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Published by Veles666
Bone Growth
Bone Growth

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Published by: Veles666 on Jan 12, 2014
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ALK2 Functions as a BMP Type I Receptor and Induces Indian Hedgehogin Chondrocytes During Skeletal Development
ABSTRACTGrowth plate chondrocytes integrate multiple signals during normal development. The type I BMP receptorALK2 is expressed in cartilage and expression of constitutively active (CA) ALK2 and other activated type IBMP receptors results in maturation-independent expression of Ihh in chondrocytes in vitro and in vivo. Thefindings suggest that BMP signaling modulates the Ihh/PTHrP signaling pathway that regulates the rate of chondrocyte differentiation.Introduction:
 Bone morphogenetic proteins (BMPs) have an important role in vertebrate limb development. Theexpression of the BMP type I receptors BMPR-IA (ALK3) and BMPR-IB (ALK6) have been more completelycharacterized in skeletal development than ALK2.
 expression was examined in vitro in isolated chick chondrocytes and osteoblasts and in vivo in thedeveloping chick limb bud. The effect of overexpression of CA ALK2 and the other type I BMP receptors on theexpression of genes involved in chondrocyte maturation was determined.
 was expressed in isolated chick osteoblasts and chondrocytes and specifically mediated BMPsignaling. In the developing chick limb bud,
 was highly expressed in mesenchymal soft tissues. In skeletalelements, expression was higher in less mature chondrocytes than in chondrocytes undergoing terminal differenti-ation. CA ALK2 misexpression in vitro enhanced chondrocyte maturation and induced Ihh. Surprisingly, althoughparathyroid hormone-related peptide (PTHrP) strongly inhibited CA ALK2 mediated chondrocyte differentiation,
expression was minimally decreased. CA ALK2 viral infection in stage 19–23 limbs resulted in cartilage expansionwith joint fusion. Enhanced periarticular expression of 
 and delayed maturation of the cartilage elements wereobserved. In the cartilage element, CA ALK2 misexpression precisely colocalized with the expression with
.These findings were most evident in partially infected limbs where normal morphology was maintained. In contrast,
 had a normal pattern of differentiation-related expression. CA BMPR-IA and CA BMPR-IB overexpressionsimilarly induced
 The findings show that BMP signaling induces
. Although the colocalization of the activated type Ireceptors and Ihh suggests a direct BMP-mediated signaling event, other indirect mechanisms may also be involved. Thus,while BMPs act directly on chondrocytes to induce maturation, this effect is counterbalanced in vivo by induction of theIhh/PTHrP signaling loop. The findings suggest that BMPs are integrated into the Ihh/PTHrP signaling loop and that a finebalance of BMP signaling is essential for normal chondrocyte maturation and skeletal development.
J Bone Miner Res 2003;18:1593–1604Key words: bone morphogenetic proteins, bone morphogenetic protein receptors, ALK2, cartilage differ-entiation, limb developmentINTRODUCTION
 important for the devel-opment of both the axial and appendicular skeleton.This process is initiated with the condensation of mesen-chymal cells and development of a cartilaginous template.Chondrocytes subsequently undergo an exquisitely regu-lated program of sequential proliferation, maturation, hy-pertrophy, and calcification that culminates in apoptosis.Vascular in-growth occurs into calcified cartilage, whichserves as a template and scaffold for primary bone forma-tion. With ongoing ossification, the midportion of the orig-
The authors have no conflict of interest.Center for Musculoskeletal Research, University of Rochester, Rochester, New York, USA.
JOURNAL OF BONE AND MINERAL RESEARCHVolume 18, Number 9, 2003© 2003 American Society for Bone and Mineral Research
inal template becomes bone, with chondrocytes movingtoward opposite ends of the long bone. During adolescentgrowth, the shaft of the bone is mineralized, leaving residualchondrocytes in the metaphyseal regions in structures re-ferred to as growth plates.
Endochondral ossi
cation con-tinues in the growth plate until skeletal maturity whenhormonal in
uence cause cessation of growth.Growth factors play a major role in controlling the eventsof limb development and endochondral bone formation.Bone morphogenetic proteins (BMPs) are regulators of cellgrowth, differentiation, apoptosis, cell lineage determina-tion, patterning, and morphogenesis, and are critical for thedevelopment of multiple organs and tissues, including theskeleton.
In early vertebrate limb development, BMPscontrol mesoderm cell proliferation, regulate the growth andregression of the apical ectodermal ridge (AER), specify theanteroposterior axis, initiate chondrogenesis, and regulateapoptosis.
Later, during endochondral ossi
cation,genes for BMPs and BMP receptors are expressed in adistinct spatial and temporal pattern. For example,
, and
 are expressed in the perichon-drium surrounding the cartilage elements,
 isexpressed in prehypertrophic chondrocytes.
Members of the transforming growth factor-
)superfamily, including TGF-
s, BMPs, activins, inhibins,nodals, and other related factors, bind to two types of transmembrane serine/threonine kinase receptors.
Thetype II receptor is a constitutively active kinase, while thetype I receptor is inactive until ligand binding results inassociation with the type II receptor, leading to phosphor-ylation at the glycerin-serine rich (GS) region. Once acti-vated, type I receptors phosphorylate receptor-associatedSmad proteins, which are released from the receptor-complex and associate with Smad4. The activated Smadcomplexes translocate to the nucleus and induce the expres-sion of target genes.
Seven type I receptors, alsocalled ALKs (activin receptor-like kinases), have been iden-ti
ed in vertebrates. BMP type IA receptor (BMPR-IA orALK3) and BMPR-IB (ALK6) are structurally similar toeach other and exclusively bind BMPs.
ALK2 bindsactivins and TGF-
s in vitro,
but recent data suggestthat it physiologically functions as type I receptor for BMPsignaling. ALK2 mimics the mesoderm ventralizing activityof BMPs but not the effect of activin or TGF-
embryo explants.
ALK2 phosphorylates the BMPreceptor-speci
c Smads 1, 5, and 8 and not the TGF-
receptor-associated Smads 2 and 3.
During vertebratelimb development,
 is expressed at low levelsthroughout the limb mesenchyme at early stages and is laterfound in chondrocytes undergoing hypertrophy, while theexpression of 
gures cartilage formation.
Although roles of BMPR-IA and -IB in skeletal develop-ment have been investigated,
less is known about theexpression and function of ALK2 receptor in skeletogen-esis.Here we show that
 is expressed in skeletal tissues.Using a constitutively active form of ALK2 that does notrequire either ligand or type II receptor for signaling, weshow that ALK2 functions as a BMP type I receptor incultured chondrocytes. Moreover, we show that ALK2 in-duces
 in cell culture and in vivo, suggesting that theBMP signaling is upstream of 
 and is integrated into theIhh/PTHrP signaling pathway.
Growth factors and DNA constructs
PTHrP was purchased from Bachem (Torrance, CA,USA) and TGF-
1 was purchased from Calbiochem (SanDiego, CA, USA). BMP-2 was kindly provided by GeneticsInstitute (Cambridge, MA, USA).The expression plasmid encoding the CA rat ALK2 re-ceptor (CA rALK2-pcDNA3.1) was a gift from Dr WylieVale.
The 2.2-kb
HI fragment containing the codingsequence for rat CA ALK2 was subcloned into a cla12shuttle vector and then inserted into the replication compe-tent avian sarcoma retrovirus RCASBP(A).
The CA hu-man ALK5 cDNA was obtained from Dr Joan Massague
and subcloned into the mammalian expression vectorpcDNA3.1 (CA hALK5-pcDNA3.1). The full-length chick ALK2 cDNA clone (chALK2-pcDNA3.1) was supplied byDr Joey Barnett,
and a 1.2-kb
I fragment of thechick ALK2 cDNA was subcloned into the KS
). The authenticity of all the constructs wasveri
ed by automated DNA sequencing. The TGF-
 re-sponsive p3TP-Lux reporter and BMP responsive Xvent2-Luc reporter were gifts from Dr Joan Massague,
and thechick Col-X promoter was obtained from Dr PheobeLeboy.
RCAS viruses that encode CA human BMPR-IAor CA chicken BMPR-IB were from Dr Lee Niswander.
Cell cultures
Chick upper sternal chondrocytes were isolated from thecephalic portion of sterna from 13-day chick embryos.
Cells were resuspended in DMEM with 10% NuSerum IV(Collaborative Biochemical, Bedford, MA, USA) andplated at 2.5 sterna per 100-mm dish. Five to seven dayslater, chondrocytes were harvested, counted, and replated in6-well plates for transient transfection experiments or60-mm dishes for Northern analysis. Chick osteoblasts wereisolated from embryonic day 18 chick calvaria,
and ad-olescent growth plate chondrocytes were isolated from 3- to5-week-old chicks.
 RNA isolation and analysis
Total RNA was puri
ed using RNeasy kit (Qiagen, SantaClarita, CA, USA) according to the manufacturer
s direc-tions. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses of chick 
 expression were performed with the Access RT-PCR System (Promega, Madison, WI, USA). RNA (0.5
g)was used as the template for the RT reaction. The RTreaction was performed at 48
C for 45 minutes, followed byPCR (94
C for 30 s, 58
C for 1 minute, 68
C for 1 minute,30 cycles). The chick 
 primers were 5
 (forward) and 5
 (reverse), resulting in a296-bp product (nt 1444
1739 in U38622). Primers used toamplify BMPR-IA, BMPR-IB, and GAPDH are identical tothose described previously.
Northern analysis was performed with denaturingformaldehyde/agarose gels, and RNA was transferred toGeneScreen Plus membranes (NEN, Boston, MA, USA).
The membranes were hybridized with a
P-labeled chick type X collagen or Ihh probe in QuickHyb solution (Strat-agene) at 68
C, washed at 60
C in 0.1
 SSC and 0.1%SDS, and exposed to Kodak XAR
Transient transfection and luciferase assay
Embryonic upper sternal chondrocytes, cultured at 30
40% con
uence in 6-well plates, were transfected on day 2after plating using the transfection reagent Superfect (Qia-gen). Cotransfection was performed with 0.5
yluciferase reporter, 0.5
g receptor expression plasmid, and5 ng renilla uniformis luciferase. Twelve hours after trans-fection, chondrocytes were incubated for 6 h in serum-freemedia (containing DMEM, hyaluronidase 4 U/ml, andpenicillin/streptomycin), followed by addition of 3 ng/mlTGF-
1 or 50 ng/ml BMP-2 as indicated. Eighteen hoursafter treatment with growth factors, chondrocytes were har-vested and assayed for luciferase activities using Dual Lu-ciferase Assay System (Promega), with renilla luciferasevalues as control for transfection ef 
Data arerepresented as the mean of triplicate samples, and error barsrepresent SEM.
 Expression of CA ALK2 in cultured chondrocytes
Chick embryonic
broblasts (CEFs) grown in DMEMcontaining 10% FBS and 0.2% fetal chick serum weretransfected with the retrovirus RCASBP(A) empty vector orRCASBP(A) containing CA rat ALK2 using Superfect re-agent (Qiagen). After transfection, cells were maintainedand passed three times in 10-cm dishes. On con
uence inthe
nal cultures, fresh DMEM with 10% NuSerum wasadded to the cultures and collected every 24 h. To infect thechondrocyte cultures with RCAS viruses, cells were treatedfor the
rst 48 h in secondary culture with a mixture of 50%fresh DMEM with 10% NuSerum and 50% medium har-vested from the RCAS-infected CEF cultures. After 2 days,chondrocytes were treated with fresh medium with or with-out BMP-2 (50 ng/ml) and/or PTHrP (10
M) for 7 days.The media, BMP-2, and PTHrP were replenished at 48-hintervals.
 In vivo expression of CA ALK2 receptor 
An established method using infected chick embryo
-broblasts (CEFs) was used to infect the developing limb budwith RCAS viruses.
y CEFs were infected withRCASBP(A) virus encoding the CA rat ALK2 receptor.Cells were passed three times, and then pelleted, stainedwith 0.01% nile blue sulfate, and approximately 1000 cells(0.1
l) were injected into the forelimb buds of Hamburger-Hamilton (HH) stage 19
23 chick embryos.
Chick em-bryos were harvested at the appropriate stages after implan-tation and
xed in 4% paraformaldehyde (PFA), followedby dehydration and embedding into paraf 
n for in situhybridization and histological analysis.For Alcian blue-Alizarin red double staining, embryoswere
xed in 95% ethanol.
Pathogen-free eggs werepurchased from SPAFAS (Norwich, CT, USA).More than 100 embryos were injected with RCAS virusexpressing CA ALK2, with approximately 80 embryos pro-cessed for morphological analysis using Alcian blue-Alizarin red double staining. More than 20 embryos wereanalyzed for gene expression by in situ hybridization. Con-sistent morphological
ndings and patterns of gene expres-sion were observed in all the embryos. Con
rmatory exper-iments were performed with embryos injected with puri
edvirus expressing CA rat ALK2 receptor and similar mor-phological and molecular
ndings were observed (data notshown).For negative controls, 10 embryos were injected withCEFs alone, 10 were injected with CEFs infected withempty RCAS viruses, and 10 others were injected withpuri
ed empty RCAS viruses. No morphological or molec-ular abnormalities were observed in any of these embryos.Additional experiments were performed using viruses tooverexpress CA BMPR-IA and CA IB. In these experi-ments, a total of 60 embryos were injected. Thirty injectionswere performed using each of the constructs (CA BMPR-IAand CA BMPR-IB), with 15 injections using infected CEFsand 15 injections using puri
ed virus. For each set of 15injections, 10 were processed for Alcian blue-Alizarin redstaining, and 5 were processed for in situ hybridization.Similar morphological
ndings and patterns of gene expres-sion were observed in each of the embryos.Some experiments were designed to cause partial or lessextensive infection of the chick limb. In these experiments,fewer infected CEFs were injected.
 In situ hybridization
In situ hybridization was performed on tissue sectionsusing
S-UTP labeled antisense RNA probes.
y,after rehydration, the tissues were re
xed in 4% PFA,subjected to 20
g/ml proteinase K treatment at roomtemperature for 5 minutes, and
xed again in 4% PFA.Hybridization was performed at 55
C in 50% formamide,and the post-hybridization washes were carried out at a
nalstringency of 50% formamide/1
 SSC at 55
C. Probeswere kindly provided by Dr Pheobe Leboy (
and Dr Cliff Tabin (
The expression plasmid for CA rat ALK2 (CA rALK2-pcDNA3.1) was digested with
I and transcribed withSP6 RNA polymerase to generate the rat ALK2 antisenseprobe. The plasmid which contains the partial chick ALK2cDNA sequence (chALK2-KS
) was digested with
Iand transcribed with T7 polymerase to generate antisenseprobe for chick ALK2 or digested with
I and transcribedwith T3 polymerase to generate the sense probe.
 ALK2 is expressed in cartilage and functions as a BMP type I receptor in cultured chondrocytes
While the ALK2 receptor is known to have a broadpattern of expression in a variety of tissues,
its expres-sion in developing skeletal tissues has not been character-ized.
 expression was identi
ed in isolated embryonicupper sternal chondrocytes (USC) and juvenile growth platechondrocytes (GPC) using RT-PCR with chick-speci

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