Heparan Sulfate-modulated, Metalloprotease-mediated Sonic Hedgehog Release from Producing Cells

Abstract

The ectodomains of numerous proteins are released from cells by matrix metalloproteases to yield soluble intercellular regulators. A disintegrin and metalloprotease (ADAM) family members have often been found to be the responsible "sheddases," ADAM17/tumor necrosis factor-␣-converting enzyme being its best characterized member. In this work, we show that ShhNp (lipidated and membrane-tethered Sonic hedgehog) is released from Bosc23 cells by metalloprotease-mediated ectodomain shedding, resulting in a soluble and biologically active morphogen. ShhNp shedding is increased by ADAM17 coexpression and cholesterol depletion of cells with methyl-␤-cyclodextrin and is reduced by metalloprotease inhibitors as well as ADAM17 RNA interference. We also show that the amount of shed ShhNp is modulated by extracellular heparan sulfate (HS) and that ShhNp shedding depends on specific HS sulfations. Based on those data, we suggest new roles for metalloproteases, including but not restricted to ADAM17, and for HS-proteoglycans in Hedgehog signaling.The proteins of the Hedgehog (Hh) 3 family are powerful morphogens that control growth and patterning during development. Establishing the molecular mechanisms that generate the Hh gradient is essential for our understanding of how the Hh signal elicits multiple responses in a temporally and spatially specific manner. The Hh spreading mechanism is especially intriguing, because all Hhs are released from the producing cells despite being synthesized as dually lipidated molecules, whereas lipid-modified peptides normally appear firmly tethered to membranes. Both in vertebrates and in Drosophila melanogaster, the Hhs are synthesized as precursor proteins consisting of the N-terminal signaling domain and a C-terminal cholesterol transferase domain. The precursor first undergoes internal cleavage between residues Gly 198 and Cys 199 (of murine Sonic hedgehog (Shh)) linked to the addition of a cholesteryl moiety to Gly 198 of the N-terminal cleavage product (1). This reaction is catalyzed by the C-terminal cholesterol transferase domain through a nucleophilic substitution resembling intein-mediated protein splicing. Inteins, also called protein introns, are parts of protein sequences that are post-translationally excised, their flanking regions (exteins) being spliced together to yield an additional protein product in a self-catalyzed manner. The second lipid adduct that modifies the Hh proteins is palmitic acid, which attaches to the N-terminal cysteine residue exposed after signal peptide cleavage (2), resulting in the formation of the processed (HhNp) protein. The molecular mechanisms through which the lipid-modified HhNp morphogen is able to diffuse long distances are currently under intense debate, and its ability to do so has been linked to oligomer formation or co-transport with lipoprotein particles (3, 4). HhNp gradient formation also depends on the presence of heparan sulfate proteoglycans (HSPGs).Heparan sulfate (HS) is produced by most cell types in vertebrates and invertebrates....

Activation of the hedgehog-signaling pathway in human cancer and the clinical implications

“…Following translation, Hh proteins enter the secretory pathway and undergo autoprocessing and lipid modifications that produce a signaling peptide modified at its both ends by palmitoyl (N terminus) and cholesteryl (C terminus) adducts (Lee et al, 1994;Porter et al, 1995Porter et al, , 1996Buglino and Resh, 2008). The movement of Hh proteins is regulated by several molecules, such as the transmembrane transporter-like protein Dispatched (Disp) (11)(12)(13)(14) and metalloproteases (Dierker et al, 2009) for release of Hh from secreting cells, the heparan sulfate proteoglycans Dally-like (Dlp) and Dally (Lum et al, 2003;Beckett et al, 2008) or their regulators (Baena-Lopez et al, 2008) for extracellular transport of Hh protein as well as enzymes such as Sulfateless and Tout velu for heparan sulfate biosynthesis (Bellaiche et al, 1998;Toyoda et al, 2000;Koziel et al, 2004).…”

Heparan Sulfate and Transglutaminase Activity Are Required for the Formation of Covalently Cross-linked Hedgehog Oligomers

“…Morphogen release from the cell surface then depends on the expression of Dispatched (3) and ADAM (a disintegrin and metalloprotease) family members. The latter mediate morphogen shedding in an HSregulated way, as has recently been shown for ShhNp in transfected Bosc23 cells, a HEK 293T-derived cell line (4).…”

“…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. In vertebrates, three closely related Hh morphogens (Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog) have been described, and a single ortholog is expressed in Drosophila melanogaster.…”

Two Distinct Sites in Sonic Hedgehog Combine for Heparan Sulfate Interactions and Cell Signaling Functions

“…To examine the biological functions of the different hShhNC24II proteins, the C3H10T1/2 osteoblast precursor cell line was utilized, which expresses alkaline phosphatase (AP) when stimulated by Shh (22). As shown in Fig.…”

“…HSPGs may be required for multimer formation (22) and might be linked to Hh by Shifted, a secreted Wnt inhibitory factor homologue. The N-terminal region of Shh contains a basic motif that is conserved across vertebrates.…”