The roles of annexins and alkaline phosphatase in mineralization process

20 Pages·2003·0.65 MB·English

Vol. 50 No. 4/2003
1019–1038
QUARTERLY
Review
The roles of annexins and alkaline phosphatase in
mineralization process(cid:1)
Marcin Balcerzak1, Eva Hamade2, Le Zhang2, Slawomir Pikula1, Gérard Azzar2,
Jacqueline Radisson2, Joanna Bandorowicz-Pikula1 and Rene Buchet2(cid:1)
1
M. Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warszawa, Poland;
2
Laboratoire de Physico-Chimie Biologique, UMR CNRS 5013, Université Claude Bernard, Lyon 1,
UFR de Chimie-Biochimie, F-69622 Villeurbanne, France
Received: 05 August, 2003; revised: 24 September, 2003; accepted: 7 October, 2003
Key words: matrix vesicle, annexin, alkaline phosphatase, hydroxyapatite, mineralization
In this review the roles of specific proteins during the first step of mineralization
and nucleation are discussed. Mineralization is initiated inside the extracellular
organelles–matrixvesicles(MVs).MVs,containingrelativelyhighconcentrationsof
2+
Ca andinorganicphosphate(P),createanoptimalenvironmenttoinducethefor-
i
mationofhydroxyapatite(HA).Specialattentionisgiventotwofamiliesofproteins
present in MVs, annexins (AnxAs) and tissue-nonspecific alkaline phosphatases
2+
(TNAPs).BothfamiliesparticipateintheformationofHAcrystals.AnxAsareCa -
2+
andlipid-bindingproteins,whichareinvolvedinCa homeostasisinbonecellsand
inextracellularMVs.AnxAsformcalciumionchannelswithinthemembraneofMVs.
Although the mechanisms of ion channel formation by AnxAs are not well under-
(cid:1)This work was supported in part bygrant No.3 P04A 007 22 from the State Committee for Scientific
Research (KBN, Poland) and by CNRS. Authors would like to thank Dr. Pascale Chavassieux from
UniversitéClaudeBernard,Lyon1,France,forfruitfuldiscussionandDr.JohnCarewforlanguagecor-
rections.
(cid:1)
Correspondingauthor:ReneBuchet,LaboratoiredePhysico-ChimieBiologique,UMRCNRS5013,
Université Claude Bernard, Lyon 1, UFR de Chimie-Biochimie, 6 rue Victor Grignard, F-69622
Villeurbanne, France; tel.: [phone]; fax: [phone]; e-mail: [email protected]
Abbreviations: 1,25-(OH) -D , 1(cid:1),25-dihydroxyvitamin D ; AnxA, vertebrate annexin; ATPase, ATP
2 3 3
hydrolase; ATRA, all-trans retinoic acid; BMP-2, bone morphogenetic protein 2; GPI,
glycosylphosphatidylinositol; HA, hydroxyapatite; MV, matrix vesicle; NTP, nucleoside triphosphate;
PE, phosphatidylethanolamine; PC-1, plasma cell membrane glycoprotein-1 (NTP pyrophosphatase
phosphodiesterase isoenzyme); PS, phosphatidylserine; RAR/RXR, receptors of retinoic acid and its
derivatives; T3, 3,5,3(cid:2)-triiodo-L-thyronine; TGF(cid:3), transforming growth factor (cid:3); TNAP, tissue-nonspe-
cific alkaline phosphatase.
1020 M. Balcerzak and others 2003
stood,evidenceisprovidedthatacidicpHorGTPcontributetothisprocess.Further-
more,lowmolecularmassligands,asvitaminAderivatives,canmodulatetheactiv-
ityofMVsbyinteractingwithAnxAsandaffectingtheirexpression.AnxAsandother
anionicproteinsarealsoinvolvedinthecrystalnucleation.Thesecondfamilyofpro-
teins,TNAPs,isassociatedwithP homeostasis,andcanhydrolyseavarietyofphos-
i
phatecompounds.ATPisreleasedintheextracellularmatrix,whereitcanbehydro-
lyzed by TNAPs, ATP hydrolases and nucleoside triphosphate (NTP) pyrophos-
phohydrolases. However, TNAP is probably not responsible for ATP-dependent
2+
Ca /phosphate complexformation.Itcanhydrolyse pyrophosphate (PP),aknown
i
inhibitor of HA formation and a byproduct of NTP pyrophosphohydrolases. In this
respect,antagonisticactivitiesofTNAPsandNTPpyrophosphohydrolasescanregu-
late the mineralization process.
SKELETAL TISSUES Growth plate chondrocytes undergo several
seriesofdifferentiationevents,includingpro-
Thetwomajorskeletaltissues,cartilageand liferation and hypertrophy. All these events
bones, are structurally and functionally dif- are required for bone formation during
ferent (Heinegard & Oldberg, 1989). Carti- endochondral ossification. Chondrocyte hy-
lage is highly hydrated and, except at the pertrophy occurs at the expense of adjacent
growth plates of long bones, rarely mineral- matrix and it requires matrix resorption.
izes, resulting in a permeable matrix of Bone formation takes place in the organism
gel-like consistency. On the other hand, bone not only during embryonic development
matrix routinely mineralizes to form a rigid (growth plate cartilage in the process of
impermeable matrix (Marks & Popoff, 1988). endochondral bone formation) and growth
Proteoglycan and type II collagen are major butthroughoutthelifeintheprocessofphysi-
matrix components in cartilage, while type I ological bone remodeling (Lian & Stein,
collagen is the major part of bone matrix 1996).
(Marks&Popoff,1988).Inbothcartilageand
bone, cellular activities include matrixforma-
tion, mineralization and resorption. CELL BIOLOGY
In each tissue, different cell types (Fig. 1)
perform distinct tasks, which sometimes Chondrocytes and osteoblasts are of
overlap each other. Bone matrix is formed mesenchymalorigin.Mesenchymalstemcells
and mineralized by osteoblasts and resorbed areabletogenerateprogenitorswithrestrict-
by osteoclasts (Fig. 1A). Osteocytes partici- ed developmental potential. From progenitor
pate in extracellular exchanges between dif- cells, various cell types can be differentiated
ferent components of osseous tissue. into fibroblasts, adipocytes, chondrocytes
Osteocytes are also involved in the and osteoblasts (Fig. 2). The two latter cell
mechanotransduction (Marks & Popoff, types under the influence of growth factors
1988). In cartilage, matrix formation results giverisetocellsabletoformcalcifiedtissues.
from the activity of chondrocytes (Marks & Hypertrophic chondrocytes and osteoblasts
Popoff, 1988). Chondrocytes express hyper- initiate the calcification process by releasing
trophic and non-hypertrophic phenotypes matrixvesicles (MVs)(Anderson, 2003).MVs
(Fig. 1B). Hypertrophic chondrocytes are of growth plate cartilage differ in lipid and
characteristic for developing bones and for protein composition from MVs produced by
so-called growth plate. Non-hypertrophic osteoblasts (Boyan et al., 1988). It has been
chondrocytes are also found in the growth suggested that MV biogenesis, from growth
plate and may participate in the formation of platehypertrophicchondrocytes,couldbethe
articular cartilage (Fig. 1C) (Poole, 2001). result of programmed cell death. This would
Vol. 50 Annexins and alkaline phosphatase in mineralization 1021
A Osteoblasts Osteocytes Boneliningcells
Osteoid
Mineralized
bone
B C
Reverse Articularsurface
zone Superficial
zone
Proliferative
zone
Middle
zone
Zoneof
maturation
Upper
Deep
hypertrophic
zone
zone
Lower
hypertrophic
zone
Calcificationofcartilage
Calcified
Vascularfront zone
Subchondral
Zoneofwovenbone bone
formation& Subchondral
angiogenesis bonemarrow
Figure1.Regionalorganizationandrelationshipsamongbone,growthplateandarticularcartilage.
PanelA.Topographicrelationshipamongbonecells.Osteoblastsarelocatedonthelininglayerofbonesurface,ac-
tivelyproducingmatrix,whichisnotyetcalcified(osteoidtissue).Osteocytesarethemostmatureorterminally
differentiatedcellsoftheosteoblastlineage.Osteocytesareembeddedinthebonematrix.PanelB.primarymam-
maliangrowthplateshowingprogressivedevelopmentofchondroblastsfromtheproliferativezonetothelowerhy-
pertrophiczone,wherematrixsynthesisstopsandtheextracellularmatrixiscalcified.PanelC.Regionalorganiza-
tion of articular cartilage. The superficial zone contains thin collagen fibrils arranged parallel with the articular
surface.Thepartlycalcifiedcartilageofthecalcifiedzoneisindicated.AdaptedfromMarks&Popoff(1988)and
Poole (2001).
appear not to be the case for MVs released requires the presence of extracellular MVs
from viable osteoblasts (Anderson, 2003). (Anderson,1995;2003),sincethefirststepof
MVs initiate mineral formation starting from mineralization is initiated inside these
embryonic ossification to bone formation in organelles.MVs(insizebetweenonehundred
adults (Hoshi & Ozawa, 2000). to several hundred nanometers in diameter)
2+
serve as a site for Ca and P accumulation.
i
MVs create a specific environment where de-
MATRIX VESICLES position of initial amorphous mineral com-
plexes (nucleation) occurs and where
Severalstagesofmineralizationwereidenti- hydroxyapatite (HA) e.g. Ca (PO )(OH) , is
10 4 2
fied.Themineralizationofboneandcartilage produced and forms needle-like crystals on
1022 M. Balcerzak and others 2003
the inner surface of the MV membrane. The 2003). Although details of the mechanism are
extracellularmatrixcontainssufficientlyhigh still unknown, assembly of mineral complexes
2+
levels of Ca and P concentrations to sus- depends probably on electrostatic, structural
i
tain the nucleation process and to propagate and stereochemical properties at the inor-
T
3-
1,25-(OH)2 D3
ATRA
TGFb
Chondrocyte Hypertrophic Boneliningcell
chondrocyte
ATRA TGFb
BMP-2 BMP-2
Multipotentstemcell Pluripotentstemcell Pre-osteoblast Osteoblast
Adipocyte Fibroblast Osteocyte
Figure 2. Lineage of osteochondroprogenitor cells.
Pluripotentstemcellsdevelopfrommultipotentialmesenchymalstemcells.Thepluripotentstemcellsareprogeni-
torsofallindigenouscellsofconnectivetissues:fibroblasts,adipocytes,osteoblastsandchondrocytes.Theinflu-
ences of several physiological factors like transforming growth factor (cid:3) (TGF(cid:3)), bone morphogenetic protein 2
(BMP-2),all-transretinoicacid(ATRA),1(cid:1),25-dihydroxyvitaminD3(1,25-(OH)2-D3),and3,5,3’-triiodo-L-thyronine
(T3)onthelineageofosteoblastsandchondrocytesareindicatedinthefigure.Osteoblastsexistintwoformsasos-
teoblastsmonolayeronthesurfaceofgrowingbonetissuewhichsynthesizeandsecreteorganiccomponentsofma-
trix and produce mineralization competent matrix vesicles and osteocytes enclosed within bone matrix.
Chondrocytesarecharacterizedbytwophenotypes:non-hypertrophiccharacteristicforarticularcartilage,andhy-
pertrophic in growth plate. Adapted from Lian & Stein (1996).
the mineralization (Anderson, 2003). Ion ganic–organicinterface.Subtleinteractionsbe-
channels and transporters present in MV tween negatively charged domains of proteins,
2+
membraneareresponsibleforCa andP up- anionic phospholipids and mineral complexes
i
take into these organelles. After reaching a arecrucialinthepropagationofarraysofcrys-
certainlength,theneedle-likeHAcrystalsare tals.AllprocessestakingplaceinMVsrequirea
released from MVs into the extracellular ma- dynamic but tightly regulated system to main-
2+
trix. The mechanisms by which the HA crys- tainCa homeostasisandP delivery.Manyac-
i
tals can break the membrane of MVs are not tors have been identified to date, among them
2+
very well understood. One possible explana- vertebrate annexin (AnxAs), Ca - and mem-
tion is that the activity of phospholipases brane-binding proteins, as well as alkaline
could be triggered, once HA crystals are phosphatase.
formed, and may affect the MV membrane
fluidity (Swain et al., 1992; Schwartz &
Boyan, 1988). ANNEXINS IN MINERALIZING
The second step of mineralization starts with TISSUES
areleaseofHAcrystals.Thesecrystalsserveas
a template for the formation of crystalline ar- Fromtwelvemembersoftheannexinfamily
rays,leadingtoatissuecalcification(Anderson, ofproteinspresentinmammalianorganisms,
Vol. 50 Annexins and alkaline phosphatase in mineralization 1023
2+
three were identified in MVs: annexin A2 and X collagens enhance Ca influx into
(AnxA2),AnxA5andAnxA6(Caoet al.,1993; MVs, promoting activity of ion channels
Kirsch et al., 1997a; 1997b; Kirsch & Claass- formed by AnxAs (Kirsch & Wuthier, 1994;
2+
en,2000).DuetothehighCa concentration Kirschetal.,1994;2000a).However,thepres-
both inside and outside MVs, and the high ence of collagen is not essential for mineral-
content of anionic phospholipids, mainly ization, as shown with knockout animals
phosphatidylserine (PS), and cholesterol in (Jacenko et al., 1993), with reconstituted sys-
MV membrane (Harder et al., 1997; Wuthier, tems (Kirsch et al., 1997a) and with purified
1975; Ayala-Sanmartin, 2001; Ayala-Sanmar- MVs (Hsu & Anderson, 1978; Kirsch et al.,
tinet al.,2001;DeDiegoet al.,2002;),AnxAs 2000a; Wang & Kirsch, 2002). Nevertheless,
can be associated with both outer and inner collagens could influence initialization and
leaflets ofMVmembrane.AnxAs affectmem- progression of mineral formation in MVs. In
2+
brane stability in a Ca -dependent manner addition,ANXA5–/–micewerenormalinre-
(Goossens et al., 1995). In addition, AnxAs spect of development of their skeletons
2+
couldbeinvolvedintheCa transport,asion (Brachvogel et al., 2003), probably because
channelsinsertedwithintheMVmembrane. otherAnxAscouldreplaceAnxA5functionin
DuringthefirstphaseofMV-mediatedcalcifi- knockout animals.
cation,mineralcomplexesappearontheinner AnxAs are specific markers of chondrocyte
surfaceofMVmembrane.Thehighaffinityfor hypertrophy. Articular cartilage cells, in con-
2+
Ca ofPSisquitestrongintheinnerleafletof trast to growth plate chondrocytes, maintain
the MV membrane enriched with anionic a stable phenotype. The upper zone of the ar-
lipids(Majeskaetal.,1979;Tayloretal.,1998). ticular cartilage (Fig. 1C) contains thin colla-
2+
Accordingly, AnxA5 exhibiting Ca -depend- gen fibrils and proteoglycan called aggrecan.
ent PS-binding property was isolated with In this zone, tensile forces connected with
2+
PS–Ca –P complexes from nucleation core daily life are maximally concentrated. In
i
of chicken growth plate MVs (Wu et al., 1993; lowerzones, asinthemiddleanddeepzones,
1996; 1997a). Smaller amounts of AnxA2 and the cell density decreases, collagen fibers are
AnxA6,aswellasotherproteins,wereco-puri- thicker and aggrecan content is higher. Calci-
fied with AnxA5 (Wu et al., 1997a; 2002a). fied zones, where chondrocytes develop an
AnxAs associated with the outer surface of hypertrophic phenotype, provide a link be-
MVandbone-derived cellmembranesmayin- tween subchondral bone and joint cartilage
teract with extracellular matrix molecules. (Poole, 2001). Articular cartilage, unlike
AnxA2andAnxA6bindchondroitinsulfatein growth plate, usually does not undergo ma-
2+
a Ca -dependent manner (Ishitsuka et al., trix calcification. However, mineralization
1998; Ishitsuka, 2000; Takagi et al., 2002). frequently occurs during osteoarthritis and
AnxA5 binds types II and X collagens and aging. In osteoarthritis, progressive damage
C-propeptide of type II collagen (Kirsch & and loss of articular cartilage matrix (espe-
Pfäffle, 1992; von der Mark & Mollenhauer, ciallyinsuperficialzone)areobserved. These
1997; Kirsch et al., 2000a). The above de- events are accompanied by cell death and
scribedinteractionsmayinfluenceMVshape, pathologicalmatrixmineralization,leadingto
thereby affecting crystal growth. Indeed, bone remodelling and to subchondral bone
chondrosarcoma cells, expressing low quanti- mass increase. In addition, an inflammatory
tiesofAnxA5, arenotabletobindtypeIIcol- process occurs, giving rise to pain and move-
lagen.ThissuggeststhatAnxA5isakeymole- ment disabilities. The amount of AnxAs is
cule to promote extracellular matrix binding, scarce in normal articular cartilage, while it
whichisessentialforcartilagefunction(King significantly increases during progression of
et al., 1997). In chicken growth plate, types II osteoarthritis (Mollenhauer et al., 1999;
1024 M. Balcerzak and others 2003
Kirsch et al., 2000b; Pfander et al., 2001). diphosphatidylglycerolandlysophospholipids
Therefore, AnxAs could be specific markers due to the difference in the rate of phos-
of differentiation during osteoarthritis. For pholipid degradation (Wuthier et al., 1977;
example, AnxA8, a protein not previously de- 1978). The anionic phospholipid content in
scribed in the growth plate, is expressed dur- calcified cartilage and bone is significantly
ing inappropriate cell differentiation in higher than in non-calcifying cartilage zones
osteoarthritis (White et al., 2002). Relatively (Wuthier, 1968; Wu et al., 2002a). This may
highannexinexpressioninarticularcartilage indicate that anionic phospholipids are in-
chondrocytes is characteristic for hypertro- volved in mineral formation. It is in agree-
phic cells and cells undergoing apoptosis mentwiththeresultsofmanyexperimentsin-
2+
(Kirsch et al., 2000b; Kouri et al., 2000) with dicating that Ca -dependent binding of
the appearance of MVs or apoptotic bodies, AnxAs to model membranes is enhanced by
respectively(Derfusetal.,1998;Hashimotoet the content of anionic phospholipids. Maxi-
2+
al., 1998; Mollenhauer et al., 1999; Kirsch et mal Ca influx mediated by AnxAs into lipo-
al., 2000b). These events lead to mineraliza- somes occurs when they are prepared from
tionofjointmatrix(Gelseet al.,2003)andex- PS and phosphatidylethanolamine (PE) mix-
pression of hypertrophy protein markers, as ture at 9:1 mole/mole (Kirsch et al., 1997a).
type X collagen and alkaline phosphatase In addition, PS clustering may be induced by
(Hoyland et al., 1991; Pullig et al., 2000; thehighcholesterolcontentinMVmembrane
Kirschetal.,2000b).MVsarepresentinartic- (Wuthier, 1975). AnxA5 interacts in a
2+
ular cartilage from healthy subjects (Einhorn Ca -dependent manner with cardiolipin in
et al., 1985; Derfus et al., 1996). In osteo- isolated mitochondria (Megli et al., 1995;
arthritis, MVs coexist in extracellular matrix 2000).SincecardiolipinispresentalsoinMV
with apoptotic bodies which are the products membrane (Wuthier, 1975), these interac-
of chondrocytes at the terminal differentia- tions may occur in MVs.
tion stage. There are no phagocytic cells in AnxA2, AnxA5 and AnxA6 are abundant in
joint cartilage, therefore, apoptotic bodies re- acidified organic extracts of MVs (25–40% of
main in the cartilage unless the extracellular extraction of AnxAs from crude preparations,
matrix becomes degraded. as reported by Genge et al., 1991), suggesting
theirpresenceinthehydrophobiccoreoflipid
bilayer.Thiswasalsoevidencedbyusingselec-
FACTORS AFFECTING ANNEXIN ION tive labeling of AnxA5 with photoactivable hy-
CHANNEL FORMATION drophobic reagent, revealing that this protein
inserts into the membrane hydrophobic core
The existence of a Ca2+ transport system in at mildly acidic pH (Isas et al., 2000). At low
MVs is not well established. Possible candi- pH, aspartate and glutamate residues of
dates are AnxAs, since ion channels formed AnxAsareprotonated.Theproteinsurfacebe-
bytheseproteinsin vitrohavebeendescribed comesmorehydrophobic,facilitatingitsinser-
in literature (Berendes et al., 1993; Arispe et tion into lipid bilayer (Kohler et al., 1997;
al., 1996; Kourie & Wood, 2000; Kirilenko et Beermann ofm cap et al., 1998; Isas et al.,
al.,2002).Tounderstand how AnxAs canme- 2000; 2003; Golczak et al., 2001a; b).
diate Ca2+ influx into MVs, factors affecting Whether, the low pH-induced annexin ion
annexinactivityinthemineralizationprocess channels in MVs may form during mineral-
shouldbeidentified,asforexamplelipidcom- ization, remains to be elucidated. In fact, it
position of MV membrane. is not clear which population of AnxAs may
MVmembraneisdistinctfromplasmamem- participate in ion channel formation:
brane (Wuthier, 1975). It is enriched in PS, AnxAs associated with the external or the
Vol. 50 Annexins and alkaline phosphatase in mineralization 1025
internal leaflet of the MV membrane. It is trix synthesis, as well as morphological
possible that annexin channels are formed changes associated with local compaction of
in plasma membrane before MV budding. matrix around the cells, may affect chon-
The pH measurements made in tissue sec- drocyte proliferation and maturation
tions indicate that intracellular pH in (Buschmann et al., 1995; Quinn et al., 1998;
chicken growth plate is dependent on the Wu&Chen,2000).Forexample,hyperosmotic
zone from which chondrocytes are derived. stimuli was reported to affect protein synthe-
2+ +
The lowest pH was observed in the periph- sisincartilage,aswellasCa andH homeo-
ery of late hypertrophic and calcifying cells stasis (Dascalu et al., 1996; Erickson et al.,
(Wu et al., 1997b). Moreover, protons are 2001).
byproducts of HA formation in MVs. Low Additional factors that may affect annexin
pHcanpreventHAformationbyincreasing ionchannelactivityduringmineralizationare
solubility of formed mineral for which the associated with their interaction with other
optimal pH for crystal formation is in the proteins. Mobasheri et al. (2002) attributed
rangeof7.4–7.8(Valhmuet al.,1990).How- perception of mechanical signals in cartilage
ever, extensive acidification during crystal to cell surface membrane mechanoreceptors.
formation is prevented by type II carbonic These receptors are composed of integrins
anhydrase (Stechschulte et al., 1992; Sauer and stretch activated ion channels. Multiple
et al., 1994). mechanosensitive ion channels were charac-
Chondrocytes in the growth plate release terizedinosteoblastsandchondrocytes.None
NTPs that may regulate cell maturation and of these channels revealed similarities with
matrix mineralization (Hatori et al., 1995; AnxAs(Davidsonetal.,1990;1996;Duncan&
Hungetal.,1997;).NTPsarealsoreleasedby Hruska, 1994; Guilak et al., 1999; Koprowski
non-stimulated (Hatori et al., 1995) and by & Kubalski, 2001; Biggin & Sansom, 2003;
stimulatedosteoblastsinresponsetomechan- Shakibaei&Mobasheri,2003).Inosteoblasts,
2+
ical activation (Romanello et al., 2001). increase in [Ca ] by oscillating fluid flow,
in
AnxAs can bind nucleotides under in vitro was attenuated by the addition of anti-AnxA5
conditions (Kirilenko et al., 2001; 2002; antibodies. This suggests that AnxA5 may be
Bandorowicz-Pikula et al., 2001; 2003) but involved in mechanotransduction in bone
probably, with the exception of AnxA7 (Yellowley et al., 2002). Recently, it was ob-
(Caohuyetal.,1996),donothydrolyzenucleo- served that AnxA5 binds to the cytoplasmic
tides. GTP in a millimolar concentration part of (cid:3)5 subunit of bovine integrin (cid:1)v(cid:3)5
range induced AnxA6 channel formation in (Andersen et al., 2002).
planarlipidbilayers(Kirilenkoetal.,2002).It Homodimeric S100A and S100B proteins
was also shown that the AnxA5 ion channel interact with AnxA5 and AnxA6 at 1 mole
activity in MV could be regulated by NTPs S100 dimer per 2 mole annexin stoichio-
(Arispe et al., 1996). However, the mecha- metry (Donato, 2003). It was previously
nism by which these channels are formed in demonstrated by co-immunoprecipitation
MVs is not yet elucidated. (Arcuri et al., 2002) and inhibition of
2+
annexin-mediatedCa fluxes(Garbugliaet
al., 1998; 2000). However, annexin–S100
INTERACTIONS OF ANNEXINS WITH interactions have not been investigated in
OTHER PROTEINS DURING cellsystemsabletoperformmineralization.
MINERALIZATION It was reported that calbindin D9k, an un-
usual monomericmemberofS100proteins,
Changes in extracellular fluid composition, is present in MVs (Balmain, 1991; 1992;
reductionsinextracellularpH,increaseinma- Balmainet al.,1989;1991;1995).Calbindin
1026 M. Balcerzak and others 2003
D9k is a vitamin D -dependent protein and ment of cells with ATRA and BAPTA-AM
3
2+ 2+
its expression affects dietary Ca accumu- (intracellular Ca chelator) or K-201, a
lation in bones (Li et al., 2001). The pres- 1,4-benzothiazepine derivative that can in-
ence of this protein is important for inter- hibitionchannelactivityofAnxAs(Kanekoet
2+
stitial Ca absorption. In rat epiphyseal al., 1997a; 1997b; Hofmann et al., 1998;
chondrocytes, calbindin D9k is highly ex- Wang et al., 2003). This may indicate that
2+
pressed only in mature and hypertrophic annexin-mediatedCa fluxesareresponsible
chondrocytes (Balmain et al., 1995). It is for events related to cell maturation, cell
postulated that calbindin D9k takes part in apoptosis and tissue mineralization. Re-
mineral nucleation (Balmain, 1991). Be- cently, we observed that precursor of ATRA,
sides, calbindin D9k reveals 47% and 37% all-trans retinol (vitamin A), binds to AnxA6
identity and 64% and 55% homology in pri- in vitro (Fig. 3), especially at acidic pH, pro-
mary structure with S100A and S100B pro- viding a possible regulatory link with an
teins, respectively. Such high similarity be- annexin-mediatedmineralizationprocess.Ad-
tweenproteinssupportsthehypothesisthat ditionofretinoidscouldpromotethemineral-
calbindin D9k can interact with AnsAs dur- ization process not only by enhancing
ing mineralization. annexin expression but by direct interaction
withAnxAsorbychangingthemembraneflu-
idity (Wang et al., 2003). It has been also
EFFECT OF RETINOIC ACID ON shown that 1(cid:1),25-dihydroxyvitamin D binds
3
THE MATURATION OF to AnxA2 of rat osteoblast-like ROS 24/1
2+
CHONDROCYTES AND ON THE cells, inducing increases in intracellular Ca
MINERALIZATION PROCESS concentration (Baran et al., 2000).
Recent findings reveal that growth plate
chondrocytes proliferate and mature faster ROLES OF ANNEXINS AND OTHER
upon treatment with all-trans retinoic acid ANIONIC PROTEINS IN THE
(ATRA) (De Luca et al., 2000). It is accompa- NUCLEATION PROCESS
nied by terminal differentiation of
chondrocytes and production of mineraliza- Most non-collagenous proteins involved in
tion competent MVs, rich in AnxAs and alka- initiationandregulationofbiologicalmineral
line phosphatase (Wang & Kirsch, 2002; formationareanionic(Boskey,1996).Among
Wangetal.,2003).ATRA,anagonistofrecep- proteinssynthesized byosteoblasts areosteo-
tors of retinoic acid and other retinoids nectin, osteopontin, osteocalcin and bone
(RAR/RXR), stimulates events leading to sialoprotein. Cartilage extracellular proteins
mineralization and matrix remodeling. In ad- are similar to bone, while both tissues differ
dition, it stimulates cell differentiation and in types of collagens. All these proteins share
apoptosis, as well expression of metallopro- a high content of aspartic and glutamic acid
teinases (Nie et al., 1998), type I collagen (ex- residues (30–40%) and multiple phosphoryl
pression of proteoglycans and type II and X andsialylgroups.Theydifferintheirabilities
collagens is inhibited), alkaline phosphatase toaffect the formationofHA in vitro(Hunter
and AnxAs (Wu et al., 1997c; Wang et al., et al., 1996). Additionally, the phosphopro-
2003). Moreover, events characteristic for teinsofboneareprocessedbylimitedproteol-
apoptosis, such as down-regulation of Bcl-2, ysis, then they are converted into more phos-
activation of capsase-3 and DNA fragmenta- phorylatedspeciesthatcouldfacilitateminer-
tion occur after treatment with ATRA. These alization (Suzuki et al., 1996). AnxAs have
events are reversed by simultaneous treat- several putative phosphorylation sites and
Vol. 50 Annexins and alkaline phosphatase in mineralization 1027
protein flexibility in comparison with wild
AnxA6 (Freye-Minks et al., 2003).
ItisnotknownhowAnxAscaninfluencethe
nucleation sites at the membrane interface
and which charged domains are responsible
forelectrostaticinteractionstakingplacedur-
ing nucleation. Crystal structures of AnxAs
suggest the importance of flexibility for
AnxA6 (Avila-Sakar et al., 2000) and AnxA5
(Olingetal.,2000;2001)intheannexin–phos-
Figure4.BindingofvitaminA(all-transretinol)
pholipid interactions. Given these findings, it
to AnxA6.
is tempting to suggest that AnxAs may influ-
TodeterminebindingofvitaminAtoAnxA6,quench- ence molecular organization during nucle-
ing of intrinsic fluorescence of the annexin was mea-
ation formation, through changes in molecu-
sured,usingthesamemethodasfortheretinolcarrier
lar flexibility or through protein–protein in-
protein(Raghuetal.,2003).PanelA.Humanrecombi-
nantAnxA6(1(cid:4)M)wasincubatedin150mMNaCl,10 teractions. Such interactions with other MV
mM Tris/HCl, pH 7.4, without (bold line) or with proteinsmayfavoraccumulationofinorganic
(dashedline)3(cid:4)MvitaminAaddedfromconcentrated material.
stock solution in ethanol (final concentration of etha-
nol did not exceed 0.5%). Asolectin liposomes were
alsoaddedinthepresenceofAnxA6(protein/lipidra-
ALKALINE PHOSPHATASE AND
tio of 1:1000, by mole) and vitamin A (dotted line).
Panel B. AnxA6 (1 (cid:4)M) was incubated in 150 mM RELATED PROTEINS IN THE
NaCl,10 mMcitricbuffer,pH 6.0,without(bold line) MATRIX VESICLES
or with (dashed line) 3 (cid:4)M vitamin A. Asolectin
liposomes were also added in the presence of AnxA6 Alkaline phosphatase is one of the most fre-
(protein/lipidratioof1:1000,bymole)andvitaminA
quently used biochemical markers of osteo-
(dottedline).Sampleswereexcitedat295nmandfluo-
blastactivity(Risteli&Risteli,1993;Garnero
rescence emission spectra were recorded at the wave-
& Delmas, 1996; Nawawi et al., 1996;
length range from 320 to 380 nm. All measurements
Magnussonetal.,1999).Fourgenesencoding
were performed on a Fluorolog 3 spectrophotometer
(JobinYvonSpexEdison,NJ)with2-nmslitsforboth human alkaline phosphatase have been
excitation and emission, at 25°C. Quenching of the cloned (Kam et al., 1985; Millán, 1986;
AnxA6intrinsicfluorescencebyvitaminAishigherat Henthorn et al., 1987; Millán & Manes, 1988)
pH 6.0 than at pH 7.4. After liposome addition, the
corresponding to three specific alkaline phos-
protein fluorescence returns to the basic level only at
phatase genes located in chromosome 2
pH7.4,probablyduetohigheraffinityofvitaminAfor
(germ-cell, placenta and intestinal) and one
lipidsthanforAnxA6(dissociationofprotein–vitamin
A complex). At pH 6.0, upon addition of liposomes, TNAP gene located in chromosome 1 (Moss,
AnxA6 inserts within the hydrophobic core of the 1992).Alkalinephosphatasesfromallsources
membranelipidbilayerwhereitisstillabletointeract arehomodimericmetalloenzymes whichcata-
withhydrophobicvitaminA.Theresultofthisexperi-
lyze the hydrolysis of almost any phospho-
ment suggests that AnxA6 binds vitamin A in vitro.
monoester with release of P and alcohol
i
(Fernley, 1971).
TNAP exists in three forms derived from
some of them are phosphorylated in vitro bone, liver and kidney and differing in carbo-
(Grima et al., 1994). In the case of AnxA6, hydrate groups. Osseous TNAP localized in
phosphorylation mimicking mutation re- plasma membrane and in MVs, is a glycosyl-
sulted in higher Ca2+-binding affinity and phosphatidylinositol (GPI)-anchored protein
conformational changes leading to increased (Nodaetal.,1987;Pizauroetal.,1994).Given
1028 M. Balcerzak and others 2003
2+
the different solubilization of TNAP from Ca and phosphate substrates (AMP, crea-
osteoblast plasma membrane, obtained from tine phosphate, glucose phosphate and (cid:3)-gly-
human primary bone cell culture, it was sug- cerophosphate). Under these conditions, ad-
gested that changes in TNAP activity result ditionofATPdoesnotpromotetheformation
from age-related modifications. These of HA (Hamade et al., 2003). This finding is
changes could be associated with the post- consistent with the fact that a specific
translational modification of TNAP or with ATPase,ratherthanTNAP,isresponsiblefor
the membrane constituents (Radisson et al., ATP-dependent mineral formation within
1996; Bourrat et al., 2000). The role of TNAP MVs isolated from bone and/or cartilage
in mineral formation was evidenced in the (Hsu & Anderson, 1995; 1996; Hsu et al.,
case of hypophosphatasia, an inheritable dis- 1999). The nature of ATPase involved in the
order leading to a defective bone formation ATP-dependent mineral formation is not
and characterized by a deficiency in TNAP known and it was proposed that a
2+
(Whyte, 1994). Mice deficient in the gene en- Ca -ATPase could fulfil this role (Hsu & An-
coding TNAP mimic a severe form of derson, 1996). Therefore, not only TNAP but
hypophosphatasia, indicating the importance also other enzymes are involved in the P ho-
i
of TNAP in hydrolyzing phosphate sub- meostasis (Fig. 4). The local concentration of
strates, including PP, during mineral forma-
i
tion (Narisawa et al., 1997). In addition, sev-
eral mutations in TNAP occur around a cal-
cium- binding site of the enzyme, not directly
associated with the metal-binding site func-
tion for hydrolysis. It is suggested that these
mutationsresultinTNAPmisfolding(Mornet
et al., 2001).
TNAP appears to be a multifunctional en-
Figure 4. Production of pyrophosphate and inor-
zyme and several of its properties may be im- ganicphosphateandtheirantagonisticeffectson
portant for the mineralization process (Bel- the mineralization process.
lows et al., 1991; Hsu, 1992a; 1992b; Rattner
PP,inhibitorsofHAformation,areproducedatleast
i
et al., 2000). Although TNAP is a well-known partly by plasma cell membrane glycoprotein-1 (NTP
biochemicalmarkerofmineralization,thena- pyrophosphatase phosphodiesterase isoenzyme, PC-1)
ture of the substrate hydrolyzed by TNAP is fromthehydrolysisofNTPs.TheactivityofTNAPmay
boost the formation of HA, by hydrolyzing PP and
notclearlyestablished.Itwasproposedalong i
eliminating its inhibitory effect on HA formation. P
time ago that TNAP may supply P by hydro- i
i arises from distinct sources, including the hydrolytic
lyzing phosphate substrates (Robison, 1924). 2+
activityofTNAP.AccumulationofP andCa canin-
i
This proposal was further substantiated by duce the formation of HA. Adapted from Hessle et al.
the observation that supplementation of cul- (2002).
ture media with (cid:3)-glycerophosphate, an exog-
enous TNAP substrate, induced osteogenesis P can be increased by the activities of ade-
i
and HA deposition (Tenenbaum, 1981;Ecaot- ninemonophosphodiesterases,ATPhydrolas-
Chevrier et al., 1983). Addition of levamisole, es (ATPases) and NTP pyrophosphohydro-
a specific inhibitor of TNAP activity, pre- lases (Bartling & Chong 1999; Anderson,
vented (cid:3)-glycerophosphate-induced mineral- 2003).
ization in vitro (Tenenbaum, 1987). Chondrocytes in the growth plate release
TNAP purified from femur of chicken em- ATP (Hung et al., 1997). ATP is also released
bryosinducestheformationofHAinmineral- by non-stimulated bone cells (Hatori et al.,
ization medium without P but containing 1995) or in response to mechanical stimula-
i