Abstract
Sonic hedgehog (Shh) signaling plays major roles in embryonic development and has also been associated with the progression of certain cancers. Here, Shh family members act directly as long range morphogens, and their ability to do so has been linked to the formation of freely diffusible multimers from the lipidated, cell-tethered monomer (ShhNp). In this work we demonstrate that the multimeric morphogen secreted from endogenous sources, such as mouse embryos and primary chick chondrocytes, consists of oligomeric substructures that are "undisruptable" by boiling, denaturants, and reducing agents. Undisruptable (UD) morphogen oligomers vary in molecular weight and possess elevated biological activity if compared with recombinant Sonic hedgehog (ShhN). However, ShhN can also undergo UD oligomerization via a heparan sulfate (HS)-dependent mechanism in vitro, and HS isolated from different sources differs in its ability to mediate UD oligomer formation. Moreover, site-directed mutagenesis of conserved ShhN glutamine residues abolishes UD oligomerization, and inhibitors directed against transglutaminase (TG) activity strongly decrease the amount of chondrocyte-secreted UD oligomers. These findings reveal an unsuspected ability of the N-terminal hedgehog (Hh) signaling domain to form biologically active, covalently cross-linked oligomers and a novel HS function in this TG-catalyzed process. We suggest that in hypertrophic chondrocytes, HS-assisted, TG-mediated Hh oligomerization modulates signaling via enhanced protein signaling activity.
Highlights
Hedgehog (Hh)4 family members are involved in tissue patterning and progenitor cell proliferation by activation of distinct target genes in a concentration-dependent manner
On the (Drosophila) cell surface, lipidated morphogen monomers are organized into suboptical oligomers that are further recruited to preexisting heparan sulfate proteoglycan (HSPG)-rich membrane subdomains to form large, visible multimeric clusters [2]
In contrast to those “labile” multimers formed from the monomeric morphogen, cultured primary chick chondrocytes endogenously secrete stable morphogen oligomers that were resistant to boiling in the presence of 2% SDS, 100 mM dithiothreitol and could, clearly be distinguished from the monomer by standard denaturing SDS-PAGE (Fig. 1, A and B)
Summary
Hedgehog (Hh)4 family members are involved in tissue patterning and progenitor cell proliferation by activation of distinct target genes in a concentration-dependent manner. These results show that Hh proteins were endogenously expressed as labile HhNp multimers consisting of UD oligomers, which probably are stabilized by covalent cross-linking.
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