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2009 Conference abstract
Understanding the role of Ihh and proteoglycans interaction during bone development
Patrick Allard, Ph.D.
Dr Clifford Tabin Laboratory Harvard Medical School Boston
Patrick Allard1, Maria Pazyra2, Rosalind Segal2, Monia Zuntini3, Luca Sangiorgi3, Clifford Tabin1 1- Genetics, Harvard Medical
School. Boston, MA. 2- Dana Farber Cancer Institute. Harvard. Boston, MA. 3- Rizzoli Orthopedic Institute, Bologna, Italy.
e-mail: pallard@genetics.med.harvard.edu.
Heparan-sulfate proteoglycans (HSPGs) have recently been recognized as mediators of intercellular signaling during development
(for review see Lin, 2004). During bone development specifically, loss of function of genes implicated in the synthesis of HSPGs,
namely Ext1 and Ext2, show a clear defect in the patterning of the growth plate. Ext1 heterozygous or hypomorphic embryos
display an increased chondrocyte proliferation and a delay in chondrocyte differentiation (Koziel et al, 2004; Hilton et al, 2005).
At the molecular level, Ext1 mutant embryos show a reduced Ihh mRNA expression but an increased Ihh protein distribution
(Koziel et al, 2004; Hilton et al, 2005). Consistent with that result, PTHrP mRNA levels, which is downstream of Ihh signaling, is
up-regulated (Koziel et al, 2004). While these studies address the role of HSPGs during growth plate development, they do not
specifically address the function of HSPGs in mediating Ihh signaling in that context. Our objective is to investigate the role of
sulfated proteoglygans (SPGs) in mediating Ihh signaling during bone development. To this aim, we will specifically abolish the
interaction of Ihh with SPGs.
The interaction of proteins with SPGs is mediated by a domain, termed the Cardin-Weintraub domain, consisting of a short
stretch of basic amino-acid (Cardin and Weintraub, 1989). Examination of Ihh amino-acid sequence revealed the presence of a
stretch of basic amino-acid highly conserved in identity and location with the described functional Cardin-Weintraub (CW)
domain of Shh. Ihh CW domain is also completely evolutionary conserved from zebrafish to human. Next, we mutated Ihh CW
domain in vitro and observed that although wild-type Ihh can bind heparin, mutation of Ihh CW domain strongly diminishes Ihh
affinity for heparin sulfate and chondroitin sulfate proteoglycans. We also showed that Ihh requires its CW domain to bind
efficiently to bone sections. Furthermore, Ihh binding to bone sections is also dependent on the presence of heparan-sulfate
chains. We are currently generating a mouse knock-in carrying a mutation of the CW domain in Ihh. Finally, in collaboration with
Dr Luca Sangiorgi, we are also screening human patients that present chondrodysplasia phenotypes consistent with a mutation
in Ihh CW domain.
Understanding the role of Ihh and proteoglycans interaction during bone development
Patrick Allard, Ph.D.
Dr Clifford Tabin Laboratory Harvard Medical School Boston
Patrick Allard1, Maria Pazyra2, Rosalind Segal2, Monia Zuntini3, Luca Sangiorgi3, Clifford Tabin1 1- Genetics, Harvard Medical
School. Boston, MA. 2- Dana Farber Cancer Institute. Harvard. Boston, MA. 3- Rizzoli Orthopedic Institute, Bologna, Italy.
e-mail: pallard@genetics.med.harvard.edu.
Heparan-sulfate proteoglycans (HSPGs) have recently been recognized as mediators of intercellular signaling during development
(for review see Lin, 2004). During bone development specifically, loss of function of genes implicated in the synthesis of HSPGs,
namely Ext1 and Ext2, show a clear defect in the patterning of the growth plate. Ext1 heterozygous or hypomorphic embryos
display an increased chondrocyte proliferation and a delay in chondrocyte differentiation (Koziel et al, 2004; Hilton et al, 2005).
At the molecular level, Ext1 mutant embryos show a reduced Ihh mRNA expression but an increased Ihh protein distribution
(Koziel et al, 2004; Hilton et al, 2005). Consistent with that result, PTHrP mRNA levels, which is downstream of Ihh signaling, is
up-regulated (Koziel et al, 2004). While these studies address the role of HSPGs during growth plate development, they do not
specifically address the function of HSPGs in mediating Ihh signaling in that context. Our objective is to investigate the role of
sulfated proteoglygans (SPGs) in mediating Ihh signaling during bone development. To this aim, we will specifically abolish the
interaction of Ihh with SPGs.
The interaction of proteins with SPGs is mediated by a domain, termed the Cardin-Weintraub domain, consisting of a short
stretch of basic amino-acid (Cardin and Weintraub, 1989). Examination of Ihh amino-acid sequence revealed the presence of a
stretch of basic amino-acid highly conserved in identity and location with the described functional Cardin-Weintraub (CW)
domain of Shh. Ihh CW domain is also completely evolutionary conserved from zebrafish to human. Next, we mutated Ihh CW
domain in vitro and observed that although wild-type Ihh can bind heparin, mutation of Ihh CW domain strongly diminishes Ihh
affinity for heparin sulfate and chondroitin sulfate proteoglycans. We also showed that Ihh requires its CW domain to bind
efficiently to bone sections. Furthermore, Ihh binding to bone sections is also dependent on the presence of heparan-sulfate
chains. We are currently generating a mouse knock-in carrying a mutation of the CW domain in Ihh. Finally, in collaboration with
Dr Luca Sangiorgi, we are also screening human patients that present chondrodysplasia phenotypes consistent with a mutation
in Ihh CW domain.
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