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Transcription factor XBP-1 is expressed during osteoblastdifferentiation and is transcriptionally regulated by

parathyroid hormone (PTH)

A. Zambelli a,b,*, E. Mongiardini b, S.N. Villegas b, N.G. Carri b,R.P. Boot-Handford a, G.A. Wallis a

a Wellcome Trust Centre for Cell-Matrix Research, 2.205 Stopford Building, School of Biological Sciences, University of Manchester,

Oxford Road, Manchester M13 9PT, UKb Instituto Multidisciplinario de Biologı́a Celular (IMBICE), CC403 (1900) La Plata, Argentina

Received 10 September 2004; revised 3 March 2005; accepted 25 March 2005

Abstract

X-box-binding protein 1 (XBP-1) is a basic-region leucine zipper protein in the cyclic AMP response element binding protein/activating transcription factor (CREB/ATF) family of transcription factors involved in different cell-differentiation processes. We

have investigated the expression of XBP-1 in differentiating MC3T3-E1 osteoblastic cells. Cultures were treated with ascorbic acid(AA) and b-glycerophosphate (BGP) to induce differentiation. Under these conditions, the basal transcription of xbp-1 increases atday 2 following induction, peaks at day 5 and decreases thereafter. This result showed that xbp-1 gene is differentially expressedduring MC3T3-E1 cell differentiation. Detection of XBP-1 by immunofluorescence at days 0 (control culture without AA and BGP),

8 and 21 showed that the protein has a major cytoplasmic perinuclear location. In addition, xbp-1 is transcriptionally upregulated byparathyroid hormone within 2.5 h of treatment and decreases thereafter.� 2005 Published by Elsevier Ltd on behalf of International Federation for Cell Biology.

Keywords: XBP-1; PTH; Osteoblast differentiation

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UNCOR1. Introduction

X-box-binding protein 1 (XBP-1) is a basic-regionleucine zipper protein of the cyclic AMP responseelement binding protein/activating transcription factor(CREB/ATF) family of transcription factors. In vitro

Abbreviations: XBP-1, X-box-binding protein 1; PTH, parathyroid

hormone; PTHrP, PTH-related protein; PTH1R, PTH/PTHrP re-

ceptor; AA, ascorbic acid; BGP, b-glycerophosphate.

* Corresponding author. Present address: Advanta Semillas, Ruta

226 Km 60.5, CC30 (7620) Balcarce, Argentina. Tel.: C54 2266

430001; fax: C54 2266 430002.

E-mail address: andres.zambelli@ar.advantaseeds.com (A.

Zambelli).

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1065-6995/$ - see front matter � 2005 Published by Elsevier Ltd on behalf

doi:10.1016/j.cellbi.2005.03.018

studies have demonstrated down-regulation of the xbp-1gene by B cell-specific activator protein (BSAP), di-merization of XBP-1 protein with c-Fos (which alsobelongs to the CREB/ATF family), and a decrease inMHC class II gene expression when antisense xbp-1sequences were introduced into Raji cells (Ono et al.,1991; Reimold et al., 1996). Disruption of the xbp-1gene in mice resulted in embryonic lethality prior toskeletogenesis and revealed that this transcription factoris essential for liver growth (Reimold et al., 2000).Furthermore, xbp-1-null B-lymphocytes fail to undergoterminal differentiation to plasma cells (Reimold et al.,2001).

Studies on the transcription of xbp-1 revealed that itis induced by activating transcription factor 6 (ATF6)

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and the mRNA is spliced by IRE1 endonuclease inresponse to stress on the endoplasmic reticulum. Hence,the spliced form of xbp-1 can be translated and theresultant protein activates the unfolded protein response(UPR) (Yoshida et al., 2001). In situ hybridization onsections of mouse embryos demonstrated that xbp-1 istranscribed at quite high levels in osteoblasts andchondroblasts (Clauss et al., 1993). However, there isno information about the role of XBP-1 during bonedevelopment.

Parathyroid hormone (PTH) and PTH-related pro-tein (PTHrP) play crucial roles in regulating bonedevelopment and homeostasis. The importance ofPTHrP and its receptor in skeletal development hasbeen emphasized by gene deletion experiments in mice(Amizuka et al., 1994). Animals in which the PTHrPgene was ablated died immediately after birth, andshowed a skeletal dysplasia characterized by short limbsdue to accelerated chondrocyte maturation (Amizukaet al., 1994). In addition to the knockout studies, furtherinformation regarding the effects of PTH/PTHrP hasbeen obtained using osteoblastic cell lines. PTH orPTHrP binds to the PTH/PTHrP receptor (PTH1R) onosteoblasts and activates protein kinase A and Cpathways (Fitzpatrick and Bilezikian, 1999), ultimatelyresulting in the regulation of genes encoding, alkalinephosphatase, type I collagen, metalloproteinase, osteo-pontin and bone sialoprotein, and dihydroxyvitamin D3expression (Fitzpatrick and Bilezikian, 1999; Yamaguchiet al., 2000).

The anabolic action of PTH has been reported to bedependent on cAMP (Fitzpatrick and Bilezikian, 1999).Interestingly, PTH and PTHrP rapidly increase thelevels of c-fos and fra-2 mRNA in osteoblastic cell lines(McCauley et al., 1997, 2001). Since XBP-1 is known todimerize with c-Fos (Ono et al., 1991) and c-Fos itselfhas been proposed to play a role in osteoblastdifferentiation and bone formation (McCabe et al.,1995; Machwate et al., 1995; Grigoriadis et al., 1995;Matsuo et al., 2000; Winchester et al., 2000), it ispossible that XBP-1 may also play an important role inbone development. Consequently, the aim of this studywas to examine the expression of the xbp-1 gene in vitroduring osteoblast differentiation, the effect of PTH (1e34) on its transcription and the intracellular distributionof the XBP-1 protein.

2. Materials and methods

2.1. MC3T3-E1 cultures

MC3T3-E1 cells, a mouse osteoblastic cell line, werecultured in D-MEM medium (Gibco BRL) supple-mented with 10% fetal calf serum (FCS), 100 U/mlpenicillin, and 100 mg/ml streptomycin. Cells were

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seeded at 2! 104 cells/cm2 and grown in 10% FCS/D-MEM containing 50 mg/ml of ascorbic acid (AA) and10 mM of b-glycerophosphate (BGP) (Green et al.,2000). PTH treatment was performed using the humanPTH (1e34) peptide (Peninsula Laboratories) at 0.1 mM.

2.2. Immunofluorescence analysis

MC3T3-E1 cells treated with AA and BGP werecultured for 0 (control), 8 and 21 days. As the cultureshad reached confluence at days 8 and 21, immunoflu-orescence detection of protein in single cells was notpossible at these time points. Thus, cells from the 0, 8and 21 day cultures were trypsinized one day before theassay, then seeded on cover slips at a density of2! 104 cells/cm2, put into a culture chamber, andcultured overnight in a humid chamber to allow cell-attachment to the surface. The following day the cellswere fixed with 4% paraformaldehyde for 20 min. Thecells were rinsed in PBS and permeabilized with TritonX-100, 0.25%, for 15 min. After rinsing in PBS, a set ofslides was either incubated overnight at 4 �C witha rabbit polyclonal antibody raised against rat XBP-1(Reimold et al., 2000) diluted 1:1000 or with a rabbitpolyclonal antibody against rat c-Fos (Santa CruzBiotechnology) diluted 1:1000. Slides were washed withPBS and incubated with secondary biotinylated goatIgG anti-rabbit IgG at 1:1000 for 1 h at roomtemperature and then washed with PBS. Thereafter,they were incubated with the fluorochrome Texas Redconjugated to avidin for 45 min at room temperature.After rinsing with PBS, cell nuclei were counterstainedwith DAPI solution, mounted with Fluoromount(Vector) and observed in an inverted fluorescencemicroscope (Zeiss). Controls with no primary antibodyand with normal rabbit serum were included in allimmunodetections. No signal was detected in any of thecontrols.

2.3. RNA isolation and northern-blot analysis

Total RNA was isolated from cells by the guanidinethiocyanate/acid phenol extraction method (Chomczyn-ski and Sacchi, 1987). RNA was electrophoresed ona 1% (w/v) denaturing agarose gel containing 2.2 M offormaldehyde and transferred onto nitrocellulose mem-brane (Hybond N). The mouse xbp-1 cDNA probe wasobtained by RT-PCR from total RNA isolated fromMC3T3-E1 cultures and using the primers 5#-AATGGTCCTTTATCAGTTTCTC-3# and 5#-GCATTGTACTTTTTAATTGCAC-3# based on the mouse xbp-1 genesequence (GenBank accession AF027963). The 272-bp-long amplification product was cloned and sequencedto confirm that it corresponded to the xbp-1 gene. In

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the same way, a c-fos cDNA probe was obtained byRT-PCR using the primers 5#-GGTTCATTATTGGAATTAAC-3# and 5#-CAACTTAAATGCTTTTATTG-3#(GenBank accession X06769). The c-fos cDNA frag-ment (445 bp long) was cloned and sequenced. A bovinepth1r probe was previously isolated from a subtractedgrowth-plate library (Hillarby et al., 1996). The clonewas sequenced confirming identity to the PTH1R.Radiolabeled probes were made using the randompriming method (Random Primers DNA LabelingSystem, Gibco BRL) using (a32P)dATP (NEN). North-ern hybridization of the filters with the xbp-1, c-fos andpth1r probes was performed at 68 �C (Sambrook et al.,1989). Blots were washed in 0.2! SSC/0.1% SDS andexposed to X-ray film at �70 �C with intensifyingscreens. Blots were stripped and reprobed using a cDNAprobe for 18S ribosomal RNA (Manuelidis and Ward,1984). A Kodak Digital Imaging system was used toquantify the signal intensity.

2.4. Statistical analysis

To measure the basal expression of xbp-1 mRNApost-mineralization, experiments were performed intriplicate and transcriptional regulation by PTH wasassayed in five independent experiments. In eachexperiment, treatments were performed in triplicate.The results were analyzed using ANOVA followed byTukeyeKramer multiple comparison test to determinestatistical differences between groups.

3. Results

Northern-blot analysis of total RNA isolated fromMC3T3-E1 osteoblastic cell cultures identified xbp-1mRNA. To evaluate whether this expression wasmodulated during osteoblast differentiation, MC3T3-E1 cells were cultured with media supplemented withAA and BGP, to induce osteoblast differentiation. TotalRNA was isolated after 0, 2, 5, 7 and 9 days of inductionand mRNA levels of xbp-1, c-fos and pth1r wereinvestigated by northern blotting. The expressionpatterns of the latter two genes have been consideredas osteoblast differentiation markers (McCabe et al.,1995; McCauley et al., 1996; Lynch et al., 1998; Purpuraet al. 2003; von Stechow et al., 2004). The analysisrevealed that, after 2 days of culture, the level of xbp-1mRNA increased about eightfold above that of thecontrol cultures (day 0, p! 0.05). The maximum xbp-1transcription was observed at day 5, although xbp-1mRNA was still significantly increased at day 7 comparedto the day 0 cells. After 9 days of culture, the xbp-1 levelsdid not differ significantly from the control (Fig. 1).

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The pattern of expression of xbp-1 was similar to thatof pth1r and c-fos. The transcription of pth1r wasincreased about fourfold after 2 days of culture withrespect to the day 0 control ( p! 0.05), and remainedsignificantly higher than the control at days 5, 7 and 9(Fig. 1, p! 0.05). The c-fos gene exhibited an increasein transcription at day 5 with respect to the control

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Fig. 1. MC3T3-E1 differentiation and xbp-1, c-fos and pth1r mRNAs

expression. (a) MC3T3-E1 cells were treated with ascorbic acid (AA)

and b-glycerophosphate (BGP). After 0, 2, 5, 7 and 9 days of culture,

total RNA was isolated for the analysis of xbp-1, c-fos and pth1r

expression by northern blot. (b) The intensity of each band was

quantified as described in Section 2 and the mean valuesG SEM of the

ratio between xbp-1, c-fos or pth1r mRNAs and their respective 18S

RNA signal intensity were plotted for each treatment day. The

autoradiograms shown are representative of three independent experi-

ments performed on different days.

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(undetectable signal), decreasing at days 7 and 9 (Fig. 1,p! 0.05).

Thereafter, the subcellular distribution of XBP-1 andc-Fos was investigated by immunofluorescence in pro-liferative MC3T3-E1 cells cultured for 0, 8 and 21 daysafter treatment with AA and BGP. Detectable levels ofc-Fos were observed following the indicated cultureperiods with the signal localized to the nucleus with noobservable differences between cultures grown fordifferent times (Fig. 2). XBP-1 was also detected at 0,8 and 21 days of culture with no observable differencesbetween cultures; but in this instance, the signal wasclearly perinuclear (Fig. 2). XBP-1 protein was in-vestigated by western blot in whole-cell extracts fromMC3T3-E1 cells after 0, 3, 7, 14 and 21 days of cultureusing the XBP-1 antibody. In all cases, a very weak bandof about 55 kDa was detected, which matched theexpected protein size (Calfon et al., 2002). The barelydetectable signal obtained did not make it possible toevaluate differences in the amount of XBP-1 protein(data not shown). The level of expression in osteoblastsis probably not enough to be quantified by western blot.For instance, XBP-1 immunoblotting from culturedmurine fibroblasts was only possible after treatment withtunicamycin or bacterial lipopolysaccharide (Calfonet al., 2002).

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To determine whether PTH influences xbp-1 mRNAlevels, we examined the effects of adding PTH toMC3T3-E1 cultures on day 5 post-induction of osteoblastmineralization. Based on a previous study of PTH-induced transcriptional regulation of c-fos in MC3T3-E1 cells, we treated cultures with 0.1 mM PTH (1e34)for periods of between 30 min (when c-fos was found tobe maximally expressed) and 4.5 h (McCauley et al.,1997). At 30 min, xbp-1 mRNA levels were not alteredcompared to unstimulated cells (time 0), whereas c-fosinduction was clearly detected (Fig. 3). After 2.5 h ofPTH treatment, the expression of xbp-1 mRNA wasupregulated significantly (twofold, p! 0.01) while thec-fos signal did not differ significantly from the control.The longest times assayed (3.5 and 4.5 h) showed thatboth xbp-1 and c-fos mRNA decreased to similar levelsas their respective controls (Fig. 3).

4. Discussion

We originally identified the xbp-1 gene as beingupregulated during growth-plate chondrocyte differen-tiation using subtractive hybridization as describedpreviously (Green et al., 2000; Hillarby et al., 1996;White et al., 2002). Furthermore, in situ hybridization

UNCORRECTE

Fig. 2. Immunofluorescence of XBP-1 (aec) and c-Fos (gei) in MC3T3-E1 cells cultured for 0 (a, g), 8 (b, h) and 21 (c, i) days after adding AA and

BGP. Cell nuclei were counterstained with DAPI (def, jel). The results presented are representative of three independent experiments performed on

different days.

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studies have shown that xbp-1 is expressed in vivo byboth chondrocytes and osteoblasts during mouseskeletal development (Clauss et al., 1993). The patternof transcription of xbp-1 was found to be very similar tothat of the genes encoding the tissue inhibitor ofmetalloproteinase (TIMP), alkaline phosphatase (ALP)and osteonectin (SPARC) throughout development inbone and cartilage (Clauss et al., 1993). XBP-1, like c-Fos, is a basic-region leucine zipper protein of theCREB/ATF family of transcription factors; in fact,these transcription factors can form heterodimers (Onoet al., 1991; Vinson et al., 2002). Mice lacking the xbp-1gene die prior to skeletogenesis and thus give no insight

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Fig. 3. Effect of PTH (1e34) on xbp-1 and c-fos mRNA expression. (a)

MC3T3-E1 cells were treated for 0.5, 2.5, 3.5, and 4.5 h with 0.1 mM

PTH (1e34), total RNA isolated and northern blotted. (b) The

intensity of each xbp-1 and c-fos band was quantified and the mean

valuesG SEM of the ratio between xbp-1 or c-fos and their 18S RNA

signal intensity were plotted for the indicated PTH-stimulation times.

The autoradiograms shown are representative of three independent

experiments performed on different days.

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into the role of this transcription factor in boneformation (Reimold et al., 2000).

We therefore looked for expression of xbp-1 in theMC3T3-E1 cell line. This cell line is a clonal osteoblast-like line, which, when cultured in the presence ofAA and BGP, undergoes a specified pattern of dif-ferentiation, which comprises: proliferation (days 1e10);extracellular matrix maturation and mineralization(days 10e20); and apoptotic mediated tissue mainte-nance (beyond day 20) (McCabe et al., 1995; McCauleyet al., 1996; Franceschi and Iyer, 1992; Lynch et al.,1998). Also, this cell line expresses pth1r (McCauleyet al., 1996) and higher levels of pth1r and c-fos havebeen observed at days 1 and 8 of culture compared topost-proliferative differentiating stages (McCabe et al.,1995; McCauley et al., 1996; Lynch et al., 1998).MC3T3-E1 cells rapidly (30e45 min) upregulated c-fosmRNA levels upon exposure to 0.1 mM PTH (1e34).This increased c-fos expression is sustained for up to 1 hand decreases thereafter (McCauley et al., 1997).

We have found that MC3T3-E1 cells also differen-tially express xbp-1 during osteoblast differentiation andthat expression increases at 2, 5 and 7 days of cultureand decreases at day 9. The pattern of expression ofxbp-1 is similar to that of pth1r and c-fos (Fig. 1)indicating that xbp-1 is also differentially expressedduring osteoblast differentiation.

We next investigated the intracellular distribution ofthe XBP-1 and c-Fos proteins by immunofluorescence.Both proteins were identified in MC3T3-E1 osteoblastsduring the proliferative and maturation stages. Stainingfor c-Fos was clearly located in the nucleus. In contrast,XBP-1 staining was predominantly perinuclear and faintwithin the nucleus. This finding was unexpectedconsidering that XBP-1 has been classified as a tran-scription factor. Recently, the upregulation and over-expression of the hXBP-1 gene has been described inprimary breast cancers (Fujimoto et al., 2003). Immu-nohistochemistry demonstrated strong staining for thehXBP-1 protein in the cytoplasm of cancer cells that wasabsent in normal breast ductal epithelial and myoepi-thelial cells; however, no explanation for the predomi-nantly cytoplasmic location of XBP-1 in the cancer cellswas given (Fujimoto et al., 2003).

So far, c-Fos (a leucine zipper transcription factor)was the only transcription factor found to have directcytoplasmic function through a mechanism independentof its genomic AP-1 activity. In quiescent fibroblastcultures stimulated with FCS, c-Fos associates with theendoplasmic reticulum, regulating synthesis/replenish-ment of phospholipid molecules required for signaltransduction pathways, and later on regulates transcrip-tion of enzymes involved in cell-growth and morpho-logical differentiation (Bussolino et al., 2001; Borioliet al., 2002; Gil et al., 2004). Our present results showthat XBP-1, like c-Fos, is expressed during osteoblast

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differentiation, which reinforces the previous finding ofFujimoto et al. (2003), constituting a new exampleof a transcription factor with a main cytoplasmiclocalization.

However, we cannot exclude that XBP-1 is alsopresent in the nucleus of osteoblasts, as the level may bebelow the detection limit of our immunofluorescentassay. It is difficult to explain the cytoplasmic functionof XBP-1 in osteoblasts. At the moment we can onlyspeculate that, as c-Fos does in growing fibroblasts,XBP-1 may modulate phospholipid degradation, affect-ing the formation of lipid second messenger productsinvolved in the signaling of osteoblast differentiation. Ithas recently been demonstrated that the phospholipidssphingosine-1-phosphate and lysophosphatidic acid pre-vent apoptosis in cultured osteoblasts (Grey et al.,2002). Thus, a possible function that remains to bedemonstrated is that XBP-1 could be associated with thesynthesis of phospholipid involved in the regulation ofosteoblast apoptosis.

PTH is known to play a critical role in osteoblastdifferentiation, in part through cAMP-dependant in-duction of c-fos. We therefore examined first whetherPTH could affect expression of xbp-1 and secondwhether the effects were similar to that seen for c-fos.We found that PTH does indeed increase xbp-1 mRNAlevels significantly. It is therefore possible that the effectsinduced by PTH are characterized by a shift in thepattern of expression of transcription factors capable offorming heterodimers that may modulate expression ofdifferent target genes in a temporal manner. However,since XBP-1 and c-Fos in osteoblasts seem to be locatedin different subcellular compartments, it would seemunlikely that XBP-1 is exerting its effect througha heterodimeric complex with c-fos. XBP-1 and c-Fosmay therefore be playing distinct and independent rolesin osteoblast differentiation.

The anabolic effect of PTH on bone and its use fortreatment of pathologies associated with bone-mass lossis well known (Neer et al., 2001; Finkelstein et al., 2003).It was proposed that the observed increased boneformation after treatment with PTH is, among othercauses, due to prevention of osteoblast apoptosis (Jilkaet al., 1999). The present results show that XBP-1 isupregulated by PTH and that the subcellular locationof the protein is mainly cytoplasmic. Based on thedocumented anti-apoptotic effects of both PTH andphospholipids on osteoblasts, the PTH/XBP-1 relation-ship shown here, and the observed cytoplasmic locationof XBP-1, we can propose that the anti-apoptotic effectof PTH could in part be produced through pathwaysinvolving the cytoplasmic activity of XBP-1.

The results reported here indicate that xbp-1 is a genedifferentially expressed during osteoblast differentiationand upregulated by PTH. Its cellular distribution mightindicate that XBP-1 acts not only as a transcription

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factor in the nucleus, but may have additional extranu-clear functions. These data suggest that XBP-1 requiresfurther investigation to address its specific role, likeother leucine zipper transcription factors, during theprogression of osteoblast differentiation.

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