Indian hedgehog and syndecans-3 coregulate chondrocyte proliferation and function during chick limb skeletogenesis.
Hedgehog proteins exert critical roles in embryogenesis and require heparan sulfate proteoglycans (HS-PGs) for action. Indian hedgehog (Ihh) is produced by prehypertrophic chondrocytes in developing long bones and regulates chondrocyte proliferation and other events, but it is not known whether it requires HS-PGs for function. Because the HS-PG syndecan-3 is preferentially expressed by proliferating chondrocytes, we tested whether it mediates Ihh action. Primary chick chondrocyte cultures were treated with recombinant Ihh (rIhh-N) in absence or presence of heparinase I or syndecan-3 neutralizing antibodies. While rIhh-N stimulated proliferation in control cultures, it failed to do so in heparinase- or antibody-treated cultures. In reciprocal gain-of-function studies, chondrocytes were made to overexpress syndecan-3 by an RCAS viral vector. Cells became more responsive to rIhh-N, but even this response was counteracted by heparinase or antibody treatment. To complement the in vitro data, RCAS viral particles were microinjected in day 4-5 chick wing buds and effects of syndecan-3 misexpression were monitored over time. Syndecan-3 misexpression led to widespread chondrocyte proliferation and, interestingly, broader expression and distribution of Ihh. In addition, the syndecan-3 misexpressing skeletal elements were short, remained cartilaginous, lacked osteogenesis, and exhibited a markedly reduced expression of collagen X and osteopontin, products characteristic of hypertrophic chondrocytes and bone cells. The data are the first to indicate that Ihh action in chondrocyte proliferation involves syndecan-3 and to identify a specific member of the syndecan family as mediator of hedgehog function.
Department of Orthopaedic Surgery, Thomas Jefferson University Medical School, Philadelphia, Pennsylvania 19107, USA.
Tsuyoshi
T
Shimo
Chiara
C
Gentili
Masahiro
M
Iwamoto
Changshan
C
Wu
Eiki
E
Koyama
Maurizio
M
Pacifici
D000818:Animals; D015152:Blotting, Northern; D001846:Bone Development; D001973:Bromodeoxyuridine / Q000494:pharmacology; D002448:Cell Adhesion; D002455:Cell Division; D002642:Chick Embryo; D019902:Chondrocytes / Q000166:cytology*; D024121:Collagen Type X / Q000096:biosynthesis; D004396:Coloring Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005121:Extremities / Q000196:embryology*; D005353:Fibronectins / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D053823:Hedgehog Proteins; D015151:Immunoblotting; D017403:In Situ Hybridization; D008562:Membrane Glycoproteins / Q000378:metabolism / Q000502:physiology*; D053495:Osteopontin; D011509:Proteoglycans / Q000378:metabolism / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012795:Sialoglycoproteins / Q000096:biosynthesis; D053670:Syndecan-3; D013997:Time Factors; D015534:Trans-Activators / Q000502:physiology*
D024121:Collagen Type X; D004396:Coloring Agents; D005353:Fibronectins; D053823:Hedgehog Proteins; D008562:Membrane Glycoproteins; D011509:Proteoglycans; D012795:Sialoglycoproteins; D053670:Syndecan-3; D015534:Trans-Activators; D053495:Osteopontin; D001973:Bromodeoxyuridine