Calcitriol mediates the activity of SGLT1 through an extranuclear initiated mechanism that involves...

ORIGINAL PAPER

Calcitriol mediates the activity of SGLT1 throughan extranuclear initiated mechanism that involvesintracellular signaling pathways

Carmen Castaneda-Sceppa & Francisco Castaneda

Received: 23 December 2009 /Accepted: 13 April 2010 /Published online: 29 April 2010# University of Navarra 2010

Abstract The present study explored whether calcitriolplays a role in the regulation of sodium-dependentglucose transporter protein 1 (SGLT1) activity. For thispurpose, alpha-methyl glucoside (AMG) uptake instable transfected Chinese hamster ovary (CHO-G6D3)cells expressing rabbit SGLT1 (rbSGLT1) was used. Theinvolvement of second messengers, intracellular signal-ing pathways, and pro-inflammatory cytokines wereexamined using specific inhibitors before incubationwith calcitriol for 15 min. The present study demon-strated the involvement of second messengers producedby phospholipase A2, phospholipase C, calmodulin,diacylglycerol kinase, and phosphoinositide 3 kinase oncalcitriol-regulated AMG uptake. Pretreatment withinhibitors of the mitogen-activated protein kinase(MAPK) signaling pathway increased calcitriol-induced AMG uptake. In contrast, inhibition of thephosphoinositide 3-kinase PI3K/Akt/mTOR signalingpathway decreased the effect of calcitriol on AMGuptake. These findings suggest that calcitriol regulatesrbSGLT1 activity through a rapid, extranuclear initiatedmechanism of action stimulated by MAPK and

inhibited by PI3K/Akt/mTOR. Another important find-ing was the effect of pro-inflammatory cytokines oncalcitriol-induced AMG uptake. Interleukin-6 increasedwhile tumor necrosis factor-α decreased calcitriol-induced AMG uptake. In conclusion, the present studydemonstrates the involvement of calcitriol in theregulation of rbSGLT1 activity. This is due to theactivation of intracellular signaling pathways triggeredby second messenger molecules and cytokines after ashort time (15 min) exposure to calcitriol.

Keywords AMG uptake . Calcitriol . SGLT.

Non-genomic effect . Second messengers .

Intracellular signaling

AbbreviationsCalcitriol 1α,25-Dihydroxyvitamin D3AACOCF3 Arachidonyl trifluoromethyl ketoneAkt Protein kinase BDAGK Diacylglycerol kinaseMAPK Mitogen-activated protein kinasemTOR Mammalian target of rapamycinPI3K Phosphoinositide 3-kinasePLA2 Phospholipase A2PLC Phospholipase C

Introduction

Vitamin D is involved in the regulation of differentbiological processes, including cell growth and

J Physiol Biochem (2010) 66:105–115DOI 10.1007/s13105-010-0015-9

C. Castaneda-SceppaBouve College of Health Sciences,Northeastern University Boston,Boston, MA, USA

F. Castaneda (*)Laboratory for Molecular Pathobiochemistry and ClinicalResearch, Max Planck Institute of Molecular Physiology,Otto-Hahn-Strasse 11,44227 Dortmund, Germanye-mail: [email protected]

differentiation [43] and apoptosis [3], as well as insome physiological mechanisms, such as hormonesecretion [2, 12], T cell proliferation and cytokineproduction [32], and immunosuppressive response[22]. The active metabolite of vitamin D, calcitriol,or 1α,25-dihydroxyvitamin D3 acts through twodifferent mechanisms. The first one mediates geneexpression and is known as the genomic action ofvitamin D. The second mechanism involves intracel-lular signaling pathways and is known as the non-genomic (extranuclear initiated) effect of vitamin D.

The genomic regulation of vitamin D, the mostextensively studied, takes place through specificreceptors localized in the nucleus or cytosol. Bindingof calcitriol to the vitamin D receptor acts as a vitaminD-dependent transcription factor and regulates geneexpression [7, 11]. Calcium and phosphate homeo-stasis represents an example of the genomic role ofvitamin D. The effect of vitamin D on insulinresistance is another example of its genomic regula-tion, which has been associated with the regulation ofinsulin secretion in pancreatic β-cells [26, 32].

In contrast to the genomic mechanism of action ofvitamin D, the extranuclear initiated mechanism ischaracterized by a rapid response that has beenobserved within a few minutes [16]. This mechanismof action is modulated by different intracellularsignaling pathways that are triggered by cell mem-brane proteins with subsequent activation of intracel-lular proteins [32]. This effect has been extensivelyevaluated in cardiac muscle cells in which calcitriolplays an important role in the regulation of thecardiovascular function [58]. It has been reported thatin cardiac muscle cells, calcitriol activates voltage-dependent Ca2+ channels following stimulation of theadenylate cyclase/cAMP/protein kinase A messengersystem [44, 48]. The other process mediated by thistype of regulation is osteogenesis. In osteoblasts,calcitriol-mediated activation of Ca2+ channels leadsto bone formation [60]. In addition to protein kinaseA (PKA), protein kinase C (PKC) has also beenassociated with the activity of calcitriol in cardiac andskeletal muscle cells [13], chondrocytes [5], as well asin colonocytes and colon cancer-derived Caco-2 cells[49] and duodenal epithelium [54]. Calcitriol directlyactivates PKC, which acts as a membrane-boundreceptor for calcitriol [50]. We previously reportedthat both PKA [51] and PKC [8] are involved in theregulation of sodium-dependent D-glucose co-transport

(SGLT1) activity. For this reason, the involvement ofcalcitriol in the regulation of SGLT1 activity can bepostulated.

Transepithelial absorption of D-glucose is mediatedby secondary active sodium-dependent glucose co-transporter proteins (SGLT) [21]. Glucose is carriedinto the cell against its concentration gradient bytransforming the energy of the sodium electrochem-ical gradient into osmotic work [15]. The SGLT co-transport system is mainly expressed in small intestine(SGLT1) and in kidney (SGLT1 and SGLT2) [29].Different methods to study the activity of SGLT havebeen used [23, 28, 33, 55]. The radioactive-labeledglucose analogue (14C-methy-α-D-glycopyranoside)combined with cell lines expressing the specificcarried is one of the most widely applied methods toexamine the activity of membrane transporter proteins[9, 10]. Chinese hamster ovary cells stably expressingrbSGLT1 (CHO-G6D3) were generated in our labo-ratory [31]. This in vitro system represents astandardized method that we have extensively usedto explore the intracellular mechanisms of biosynthe-sis and functional regulation of SGLT1 [41, 42, 59].

In vitro studies using chicken SGLT1 havedemonstrated that calcitriol increases glucose uptakeby a direct interaction with SGLT1 itself, which isobserved by an increased maximal velocity (Km) [39].Such effect is mediated by increasing the intracellularamount of sodium [18]. In addition, studies performedin brush border membrane vesicles (BBMV) fromembryonic chick jejunum suggested that calcitriolmodulates the activity of SGLT1 through an incre-ment in the number of SGLT1 transporter protein inthe BBMV [14]. Moreover, the effect of calcitriol onrapid and transit activation of intracellular signalingpathways, including PI3K/Akt/mTOR, p38/mitogenactivated protein kinase (MAPK), and ERK/MAPK,has already been reported in different cell culturessystems, including rat intestinal cells [38] as well asCHO cells [25], which is the cell line used to expressrbSGLT1 in the present study.

Given the associations between activated/hormonalvitamin D (calcitriol) and secondary active SGLT co-transport proteins, the aim of the present study was toevaluate whether calcitriol plays a role in the regulationof rabbit SGLT1 (rbSGLT1). For this purpose, weexamined calcitriol-mediated alpha-methyl glucoside(AMG) uptake in stable transfected Chinese hamsterovary (CHO-G6D3) cells expressing rbSGLT1.

106 C. Castaneda-Sceppa, F. Castaneda

Materials and methods

Drugs and reagents

All chemicals were of the highest purity available fromSigma (Deisenhofen, Germany), unless other sourcesare indicated. [14C] α-methyl D-glucopyranoside([14C]-AMG; specific radioactivity 300 mCi/mmol)was purchased from Perkin-Elmer (Boston, MA).

Cell culture

Chinese hamster ovary cells stably expressingrbSGLT1 (CHO-G6D3) generated in our laboratory[31] were used. CHO-GD3 cells were grown in 25-cm2 flasks (Falcon, Heidelberg, Germany) under7.5% CO2 at 37°C. The cells were cultured to 80%confluence in Dulbecco’s modified Eagle’s medium(PAN Biotech, Aidenbach, Germany) containingglucose (5 mM) supplemented with 5% fetal calfserum, 1 mM sodium pyruvate, 2 mM glutamine and50 µM β-mercaptoethanol, and 400 µg/ml paneticinG420.

[14C] α-Methyl-D-glucopyranoside uptake

Sodium dependent D-glucose co-transport activity inCHO-G6D3 cells was determined by measuring theuptake of AMG, a substrate specific for SGLT1. Cellswere grown to confluence in 24-well plates at aconcentration of 2×104cells/well and maintained inculture for 2 days to allow the cells to form aconfluent monolayer [10]. Prior to the transport assay,the cells were incubated in a D-glucose free medium,but not glucose depleted, for 2 h at 37°C to removethe extracellular D-glucose and to reduce the intracel-lular glucose concentration [31]. For the purpose ofthis study, Krebs–Ringer–Henseleit (KRH) solutioncontaining 120 mM NaCl, 4.7 mM KCl, 1.2 mMMgCl2, 2.2 mM CaCl2, and 10 mM HEPES (pH 7.4with Tris) was used to assess sodium-dependentD-glucose transport. For sodium-free conditions, KRHsolution containing 120 mM N-methyl-glucamine(NMG) instead of NaCl (Na+) was used.

Briefly, cells were pretreated with the stated drugs(inhibitors or cytokines) for 15 min at 37°C beforeincubation. Subsequently, cells were exposed tocalcitriol (10 nM) for another 15 min while the drugwas maintained for calcitriol-treated cells with the

drug. Drug-treated cells without calcitriol (negativecontrols), as well as calcitriol-treated cells without thedrug, underwent the same incubation steps whilebeing exposed (or not) to the appropriate substance.In all cases, cells were followed by 30 min with500 µl transport buffer containing KRH-Na+ or KRH-NMG plus [14C]-AMG (0.1 µCi/µl). At the end of theuptake period, 500 µl of ice-cold stop buffer (KRH-Na+, containing 0.5 mM phlorizin) was added. Then,cells were solubilized in 2% SDS, the amount ofradioactive AMG taken up was counted, and thenprotein content determined as described by Bradford[6]. Finally, sodium-dependent uptake was calculatedby subtracting the uptake values obtained in theabsence of sodium (KRH-NMG) from the values ofKRH-sodium and expressed as picomoles per milli-gram protein per 30 min.

Quantitative real-time reversetranscription-polymerase chain reaction

Quantitative real-time PCR was used to evaluate theeffect of calcitriol on the expression of transcriptionfactors. Total RNA was extracted using RNase kit(Qiagen, Hilden, Germany) and its quality wasconfirmed by electropherograms using a 2100 Bio-Analyzer (Agilent, Santa Clara, CA). Real-time PCRwas performed using the QuantiTect SYBR green RT-PCR kit (Qiagen). A quantitative real-time PCRdetermination using Optical System Software (iQ5,version 1.0) provided with the BioRad iQ5 cycler(BioRad, Munich, Germany) was performed. Specificprimers for c-Fos forward (5′-AGG CCC AGA GCAAGA GAG GTA-3′) and c-Fos reverse (5′-CCA TGTCGT CCC AGT TGG TAA-3′), Elk-1 forward (5′-CTA CCA GGT GTC GGA GTC TGA-3′) and Elk-1reverse (5′-CCA GAT GAT GCT GTC AGG AACA-3′), STAT3 forward (5′-CTG GCT CTC GGG CCTAC-3′) and STAT3 reverse (5′-CCC CGA CAT TCCCAA GGA-3′), Foxo1 forward (5′-GTG AAC ACCAAT GCC TCAC AC-3′) and Foxo1 reverse (5′-CACAGT CCA AGC GCT CAA TA-3′), and Foxo4forward (5′-CAA GAA GAA GCC GTC TGT CC-3′)and Foxo4 reverse (5′-CTG ACG GTG CTA GCATTT GA-3′) were used. All primers were synthesizedby MWG Biotech AG (Ebersberg, Germany).Samples were prepared in triplicate, and real-timePCR measurement for each sample was done induplicate. The analysis of relative RT-PCR quanti-

Calcitriol mediates SGLT1 activity 107

fication was calculated using the threshold cyclemethod [40]. For normalization, primers for humanβ-actin forward (5′-GCG CATGGGTCA GAA GGACT-3′) and β-actin reverse (5′-TCG TCC CAG TTGGTG ACG AT-3′) were used. The average oftriplicate real-time PCR measurements was used tocalculate the mean induction ratio and standard error(SE).

Membrane preparation and Western blot analysis

CHO-G6D3 cells grown in Petri dishes were treatedwith calcitriol (10 nM) for 15 min at 37°C. Then, thecells were scraped from the plates and centrifuged at500×g for 10 min at 4°C. The pellet was suspended in1 ml of ice-cold hypotonic buffer (10 mM HEPES–Tris, 5 mM EGTA-Na+, and 0.01% thimerosal, pH7.4) supplemented with protease and phosphataseinhibitors. The cells were lysed by two freeze–thawcycles and centrifuged at 100,000×g for 60 min at 4°C to remove the cytosolic proteins. The pellets weresolubilized in hypotonic buffer containing 1% TritonX-100 and incubated at 4°C for 30 min. The non-solubilized material was centrifuged again to excludeany non-solubilized material remaining. The super-natants, which represent the membrane proteinfraction, were analyzed by Western blotting withanti-SGLT1 antibody QIS-30 (1:1,000). Quantifica-tion of the amount of rbSGLT1 was performed usingImageJ v. 1.34 software for Windows (NationalInstitutes of Health, Bethesda, MD, USA) by theintegrated optical density of bands and expressed asfold values of the average optical density of untreated,control CHO cells. RbSGLT1 signal was correctedrelative to β-actin, which was determined using anti-β-actin monoclonal antibody.

Statistical analysis

Data are shown as mean ± SE from a number (n) of atleast three independent experiments each performedin triplicate. Statistical comparisons using indepen-dent t test analysis were made among calcitriol-treatedvs. untreated (control cells), inhibitor-treated cells vs.negative controls, or inhibitor-treated cells withoutcalcitriol, calcitriol-treated cell with and without in-hibitors, and calcitriol-treated cell with and withoutcytokines. P values <0.05 (two-tailed) were consid-ered statistically significant.

Results

Calcitriol modulates rbSGLT1 uptake in CHO-G6D3cells

CHO-G6D3 cells exposed to calcitriol showed anincrease in sodium-dependent AMG uptake. Asshown in Fig. 1, calcitriol treatment (10 nM) for15 min at 37°C resulted in an AMG uptake meanvalue of 1,651±42 pmol/mg protein/30 min. Thisvalue was significantly higher than that obtained inuntreated, control cells in which a mean value of 1,176±48 pmol/mg protein/30 min was found (P<0.05).These data represent an increase in sodium-dependentAMG uptake equivalent to 40% after calcitrioltreatment. AMG uptake on mock-transfected CHOcells demonstrated a value of 103±38 pmol/mgprotein/30 min. A similar AMG uptake was obtainedby calcitriol-treated mock-transfected cells with avalue of 117±35 pmol/mg protein/30 min. AMGuptake on mock-transfected cells represent a ten timeslower value compared to rbSGLT1-transfected CHOcells.

Involvement of second messenger-producingmolecules in calcitriol-regulated activity of rbSGLT1

To analyze further the mechanism of action involvedin the modulation of rbSGLT1 activity by calcitriol,the effect of specific inhibitors of second messenger-producing molecules (diacylglycerol kinase (DAGK),phospholipase A2 (PLA2), phospholipase C (PLC),and a calmodulin inhibitor) on AMG uptake wasanalyzed. For this purpose, CHO-G6D3 cells werepretreated with the specific inhibitors for 15 min andsubsequently exposed to calcitriol (10 nM) for15 min. As shown in Fig. 2, treatment with DAGKinhibitor (R59002, 30 µM) increased AMG uptakemean value to 1,896±59 pmol/mg protein/30 mincompared to negative controls (inhibitor-treated cellswithout calcitriol) in which an AMG uptake value of1,176±48 pmol/mg protein/30 min was found (P<0.05). In addition, treatment with DAGK inhibitorsignificantly increased AMG uptake of calcitriol-treated cell with the inhibitor as compared tocalcitriol-treated cells without the inhibitor (1,643±45 pmol/mg protein/30 min, P<0.05; Fig. 2, whitebar). This effect represented a 15% increase in AMGuptake induced by calcitriol in the presence of


R59002 (DAGK inhibitor). In contrast, CHO-G6D3cells treated with the PLA2 inhibitor AACOCF3(arachidonyl trifluoromethyl ketone, 10 µM) and thePLC inhibitor neomycin (100 µM) did not show anyeffect on rbSGLT1 activity when compared to theirrespective negative controls. Additionally, this effectrepresented a significant decrease in AMG uptakewhen compared to calcitriol-treated cells withoutinhibitors (with bar, P<0.05). Specifically, theseeffects represented a 29% and 24% decrease inAMG uptake induced by calcitriol-treated cells inthe presence of AACOCF3 (PLA2) and neomycin(PLC), respectively (Fig. 2).

Finally, under these conditions, pretreatment withcalmodulin inhibitor (W13, 50 µM) abolished theeffect of calcitriol on AMG uptake, as shown by asignificant reduction in AMG uptake (508±25 pmol/mg protein/30 min) compared to its respectivenegative control (P<0.001, Fig. 2). This effectrepresented a 69% decrease in AMG uptake inducedby calcitriol-treated cells in the presence of W13.Taken together, these data suggest the complementaryeffect of different second messenger molecules in theregulation of rbSGLT1.

Involvement of the MAPK signaling pathwayon calcitriol-regulated activity of rbSGLT1

To determine the involvement of the MAPK signalingpathway on calcitriol-regulated AMG uptake, CHO-G6D3 cells were incubated with the following

inhibitors: SB203580 (a p38 MAPK inhibitor,1 µM), PD98059 (an ERK/MAPK inhibitor,10 µM), and SP600125 (a Jun kinase (JNK) inhibitor,1 µM) for 15 min each and compared to negativecontrols (inhibitor-treated cells without calcitriol). Asshown in Fig. 3, pretreatment with SB203580 andPD98059 increased rbSGLT1 activity, as demonstrat-ed by AMG uptake mean values of 2,308±51 and2,932±47 pmol/mg protein/30 min, respectively,compared to the respective negative controls in whichAMG uptake values ranging from 1,124 to1,224 pmol/mg protein/30 min (P<0.05) were seen.In addition, calcitriol-treated cells with SB203580 andPD98059 inhibitors significantly increased AMGuptake as compared to calcitriol-treated cells withoutthese inhibitors (with bar, 1,651±42 pmol/mg protein/30 min, P<0.05). This represented significantincreases in AMG uptake equivalent to 40% and78% for SB203580 and PD98059, respectively(Fig. 3).

In contrast, pretreatment with SP600125 did notshow a change in AMG uptake, as demonstrated by amean value of 1,685±21 pmol/mg protein/30 mincompared to calcitriol-treated cells without inhibitors(with bar, P<0.05). This suggests that the JNK

Fig. 2 Effect of DAGK, PLA2, PLC, and calmodulin inhibitorson calcitriol-regulated sodium-dependent AMG uptake inCHO-G6D3 cells. Cells were pre-incubated at 37°C withspecific inhibitors of DAGK (R59002, 30 µM), PLA2

(AACOCF3, 10 µM), PLC (neomycin, 100 µM), and calmod-ulin (W13, 100 µM) for 15 min before incubation withcalcitriol (10 nM, black bars) and compared to their respectivenegative controls (inhibitor-treated cells without calcitriol, graybars). The white bar represents AMG uptake obtained fromcalcitriol-treated cells without inhibitors. AMG uptake wasmeasured after 30 min. Results are mean ± SE, n=24. *P<0.05or ‡P<0.001 inhibitor-treated vs. negative controls. §P<0.05calcitriol-treated cells with inhibitors vs. calcitriol-treated cellswithout inhibitors

Fig. 1 Effect of calcitriol on sodium-dependent AMG uptakein CHO-G6D3 and mock CHO transfected cells. Cells wereincubated at 37°C with calcitriol (10 µM) for 15 min prior touptake experiments and compared to untreated CHO-G6D3cells (control). The chemical concentration of AMG was 3 µM.AMG uptake was measured after 30 min and expressedas pmol/mg protein/30 min. Results are mean ± SE, n=24.*P<0.05 treated vs. control cells


pathway may not be related with the regulation ofrbSGLT1 induced by calcitriol. AMG uptake inSP600125-treated cells, however, was significantlyincreased as compared to negative controls (P<0.001,Fig. 3).

Involvement of the PI3K/Akt/mTOR signalingpathway on calcitriol-regulated activity of rbSGLT1

To examine the role of the PI3K/Akt/mTOR signalingpathway in calcitriol-regulated AMG uptake, CHO-G6D3 cells were pretreated with wortmannin (a PI3Kinhibitor, 25 nM), an Akt inhibitor (KP372-1,10 µM), and rapamycin (an mTOR inhibitor,25 nM) for 15 min before calcitriol treatment. Figure 4shows that pretreatment with wortmannin, Akt inhib-itor, and rapamycin abolished the effect of calcitriolon AMG uptake, as shown by a significant reductionin AMG uptake mean values equivalent to 865±55,918±38, and 948±36 pmol/mg protein/30 min, re-spectively, compared to calcitriol-treated cells withoutinhibitors (white bar, 1,651±42 pmol/mg protein/30 min, P<0.05). The reductions seen resulted in

significant decreases in AMG uptake of 52%, 55%,and 57% for wortmannin, Akt inhibitor, and rapamy-cin, respectively (Fig. 4). These data suggest theinvolvement of PI3K/Akt/mTOR pathway oncalcitriol-regulated AMG uptake.

Calcitriol-induced expression of transcriptionfactors with subsequent transcriptional regulationof rbSGLT1

To evaluate the effect of calcitriol on transcriptionalregulation, the mRNA levels of transcription factorsactivated by p38/MAPK, ERK/MAPK, and PI3K/Akt/mTOR signaling pathways were analyzed.Figure 5 shows the effect of calcitriol on mRNAlevels of different transcription factors as well as theeffect of calcitriol on transcriptional regulation.Figure 5a shows the relative expression of transcriptionfactors associated with the MAPK signaling pathway(such as c-Fos, Elk-1, and STAT3) as well as theexpression of transcription factors associated with thePI3K/Akt/mTOR signaling pathway (Foxo1 andFoxo4). The relative expression of these transcriptionfactors was more than ten times that of control cellsnot treated with calcitriol. In order to evaluate further

Fig. 4 Involvement of the PI3K/Akt/mTOR signaling pathwayon calcitriol-regulated AMG uptake. CHO-G6D3 cells werepre-incubated at 37°C with the following inhibitors: wortman-nin (a PI3K inhibitor, 25 nM), an Akt inhibitor (KP372-1,10 µM), and rapamycin (an mTOR inhibitor; 25 nM) for15 min before incubation with calcitriol (10 nM, black bars)and compared to negative controls (inhibitor-treated cellswithout calcitriol, gray bars). The white bar represents AMGuptake obtained from calcitriol-treated cells without inhibitors.AMG uptake was measured after 30 min. Results are mean ±SE, n=24. §P<0.05 calcitriol-treated cells with inhibitors vs.calcitriol-treated cells without inhibitors

Fig. 3 Involvement of intracellular signaling pathways oncalcitriol-regulated AMG uptake. CHO-G6D3 cells were pre-incubated at 37°C with the following inhibitors: SB203580(a p38 MAPK inhibitor, 1 µM), PD98059 (an ERK/MAPKinhibitor, 10 µM), and SP600125 (a Jun kinase (JNK) inhibitor,1 µM) for 15 min before incubation with calcitriol (10 nM,black bars) and compared to their respective negative controls(inhibitor-treated cells without calcitriol, gray bars). The whitebar represents AMG uptake obtained from calcitriol-treatedcells without inhibitors. AMG uptake was measured after30 min. Results are mean ± SE, n=24. *P<0.05 or ‡P<0.001inhibitor-treated vs. negative controls. §P<0.05 calcitriol-treated cells with inhibitors vs. calcitriol-treated cells withoutinhibitors. ns non-statistically significant P value


the role of calcitriol on transcriptional regulationactinomycin D, an inhibitor of transcription was used.As shown in Fig. 5b, pretreatment of CHO-G6D3 cellswith actinomycin D (0.1 µM for 15 min) abolishedthe effect of calcitriol on AMG uptake as shown byAMG uptake values of 1,245±39 compared to 1,569±

48 pmol/mg protein/30 min found in calcitriol-treatedcells without actinomycin D (P<0.05).

Furthermore, we estimated the amount of rbSGLT1found in the plasma membrane using Western blots todetermine calcitriol-regulated effects on rbSGLT1.Figure 5c shows the amount of rbSGLT1 blottedfrom the plasma membrane fraction in CHO-G6D3cells treated with calcitriol as compared to control.The amount of rbSGLT1 protein in calcitriol-treatedcells was higher than that seen in untreated, controlcells (P<0.05), as shown by integrated densities of4.6±0.4 and 2.1±0.3, respectively.

Effect of IL-6 and TNF-α on calcitriol-regulatedactivity of rbSGLT1

To evaluate the effect of interleukin-6 (IL-6) andtumor necrosis factor (TNF)-α on calcitriol-regulatedrbSGLT1 activity, CHO-G6D3 cells were pretreatedwith three different concentrations of either IL-6(100, 150, and 200 ng/ml) or TNF-α (100, 250, and500 ng/ml) for 15 min before calcitriol treatment anddetermination of AMG uptake were performed. Asshown in Fig. 6, pretreatment of CHO-G6D3 cellswith IL-6 increased the effect of calcitriol on AMG

Fig. 5 Effect of calcitriol on transcriptional regulation. aMedian relative expression ratios (2ΔΔCt) for mRNA levels oftranscription factors in CHO-G6D3 cells after calcitriol treat-ment (10 nM). Results are mean ± SE, n=18. b AMG uptake incalcitriol-treated cells after pretreatment with actinomycin D(0.1 µM) for 15 min. Results are mean ± SE, n=18. *P<0.05calcitriol-treated cells with and without actinomyocin D. cEffect of calcitriol on the amount of SGLT1 in the plasmamembrane. Plasma membrane fractions from both calcitriol-treated and untreated, control cells (20 µg protein/lane) wereblotted with an anti-SGLT1 antibody (QIS-30; 1:1,000),corrected to β-actin and quantified using integrated opticaldensity of bands, and expressed as arbitrary units of theaverage optical density of CHO cells. Values are mean ± SE,n=6. *P<0.05 treated versus untreated, control cells

Fig. 6 Effect of IL-6 and TNF-α on calcitriol-regulated AMGuptake. CHO-G6D3 cells were pre-incubated at 37°C with thepro-inflammatory cytokines interleukin-6 (IL-6) at concentra-tions of 100, 150 and 200 ng/ml and tumor necrosis factor(TNF)-α at concentrations of 100, 250 and 500 ng/ml for15 min before incubation with calcitriol (10 nM; balck bars)and compared negative controls (inhibitor-treated cells withoutcalcitriol; gray bars). The white bar represents AMG uptakeobtained from calcitriol-treated cells without inhibitors. AMGuptake was measured after 30 min. Results are mean ± standarderror (SE), n=24. *P<0.01 inhibitor-treated vs. negativecontrols. §P< 0.01 calcitriol-treated cells with cytokines vs.calcitriol-treated cells without cytokines


uptake as shown by values of 1,997±52, 1,951±49,and 1,910±51 pmol/mg protein/30 min for eachincreasing IL-6 concentration, respectively, comparedto calcitriol-treated cells without IL6 (white bar,1,651±42 pmol/mg protein/30 min, P<0.05). Incontrast, TNF-α abolished the effect of calcitriol onAMG uptake, as demonstrated by mean uptake valuesof 1,204±47, 1226±39, and 1,012±42 pmol/mgprotein/30 min for each increasing TNF-α concentra-tion, respectively, compared to calcitriol-treated with-out TNF-α (white bar, P<0.05).

Discussion

The effect of calcitriol on rbSGLT1 in stable CHO cellsexpressing this transporter (CHO-G6D3) demonstratesthat the regulatory effect of this vitamin is mediated bya non-genomic (extranuclear initiated) mechanism ofaction. This statement is based on the short-termeffects of calcitriol treatment on AMG uptake weobserved after 15 min of incubation with calcitriol.

The extranuclear initiated effect of calcitriol foundin the present study revealed the involvement ofenzymes producing second messenger moleculesincluding phospholipase A2, phospholipase C, cal-modulin, diacylglycerol kinase, and phosphoinositide3-kinase. As a consequence, triggering of intracellularsignaling pathways, specifically p38/MAPK, ERK/MPAK, and PI3K/Akt/mTOR, were shown to beinvolved. This activation stimulated the expression oftranscription factors, as shown by increased mRNAexpression in calcitriol-treated cells, with subsequentincrement in the amount of transcription of rbSGLT1.Subsequently, rbSGLT1 was shown to be translocatedinto the plasma membrane, which is suggestive offunctional activity based on the increased amount ofAMG uptake observed.

The extranuclear initiated effect of calcitriol resultsfrom the mediation of receptors localized in theplasma membrane [35–37]. In the present study,calcitriol-induced activation of second messengermolecules represented the initial step in the extranu-clear initiated effect of calcitriol on rbSGLT1 regula-tion. This effect was similar to the reported effect ofcalcitriol observed in chondrocytes, which depends onPLA2 [46] and PLC [45, 47, 52]. In addition, theinvolvement of calmodulin, a calcium-binding pro-tein, has also been reported in the extranuclear

initiated effect of calcitriol in muscle cells andosteoblasts. In cardiac muscle cells, calcitriol activatesvoltage-dependent Ca2+ channels following activationof guanine nucleotide binding (G) protein-mediatedstimulation of the adenylate cyclase/cAMP/proteinkinase A messenger system [44, 48]. In osteoblasts,calcitriol-mediated activation of Ca2+ channels leadsto bone mass formation [60]. Similarly, our findingsdemonstrate the involvement of phospholipase A2,phospholipase C, and calmodulin in calcitriol-inducedAMG uptake. These enzymes producing secondmessenger molecules increased the activity ofrbSLT1, as shown by a decrease in AMG uptake inthe presence of specific inhibitors for each of theseenzymes. Specifically, we found that pretreated cellswith the PLA2 inhibitor AACOCF3 (arachidonyltrifluoromethyl ketone) and the PLC inhibitor (neo-mycin) significantly decreased AMG uptake. Incontrast, DAG kinase decreased the activity ofrbSGLT1, as shown by increased AMG uptake inthe presence of the DAGK inhibitor R59002.

The reported extranuclear initiated effect of calci-triol has been associated with the involvement ofPKC. A calcitriol-dependent signaling pathway me-diated by PKC has been reported in cartilage andbone [5, 53]. In addition, PKC also plays a role inmodulating calcitriol signal transduction in musclethrough intracellular second messengers [13]. Anumber of studies have implicated PKC in theextranuclear initiated actions of calcitriol via adiacylglycerin-dependent and diacylglycerin-independent pathways [50]. Our data suggest thatcalcitriol-regulated rbSGLT1 activity involves adiacylglycerin-independent pathway, which was dem-onstrated when using the DAGK inhibitor.

Studies using cultured chondrocytes have demon-strated that calcitriol-dependent PKC signaling sharessome characteristics with estrogen-dependent PKCsignaling [5, 53]. This finding suggests that calcitriol,like other steroid hormones, exerts its biologicalresponse via extranuclear initiated mechanisms, there-by acting as a messenger in a wide number of speciesand target tissues. However, despite the reportedcalcitriol-mediated PKA and PKC activation reportedin different cell types, a direct association betweencalcitriol-regulated SGLT1 activity and PKA or PKChas not been reported. Interestingly, the mechanismsof action of calcitriol are similar to those seen withregulation of rbSGLT1 by PKC [8].


The effect of calcitriol on rapid and transit activa-tion of intracellular signaling pathways, specificallyp38/MAPK, ERK/MAPK, and PI3K/Akt/mTOR, hasalready been reported in rat intestinal cells [38] as wellas in CHO cells [25] under cell culture conditions.The data obtained in the present study demonstratethe participation of the MAPK pathway. A similareffect has been reported in skeletal muscle cells [34].We found that the regulation of AMG uptake inducedby calcitriol was mediated by MAPK (p38/MAPKand ERK/MAPK) and not by JNK pathway. Interest-ingly, inhibition of p38/MAPK and ERK/MAPKresulted in a significant increase in AMG uptake.This finding suggests an inhibitory effect of MAPKon rbSGLT1 activity and corroborates the reportedinvolvement of MAPK signaling in SGLT1 inprimary cultured rabbit renal proximal tubule cells[19, 27]. In these cells, MAPK signaling resulted in areduction of SGLT1 activity. Similarly, in the presentstudy, the inhibitory effect of MAPK (p38/MAPK andERK/MAPK) on SGLT1 activity was confirmed bythe abolishment of AMG uptake by the calmodulininhibitor W13. This finding is in accordance with theactivation of p38/MAPK and ERK/MAPK seen inother studies using NIH 3T3 [4] and PC12 [30] cells.

There is a report suggesting the involvement ofPI3K/Akt/mTOR signaling pathway on the extranu-clear initiated effect of calcitriol [35]. Our dataexpand that report by showing that there is also aninvolvement of this signaling pathway in calcitriol-induced rbSGLT1 activity. This is based on theabolishing effect of wortmannin on calcitriol-inducedAMG uptake. We also found that PI3K/Akt/mTORsignaling played an important role in the regulation ofrbSGLT1 mediated by calcitriol. Pretreatment withspecific inhibitors demonstrated that calcitriol-induced rbSGLT1 activity depends on the PI3K/Akt/mTOR signaling pathway. The regulatory effect ofcalcitriol on rbSGLT1 activity was corroborated by asignificant increase in transcript levels of specifictranscription factors including c-Fos, Elk-1, STAT3,Foxo1, and Foxo4. Additionally, Western blotsanalysis suggests translocation and insertion ofrbSGLT1 into the plasma membrane and, consequent-ly, the effect of calcitriol on the regulation ofrbSGLT1. Functional rbSGLT1 must be localized inthe plasma membrane.

The data presented in this study, using our cellculture system (rbSGLT1 transfected CHO cells),

demonstrate the involvement of calcitriol in theregulation of SGLT1 activity. This effect, in additionto our previously reported regulatory effect of PKAand PKC, suggests the complexity in the regulation ofrbSGLT1 activity. Similar complexity due to theinvolvement of multiple factors is observed underpathological conditions such as diabetes. Insulinresistance and type 2 diabetes are associated withincreased levels of pro-inflammatory cytokines suchas tumor necrosis factor-α (TNF-α) [17, 56] andinterleukin-6 (IL-6) [24, 57]. TNF-α inhibits SGLT1activity by decreasing the number of SGLT1 mole-cules at the plasma membrane through a mechanismin which several protein-like kinases, including PKC,PKA, and MAP kinases, are involved [1]. For thisreason, we analyzed the effect of calcitriol in thepresence of varying concentrations of the pro-inflammatory cytokines IL-6 and TNF-α. We foundan inhibitory effect of TNF-α on calcitriol-regulatedSGLT1 activity while IL-6 increased AMG uptake.This finding is interesting based on the reportedassociation between IL-6 and SGLT1 [20]. However,these findings, albeit interesting, need to be investi-gated further.

In conclusion, the present study demonstrates theinvolvement of activated vitamin D (calcitriol) in theregulation of rbSGLT1 activity. This is due to the ac-tivation of intracellular signaling pathways triggeredby second messenger molecules that takes places aftera short exposure (15 min) to activated/hormonalvitamin D. Additionally, calcitriol induced the expres-sion of transcription factors with subsequent incre-ment in the amount of rbSGLT1, resulting in anincreased AMG uptake. This work demonstrates forthe first time an interrelationship between extranucle-ar initiated (non-genomic) and the genomic pathwaysof rbSGTL1 calcitriol-induced activity and representsan additional step in our understanding of themechanisms of regulation of SGLT.

Acknowledgments We acknowledge the financial support ofthe International Max Planck Research School in ChemicalBiology, Dortmund, Germany.

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