Parathyroid Hormoe-related Protein Regulates Calcium Absorption In Rat Intestinal Cells

Calcium is essential for several physiological processes including muscle contraction, neuromuscular excitability, blood coagulation, enzyme activation, hormone secretion, cell division, and cell membrane stability developed special vitamin D dependent mechanisms to ensure absorption of calcium with adequate adaptation to variations in demands and dietary supply. Calcium can be transferred from intestinal lumen to the circulatory system by paracellular and transcellular paths. Paracellular calcium transport is passive, non-saturable, concentration-dependent, and poorly regulated, is more important in distal parts of the small intestine. In contrast, transcellular transport is active, saturable, energy-dependent and highly regulated by various factors, including PTH and the complex process of transcellular calcium absorption is energy-dependent and predominant in duodenum.1,25(OH)2D3. In the gastrointestinal tract, transcellualr calcium transport takes place primarily in the proximal small intestine (duodenum and proximal jejunum).The process of transcellular calcium transport involves three steps:calcium entry at the apical side cell of the membrane through calcium channels, calcium movement across the cytoplasm by calcium binding proteins and calcium extrusion at the basolateral side by calcium transporters. Apical membrane channels include the transient receptor potential vanilloid member5(TRPV5) and member6(TRPV6), it is the limiting step in transcellular calcium transport]. Cytoplasmic calcium binding proteins include calbindin D9k (CB9) and calbindin D28k (CB28)(Basolateral transporters consist of the sodium calcium exchanger1(NCX1) and the plasma membrane calcium ATPase1(PMCA1). These proteins are regulated bya homeostatic system that includes1,25(OH)2D3, PTH, estrogens, glucocorticoids and other steroid hormones.Parathyroid hormone-related protein(PTHrP, PTHLH) was discovered in1987in humans as the circulating peptide responsible for the syndrome of humoral hypercalcemia of malignancy(HHM). PTHrP and parathyroid hormone(PTH), the main hypercalcemic hormone in vertebrates,evolved from a common, ancestral gene. The two protein share a high, about70%N-terminal amino acid homology and bind with similar affinity to a shared PTH/PTHrP receptor. There are three different pthrp mRNA transcripts, encoding for isoforms of139,141and173aa. These isoforms have different physiological functions, have three bioactive subdomains(N-terminal, mid segment and C-terminal sequences) and mostly act in an intra-, auto-or paracrine fashion. Besides the pathological effects of overproduction of PTHrP in HHM, the main functions of PTHrP in’nomal physiology’ are regulation of smooth muscle tonus and tissue growth and differentiation, as well as cell proliferation. These findings would suggest that N-terminal (1-34) of PTHrP stimulates an osteoclastic activity in sea bream scales through the PTHIR via a cAMP/AC (adenylate cyclase) intracellular pathway in higher vertebrates, both PTH and PTHrP bind to PTH receptor1(PTHR1), but in teleost fish they also share a receptor designated as PTH3R,encoded by a separate gene and closely related to PTHIR. In sea bream, enterocyte PTHrP binds PTH3R, activating the adenylate cyclase/protein kinase A intracellular signaling pathway. But in the placenta and uterus, studies has shown that PTHrP (67-86) not (1-34) stimulate calcium transport. But in the vertebratea, the specific mechanism of PTHrP promoting calcium absorption are unkown present.Accordingly, in this study, we intended to test whether PTHrP could promote calcium absorption in rat duodenum, and which active part produce the effect, and which receptor was that PTHrP binds to.MATERIALS AND METHODSChemicalsRat PTHrP(1-40),(67-86),(134-143) were purchased from Bachem Americans, Inc (Torrance, CA, USA), H-89were got from CST, and other chemical synthetics if not mentioned especially were purchased from Sigma-Aldrich.)Cell cultureIEC-6cells were grown by routine methods in monolayer culture at37℃in an atmosphere of5%CO2/95%air. Dulbecco’s modified Eagl’s medium(DMEM; life technologies, USA) supplemented with8%fetal bovine serum(FBS, gibco, life technologies, USA) and antibiotics(100U/ml penicillin and100ug/ml streptomycin) was used. Cell cultures were passaged twice per week. At70%confluence, the culture medium was replaced for24h with serum-free medium. Cells were then exposed to a test substance such as various time-periods or concentration-periods with PTHrP in DMEM with0.5%FBS for the duration indicated. In studies of inhibitors, serum-deprived cells were treated with20uM H89, a PKA inhibitor;20uM GO1918, a PKC inhibitor; nitrendipine lOuM; SEA040020uM, a NCX inhibitor or Trifluoperazine10nM, a PMC A inhibitor for30minutes preceding incubation for6nM PTHrP(1-40).Western blottingConfluent cells monolayer from IEC-6cells were harvested by a modification of the method RIPA(50mM Tris-HCL, pH8.0,), followed by dounce homogenization. Cytosolic supernatants were prepared by centrifuging the extracts at14000g for30minutes. The cytosolic fractions concetration were assayed for protein by the method of BCA method. All samples were diluted in5X SDS-loading buffer(Tris-HCL250mM,10%SDS,0.5%brompheno blue,50%glycerol and5%β-mercaptoethanol), heat denatured at100℃for10min, and size-fractionated on10%SDS-polyacrylamide gels. The gels wereelectrotransferred topolyvinylidene difluoride(PVDF)membrane at150mA for2hat4℃. The filters were blocked for1h at room temperature in TBS-T(50mM Tris-HCL, pH7.4,200mM NaCl,0.5%Tween-20containing5%nonfat dry milk). Then the filters were incubated with the different antibody (TRPV6, Cabp, NCX, PMCA and GAPDH, the calcium transport antibody were got from santa cluz, diluted with1:200) at4℃overnight. Next, the membranes were washed three times in TBS-T, incubated with a1:3000dilution of peroxidas-conjugated anti-mouse or anti-rabbit secondary antibody for2hour at room temperature and washed three times with TBS-T. The membranes were then visualized using an enhanced chemiluminiscent technique (ECL, Millipore, Merk), according to the manufacture’s instructions. Images were obtained with a model GS-700Imaging densitomer from bio-rad. Bands were quantified using the Molecular Anylyst program(Bio-Rad).RT-PCRTotal RNA was extracted from IEC-6cells by using Trizol reagent (Takara, China), purity and integrity of RNA were determined by260/280nm absorbance and denaturing agarose gel electrophoresis, respectively, quantitated, and stored in formazol at-80℃. Total RNA (2μg) was reverse transcribed using first strand cDNA synthesis kits (Fermentas, Thermo scientific)according to the manufacurer’s instructions. PCR was performed using the following sets of specific primers:Each reaction mixture (25μl) contained the following:2.0μl RT reaction products, the pair of primers (0.5μmol/l each),12.5μl taq DNA polymerase (Takara syuzo), and8.5μl deionisation water. The thermocycling protocol comprised35cycles of denaturation at94℃for30sec, annealing at55-62℃for1min and extention at72℃for1min20second. The PCR products were then separated on1.5%agarose gels containing ethidium bromide, and visualized under a UV illuminator. Amplification of expected fragments was confirmed using an automated sequencer.Synthesis and transfection of siRNA for TRPV6and calbindin-9kThe21-nucleotide siRNA sequences specifically targeting rattus TRPV6and calbindin-9k were designed and synthesized by GenePharma. The specific sequences used in this study were Trpv6:sense:5’GGUGGAAGAUAGACAAGAUTT3’, antisense:5’AUCUUGUCUAUCUUCCACCTT3’; calbindin-9k:sense:5’ CUCUAGACAAUCUCUUUAATT3’, antisense:5’ UUAAAGAGAUUGUCUAGAGTT3’. Cells (0.5x106well/ml) were seeded into6-well plates in2ml of antibiotic-free DMEM medium18h prior to transfection. During transfection, cells were incubated at37℃in Opti-MEM(?) serum and serum-free medium for6h.6hours later, fresh antibiotic-free DEMEM medium was then added. All experimental measurements were performed after the confluence reached at70%. The estimated percentage of transfection was>90%. The optimal concentration of siRNA used to transfect cells was100pM.Calcium uptake measurementsFor calcium-imaging measurements,5X105/ml cells per well were seeded in the35mm glass-bottom dish photo, after the confluence reached60%, IEC-6cells were starved overnight, the cells were incubated in5μM fluo-4acetoxymethyl ester (fluo-4am) with0.01%pluronic F-127in HBSS for1hour in the dark at37℃, after washed three times with D-Hanks, HBSS or different agonist were added to the dishes which dilulted by HBSS. Measurements of Ca2+concentration were made using Cofocal Laser Scanning Microscope (FV1000, Olympus). The concentration of intracellular Ca2+was expressed as the ratio of fluorescence emission recorded at494nm, following atternate excitation at516nm. In each measurements, selecting12cells and got and analyze the fluorescence intensity of before and after adding the stimulators.Statistical analysisThe data are shown as means±S.E.M, data were analysed with paired t test for individual calcium absorption treatments or One-way ANOVA for multiple groups. Unless otherwise indicated, using SPSS13.0.ResultsPTHrP (1-40) could promote enterocyte calcium absorption and increased calcium transport proteinThree bioactive subdomains of PTHrP were added to the glass-bottom dishes under cofocal laser scanning microscope, only adding the part1-40could resulted in significant calcium increased (P<0.01), but the segments67-86or134-173resulted in no significant changes. The actions is fast and transient, the increasement emerged as soon as adding the stimulators, and decreased to the baseline after200s. The changes presented parabola form.Effect of PTHrP on calcium transform protein induction with different segements. Western-blot analysis on protein isolated from IEC-6cells and RT-PCR analysis on RNA isolated from IEC-6cells was performed as described in materials and methods. The results was in agreement with the calcium absorption observations. Western-blot and PCR results showed that only the segment1-40could significantly increase the calcium transport protein, mainly the transcellular calcium transport protein, but the L-type transport protein cav1.3was not changed significantly. The other two active part have no action on these transport proteins. In the study, PTH and1,25(OH)2D3 was used as positive control.PTHrP promote calcium absorption in a dose-response and time-course manner Increasing concetrations (0.1nM,1nM,3nM,6nM,10nM) of PTHrP(1-40) applied to IEC-6cells, a smaller increase was found with3nM, which nevertheless was significant (P<0.05, One-Way ANOVA), the peak effect was observed at6nM and10nM(P<0.01, One-Way ANOVA, compared with control). So the concentration of6nM (PTHrP,1-40) was chosen for further experiments.PTHrP (1-40) increased the expression of calcium transport peotein mRNAs and proteins in a dose-dependent manner (0.1nM to lOnM), with significant expression at6nM(P<0.05, One-Way ANOVA).A time-course study showed that the mRNA and protein expression of calcium transform protein peaked at6h(P<0.01, one way ANOVA), was essentially of the same magnitude after12h, and was reduced gradually after24h. All expression values were normalized to GAPDH.PTHrP promote calcium absorption through PTHR1, not by PTHR2or1,25D3-MARRSThe PTHrP receptor PTHR2was not detected in IEC-6cells at mRNA and ptotein level. Meanwhile, PTHrP can increased the expression of PTHR1proteins in a dose-dependent manner (0.1nM to10nM), with the highest expression at6nM (P<0.05, One-Way ANOVA, compared with control). A time-course study showed that the expression of PTHR1protein was up-regulted9hour after the addition of PTHrP (6nM).The mRNA and protein expression of1,25D3-MARRS appeared similar in those groups incubating with the three PTHrP segments(P>0.01). The hormones PTH also could not increase the1,25D3-MARRS expression, only1,25(OH)2D3could increase the1,25D3-MARRS protein and mRNA level. Through the calcium absorption test, pre-incubation with the PTHrP receptor PTHR1antibody(1:200)5minutes, then added6nM PTHrP(1-40), the calcium intensity showed no significantly different(P>0.05, paired t-test). At the same time, pre-incubation with PTHR2or1,25D3-MARRS antibody (1:200) before adding6nM PTHrP, the calcium absorption was increased significantly(.P<0.05, paired t-test).PTHrP promote calcium absorption mainly through transcellular calcium channels, not by L-type calcium channels or through paracelluar pathwayThe expression of cavl.3(one of the L-type calcium channels protein)was no significant increase among induction with the three segments of PTHrP.Pre-incubation with Nitrendipine for30min, calcium absorption increased immediatedly and markedly after adding6nM PTHrP.Paracelluar pathway usually contains claudin and zo-1, western-blot and RT-PCR showed that PTHrP had no influenced on the expression of claudin and zo-1.The role of calcium channel protein on PTHrP promote calcium absorptionIn the calcium absorption studies, pre-incubation with TRPV6and Calbindin-9k antibody5minutes, PTHrP could not increase calcium absorption markedly(P>0.05, paired t-test). In agreement with the results, after gene knockout rat TRPV6and Calbindin-9k, the calcium intensity was not significantly changed after adding6nM of PTHrP(1-40). So, knockout of TRPV6and calbindin-9k by gene and protein levels, could inhibit calcium absorption induced by PTHrP. On the other hand, pre-incubation with NCX or PMCA antibody (1:200) could not inhibit PTHrP induced calcium absorption. Pre-incubation with NCX inhibitor SEA04000and PMCA inhibitor Trifluoperazine for30minute, PTHrP also promote calcium absorption significantly.PTHrP influence calcium absorption through PKC not PKA signaling pathwayIEC-6cells in culture plates were stimulated with6nM PTHrP(l-40), at different time(0,1m,3m,5m,7m,10m,15m,20m,30m,60m,90m,120m), overdose of cold-PBS were added to terminate reaction, total proteins were harvested at the indicated time. Western-blot showed that PKA(c-aPKA type) was significantly higher at20minute, and gradually reduced to the baseline. Meanwhile, the PKC protein (P-PKCα/β) was significantly increased after stimulate3min, and the peak effect was observed at10min.In the calcium absorption test, pre-incubation with PKA inhibitor H-8920μM30minute, then IEC-6cells was stimulated with PTHrP1-40, Ca2+absorption was increased immediately, this showed that H-89could not inhibit the action of PTHrP. Pre-incubation with PKC inhibitor GO191820μM30minute, PTHrP1-40could not induce calcium uptake, means that PTHrP may promote calcium absorption through PKC signaling pathway.