Int..I. De,.. Bioi. 39; 257.262 (1995)

Ameloblasts and odontoblasts, target-cells for1,25-dihydroxyvitamin D3: a review

ARIANE BERDAL', PETROS PAPAGERAKIS, DOMINIQUE HaTTON,ISABELLE BAILLEUL-FORESTIER, JEAN LUC DAVIDEAU

INSERM U120, H6pitaf Robert Debre. Universite Paris VI and Universite Paris VII: Paris, France

ABSTRACT The basic features on the vitamin D endocrine system, synthesis afthe main metabolite1,25 - dihydroxyvitamin 0] (1,251 and its genomic action mediated via the vitamin D receptor (VCR),are reviewed. Calbindin-D9k, calbindin-D28k and osteocalcin are presented as the most-extensivelyinvestigated vitamin D-dependent calcium.binding proteins. The action of 1,25 on the basic processof proliferation and differentiation is introduced. Then, the basis of the systemic theory of vitamin Daction on teeth (clinical and experimental data and the dissimilar distribution of VDR and of potentialvitamin D-dependent proteins in dental cells) are exposed. Finally, the data obtained with calbindin-D9k,calbindin-D28k,osteocalein and VDR, which supports the theory that ameloblasts and odontoblastsare target-cells for 1,25 is presented. As a perspective, a cross-survey of the 1.25 and tooth-relatedliterature is proposed which may indicate potential target-genes for 1,25 in teeth as done previouslyfor calbindins-D.

KEY WORDS: tooth, b011e,vitamin n, wlbiTUlin-lJ, vitamin D receptor

Introduction

Since the early studies on the impact of vitamin D on toothdevelopment (Mellanby, 1928), evidence has been accumulatedconcerning the role of vitamin D on teeth, in close relation with thedecisive steps of the basic and clinical knowledge on this steroid:determination of the active hormonal metabolites (principally 1,25-dihydroxyvitamin 03) and their anabolic and catabolic pathways(for review see Kumar, 1986), identification olthe hormonal receptor(VDR) and at vitamin D-controlled genes (for review see Lowe etal., 1992), and finally understanding of the molecular basis ofclinical disorders related to the vitamin D-endocrine system (forreview see Glorieux ela1..1991).

Basic features of the vitamin D endocrine system

1,25 (OH)2 vitamin D3 and vitamin D receptorVitamin 0 is a secosteroid which can be obtained from the diet

or endogenously produced in the skin in response to UV irradialion(Bikle and Pilla;' 1993). Vitamin 0 and its metabolites circulate inthe blood bound to a vitamin 0 binding globulin DBP (Walters,1992)_ The major metabolite of vitamin 03 is hydroxylated in the

liver in position 25 and then in the kidney and other target-organsand cells in position 1alpha (for review see Kumar, 1986; De Lucaet a1., 1990). The actions of 1,25-dihydroxyvitamin 03 [1,25] aremediated (forreview see Haussler, 1986; Pike, 1991; Lowe et al.,1992) by a nuclear receptor [VDR]. Apart from the genomic effects

of 1,25, this steroid is able to generate biological actions via Ca++,protein kinase C- and cAMP-dependent protein kinase. pathways(Lowe et al., 1992; Walters, 1992).ln the nuclei, VDRs bind to DNAsequences called vitamin 0 responsive elements [VDREJ locatedin the promoter region of target-genes and control their transcrip-tion (Lowe et a1., 1992). VDRs may function as homodimers butalso as heterodimers (Cheskis and Freedman, 1994) with RARs,RXRs and, as described more recently, with T3R (Shrader et a1.,1994). Therefore, the signalling pathways of 1,25 for the control ofgene expression may include other hormonal metabolites (9 cis-and trans-retinoic acids, T3). VDREs were initially discovered inosteocalcin and osteopontin genes, which were first identified inmineralized tissues (forreview see Pike, 1991; Lowe et al., 1992).Theywere then found in other genes related to calcium homeostasis:25-hydroxyvitamin 03 24-hydroxylasegene (Ohyama et a1.. 1994).

Cafbindins-D and osteocafcinMany investigations have focused their attention on intestine,

bone and kidney in order to understand the physiological funclionsof vitamin 0 in the metabolism of calcium and phosphate (Suda eta1., 1991). Among the proteins Ihat are controlled by 1,25, threecalcium.binding proteins, calbindin-D9k and calbindin-D28k, as

Ahbrt't,jfltjOnl fllm;1/ this lml'l'r: 1.25. 1.15 dihydrox}'\ltal11in D:\; \ 'DR. ,ilaminD; DBP, vilamin 0 bindinK globulin; \TIRE. vilamin D responsive clemen I;

RAR. reunoic acid receptor; RxR. retinoic X reet'plor; T3R. th~TOid hor-

mOIlt" reccplOr; T3. :\.5.::r.lriiodOlh~Tonine.

"Address for reprints: INSEAM U120, H6pital Robert Debre. 48 bid Serurier, 75019 Paris, France.FAX:33.1.40031903.

0214-6282/95/$03.00c cae PreuPrinlcdinSplin

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--

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Fig. 1. Parallel expression pattern of VDR and a vitamin O-dependentcalciprotein: calbindin-D9k, in a classical target-cell. the enterocyte.x660. IAI In Situ hybridization of VDR mRNAs in the rat duodenum.Anrisense riboprobes were obtained by in vitro transcription with 1535/-UTPof VDRcDNA inserted in Gemini plasmid (c. Perret ./NSERM UI20).cDNA of chick VDR was provided by J. W. Pike (Ligand, San Oi"ego, CA,USAl. (8) In Situ hybridization of calbindin-D9k mRNAs in a serial section ofrat duodenum. Antisense riboprobes were transcnbed (rom rat calbmdin-D9k cDNA cloned andsub-clonedin Geminiplasmid(c. Perret-INSERMU120). ICI In situ hybridization with the corresponding calbindin-D9k senseriboprobes in a serial secrion of rat duodenum.

well as the bone gla-protein, osteocaicin, have been the mostextensively studied (for review see Lowe et a/., 1992). Calbindins-D are characterized by their different molecular weight, 9 kDa forcalbindin-D 9k and 30 kDa for calbindin-D28k. They belong to thecalcium-binding protein superfamily of parvaibumin,like calmodulinand S100 proteins (for review see Christakos et a/., 1989). Their

tissue-specific expression pattern has been extensively investi-gated: they are used as a tool to map various nuclei in the nervoussystem (for review see Celio, 1990). They are involved in calcium-handling by acting as a calcium-shuttle, or buffer or interacting withvarious enzymes (for review see Christakos et a/.. 1989). Theirpresence in a cell-type does not automatically imply their vitamin D-dependency. For instance, calbindin-D9k is vitamin D-dependentin duodenum, 178 estradiol-dependent in uterus and unresponsiveto both steroids in lung (Dupret et a/" 1992). In a classical target-cell, the enterocyte, a close relationship between ca/bindin-D andVDRgene expression is observed during the cell life-cycle, alignedfrom the crypt along the villus axis (Fig. 1, in situ hybridization ofcalbindin-D and VDR mRNAs in rat duodenum). This paralleldevelopmental pattern illustrates the tact (Arbour et a/., 1993) thatthe amount of VDR controls the responsiveness to 1,25 (in thiscase control of calbindin-D9k expression). Osteocalcin is a weli-known non-collagenous protein of bone matrix and is classicallyused as a marker of osteoblast differentiation in vitro (Lian et a/.,,992). This protein is characterized by the presence of y-carboxylgroups on their glutamic acid residues. This addition confers theability to link ionic calcium and hydroxyapatite to the protein.However, its precise role in the mineralization process is stillunclear. The VDREs of osteoca/cin gene have been identified inrodent and human species (for review see Lowe et a/., 1992).

Other actions of 1,25 (OH)2 vilamln 03

The observation of VDR in many diverse cells (Stumpf, 1988)and the identification of numerous target-genes (Walters. 1992) intissues not related to calcium homeostasis progressively showedthe wider spectrum of action of this hormone. indeed, 1,25 controlsbasic developmental processes. It modulates proliferation andinduces differentiation in many cell-types pertaining to systems asdiverse as the immune system and the skin, as well as the tissuesinvolved in phospho-calcium metabolism, intestine and bone (Abeet a/.. 1986; Suda et a/., 1991; Bickle and Pillai, 1993). Theseeffects have been related to the control of a set of vitamin D-dependent molecules such as proto-oncogenes: c-myc and c-fos(for review see Lowe et al., 1992), homeobox containing genes:Msx II (Hodgkinson et al., 1993), various growth factors (NGF:Wion et al., 1991) and theirreceptors, (EGF receptor: Petkovich eta/., 1987) and differentiation agents: (TGF-beta, Sato et al., 1993).Converseiy. in highly specialized cells when the cells are overtiydifferentiated, 1,25 controls the synthesis of peptides such ashormones (preproparathyroid hormone, prolactin, thyrotropin andcalcitonin; for review see Lowe et al., 1992).

The systemic theory of vitamin D action on teeth

Biological effects of vitamin D on developing teethClinical investigations constantly report the existence of enamel

and dentin alterations in vitamin D-deficient children and in casesof hereditary vitamin D-resistant rickets types I and II (for reviewsee Nikiforuk and Fraser, 1979). Experimental dental featuresinduced by hypovitaminosis (Berdal et al., 1987; Limeback et al.,1992) and hypervitaminosis (Pitaru et al" 1982; Matsumoto et al.,1990) confirmed these clinical observations. In rats raised invitamin D-free conditions. enamel and dentin matrix as well theameloblasts and odontoblasts ultrastructure are abnormal (Fig. 2).However, these disorders were proposed to be secondary to thesystemic effects of 1,25: clinical enamel dysplasia has been

---- - --- - - ---

Fig. 2. Ultrastructure of the apical pole of odontoblasts in 9 day-oldvitamin D-deficient rat. x37.500. In the areas of the first cuspal rowlocated at the cusp tip of the first ratmolars. the secretorypoleis disturbed.It appears tobe fragmented into numerous cytoplasmic patches (Star). VonKorff fibers are present. Other colfagen fibers (diameter: 40-80 nm) showa regular size throughout the widened predentin.

described to be related to hypocalcemia and dentinhypomineralization to hypophosphatemia (Nikiforukand Fraser,1979). Itwas therefore suggested that 1,25 acts on teeth only bycontrolling serum calcium and phosphate. This clinical conceptwas supported by experimental data: 1) enamel mineralization isobtained in a chemically defined medium without any supplemen-tation of vitamin D (Bringas et al., 1987); 2) in vitro caicium andphosphate controi enamel and dentin formation (Woltgens et al"1987); 3) the active metabolites 1,25 and 24,25-dihydroxyvitamin03 do not increase calcium incorporation in tooth germ in vitro(Bawden et al., 1983, 1985).

VDR and vitamin D-dependent molecules were observed indifferent cell-types

Initial investigations on the vitamin O.endocrine system in teethprovided a distribution pattern which tends to confirm the systemictheory ot vitamin D action. Vitamin D receptors were mapped byautoradiography (Kim et al., 1983, 1985; Clark et al., 1985; Stumpf,1988) and described as present in dental cells except in ameloblastsand odontoblasts. These same cells are directly involved in matrixdeposition and mineralization and contain vitamin O-dependentmolecules such as immunoreactive calbindin-D9k (Tayior et al.,1984) and calbindin-D28k (Celio et al., 1984; Taylor, 1984; Elms

\'iramin D alld odollfOg£,lle.<;;.\' 259

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and Taylor, 1987; Magloire et al" 1988) for ameloblasts andosteocalcin (Helder et al., 1993; Bronckers et al., 1994) forodontoblasts, respectively. In view of this conflicting data, thefollowing theory could be proposed: the organization of the promot-ers of vitamin O-responsive genes in bone, duodenum and kidneywould result in their tissue.specific unresponsiveness in teeth, asshown for the third component 01 complement (C3), vitamin D-dependent in bone and unresponsive to the hormone in liver (Jinet al., 1992) respectively. But this situation appeared quite puzzlingbecause ot the strong functional analogy between tooth (for reviewsee Slavkin, 1991) and bone (forreview see Suda et al., 1991), andalso between enamel organ, duodenum and kidney for ionictransfer (for review see Bawden, 1989). Indeed, dental cells andosteoblasts (for review see Table 1 and Suda et al" /991) expressmore than 1a common proteins which are all vitamin O-dependentin bone (for review see Lowe et al., 1992). Since auto radiographicdata on VOR in teeth was obtained in vitamin D-deficient animals(Kim et al., 1983, 1985, in order to obtain unbound receptors),another interpretation of the absence ot the VDR in ameloblastsand odontoblasts was proposed. Vitamin D-free conditions wereconsidered to decrease the receptor quantities, specifically inameloblasts and odontoblasts (Berdal et al" 1993). Indeed, apositive autoregulation of VDR levels (also via a stabilization of theprotein) is observed in rat osteosarcoma cells (Arbour et al., 1993)and may control vitamin 0 responsiveness of the cells. Vitamin 0receptors could be expressed and functional in ameloblasts andodontoblasts at a concentration not detected by auto radiographicexperiments. Therefore. in order to investigate the potential VOR.mediated action of vitamin 0 on ameloblasts and odontoblasts, thebasic concept consisted of using markers of the hormonal action:VDR as well as the widely studied vitamin D-dependent molecules,calbindin-D9k, calbindin-D28k and osteocalcin.

E MEMEM E M

ETB

,II'.. !

9K 28K DC

285

I.

Fig. 3. Northern-blot analysis of enamel organ and dental mesenchymemRNAs. Tota' RNAs extracted from dental epithelium iE) and dentalecromesenchyme (M) are fractioned under denatured conditions andstamed bVethidum bromide (ETB). Calbmdin-D9k mRNAs 19k)aredetectedin E and not in M while both E and M contain calbindin-D28k (28k).OsteocaJcin mRNAs (DC) are restricted to M.

260 A. Bl'rda/ el a/.

TABLE1

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Involvement in tooth germ

Cell growth and differentiationExpression in teeth

POTENTIAL TARGET-GENES FOR 1,25 IN DEVELOPING TOOTH

Control by 1.25 in other tissues

Growth factorsand receptors

EGFrNGFTGFaBMP

c-fosc.myc

Msx-2VDR

Fibronectin

Davideau et al., in pressMitsiadis and Byers, this issue

D'Souza et al., 1990Begue-Kim et al., 1992

Caubet and Bernaudin, 1988Hirning et al.. 1992

McKenzie et al., 1992Serdal et al., 1993

review: Lesot. 1986Thesleff et al.. 1991Andujar et al., 1991

Proto-oncogenes

Factors controlling transcription

Extracellular matrix

a 1(I) collagen

Hard tissue formationa 1(1)collagen

Matnx secretion OsteocalcinOsteopontin

Ionic handling during mineralization CalbindinsCalcium pumpAlkaline phosphatase

breast carcinomaFibroblast L.292Bone

Desprez etal., 1991Wion et al., 1991Finkelman et al.. 1991

Osteoblastic cellsKeratinocytes

BoneBone

Fibroblasts

51. Arnaud Bt al., 1994Hanafin et al.. 1994

Hodgkinson et al., 1993Arbour et aJ., 1993

Franceschi et al., 1987

lichtler et al., 1989

Pavlin at al., 1992Bronckers et al., 1994

BoneBone

Lichtler et al., 1989Ozono et al., 1991Old berg at al., 1990

for review: Lowe at al., 1992Wasserman et al.. 1994Kyeyune-Nyombi at al., 1989

Berdal et al., 1993Barke et al., 1993Engstrom et al., 1977

VDRand control of dental gene expression by 1,25(OH)2vitamin D3

Ca/bindin.D9k, ca/bindin.D28k, as/eoca/cin and VDRThe effects of 1,25 were analyzed in vitamin D-deficient and

control rat teeth. The continuously erupting rat incisor provided auseful experimenfal model since biochemical analysis (RIA, West-ern-blotting and Northern-blotting) can be performed separately onmicrodissecfed enamei organ and dentai ectomesenchyme (Berdaiet al., 1989, 1991 c, 1993). Moreover, since the enamel presecretion,secretion and maturation stages are arranged along the incisoraxis, the developmental pattern of calbindin-D9k and calbindin-D28k expression could be easily followed throughout the ameioblastlife-cycle (Berdal et al., 1991 b,c; Hatton et al.. in press). Target-cells for VDR were identified by light microscopy immunolocali2ationin rat (Berdal et al., 1993) and human developing teeth (Bailleul-Forestier et al., unpublished). VDR visualization in epithelial as wellas ectomesenchymal progenitor cells indicates that 1,25 may playa part in the initial stages of ameloblast and odontoblast life-cycles,as described in various other cell-types (Suda et al., 1991; Bikleand Pillai, 1993). Indeed, vitamin D-deficient rat molars show majordisturbances in the process of morphogenesis, histodifferentiationand terminal differentiation of ameloblasts and odontoblasts (Berdalet al., 1987). 1,25 effects on cell proliferation shown in tooth germin vitro (Sakakura et a/., 1988) may be a key mechanism in vitaminD control of early development.

When cells are overtly differentiated, 1,25 up-regulates the VDRdetected in the ameloblasts and odontoblasts (Berdai el al., 1993).The distribution pattern of the investigated calciproteins variesdepending on the cell-type. Ca/bindin-D9k and calbindin-D28kgenes are both expressed by ameloblasts (Berdal et a/., 1993). Thequantities of cytoplasmic proteins and mRNAs co-vary dependingon the developmental stage (Berdal et a/., 1991 b,c; Hatton et a/.,in press). Calbindin-D28k protein and mRNAs are present indifferentiated odontoblasts (Berdal et al., 1993, 1994). Thesecalciproteins show a cellular distribution very similar to that de-

Intestine. kidneyIntestineBone

scribed for active transcellular calcium transport (for review seeBawden, 1989). Osteocalcin (Helder el a/., 1993; Bronckers et al.,1994) is selectively restricted to the odontoblasts in dentalectomesenchyme. Northern-blot illustrates this selective pattern ofgene expression in the enamel organ and the dental ecto-mesenchyme of the rat incisor (Fig. 3). These three calcium-binding proteins could therefore be used as markers of 1,25genomic action, specifically in ameloblasts and odontoblasts. Anincrease in the steady-state levels of calbindins-D mRNAs in theenamel organ and dental mesenchyme induced by a single injec-tion of 1,25 indicates that these genes are also under the control ofvitamin D in teeth. This data, supported by the immunodetection ofVDR, shows that these cells may be considered as target-cells for1,25 as classically accepted for duodenum, kidney and bone cells(for review see Lowe el a/., 1992). This assertion is supported bythe apparent decrease of immunoreactive osteocalcin in vitamin D-deficient rat molar dentin and odontobiasts (which, however,appears to contain dental constitutive proteins: dentin phospho-proteins, Berdal et a/., 1991 a). Therefore, 1,25 would controlenamel and dentin formation, at least in part, by acting on theexpression of dental genes important for matrix secretion(os/eoca/cin) and mineralization /ca/bindins-D). Further charac-terization of the respective role for 1,25 and calcium and thedifferent transcriptional and/or post-transcriptional steps in the1,25-control of gene expression, as described in other systems (forreview see Lowe el a/., 1992), are critically needed in teeth. Otherroles for vitamin D (control of the exocytosis) were also suggestedby the absence of the dentin phosphoproteins in dentin and theirpresence in odontoblasts of vitamin D-deficient rat molars (Berdalet al., 1991a).

Conclusion and perspectives

Other potential target-genes for 1,25 in developing tooth germmay be identified, as done previously for calbindins-D, by thecross.control in the literature of their expression in teeth and

vitamin D-dependency in other tissues (Table 1). However, theirvitamin D-dependency should be characterized since there mayexist tissue.specific differences in the responsiveness to 1.25(Dupret et ai" 1992; Jin et al" 1992).

Furthermore, an interesting issue would be to investigate thecontrol of 1,25 on the expression of dental-specific genes;amelogeninsand non-amelogeninsfor ameloblasts (for review seeSlavkin et ai" 1991; Brookes et aI" 1995), dentin phosphoproteins(MacDougall et ai" 1992; George et aI" 1994) and sialoprotein(Ritchie et ai" 1993) for odontoblasts. Investigations of the up-stream region of X chromosome-amelogeningene may provide theidentification of the VDRE consensus sequence while its effectivefunction could be established in transgenic mice containing 5'.located promoter combined with a reporter gene as initiatedrecently for the developmentally-controlled consfitutive expressionof this protein (Chen et al., 1994)_ Finally, the interactions of retinoicacids which act on similar stages of development when comparedto 1,25 (Bloch-Zupan et al., 1994 a,b) and 1,25 effects on toothgerm formation, may clarify the complex cross-pathway of thesesteroids (Cheskis and Freedman, 1994) on the control of geneexpression important in the basic processes of development.

AcknowledgmentsInvestigations on the action of vitamin 0 in teeth were supported by

faboratoires CRfNEX, INSERM (UI20) and UniversityRene Diderot{ParisVII} funds. I. B-F. is a recipient of Fonds d'Etudes ef de Recherches duCorps Medical des H6pitaux de Paris. Prof. J. E/ion and the members of hisresearch unit INSERM U 120 are acknowledged for their kind and carefulattention towards dental research.

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