Lipid Modifications of Sonic Hedgehog Ligand Dictate Cellular Reception and Signal Response

Abstract

BackgroundSonic hedgehog (Shh) signaling regulates cell growth during embryonic development, tissue homeostasis and tumorigenesis. Concentration-dependent cellular responses to secreted Shh protein are essential for tissue patterning. Shh ligand is covalently modified by two lipid moieties, cholesterol and palmitate, and their hydrophobic properties are known to govern the cellular release and formation of soluble multimeric Shh complexes. However, the influences of the lipid moieties on cellular reception and signal response are not well understood.Methodology/Principal FindingsWe analyzed fully lipidated Shh and mutant forms to eliminate one or both adducts in NIH3T3 mouse embryonic fibroblasts. Quantitative measurements of recombinant Shh protein concentration, cellular localization, and signaling potency were integrated to determine the contributions of each lipid adduct on ligand cellular localization and signaling potency. We demonstrate that lipid modification is required for cell reception, that either adduct is sufficient to confer cellular association, that the cholesterol adduct anchors ligand to the plasma membrane and that the palmitate adduct augments ligand internalization. We further show that signaling potency correlates directly with cellular concentration of Shh ligand.Conclusions/SignificanceThe findings of this study demonstrate that lipid modification of Shh determines cell concentration and potency, revealing complementary functions of hydrophobic modification in morphogen signaling by attenuating cellular release and augmenting reception of Shh protein in target tissues.

Expression of Hedgehog ligand and signal transduction components in mutually distinct isocitrate dehydrogenase mutant glioma cells supports a role for paracrine signaling

“…The cytosolic expression pattern of SHH in neoplastic cells by immunofluorescence staining (Figure 1C) is consistent with ligand expression in the secretory pathway of a producing cells, as in prior studies SHH staining could not be detected in the cytosol of receiving cells [42]. To test this hypothesis and gain further insight into potential autocrine and paracrine Hh signaling modes, in situ hybridization for SHH transcript was performed in conjunction with GLI1 and PTCH1 expression.…”

Arsenic inhibits hedgehog signaling during P19 cell differentiation

“…SHH protein is processed by cleavage, and the final product modified with choleseterol and palmitate moieties, as well as with heparin sulfate (Beachy et al , 2010). The modifications allow SHH to form multimeric complexes, to be anchored to the cell membrane, or to vesicle membranes, and the modifications also correlate with its signaling potency (Chen et al , 2004; Grover et al , 2011). Our results show that SHH protein expression is very robust in both the P19 cells themselves as well as in secreted, exosomal vesicles, although arsenic exposure does not alter SHH protein expression.…”

GALNT1-Mediated Glycosylation and Activation of Sonic Hedgehog Signaling Maintains the Self-Renewal and Tumor-Initiating Capacity of Bladder Cancer Stem Cells

“…Primary tumor tissues were digested with 200U type I collagenase, 20U type IV collagenase (Sigma, C-0130, C-5138), and DNase at 37 C for 2 to 6 hours. Then, the cancer cells were stained with FITC-conjugated BCMab1 and PE-conjugated anti-CD44 antibody or the same isotype FITC/PE-conjugated antibody for 30 minutes on ice.…”

“…The N-terminal cysteine is catalyzed by the acyl-transferase Skinny hedgehog to form the amide linkage of palmitate (34,35). Hydrophobic modifications confer membrane affinity such that the signaling domain is successfully associated with its membrane receptors, leading to a signaling cascade (36,37). Here we defined a novel glycosylation modification for SHH in BCSCs, which is uniquely mediated by GALNT1.…”