Secretion and Signaling Activities of Lipoprotein-Associated Hedgehog and Non-Sterol-Modified Hedgehog in Flies and Mammals

<div><p>Hedgehog (Hh) proteins control animal development and tissue homeostasis. They activate gene expression by regulating processing, stability, and activation of Gli/Cubitus interruptus (Ci) transcription factors. Hh proteins are secreted and spread through tissue, despite becoming covalently linked to sterol during processing. Multiple mechanisms have been proposed to release Hh proteins in distinct forms; in <i>Drosophila</i>, lipoproteins facilitate long-range Hh mobilization but also contain lipids that repress the pathway. Here, we show that mammalian lipoproteins have conserved roles in Sonic Hedgehog (Shh) release and pathway repression. We demonstrate that lipoprotein-associated forms of Hh and Shh specifically block lipoprotein-mediated pathway inhibition. We also identify a second conserved release form that is not sterol-modified and can be released independently of lipoproteins (Hh-N*/Shh-N*). Lipoprotein-associated Hh/Shh and Hh-N*/Shh-N* have complementary and synergistic functions. In <i>Drosophila</i> wing imaginal discs, lipoprotein-associated Hh increases the amount of full-length Ci, but is insufficient for target gene activation. However, small amounts of non-sterol-modified Hh synergize with lipoprotein-associated Hh to fully activate the pathway and allow target gene expression. The existence of Hh secretion forms with distinct signaling activities suggests a novel mechanism for generating a diversity of Hh responses.</p> </div

Shh is secreted in lipoprotein-associated and lipoprotein-free forms.

<p>(A) Density of human Shh secreted by HeLa cells in the absence or presence of fetal bovine serum (FBS), analyzed by Optiprep density gradient centrifugation, and Western blotting (WB). HeLa cells transfected with Shh were grown in serum-free medium or in the presence of 10% FBS, and equal volumes of supernatants analyzed. Colors indicate fractions corresponding to bovine Very Low-, Low-, and High-Density Lipoproteins (VLDL, LDL, and HDL) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001505#pbio.1001505-Chapman1" target="_blank">[68]</a>. (B) Density of non-lipid-modified Shh-N^C24S, analyzed by Optiprep density gradient centrifugation and WB. Supernatants were derived from HeLa cells transfected with Shh-N^C24S and grown in the presence of FBS. (C) Shh levels in cell lysates and supernatants derived from HeLa cells transfected with Shh, grown in serum-free medium supplemented with individual human lipoprotein classes. Equal protein amounts (cell lysates) or volumes (supernatants) were analyzed. (D) Density of Shh in HeLa cell supernatants shown in (C), analyzed by Optiprep density gradient centrifugation and WB. Colors indicate fractions corresponding to human VLDL, LDL, and HDL <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001505#pbio.1001505-Vance1" target="_blank">[43]</a>. (E) Co-Immunoprecipitation (Co-IP) of secreted Shh with different lipoprotein classes, analyzed by WB. Supernatants were derived from HeLa cells transfected with Shh or Shh-N^C24S, grown in serum-free medium supplemented with individual human lipoproteins classes. (F) Shh levels in supernatants derived from MIA PaCa-2 cells grown in serum-free medium supplemented with individual human lipoprotein classes. Equal volumes were used for WB. (G) Density of Shh in MIA PaCa-2 cell supernatants shown in (F), analyzed by Optiprep density gradient centrifugation and WB. (H) Density of Shh in supernatants from Shh-expressing HeLa cells grown in serum-free medium supplemented with hemolymph from <i>Drosophila</i> larvae, analyzed by Optiprep density gradient centrifugation and WB. Purple indicates fractions corresponding to <i>Drosophila</i> Lpp.</p

Signaling properties of Lpp-associated Hh and Hh-N*.

<p>(A) Cartoon depicting the fat body to wing disc signaling assay of secreted Hh. (B) IF of wing discs from larvae secreting Hh from the fat body, and Lpp RNAi larvae, stained for Hh, Ci₁₅₅, and Lpp. Scale bar = 50 µm. In all wing discs, A denotes the anterior compartment, P the posterior compartment; yellow lines indicate the compartment boundary. Scale bar = 50 µm. (C and D) Quantification of (C) Hh and (D) Ci₁₅₅ staining of wing discs shown in (B). Translucent lines indicate ±SD (<i>n</i> = 12). (E) Phosphorylation status of Fused in wing discs of larvae secreting Hh from the fat body and Lpp RNAi larvae, analyzed by WB. (F) Ci₇₅ repressor levels in wing discs of larvae secreting Hh from the fat body, and Lpp RNAi larvae, analyzed by WB. (G) IF of wing discs from larvae secreting Hh or Hh-N* from the fat body, stained for <i>dpp</i>LacZ and, to mark cell boundaries, with phalloidin. Hh-N* was generated by expressing Hh in the fat body of Lpp RNAi animals. Scale bar = 50 µm. (H) Wing disc anterior to posterior compartment ratio of larvae secreting Hh or Hh-N* from the fat body and Lpp RNAi larvae. Error bars indicate ± SEM (<i>n</i> = 20). ***<i>p</i><0.0005; ****<i>p</i><0.00005.</p

Imaginal discs produce Hh-N*.

<p>(A) Immunofluorescence (IF) of wing imaginal discs of MTP/Lpp co-RNAi larvae, stained for Hh, Lpp, and Ptc. A denotes the anterior compartment, P the posterior compartment; yellow lines indicate the compartment boundary. Scale bar = 20 µm. (B) Experimental scheme for the purification of Hh-N* from imaginal discs. Samples for experiments shown in (D–F) were prepared in 100 mM Na₂CO₃, pH 9. (C) WB showing the levels of Hh recovered from imaginal discs with PBS or 100 mM Na₂CO₃, pH 9 after 16,000 <i>g</i> (S1) and 100,000 <i>g</i> (S2) centrifugation steps. Sample amount in each lane corresponds approximately to the imaginal discs of two third instar larvae. (D) Density of Hh in 100,000 <i>g</i> supernatant of imaginal discs dissociated in pH 9 buffer, analyzed by KBr density gradient centrifugation and WB. Hh is present mainly in high-density fractions; in some experiments, minor amounts of Hh can also be detected in low-density fractions. (E) Hydrophobicity of low-density and high-density Hh recovered from imaginal discs, analyzed by Triton X-114 phase separation and WB. (F) Size of high-density Hh recovered from imaginal discs, analyzed by gel filtration chromatography and WB.</p

Signaling properties of lipoprotein-associated Shh and Shh-N* in Shh-LIGHT2 cells.

<p>(A, B) Concentration-dependent signaling activity of (A) lipoprotein-associated Shh and (B) Shh-N*. Lipoprotein concentration in (A) was kept constant, and only the fraction carrying Shh increased. Shh and Shh-N* levels used for signaling assays were assessed by WB. (C,D) Shh pathway activity in cells stimulated by Shh-N* in the absence or presence of lipoproteins, or cells stimulated with lipoprotein-associated Shh. Lipoproteins, where added, were kept at a constant level. (C) Mammalian lipoproteins, (D) <i>Drosophila</i> Lpp. (E) Synergistic signaling activity of Shh-N* and lipoprotein-associated Shh. Shh-N* and lipoprotein-associated Shh were applied to cells alone or in combination. Predicted additive values represent the combined activity of lipoprotein-associated Shh and Shh-N* in the presence of lipoproteins, minus the basal assay activity measured in unstimulated cells. Note that the same batch of samples was used for assays shown in (A) and (B). For (A–E), error bars indicate ± SD (<i>n</i> = 3; **<i>p</i><0.005; ***<i>p</i><0.0005) of one representative experiment. Experiments were repeated at least twice.</p

Signaling properties of Hh-N.

<p>(A) Hemolymph Hh levels of larvae secreting Hh, Hh-N^Med, Hh-N^Low, or Hh+Hh-N^Low from the fat body, analyzed by WB. (B) Phosphorylation status of Fused in wing discs from larvae secreting different combinations of Hh and Hh-N from the fat body, analyzed by WB. (C) Levels of Ci repressor (Ci₇₅) and Ci₁₅₅ (full-length) in wing discs of larvae secreting different combinations of Hh and Hh-N from the fat body, analyzed by WB. (D) Wing disc anterior to posterior compartment ratio of larvae secreting different combinations of Hh and Hh-N from the fat body. Error bars indicate ± SEM (<i>n</i> = 20). *<i>p</i><0.05; **<i>p</i><0.005; ***<i>p</i><0.0005; ****<i>p</i><0.00005. (E–G) Quantification of (E) Hh, (F) Ci₁₅₅, and (G) Collier staining of wing discs shown in (H). Translucent lines indicate ± SD (<i>n</i> = 12). (H) IF of wing discs from larvae secreting different combinations of Hh and Hh-N from the fat body, stained for Hh, Ci₁₅₅, and Collier. A denotes the anterior compartment, P the posterior compartment; yellow lines indicate the compartment boundary. Scale bar = 50 µm.</p

<i>Drosophila</i> Hh is secreted in Lpp-associated and Lpp-free forms.

<p>(A) Hh levels in hemolymph and whole extracts of wild-type and <i>hh^TS</i> larvae at restrictive temperature, analyzed by WB. Hemolymph loading control is a secreted GFP expressed from the tubulin promoter. (B) Hemolymph Hh levels in wild-type and <i>disp</i> mutant larvae, analyzed by WB. Loading control is Cv-d. (C) Density of Hh in hemolymph of wild-type and Lpp RNAi larvae, analyzed by Optiprep density gradient centrifugation and WB. Equal amounts of hemolymph (normalized by protein) were analyzed. (D) Hemolymph Hh levels in larvae overexpressing Hh in imaginal discs with <i>en105</i>-GAL4, analyzed by WB. Loading control is Cv-d. (E) Density of hemolymph Hh in larvae overexpressing Hh in imaginal discs, analyzed by Optiprep density gradient centrifugation and WB. (F) Density of Hh lipid modification mutants (Hh^C85S, Hh-N, Hh-N^C85S), secreted into the hemolymph from the fat body (FB) with <i>lpp</i>-GAL4, analyzed by Optiprep density gradient centrifugation and WB.</p

Comparison of the lipid profiles obtained by three independent analytical methods.

<p>Total lipid extract from <i>E.coli</i> was analyzed on the QSTAR and LTQ Orbitrap Velos mass spectrometers in DDA mode and on the TSQ Vantage triple quadrupole mass spectrometer by precursor ion scanning for acyl anion fragments. The same MFQL queries were employed to identify and quantify lipids of PE and PG classes. Cardiolipins, another major component of the <i>E.coli</i> lipidome, were omitted from the comparative test because their precursors were detected in two charge states and the interpretation might be biased by the instrument interface settings and mass resolution. Relative abundances of individual species were normalized to the total abundance of all species of each class. Error bars represent standard deviations (SD, n = 3 for experiments on the TSQ Vantage and n = 4 on the QSTAR and LTQ Orbitrap mass spectrometers). Relative abundances determined on LTQ Orbitrap and QSTAR correlated with r2 and slope of 0.99 and 0.94, respectively; on LTQ Orbitrap and TSQ Vantage: r2 = 0.98 and slope 0.93; QSTAR and TSQ Vantage r2 = 0.98 and slope 0.98.</p

Comparison of the lipid profiles obtained by precursor ion scanning and neutral loss scanning on a triple quadrupole mass spectrometer.

<p>Total lipid extract from <i>E.coli</i> was analyzed in negative mode on the TSQ Vantage triple quadrupole mass spectrometer by precursor ion scanning for acyl anion fragments (profiles are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029851#pone-0029851-g003" target="_blank">Figure 3</a>). The same extract was analyzed in positive mode by lipid-class specific neutral loss scanning for the loss of head groups of PE and PG: Δ <i>m/z</i> 141.02 for [M+H]⁺ molecular ions of PE and Δ <i>m/z</i> 189.04 for ammonium adducts [M+NH₄]⁺ of PG. Relative abundances of individual species were normalized to the total abundance of all species of each class. Error bars represent standard deviations (SD, n = 3 for experiments on the TSQ Vantage). Relative abundances of species determined on TSQ Vantage by precursor ion scanning and neutral loss scanning correlated with r2 and slope of 0.98 and 0.94 for PE and r2 = 0.98 and slope 1.03 for PG.</p

DDA-driven MS/MS and Precursor Ion Scanning (PIS) Spectra.

<p>The scheme explains how data-dependent acquisition of full MS/MS spectra (DDA-driven MS/MS) and precursor ion scanning spectra are related. In DDA mode (panel A) a tandem mass spectrometer first acquires a survey spectrum that determines masses of intact lipids (here we are showing precursors with <i>m/z</i> 660.46; 688.49; 728.52 and 773.53 as an example) and then acquires full MS/MS spectra (from <i>m/z</i> of a lowest expected fragment till <i>m/z</i> of the intact precursor) from all plausible precursors. In MS/MS spectra (panel A) we designated <i>m/z</i> of characteristic acyl anion fragments (<i>m/z</i> 227.2; 281.1; 283.3) produced from fatty acid moieties of molecular anions of glycerophospholipids. In PIS spectra (panel B) a mass spectrometer registers the intensity of one pre-selected fragment (in this example, one of the acyl anion fragments) produced from all precursor masses within the specified <i>m/z</i> range. Hence, only precursors yielding the specific fragment will produce a peak, while others will not. Usually, on triple quadrupole mass spectrometers PIS spectra for a large number of fragments (like, all acyl anions of all major fatty acids) are acquired successively. Subsequent alignment of PIS spectra reveals what expected fragments were produced from each precursor (dotted line). For example, a lipid with <i>m/z</i> 660.46 produced acyl anions with <i>m/z</i> 227.2 and 283.3 that correspond to 14∶0 and 18∶0 fatty acids. The scheme exemplifies that DDA-driven MS/MS and PIS produce complementary structural evidence, although they originate from two completely different modes of spectra acquisition.</p