT on IPEC-1 cells proliferation
The morphology of IPEC-1 cells in 0 mol/L, 1 × 10-12 mol/L, 1 × 10-11 mol/L, 1 × 10-10 mol/L, 1 × 10-9 mol/L and 1 × 10-8 mol/L CT group are shown in . After a 24 h of culture, all cells nearly had same cell morphology. It suggested that different concentration CT had no effects on the proliferation of cells. The growth curves of IPEC-1 cells are shown in . There are no differences of OD value among 0 mol/L, 1 × 10-12 mol/L, 1 × 10-11 mol/L, 1 × 10-10 mol/L, 1 × 10-9 mol/L and 1 × 10-8 mol/L CT groups in 24, 48, 72 or 96 h. It indicted that CT had no effect on proliferation of IPEC-1 cells.
Effects of CT on phosphorus absorption
Effect of CT on phosphorus absorption was shown in . The results showed that groups of 1×10-9 mol/L, 1×10-10 mol/L and 1×10-11mol/L had a lower phosphorus absorption compared with control group (P < 0.05). 1×10-8 mol/L and 1×10-12 mol/L groups have no difference compared with control group (P > 0.05). What else, there was a tendency of linear relationship between phosphorus absorption to CT concentration (P=0.073), and there was a significant quadratic relationship between phosphorus absorption to CT concentration (P< 0.05). The results indicated that 1×10-11mol/L to 1×10-9 mol/L CT had an inhibition effect on phosphorus absorption.
Effect of CT on NaPi-IIb mRNA expression
After 24 h of culture, the cell total RNA was extracted, and the concentration total RNA was detected, all samples had an OD260/OD280 value between 1.9 -2.1, then 1% agarose gel electrophoresis showed that all samples had a brightness 28s and 18s band (). The NaPi-IIb gene expression in different levels of CT treated was showed in . Compared with control group, adding 1×10-12, 1×10-11, 1×10-10, 1×10-9, 1×10-8 mol/L CT decreased the mRNA level of NaPi-IIb for 12.84%, 44.28%, 22.98%, 5.07%, 32.54%, respectively, but the difference was not significant.
Effect of CT on NaPi-IIb protein expression
The NaPi-IIb protein expression was showed in . Compared with control group, adding 1×10-12, 1×10-11, 1×10-10, 1×10-9, 1×10-8 mol/L CT decreased the NaPi-IIb protein expression for 1.44%, 1.38%, 0.53%, 0.81%, 2.95%, respectively, but the difference was not significant. It suggested that CT had no effects on NaPi-IIb protein expression.
Effect of CT on intestinal epithelial cells proliferation
Cell morphology and growth trend could reflect the effects of drugs on cell differentiation and cell vitality. In present study, we treated the cells with different levels of CT for 24 h, observed under an inverted microscope, and found that cells were in a good condition all with a clear edge. MTT colorimetric method was usually used to measure cell proliferation, to detect cell survival and growth, and to investigate whether drugs treated have impacts on cell proliferation (). MTT was a yellow dyestuff which can react with mitochondria succinate dehydrogenase of viable cells to generate violet crystal formazan, and deposited in cells, while dead cells have no this feature. Formazan was water-insoluble, but can dissolve in dimethyl sulfoxide (DMSO). The optical density value measured at the wavelength of 490 nm (or 570 nm) can serve as an estimation of the number of living cells indirectly. In present study, there are no differences of OD value among 0 mol/L, 1 × 10-12 mol/L, 1 × 10-11 mol/L, 1 × 10-10 mol/L, 1 × 10-9 mol/L and 1 × 10-8 mol/L CT groups in 24, 48, 72 or 96 h. The results suggested that there were no significant effects of CT on porcine intestinal epithelia cell morphology and proliferation, and it also can explain the changes of phosphorus content in cell supernatants was not caused by cell viability and quantities in subsequent experiment.
Effects of CT on phosphorus absorption
As an important component of the body, phosphorus is one of main element in bone, and involved in a variety of physiological functions (, ). CT was a 32 peptide hormone secreted by para-follicular cells (C cells) in thyroid, which involved in calcium and phosphate regulation, osteoclast activity inhibition and blood calcium reduction (); inhibiting the absorption of calcium and phosphate in intestine (). CT was an important hormone for calcium and phosphorus regulation, as well as an effective drug for the treatment of bone hyperplasia. In this study, the absorption of phosphorus can be measured via detecting the content of phosphorus in culture supernatant, which the content of phosphorus in medium was known (0.902 mmol/L). The results showed that 1×10-9 mol/L, 1×10-10 mol/L and 1×10-11 mol/L of CT can inhibit the phosphorus absorption.
Effects of CT on NaPi-IIb expression
As the major sodium-dependent phosphate transporter protein in intestine, NaPi-IIb plays an important role in Pi active absorption (), and it was responsible for intracellular Pi accumulation and phosphate homeostasis (). The expression of NaPi-IIb was related to transcription factors, protein translation and intracellular transport, and influenced by many physiological factors, such as age (), diets phosphorus levels (), intestinal segment (), estradiol (), glucocorticoid (), 1α-OHD3 (), EGF (; ), B-RAF () and neuropeptides () and so on. Early studies about CT were focused on the influence of osteoclasts and calcium metabolism (; ), only suggested that in the absence of PTH and cAMP, CT reduced the sodium dependent phosphate transporter protein on the proximal tubule brush border membrane by increasing the intracellular concentration of calcium. In present study, porcine intestinal epithelial cells was used as cell model to detect the effects of CT on expression of NaPi-IIb protein and NaPi-IIb mRNA. The results suggested that there was no effect on expression of NaPi-IIb protein and NaPi-IIb mRNA. It suggested that the absorption of phosphorus in cells regulated by CT was not mediated by NaPi-IIb. In body, CT was usually worked with PTH and 1,25-(OH)2D3 to maintain the balance of blood calcium and phosphorus (). PTH reduced the absorption of phosphorus by inhibiting the NaPi-IIb expression in intestine epithelium (). 1,25-(OH) 2D3 can simulate brush border NaPi-IIb expression to promote the absorption of phosphorus (). Hence, CT may inhibit intestine phosphorus absorption by changing PTH and 1, 25-(OH)2D3 expression indirectly.
The results of this research suggested that the concentration of CT range from 1×10-12mol/L to 1×10-8mol/L has no effects on porcine intestinal epithelial cells growth; and 1×10-11mol/L CT to 1×10-9mol/L CT can inhibit the phosphorus absorption; also there was no effects of the concentration of CT range from 1×10-8mol/L to 1×10-12mol/L on NaPi-IIb expression. The present study suggested that CT can inhibit the absorption of phosphorus in porcine intestinal epithelial cells without affects NaPi-IIb expression.
This research was supported by grants from the Natural Science Foundation of Hunan Province (No. 2018JJ2163), and the National Natural Science Foundation of China (No. 31572419). We also thank Professor Olayiwola Adeola from Purdue University of America for his kindness revised of this manuscript.
Statement of conflict of interest
The authors have declared no conflict of interest.
Arima, K., Hines, E.R., Kiela, P.R., Drees, J.B., Collins, J.F. and Ghishan, F.K., 2002. Glucocorticoid regulation and glycosylation of mouse intestinal type IIb Na-Pi cotransporter during ontogeny. Am. J. Physiol. Gastrointest. Liver Physiol., 283: G426-G434.
Chang, Z., Zhang, H., Mehmood, K., Rehman, M.U., Yuan, X., Li, Z., Nabi, F., Wu, X., Tian, X., Liu, X., Xu, J. and Zhou, D., 2017. Protective effects of intestinal trefoil factor against endotoxin-induced injury of intestinal mucosal epithelial cells in piglets. Pakistan J. Zool., 49: 2249-2255.
Dermaku-Sopjani, M., Sopjani, M., Saxena, A., Shojaiefard, M., Bogatikov, E., Alesutan, I., Eichenmuller, M. and Lang, F., 2011. Downregulation of NaPi-IIa and NaPi-IIb Na+-coupled phosphate transporters by coexpression of Klotho. Cell Physiol. Biochem., 28: 251-258.
Fang, R., Xiang, Z., Hu, L., Su, W., Tang, X. and Wang, X., 2016. Effects of mechanistic target of rapamycin signaling pathway on the estrogen-mediated NaPi-IIb protein expression in pig small intestinal epithelial cells. J. Anim. Sci., 94: 303-306.
Hu, L. and Fang, R., 2014. Effect of neuropeptide Y on expression of NaPi-IIb protein and absorption of inorganic phosphorus in porcine small intestinal epithelial cells. Acta Vet. Zootech. Sin., 45: 1640-1647.
Marks, J., Srai, S.K., Biber, J., Murer, H., Unwin, R.J. and Debnam, E.S., 2006. Intestinal phosphate absorption and the effect of vitamin D: A comparison of rats with mice. Exp. Physiol., 9: 531-537.
Radanovic, J., Wagner, C.A., Murer, H. and Biber, J., 2005. Regulation of intestinal phosphate transport I. Segmental expression and adaptation to low-pi diet of the type IIb Na-Pi cotransporter in mouse small intestine. Am. J. Physiol. Gastrointest. Liver Physiol., 288: G496-G500.
Reining, S.C., Liesegang, A., Betz, H., Biber, J., Murer, H. and Hernando, N., 2010. Expression of renal and intestinal Na/Pi cotransporters in the absence of GABARAP. Pflugers Arch. Eur. J. Physiol., 460: 207-217.
Rosario, F.J., Kanai, Y., Powell, T.L. and Jansson, T., 2013. Mammalian target of rapamycin signalling modulates amino acid uptake by regulating transporter cell surface abundance in primary human trophoblast cells. J. Physiol., 591: 609-625.
Sabbagh, Y., O’Brien, S.P., Song, W., Boulanger, J.H., Stockmann, A., Arbeeny, C. and Schiavi, S.C., 2009. Intestinal Npt2b plays a major role in phosphate absorption and homeostasis. J. Am. Soc. Nephrol., 20: 2348-2358.
Segawa, H., Kaneko, I., Yamanaka, S., Ito, M., Kuwahata, M., Inoue, Y., Kato, S. and Miyamoto, K.I., 2004. Intestinal Na-Pi cotransporter adaptation to dietary Pi content in vitamin D receptor null mice. Am. J. Physiol. Renal., 287: F39-F47.
Xiang, Z., Fang, R., Hu, L. and Su, W., 2012. Molecular cloning and functional characterization of swine sodium dependent phosphate cotransporter type IIb (NaPi-IIb) gene. Mol. Biol. Rep., 39: 10557-10564.
Xing, T., Tan, X., Yu, Q., Yang, T., and Fang, R., 2017. Identifying the location of epidermal growth factor-responsive element involved in the regulation of type IIb sodium-phosphate cotransporter expression in porcine intestinal epithelial cells. J. Anim. Physiol. Anim. Nutr., 101: 1249-1258.
Xu, H., Inouye, M., Hines, E.R., Collins, J.F. and Ghishan, F.K., 2003b. Transcriptional regulation of the human NaPi-IIb cotransporter by EGF in Caco-2 cells involves c-myb. Am. J. Physiol. Cell Physiol., 284: C1262-C1271.