Two distinct sites in sonic Hedgehog combine for heparan sulfate interactions and cell signaling functions

Abstract

Hedgehog (Hh) proteins are morphogens that mediate many developmental processes. Hh signaling is significant for many aspects of embryonic development, whereas dysregulation of this pathway is associated with several types of cancer. Hh proteins require heparan sulfate proteoglycans (HSPGs) for their normal distribution and signaling activity. Here, we have used molecular modeling to examine the heparin-binding domain of sonic hedgehog (Shh). In biochemical and cell biological assays, the importance of specific residues of the putative heparin-binding domain for signaling was assessed. It was determined that key residues in human (h) Shh involved in heparin and HSPG syndecan-4 binding and biological activity included the well known cationic Cardin-Weintraub motif (lysines 32-38) but also a previously unidentified major role for lysine 178. The activity of Shh mutated in these residues was tested by quantitation of alkaline phosphatase activity in C3H10T1/2 cells differentiating into osteoblasts and hShh-inducible gene expression in PANC1 human pancreatic ductal adenocarcinoma cells. Mutated hShhs such as K37S/K38S, K178S, and particularly K37S/K38S/K178S that could not interact with heparin efficiently had reduced signaling activity compared with wild type hShh or a control mutation (K74S). In addition, the mutant hShh proteins supported reduced proliferation and invasion of PANC1 cells compared with control hShh proteins, following endogenous hShh depletion by RNAi knockdown. The data correlated with reduced Shh multimerization where the Lys-37/38 and/or Lys-178 mutations were examined. These studies provide a new insight into the functional roles of hShh interactions with HSPGs, which may allow targeting this aspect of hShh biology in, for example, pancreatic ductal adenocarcinoma.

FIGURE 1.

Modeling of heparin-murine Shh interactions. The three lowest energy predictions for the heparin-mShh interaction from docking calculations are shown, with the protein structure exactly overlaid. The proteins are shown as ribbon diagrams coded red for helix, blue for β-strand, and gray for loops and sequences lacking secondary structure. Heparin molecules are shown in stick form, colored by element as follows: gray for carbon, red for oxygen, blue for nitrogen, yellow for sulfur, and white for hydrogen. A, predicted complex between mShh (PDB code 1VHH) and a heparin undecasaccharide, with heparin-interacting residues Lys-39 and Lys-179 shown in green stick form. B, predicted complex between hShh (PDB code 3M1Nb) and a heparin undecasaccharide, with heparin interacting residues Arg-28, Lys-32, and Lys-34 shown as green sticks; Lys-38 and Lys-178 are shown as orange sticks.

FIGURE 2.

Characterization of purified wild type and mutant hShh proteins by heparin affinity chromatography. Each of the five purified Shh proteins were analyzed by Western blot (A) and dot blot (B) with two Shh antibodies, H160 and 5E1. The latter confirms that all mutant hShh proteins retain appropriate conformation. Heparin binding efficacy of all five proteins was determined by heparin-Sepharose chromatography (C) with a linear elution gradient of 0–0.7 m NaCl in phosphate buffer (2.7 mm KCl, 10 mm Na₂HPO₄, 1.8 mm KH₂PO₄, pH 7.4).

FIGURE 3.

Differentiation of C3H10T1/2 osteoblast precursor cells requires hShh with heparin-binding properties. C3H10T1/2 osteoblast precursor cells were cultured in the presence of wild type hShh and hShh mutants at 300–2000 ng/ml for 5 days (A). The relative amount of Shh-induced alkaline phosphatase activity was measured as described under “Experimental Procedures” to determine biological activity of the morphogen. As a control, Shh-treated cells were incubated with 5E1 anti-Shh to demonstrate specificity of Shh-induced differentiation (B). Values are means ± S.D., n = 9 in each group; *, p value <0.001 compared with control group ShhNC24II; **, p value <0.001 compared with 5E1 treatment group; ‡, p value <0.01 compared with 5E1 treatment group.

FIGURE 4.

Signaling in human PDAC cells is mediated optimally by Shh with heparin-binding properties. Expressions of Ptc and Gli1 were examined by both RT-PCR and Western blot in PANC1 cells after treatment of cells with recombinant wild type and mutant hShh. The PCR analysis and immunoblotting data are shown in A and C, respectively, and are quantified in B and D. Values are means ± S.D., n = 9 in each group in RT-PCR experiments (B). *, p value <0.0001 compared with untreated group. ‡, p value <0.001 compared with ShhNC24II group. In Western blotting assay (D, n = 4). *, p value <0.0001 compared with untreated group. ‡, p value <0.001 compared with ShhNC24II group.

FIGURE 5.

Mutant hShh proteins that lack heparin affinity promote lower levels of PANC1 cell proliferation. Both 5E1 blocking antibody treatments on day 1 (5E1 D1) and siRNA depletion of endogenous Shh (Shh KD) confirmed that PANC1 proliferation is Shh-responsive. 5E1 treatment on day 4 (5E1 D4) was used to mimic in vivo RNAi KD kinetics. The maximum labeling was determined by treating cells with carboxyfluorescein, succinimidyl ester, just before flow cytometer assessment. To study the effect of the wild type and mutant Shh proteins in PANC1 proliferation 8 days after Shh siRNA transfection/hShh treatment, the cells were analyzed by flow cytometry. The y axis shows the level of CF intensity observed from 30,000 cells. The experiments were repeated three times in triplicate, and statistical significance was assessed with the two-tailed t test (**, p < 0.0001 compared with untreated group; ‡, p < 0.0001 compared with the Shh KD group).

FIGURE 6.

PANC1 cell invasion in response to hShh requires the morphogen to possess heparin-binding properties. To study the effect of wild type and mutant Shh proteins on PANC1 invasion, 72 h after Shhs treatment/RNAi transfection the cells were plated onto Matrigel invasion chambers for 24 h. Cells that migrated from the upper to the lower side of the filter were photographed (A) and counted with a light microscope (10 fields/filter, B). The experiments were repeated four times, and statistical significance was calculated with the two-tailed t test (*, p < 0.001 compared with untreated samples; ‡, p < 0.0001 compared with ShhNC24II/Shh KD samples).

FIGURE 7.

Multimerization of hShh proteins is reduced in parallel with decreased heparin affinity. 72 h after RNAi transfection, PANC1-conditioned media were TCA-precipitated and examined by Western blot for Shh (A). To examine the ability of mutated hShh proteins to form multimers, endogenous Shh was depleted by 16 h of siRNA treatment. Ectopically expressed wild type or mutant hShh was achieved by cDNA transfection. After 48 h of incubation, conditioned media were analyzed by gel filtration chromatography (Superdex 200 10/300 GL column). After TCA precipitation, each fraction was probed by Shh dot blot (B). The elution of molecular mass standards is shown across the top of the dot blot, in kDa. Monomerization factor (%) = (monomer/monomer + multimer) × 100%.