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.
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,
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-
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.
MATERIALS AND METHODS
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.
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.
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
ALK2, BMPR-IA, BMPR-IB,
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.
1594 ZHANG ET AL.