Proteolytic processing of palmitoylated Hedgehog peptides specifies the 3-4 intervein region of the Drosophila wing.

Cell fate determination during development often requires morphogen transport from producing to distant responding cells. Hedgehog (Hh) morphogens present a challenge to this concept, as all Hhs are synthesized as terminally lipidated molecules that form insoluble clusters at the surface of producing cells. While several proposed Hh transport modes tie directly into these unusual properties, the crucial step of Hh relay from producing cells to receptors on remote responding cells remains unresolved. Using wing development in Drosophila melanogaster as a model, we show that Hh relay and direct patterning of the 3-4 intervein region strictly depend on proteolytic removal of lipidated N-terminal membrane anchors. Site-directed modification of the N-terminal Hh processing site selectively eliminated the entire 3-4 intervein region, and additional targeted removal of N-palmitate restored its formation. Hence, palmitoylated membrane anchors restrict morphogen spread until site-specific processing switches membrane-bound Hh into bioactive forms with specific patterning functions.

Sonic hedgehog processing and release are regulated by glypican heparan sulfate proteoglycans.

All Hedgehog morphogens are released from producing cells, despite being synthesized as N- and C-terminally lipidated molecules, a modification that firmly tethers them to the cell membrane. We have previously shown that proteolytic removal of both lipidated peptides, called shedding, releases bioactive Sonic hedgehog (Shh) morphogens from the surface of transfected Bosc23 cells. Using in vivo knockdown together with in vitro cell culture studies, we now show that glypican heparan sulfate proteoglycans regulate this process, through their heparan sulfate chains, in a cell autonomous manner. Heparan sulfate specifically modifies Shh processing at the cell surface, and purified glycosaminoglycans enhance the proteolytic removal of N- and C-terminal Shh peptides under cell-free conditions. The most likely explanation for these observations is direct Shh processing in the extracellular compartment, suggesting that heparan sulfate acts as a scaffold or activator for Shh ligands and the factors required for their turnover. We also show that purified heparan sulfate isolated from specific cell types and tissues mediates the release of bioactive Shh from pancreatic cancer cells, revealing a previously unknown regulatory role for these versatile molecules in a pathological context.

Sweet on Hedgehogs: regulatory roles of heparan sulfate proteoglycans in Hedgehog-dependent cell proliferation and differentiation.

Morphogens exert their effects over long distances, typically by spreading from cell to cell to activate signal transduction in surrounding tissues in concentration-dependent manner. One example of a morphogen is the signaling molecule Hedgehog (Hh), which controls growth and patterning during development and has also been implicated in the progression of numerous cancers. To this end, accessory mechanisms that release, transport, and receive Hhs are required to elicit temporally and spatially specific responses in cells and tissues. The Hh spreading mechanism is especially intriguing, because all Hhs are released from the producing cells despite being synthesized as dually lipidated, membrane-tethered molecules. In addition to this cellular association, Hhs bind strongly to extracellular heparan sulfate proteoglycans (HSPGs), which is expected to further reduce their spreading. Paradoxically, several lines of evidence suggest that Hh gradient formation actually requires HSPG expression, and that HSPGs act as both positive and negative regulators of Hh function. This article reviews the multiple roles that HSPGs play in Hh morphogen function, and discusses their congruity with proposed mechanisms of Hh solubilization, transport, and signal reception in vertebrate and invertebrate tissues.

Scube2 enhances proteolytic Shh processing from the surface of Shh-producing cells.

All morphogens of the Hedgehog (Hh) family are synthesized as dual-lipidated proteins, which results in their firm attachment to the surface of the cell in which they were produced. Thus, Hh release into the extracellular space requires accessory protein activities. We suggested previously that the proteolytic removal of N- and C-terminal lipidated peptides (shedding) could be one such activity. More recently, the secreted glycoprotein Scube2 (signal peptide, cubulin domain, epidermal-growth-factor-like protein 2) was also implicated in the release of Shh from the cell membrane. This activity strictly depended on the CUB domains of Scube2, which derive their name from the complement serine proteases and from bone morphogenetic protein-1/tolloid metalloproteinases (C1r/C1s, Uegf and Bmp1). CUB domains function as regulators of proteolytic activity in these proteins. This suggested that sheddases and Scube2 might cooperate in Shh release. Here, we confirm that sheddases and Scube2 act cooperatively to increase the pool of soluble bioactive Shh, and that Scube2-dependent morphogen release is unequivocally linked to the proteolytic processing of lipidated Shh termini, resulting in truncated soluble Shh. Thus, Scube2 proteins act as protease enhancers in this setting, revealing newly identified Scube2 functions in Hh signaling regulation.