Gain-of-function Shh mutants activate Smo cell-autonomously independent of Ptch1/2 function

Casillas, Catalina; Roelink, Henk

doi:10.1016/j.mod.2018.08.009

Cited by 9 publications

(14 citation statements)

References 75 publications

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“…While Hh canonical Hh signaling is typically induced in a "mesemchymal type" cell by a fully process 19 kDa N-Shh form produced by an "epithelial like" cells, Hh signaling in ECs occur through a noncanonical autocrine signaling. Consistent with the present finding such signaling was shown to be induced by full length-unprocessed Hh ligands 31 . However the molecular mechanisms by which 43 kDa FL-Dhh promotes endothelial barrier function remains to be characterized.…”

Section: Discussionsupporting

confidence: 93%

Endothelial cell response to Hedgehog ligands depends on their processing

Hollier

Chapouly

Diop

et al. 2020

Preprint

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Rationale:The therapeutic potential of Hedgehog (Hh) signaling agonists for vascular diseases is of growing interest. However, molecular and cellular mechanisms underlying the role of the Hh signaling in vascular biology remain poorly understood. Objective: The purpose of the present paper is to clarify some conflicting literature data. Findings: With this goal we have demonstrated that, unexpectedly, ectopically administered N-terminal Sonic Hedgehog (N-Shh) and endogenous endothelial-derived Desert Hedgehog (Dhh) induce opposite effects in endothelial cells. (ECs). Notably, endothelial Dhh acts under its full-length soluble form (FL-Dhh) and activates Smoothened in ECs, while N-Shh inhibits it. At molecular level, N-Shh prevents FL-Dhh binding to Patched-1 demonstrating that N-Shh acts as competitive antagonist to FL-Dhh. Besides, we found that even though FL-Hh ligands and N-Hh ligands all bind Patched-1, they induce specific Patched-1 localization. Finally, we confirmed that in a pathophysiological setting i.e. brain inflammation, astrocyte-derived N-Shh act as a FL-Dhh antagonist. Conclusion: The present study highlights for the first time that FL-Dhh and N-Hh ligands have antagonistic properties especially in ECs, and demonstrates that Hh ligands or forms of Hh ligands cannot be used instead of another for therapeutic purposes.

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Section: Discussionsupporting

confidence: 93%

Endothelial cell response to Hedgehog ligands depends on their processing

Hollier

Chapouly

Diop

et al. 2020

Preprint

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“…First we tested whether Gas1 or Cdon modulate Dhh interaction with Ptch1 and Smo. Notably, Smo has been recently shown to be a receptor for Hh ligands especially in the case of cell autonomous signaling (Casillas et Roelink 2018). Interestingly, we found that Gas1 prevents Dhh interaction with Ptch1 but promotes Dhh interaction with Smo.…”

Section: Gas1 Promotes Dhh Interaction With Smo While Cdon Prevents Dmentioning

confidence: 58%

Desert Hedgehog-driven endothelium integrity is enhanced by Gas1 but negatively regulated by Cdon

Chapouly

Hollier

Guimbal

et al. 2020

Preprint

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Evidences accumulated within the past decades, identified Hedgehog (Hh) signaling as a new regulator of micro-vessel integrity. More specifically, we recently identified Desert Hedgehog (Dhh) as a downstream effector of Klf2 in endothelial cells (ECs). Objective: The purpose of this study is to investigate whether Hh co-receptors Gas1 and Cdon may be used as therapeutic targets to modulate Dhh signaling in ECs. Methods and results: We demonstrated that both Gas1 and Cdon are expressed in adult ECs and relied on either siRNAs or EC specific conditional KO mice to investigate their role. We found that Gas1 deficiency mainly photocopies Dhh deficiency especially by inducing VCAM-1 and ICAM-1 overexpression while Cdon deficiency has opposite effects by promoting endothelial junction integrity. At a molecular level, Cdon prevents Dhh binding to Ptch1 and thus acts a decoy receptor for Dhh, while Gas1 promotes Dhh binding to Smo and as a result potentiates Dhh effects. Since Cdon is overexpressed in ECs treated by inflammatory cytokines including TNFα and Il1β, we then tested whether Cdon inhibition would promote endothelium integrity in acute inflammatory conditions and found that both fibrinogen and IgG extravasation were decreased in association with an increased Cdh5 expression in the brain cortex of EC specific Cdon KO mice administered locally with Il1β.

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“…Mutating the residues directly involved in the coordination of zinc are obvious candidates to assess a role for the putative peptidase activity. However, Shh mutants in the zinc coordination domain could barely be detected the N-terminal processed form (ShhNp) on Western Blots, despite normal detection of the Shh pro-protein (Casillas and Roelink, 2018). We and others (Traiffort et al, 2004) initially incorrectly interpreted this as a failure of auto-processing, but the same low levels were observed analyzing these mutants in Shh-C199*, a Shh mutant truncated at the auto-processing site (ShhN) (Roelink et al, 1995).…”

Section: Mutations In the Zinc Coordination Domain Reduce The Stabilimentioning

confidence: 93%

“…The Dynamin inhibitor Dynasore (that inhibits endocytosis) (Macia et al, 2006) causes strong accumulation of Shh-C199*, but not of Shh-C199*/H183A, further indicating that the destabilization of Shh-C199*/H183A occurs before it reaches the plasma membrane ( Figure 2E). We found that other zinc coordination mutations as well as several holoprosencephaly-associated point mutations in Shh cause its destabilization, indicating a role for increased ShhN degradation in this birth defect (Casillas and Roelink, 2018). In general, we will not use these mutants with a reduced half-life.…”

Section: Mutations In the Zinc Coordination Domain Reduce The Stabilimentioning

confidence: 99%

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Association of Sonic Hedgehog with the extracellular matrix requires its zinc-coordination fold

Jaegers

Roelink

2019

Preprint

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Sonic Hedgehog (Shh) has a catalytic cleft characteristic for zinc metallopeptidases. Nevertheless, the putative peptidase activity of Shh is dispensable for activation of the response in vitro and was deemed "pseudo-active". Still, Shh has significant sequence similarities with some bacterial peptidoglycan metallopeptidases defining a subgroup within the M15A family that, besides having the characteristic zinc coordination domain, can bind two calcium ions. Extracellular matrix (ECM) components in animals include heparan-sulfate proteoglycans, which are analogs of bacterial peptidoglycan and thus potential peptidase substrates. We found that the putative metallopeptidase activity of Shh is required for its association with ECM as well as for non-cell autonomous signaling. The putative peptidase requires the presence of at least 0.1 µM zinc and is inhibited by mutations affecting critical catalytic residues as well as extracellular calcium. Our results indicate that Shh is a calcium-regulated zinc metallopeptidase.

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Gain-of-function Shh mutants activate Smo cell-autonomously independent of Ptch1/2 function

Cited by 9 publications

References 75 publications

Endothelial cell response to Hedgehog ligands depends on their processing

Endothelial cell response to Hedgehog ligands depends on their processing

Desert Hedgehog-driven endothelium integrity is enhanced by Gas1 but negatively regulated by Cdon

Association of Sonic Hedgehog with the extracellular matrix requires its zinc-coordination fold

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