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

<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

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

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

Fast, Potent Pharmacological Expansion of Endogenous Hes3+/Sox2+ Cells in the Adult Mouse and Rat Hippocampus

<div><p>The adult hippocampus is involved in learning and memory. As a consequence, it is a brain region of remarkable plasticity. This plasticity exhibits itself both as cellular changes and neurogenesis. For neurogenesis to occur, a population of local stem cells and progenitor cells is maintained in the adult brain and these are able to proliferate and differentiate into neurons which contribute to the hippocampal circuitry. There is much interest in understanding the role of immature cells in the hippocampus, in relation to learning and memory. Methods and mechanisms that increase the numbers of these cells will be valuable in this research field. We show here that single injections of soluble factors into the lateral ventricle of adult rats and mice induces the rapid (within one week) increase in the number of putative stem cells/progenitor cells in the hippocampus. The established progenitor marker Sox2 together with the more recently established marker Hes3, were used to quantify the manipulation of the Sox2/Hes3 double-positive cell population. We report that in both adult rodent species, Sox2+/Hes3+ cell numbers can be increased within one week. The most prominent increase was observed in the hilus of the dentate gyrus. This study presents a fast, pharmacological method to manipulate the numbers of endogenous putative stem cells/progenitor cells. This method may be easily modified to alter the degree of activation (e.g. by the use of osmotic pumps for delivery, or by repeat injections through implanted cannulas), in order to be best adapted to different paradigms of research (neurodegenerative disease, neuroprotection, learning, memory, plasticity, etc).</p> </div

Soluble factors increase the numbers of Hes3+ cells in the adult mouse hippocampus.

<p>(a) A schematic diagram shows the site of a single injection (lateral ventricle) of soluble factors (a combination of Delta4 and Angiopoietin 2) in adult mice. Analysis was performed 7 days following the injection. (b) Quantification of the number of Sox2+ and Hes3+ cells in different areas of the hippocampus (numbers from control animals are shown as 100%).</p