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Originally published In Press as doi:10.1074/jbc.M605905200 on August 30, 2006

J. Biol. Chem., Vol. 281, Issue 45, 33860-33870, November 10, 2006
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Molecular Pathways Regulating Pro-migratory Effects of Hedgehog Signaling*Formula

Eldar Hochman1, Asher Castiel, Jasmine Jacob-Hirsch, Ninnette Amariglio, and Shai Izraeli2

From the Research Section of Childhood Malignancies, Sheba Cancer Research Center, Safra Children Hospital, Sheba Medical Center and Faculty of Medicine, Tel-Aviv University, Tel Hashomer 52621, Israel

Received for publication, June 20, 2006 , and in revised form, August 29, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Hedgehog proteins play a crucial role in metazoan embryo development. Constitutive activation of the pathway is associated with multiple types of cancer. Recent experimental data suggest involvement of Hedgehog signaling in vascular remodeling, germ cell migration, and axon guidance. The molecular mechanisms underlying these effects remain elusive. Here we show that yolk sac-derived endothelial cells and embryonic fibroblasts can directly respond to the Hedgehog signal by increased migration in an in vitro scratch (wound) assay. We also identify Hedgehog transcriptional target genes in these cells, many of which participate in cell migration, axon guidance, and angiogenesis processes. Inhibition of one such molecular pathway, neuropilin-flavomonooxygenase, blocks Hedgehog-induced cell migration. These findings suggest that Hedgehog signaling directly affects embryonic endothelial and fibroblast cell migration via molecules and pathways known to regulate cell migration in response to a variety of environmental cues.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Hedgehog family of signaling molecules is conserved throughout evolution with three secreted proteins that have been identified in mammals: Sonic Hedgehog (Shh),3 Indian Hedgehog, and Desert Hedgehog. Although these proteins differ in their tissue specificity, they all activate the same downstream signaling cascade, including two transmembrane receptors Patched1 and Smoothened, the family of Gli transcription factors, and other molecules involved in the signal transduction such as Fused, Suppressor of Fused, and Costal2 (1).

The Hedgehog pathway plays a crucial role in metazoan embryo patterning (1). The most intensively investigated aspects of the Hedgehog pathway to this day include the following: establishment of ventral cell identity in the neural tube (2), establishment of posterior identity in the developing limb bud (3), and the roles of the pathway in neoplastic transformation.

Inherited or acquired aberrations in components of the Hedgehog cascade result in various phenotypes, including congenital anomalies (e.g. holoprosencephaly and Pallister-Hall syndrome) and various cancers, most frequently medulloblastoma, basal cell carcinoma, gastrointestinal cancers, and rhabdomyosarcoma (4, 5).

Recently, observations from different systems have suggested a role for Hedgehog signaling in the control of motility and migration of multiple cell types. Hedgehog has been shown to act as both a stimulant and an inhibitor of axonal migration (6, 7). Hedgehog signaling blocks migration of neural crest cells in chick but is required for migration of germ and tracheal cells in Drosophila (8–10). Similarly, Hedgehog signaling is essential for yolk sac and embryonic vasculogenesis (11, 12) and enhances endothelial progenitor cell-mediated microvascular remodeling during wound healing (13). Transfer of naked Shh cDNA into myocardial cells also enhanced the contribution of bone marrow-derived endothelial progenitor cells to myocardial neovascularization after acute and chronic myocardial ischemia (14). Other studies, however, have failed to show a direct effect on endothelial cells in vitro (15), implying that endothelial cell response to Hedgehog may be dependent on their differentiation status, developmental age, and organ origin.

To elucidate the direct molecular consequences of Hedgehog signaling in endothelial cells, we studied endothelial cells derived from the mouse embryo yolk sac at the time the Hedgehog signal is secreted from the visceral endoderm. We show that these endothelial cells as well as embryonic mouse fibroblasts respond directly to Hedgehog by transcriptional activation of multiple genes coding for proteins involved in migration and angiogenesis. We further show that Hedgehog signaling enhances in vitro wound healing and that the neuropilin flavoprotein monooxygenase pathway mediates this effect. These findings suggest a molecular pathway mediating direct pro-migratory effect of Hedgehog on embryonic endothelial and fibroblast cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Cyclopamine was kindly provided by Dale R. Gardner (Poisonous Plant Research Laboratory, U. S. Department of Agriculture) and was suspended in 96% ethanol. It was used at 10 µM concentration in all assays. Epigallocatechin gallate (EGCG) (E4143; Sigma) was suspended in PBS.

Cell Culture—C166 endothelial cell line was isolated from the yolk sac of 12-day-old transgenic mouse embryos expressing multiple copies of the activated allele of the human c-fes proto-oncogene (16). The human embryonic kidney epithelial cell line 293T and mouse embryonic fibroblast cell lines NIH 3T3 and C3H/10T1/2 Clone 8 were obtained from the ATCC (Manassas, VA; product numbers CRL-11268, CRL-1658, and CCL-226, respectively). The mouse embryonic fibroblast cell line BALB/c 3T3 clone A31 was kindly provided by Professor Gera Neufeld (Technion-Institute of Technology, Haifa, Israel). All cell lines were cultured in Dulbecco's modified Eagle's medium (41965-039; Invitrogen) supplemented with 10% fetal bovine serum (10270-106; Invitrogen). Primary mouse embryonic fibroblasts (MEFs) were isolated directly from embryonic day 12 to embryonic day 14 HS:ICR mouse embryos and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells were used up to six passages.

Generation of Conditioned Media—Shh-conditioned media were produced as reported previously with few modifications (17). Briefly, 293T cells were transiently transfected with Shh-N encoding plasmid (Shh-N is active secreted Shh), kindly provided by Philip A. Beachy, or with control pcDNA3.1 plasmid (V79020; Invitrogen) The growth media were collected 3 days post-transfection, centrifuged at 3000 x g for 10 min, and transferred through a 0.45-µm filter to remove cell debris. The conditioned media potency was routinely checked on NIH 3T3 cells by studying the expression of Shh known transcriptional targets Gli1 and Patched by quantitative real time PCR. When used to stimulate the Hedgehog pathway, the conditioned media were diluted in the maintenance media of the cells in different ratios as indicated in the text.

RNA Interference and Retroviral Infections—Virus-mediated RNA interference was accomplished using the pRETRO-SUPER (pRS) construct kindly provided by Reuven Agami (18). To generate pRETRO-SUPER targeting mouse neuropilin1 (pRS-Nrp1), pRS was digested with BglII and HindIII, and the annealed oligonucleotides (5'-gatccccGGAATGTTCTGTCGCTATGttcaagagaCATAGCGACAGAACATTCCtttttggaaa-3' and 5'-agcttttccaaaaaGGAATGTTCTGTCGCTATGtctcttgaaCATAGCGACAGAACATTCCggg-3') were ligated into the vector. The 19-bp neuropilin1 target sequences are indicated in capitals in the oligonucleotide sequence. To create mouse neuropilin1 control RNA interference, mutations were generated in the target Nrp1 sequences, and the new annealed oligonucleotides (5'-gatccccGTAAGGTTCTATCGCCATCttcaagagaGATGGCGATAGAACCTTACtttttggaaa-3' and 5'-agcttttccaaaaaGTAAGGTTCTATCGCCATCtctcttgaaGATGGCGATAGAACCTTACggg-3') were ligated into pRS to generate pRS-CONTROL.

293T cells were transiently transfected with pRS-CONTROL or pRS-Nrp1 retroviral plasmids, in combination with vectors coding for ecotropic retrovirus envelope and the Gag and Pol proteins. Forty eight hours post-transfection, the culture medium was filtered through a 0.45-µm filter, and the viral supernatant was used for infection of C166 cells after addition of 8 µg/ml Polybrene (H9268; Sigma). C166 cells were infected for 24 h and allowed to recover for another 24 h with fresh medium. Infected cells were selected with 10 µg/ml puromycin (ant-pr-1; InvivoGen) for a week, and polyclonal puromycin-resistant populations infected by Prs-CONTROL ("Nrp1 CONTROL" line) or pRS-Nrp1 ("Nrp1 knockdown" line) were established.

Quantitative Real Time PCR—Total RNA was extracted using TRIzol reagent (15596-018; Invitrogen). cDNA was synthesized using Reverse-iT 1st strand synthesis kit (AB-0789/B; ABgene) with random decamers following the manufacturer's instructions.

The quantitative real time PCRs were performed using SYBER Green Master Mix (RT-SN2X-075+; EST Group) with cDNA equivalent to 100 ng of RNA (for each sample) using Applied Biosystems 7900HT prism real time PCR instrument (Taqman; PerkinElmer Life Sciences). PCR amplification included a first step of 10 min at 95°C followed by 40 cycles of amplification (95°C for 15 s and 60°C for 60 s). Ratio was calculated by dividing each gene expression with that of an internal standard in each sample.

Forward and reverse primers were designed in exon-intron junctions in order to avoid amplification from contaminating DNA. For all the reactions primers designed to amplify beta2-microglobulin gene were used as an internal control. Primers (Sigma) used are as follows: beta2-microglobulin forward primer, 5'-GAG ACT GAT ACA TAC GCC TGC AGA-3', and beta2-microglobulin reverse primer, 5'-TCA CAT GTC TCG ATC CCA GTA GA-3'; Gli1 forward primer, 5'-CAA GTG CAC GTT TGA AGG CT-3', and Gli1 reverse primer, 5'-CAA CCT TCT TGC TCA CAC ATG TAA G-3'; Patched1 forward primer, 5'-AGG CTC TCC TGC AAC ACC TG-3', and Patched1 reverse primer, 5'-GTA ACC TGT CTC CGT GAT AAG TTC C-3'; neuropilin1 forward primer, 5'-CAA GCG CAA GGC TAA GTC G-3', and neuropilin1 reverse primer, 5'-CCG AAG CTC AGG TGT GTC ATA G-3'; neuropilin2 forward primer, 5'-CTG CAG CTT TGA GGA TGA CA-3', and neuropilin2 reverse primer, 5'-CAC CCA ACC ACA GGT CTC TT-3'; and Mical2 forward primer, 5'-GTT ACC AGC ACG TCA GAG TCA CTG-3', and Mical2 reverse primer, 5'-CAT TCA GCG AGT CAA AGT TGA TC-3'.

Microarray Design, Hybridization, and Analysis—Seven plates of C166 cells with initial confluence of 2 x 104 cells/cm2 were serum-starved for 12 h before initiation of the experiment with control conditioned media diluted 1:10 in media without serum (resulting in media containing 1% serum). One plate was then harvested and served as a base-line sample, whereas the rest of the plates were washed and received fresh control conditioned media or an Shh conditioned media, again diluted 1:10 in media without serum, for an additional 12 or 24 h. RNA was generated from the base-line sample and afterward from the 12- and 24-h time points. Each time point included one control-treated sample and two Shh-treated samples.

All experiments were performed using Affymetrix MOE430A oligonucleotide arrays, as described oline. Total RNA from each sample was used to prepare biotinylated target RNA, with minor modifications from the manufacturer's recommendations.

Five µg of mRNA was used to generate first strand cDNA by using a T7-linked oligo(dT) primer. After second strand synthesis, in vitro transcription was performed with biotinylated UTP and CTP (Enzo Diagnostics), resulting in ~300-fold amplification of RNA. The target cDNA generated from each sample was processed as per the manufacturer's recommendation using an Affymetrix GeneChip Instrument System. Briefly, spike controls were added to 15 µg of fragmented cRNA before overnight hybridization. Arrays were then washed and stained with streptavidin-phycoerythrin, before being scanned on an Affymetrix GeneChip scanner. Additionally, quality and amount of starting RNA was confirmed using an agarose gel. After scanning, array images were assessed by eye to confirm scanner alignment and the absence of significant bubbles or scratches on the chip surface. 3':5' ratios for glyceraldehyde-3-phosphate dehydrogenase and beta-actin were confirmed to be within acceptable limits (1.03–1.14 and 0.78–0.86, respectively), and BioB spike controls were found to be present on all chips, with BioC, BioD, and CreX also present in increasing intensities. When scaled to a target intensity of 150 (using Affymetrix MAS 5.0 array analysis software), scaling factors for all arrays were within acceptable limits (0.63–0.84), as were background Q values and mean intensities (supplemental Table 1). Details of quality control measures can be found online.

Genes were filtered using MAS 5 algorithm. A list of 13,482 "valid" probe sets, representing probe sets with signal higher then 100 and detected as present (P) in at least one sample, was obtained (supplemental Table 2; the data are represented to comply with "minimum information about a microarray experiment" standards). The two samples treated with Shh for 24 h were compared with the 24-h control sample. The comparison generated a list of "active genes" in the Shh-treated samples for 24 h. Genes with an increased expression by at least in 1.5-fold compared with control were marked as "Increase" (I, p value 0.0025), or "Marginal Increase" (MI, p value 0.003) if less than 1.5-fold. Similarly, lower expression in the Shh group was labeled as "Decrease" (D, p value 0.0025) or "Marginal Decrease" (MD, p value 0.003). Hierarchical clustering was performed using Spotfire DecisionSite for Functional Genomics (Somerville, MA). Calculation of over-represented functional classes were done using Ease (19). Functional classifications with an "Ease score" lower than 0.05 were marked as over-represented.

Protein Analysis—For the purpose of detecting the Shh-N protein in the cell media, 25 µl from Shh-N or control conditioned media were mixed with Laemmli buffer and separated by 12% SDS-PAGE. After electronic transfer onto nitrocellulose membranes (401385; Schleicher and Schuell), the blots were probed with anti-Shh, 5E1 hybridoma supernatant (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City).

Neuropilin1 Immunoprecipitation and Immunoblotting—Cells were lysed in a lysis buffer containing 50 mM Hepes, pH 7.4, 4 mM EDTA, 1% Triton X-100, and fresh protease inhibitor mixture (1-836-145; Roche Applied Science). 500 µg of sample lysate were incubated with goat anti-neuropilin1 antibody (c-19; Santa Cruz Biotechnology), and immune complexes were further precipitated using agarose-conjugated protein A/G (sc-2003; Santa Cruz Biotechnology). Immunoprecipitated proteins were separated by 6% SDS-PAGE and transferred to nitrocellulose membranes. The blots were probed with the same anti-neuropilin1 antibody used for immunoprecipitation. The detection of the anti-neuropilin1 antibody heavy chain was used as a conformation for equal addition of the antibody to the samples during precipitation. 293T cells transiently transfected with neuropilin1 expressing vector (kindly provided by Gera Neufeld, Technion-Institute of Technology, Haifa, Israel) served as a positive control for the immunoblotting.

Expression and Purification of Recombinant Shh-N Protein—Mouse Shh-N cDNA subcloned into a pET11d expression vector, forming a 6 histidine-tagged fusion protein, was kindly provided by Andrew P. McMahon. This plasmid was transformed into Escherichia coli strain BL21 (DE3) PlysS (C6020-03; Invitrogen). Transformed bacteria were grown to an A550 of 0.5 and induced with 1 mM isopropyl 1-thio-beta-D-galactopyranoside (I6758; Sigma) for a further 2 h at 30°C. The bacteria were collected by centrifugation and stored at –70 °C. Bacterial pellets were resuspended in lysis buffer (25 mM sodium phosphate, pH 8, 150 mM NaCl, 1 mM EDTA, 0.5 mM DTT, 10 mM imidazole (I5513; Sigma), 1 mg/ml lysozyme (1-243-004; Roche Applied Science) and protease inhibitor mixture). Afterward, the lysate was sonicated (6 times with 10-s bursts), and the homogenate was centrifuged at 14,000 x g for 60 min at 4 °C. 50% nickel-nitrilotriacetic acid-agarose slurry (30210; Qiagen) was added to the homogenate (in 1:4 ratio) and mixed gently by shaking at 4 °C for 60 min at 200 rpm. After a series of washes (25 mM sodium phosphate, pH 8, 300 mM NaCl, 0.5 mM DTT, and 20 mM imidazole), the His-Shh-N protein was eluted (25 mM sodium phosphate, pH 8, 300 mM NaCl, 0.5 mM DTT, and 200 mM imidazole) and dialyzed (against 5 mM sodium phosphate, pH 5.5, 150 mM NaCl, and 0.5 mM DTT). Finally, glycerol was added to give 20% of the final volume, and the protein was stored at –70 °C. His-Shh-N was pure as characterized by Coomassie Blue staining (B0149; Sigma), and the immunoblot studies done were similar as for the detection of Shh-N in the conditioned media. As reported before (20), a dimeric form of the recombinant protein was formed spontaneously, probably by formation of disulfide bonds, giving an ~38-kDa protein. His-Shh-N protein concentration was determined by bicinchoninic acid (BCA) quantification assay (23227; Pierce). The potency of the His-Shh-N protein was checked similar to Shh-N conditioned media.

Scratch (Wound) Assay—Migration through a wound introduced in cell monolayer was done similar to previous reports (21). Briefly, C166, NIH 3T3, or MEF cells were grown to confluence and formed a monolayer covering the surface of the entire plate. Cells were serum-starved and treated with Shh or control conditioned media, with or without cyclopamine (10 µM), according to expression studies. After 24 h of treatment, the wound was created in the center of the cell monolayer by the gentle removal of the attached cells with a sterile plastic pipette tip. Debris was removed by PBS wash, and the cells received fresh media with the same treatments as before. The ability of the cells to migrate into the wound area was assessed after 24 h by comparing the 0- and 24-h phase-contrast micrographs of several marked points along the wounded area at each plate. Chemical inhibitors, when used, were applied 30 min before wounding the cells. The percentage of nonrecovered wound area was calculated by dividing the nonrecovered area after 24 h by the initial wound area at zero time.

Trypan Blue Viability Assay—C166, treated with control or Shh conditioned media (diluted 1 to 10 in serum free media) for 48 h, were incubated in the presence of 0.4% trypan blue solution (T8154; Sigma) in PBS for 60 s, and the number of viable cells was quantified. The experiments were repeated twice.


Figure 1
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  		FIGURE 1.
C166 endothelial line response profile to Shh treatment. A, immunoblot analysis. Anti-Shh, 5E1 hybridoma supernatant, detects the 19-kDa Shh-N protein in Shh-conditioned media (CM) and not in the control CM. B, quantitative real time PCR. Representative graphs show the fold increase in Gli1 and Patched1 expression upon Shh treatment relative to control treatment. In all experiments the conditioned media dilution ratio is 1:5; initial C166 seeding density is 1 x 104 cells/cm2; exposure time to Shh is 48 h, and C166 maintenance media is supplemented with 10% serum. One parameter becomes a variable in each experiment as follows: panel 1, the conditioned media dilution ratio in C166 maintenance media (marked as X/X); panel 2, C166 initial seeding density; panel 3, the exposure time of C166 to Shh treatment; and panel 4, the percentage of serum in C166 maintenance media (reproducible results were obtained from two independent experiments for each assay).

 
Statistical Analyses—Student's t test was carried out for comparison of paired mean experimental values.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
C166, a Mouse Yolk Sac Endothelial Cell Line, Responds to Sonic Hedgehog—The response of the yolk sac-derived endothelial cell line C166 to Hedgehog signal was studied by monitoring the expression of well characterized primary target genes of the pathway, Gli1 and Patched1, using quantitative real time PCR technique. To stimulate the pathway, both Shh and control conditioned media were generated using 293T cells (Fig. 1A). Exposure to Shh induced the expression of Gli1 and Patched1 (Fig. 1B). C166 cells response to Shh was dose-dependent, increased with the length of exposure and with the degree of cell confluence, and was further augmented by serum starvation (Fig. 1B). As reported earlier, the transcriptional response to Shh was rather delayed, occurring at least 24 h after exposure under these conditions (17).

Shh Induces Migration of Embryonic C166 and 3T3 Cells in Vitro—The yolk sac phenotype of Smo–/– embryos suggests a defect in endothelial cell organization and migration (22). To examine the functional effects of Shh on C166 cells, we chose the in vitro scratch (wound) assay. This assay is commonly used for testing the effects of pro- and anti-migratory agents on cultured cells (21). Another advantage of this assay is that it is performed on a confluent cellular layer, a pre-condition for Hedgehog activity in vitro (17, 23). C166 cells were grown to confluence and treated with control or Shh-conditioned media for 24 h, in the presence or absence of the specific Hedgehog signaling inhibitor cyclopamine (24). After 24 h, a wound was introduced into the cell monolayer, and fresh media, treated as before, were applied again. The migration of the cells into the wound area was assessed after 24 h. Shh significantly enhanced the migration of C166 cells into the wounded area. This effect was eliminated by Hedgehog pathway-specific inhibitor, cyclopamine (Fig. 2A). Similar results were obtained on NIH 3T3 fibroblasts, a known Hedgehog-responsive cell line (23) (Fig. 2B), suggesting that the Shh promigratory effect is not restricted to endothelial cells.

The Hedgehog signal can also act as a mitogen in some systems (25). Although the wound assay was performed on confluent cells during serum starvation, we wished to rule out the possibility that the enhanced wound closure by Shh was not the result of a proliferative advantage conferred by Shh. Trypan blue viability counts showed no difference in the number of living cells between plates treated with Shh and control media (living cells counts, at the end of the experiment, were 2.5 x 106 and 2.8 x 106 in control and Shh treatments, respectively (p > 0.05)). These results were confirmed independently by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (supplemental Fig. 2). Thus, the enhanced wound closure induced by Shh was caused by increased cell migration and not by increased cell number.


Figure 2
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  		FIGURE 2.
Shh induces migration of C166 and NIH 3T3 cells in an in vitro scratch (wound) assay. A, C166 in vitro scratch assay. Wound was applied to cell monolayer treated with Shh or control conditioned media in the presence of 10 µM cyclopamine (Cyclop.) or its dissolvent ethanol (Etoh) as described in detail under "Experimental Procedures." Panel 1, representative fields showing the wounded area and cell migration into the wound at zero time and after 24 h, respectively. Both low (x10) and high (x20) magnifications (Magnif.) are shown at 24 h. Scale bar (for low magnification) = 500 µm. Panel 2, graph shows the percentage of nonrecovered wound area from the intial wound area after 24 h of migration. B, NIH 3T3 in vitro scratch assay. Experiments were conducted the same as for C166 cells. Graph shows the percentage of nonrecovered wound area from the initial wound area after 24 h of migration. Error bars indicate S.D. based on two independent experiments. *, p < 0.0005; **, p < 0.05 versus control.

 
Characterization of the Transcriptional Response of C166 Cells to Shh—To identify the transcriptional response to Shh in C166 cells, an oligonucleotide microarray gene expression analysis was performed 12 and 24 h after exposure (Fig. 3A). These time points were chosen after optimizing the exposure conditions of C166 to Shh according to the parameters studied in Fig. 1. Gli1 and Patched1 expression, as detected by both quantitative real time PCR and the GeneChip hybridization, was increased by treatment with Shh compared with control and served as an internal control for activation of the Hedgehog pathway in the gene expression array (Fig. 3B). Nonsupervised hierarchical clustering performed with Spotfire DecisionSiteTM revealed clear differences in the gene expression profiles of samples treated with Shh compared with controls after 24 h. Consistent with the delayed transcriptional activity of Hedgehog signaling (Fig. 1), the difference was less marked after 12 h (not shown), and thus comparisons were made between the 24-h treated samples and their control. Sixty one probe sets (representing 54 genes) changed by at least 1.5-fold between the treated samples and their control. Of the 54 genes, 41 were up-regulated and 13 down-regulated in the Shh-treated samples (Table 1).


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  		TABLE 1
Genes regulated by Shh after 24 h of treatment

Fifty-four genes (represented by 61 probes), changed at least in 1.5-fold (as calculated from the signal values) between the 24-h Shh-treated samples and the 24-h control sample, are listed in order of-fold change (FC). The FC of the genes between the 12-h Shh-treated samples and 12-h control-treated sample is also shown. Mean signal values were calculated for the two Shh-treated samples in each time point. Genes belong to over-represented biological themes (Ease score lower than 0.05) are in boldface. LHRH indicates luteinizing hormone-releasing hormone.

 
We next searched for over-represented biological themes among the 54 genes using the "Ease" program (19). Functional classifications with an Ease score lower than 0.05 were marked as over-represented. In addition to the "Smoothened receptor (Hedgehog) signaling pathway," the most significant over-represented biological themes were related to cell motility, angiogenesis, and cytoskeletal organization (Table 2). Genes representing these categories are shown in boldface in Table 1.


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  		TABLE 2
Identification of over-represented biological themes (GO biological process) using Ease score

Selected functional classifications with an Ease score lower than 0.05 are shown. GO indicates gene ontology.

 
The two genes mostly induced by Shh (after Gli1 and Patched1) were the flavoprotein oxidoreductase Mical2 and Nrp1 (Table 1). Neuropilin1 is a cell surface glycoprotein expressed in both axons and endothelial cells. Its known ligands are semaphorin3A and VEGFs. Knock-out of neuropilin1 in the mouse results in abnormal blood vessel formation as well as insufficient development of the yolk sac vascular network (26), which interestingly resembles the Smo–/– embryo phenotype. Mical2 belongs to a family of multidomain cytosolic proteins with a flavoprotein monooxygenase domain (27, 28). This family has recently been linked to semaphorin signal transduction, where they are thought to transmit the signal from the Plexin family transmembrane receptors into the cell and provide the link for regulation of cytoskeletal dynamics (28). Thus the most significantly induced genes participate in the same pathway which regulates cell migration in response to variety of environmental cues.

We validated the increase in expression of neuropilin1 and Mical2 by quantitative real time PCR and by immunoprecipitation with RNA and protein derived from new independent experiments (Fig. 4, A and B). The Shh protein used in our experiments is in the form of conditioned media derived from 293T cells transfected with the active N-terminal peptide of Shh. Although this approach ensured the formation of properly processed and active Shh protein (29), the media might have contained other factors that could induce the expression of neuropilin1 and Mical2 in C166 cells, such as VEGF-A (30). To rule out this possibility, we added 10 µM cyclopamine, a specific inhibitor of the Smoothened receptor, the central transducer of all Hedgehog proteins (24) to our assay. The addition of cyclopamine abolished the increased expression of neuropilin1 and Mical2 (Fig. 4A), proving they were induced by Shh. To further determine the specificity of the response to Shh, we generated a bacterially expressed His-tagged Shh-N protein (supplemental Fig. 1 and "Experimental Procedures"). This recombinant protein is known to be less active than the protein prepared in mammalian cells, probably because of the lack of post-translational modifications (29). As expected, His-Shh-N was less potent in activating the Hedgehog cascade (as exemplified by the limited increase in Patched1 gene expression), but still resulted in an increase of 2-fold in the expression of neuropilin1 and Mical2 compared with control (Fig. 4C). Gene expression array results excluded the possibility that C166 cells themselves express VEGF-A in response to Shh treatment. Expression of VEGF-A transcripts was very low in C166 and showed minimal if any increase in response to Hedgehog stimulation. Together these results confirm that Shh is the only protein in the conditioned media mediating the increased expression of neuropilin1 and Mical2 in C166 cells.


Figure 3
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  		FIGURE 3.
Oligonucleotide microarray outlines. A, schema representing microarray design. Base line, 12-, and 24-h time points, in which RNA was collected from control and Shh-treated samples, are illustrated. The microarray methodology is described in detail under "Experimental Procedures." B, microarray internal controls. Graphs show the fold increase in Gli1 and Patched1 expression upon treatment with Shh (duplicate) relative to control. Gli1 and Patched1 expression is detected both by quantitative real time PCR (panel 1) and by the GeneChips hybridization signal (panel 2). y axis is in logarithmic scale. The difference in the magnitude of Gli1 fold induction between both techniques is because of the high threshold of GeneChips (low sensitivity) as compared with quantitative real time PCR and the fact that Gli1 basal expression is very low.

 
We next asked whether neuropilin1 and Mical2 are the endothelial lineage-specific transcriptional targets for Hedgehog, or do they serve as Hedgehog pathway targets in a wider range of cell types? C3H/10T1/2, NIH 3T3, and BALB/c 3T3 are all mouse embryonic fibroblast cell lines known to respond to the Hedgehog signal (17, 23). In all these cell lines Shh induced the expression of neuropilin1 and Mical2 (Fig. 4D), suggesting that neuropilin1 and Mical2 are induced by Hedgehog in cell lineages other than endothelium.

Shh Induces Migration of Primary Cells in Vitro—As C166 and NIH 3T3 are both immortalized cell lines, we decided to repeat our assays on primary cells that better reflect the in vivo physiology. Primary MEFs were tested for Shh-induced migration in the wound assay. Not only did stimulation with Shh enhance the migration of MEFs, but inhibition with cyclopamine, in 10 µM concentration, depressed the migration of MEFs to levels below control (Fig. 5A). This finding implies that the Hedgehog signaling is partially activated in MEFs and is supported by the Gli1 expression studies (done on Shh and cyclopamine-treated MEFs) in which cyclopamine lowered Gli1 expression below control treatment (Fig. 5B). In correlation with these findings, cyclopamine treatment reduced Mical2 and neuropilin1 expression levels relative to control treatment, whereas Shh treatment increased them (Fig. 5B). Thus, the revealed Hedgehog pro-migratory effect and transcriptional regulation are observed in primary embryonic fibroblasts.

Molecular Pathways Mediating Hedgehog-induced Cell Migration—To decipher the role of the neuropilin-flavomonooxygenase pathway in mediating the effects of Shh on cell migration, we repeated the in vitro wound assay using gene silencing and a chemical inhibitor of this pathway.

To study the effects of neuropilin1 inhibition on Shh-induced migration, we applied a virally mediated RNA interference technique (18) for efficient delivery of short interfering RNAs targeting the neuropilin1 gene. A polyclonal population of C166 cells was generated and infected with neuropilin1 (Nrp1), silencing retrovirus (Nrp1 knockdown), or control retrovirus (Nrp1 CONTROL). To ensure the specificity of the neuropilin1 short hairpin RNA, the control short hairpin RNA differed only in five nucleotides from the short hairpin RNA targeting neuropilin1. Quantitative real time PCR analysis showed markedly suppressed expression of neuropilin1 in the Nrp1 knockdown population compared with Nrp1 CONTROL, whereas the expression of Mical2 and the closely related molecule neuropilin2 (Nrp2) was similar in both cell types (Fig. 6A). We next tested if neuropilin1 is crucial for the transcriptional regulation seen in C166 following Hedgehog stimulation. Target gene expression analysis indicates that Hedgehog transcriptional regulation was not affected in cells with reduced neuropilin1 expression (Fig. 6B); however, knockdown of neuropilin1 dramatically reduced the Shh induction of in vitro wound repair (Fig. 6B).

As mentioned before, Mical2, a flavoprotein monooxygenase, has recently been shown to mediate semaphorin signal transduction involving transmembrane Plexin receptors and resulting in cytoskeletal remodeling (28). EGCG, a potent inhibitor of flavoprotein monooxygenases (31, 32), was shown to inhibit the semaphorin-repulsive effect through blocking Mical activity (28). Based on this information, we asked if EGCG can inhibit Shh-induced migration in the wound assay. EGCG in concentrations of up to 2.5 µM, shown before to have an inhibitory effect on VEGF-induced migration (33), had no effect on C166 proliferation or survival rates (data not shown). EGCG inhibited Shh-induced migration in the in vitro wound assay in a dose-dependent manner (Fig. 7A) without affecting the differential expression of Hedgehog target genes (Fig. 7B). Thus, the promigratory effect of Hedgehog signaling requires both neuropilin1 and flavoprotein monooxygenases.


Figure 4
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  		FIGURE 4.
Shh, through Smoothened, regulates neuropilin1 and Mical2 expression in C166 and other Hedgehog-responsive cell lines. A, quantitative real time PCR. Graph shows the fold increase in Mical2, neuropilin1 (Nrp1), and Patched1 expression upon treatment with Shh. Shh transcriptional activity is inhibited by 10 µM cyclopamine (Cyclop.) but not by its dissolvent ethanol (Etoh). The assay was done in the same conditions as the microarray with 24 h as the end point. B, Nrp1 protein analysis. Neuropilin1 protein expression, studied by immunoprecipitation and immunoblotting (as described in details under "Experimental Procedures"), in C166 cells treated with Shh or control conditioned media for 40 h (in the same conditions as in the microarray). A base-line (zero) sample after initial starvation was also analyzed. The neuropilin1 protein is detected around 130 kDa. Anti-neuropilin1 antibody's heavy chain is also detected. C, quantitative real time PCR. Graph shows the fold increase in Mical2, Nrp1, and Patched1 expression upon His-Shh-N treatment (5 µg/ml) relative to control treatment (Dialysis buffer). The treatment was given for 24 h in the presence of 1% serum in the media. D, quantitative real time PCR. Graph represents the fold increase in Nrp1 and Mical2 expression upon treatment with Shh-conditioned media relative to control condition media treatment in C3H/10T1/2, NIH 3T3, and BALB/c 3T3 cell lines. Error bars indicate S.D. based on two independent experiments.

 
Finally, we explored the gene expression data to identify the possible ligand that may bind to neuropilin1 and mediate the pro-migratory response to Shh. Analysis of the expression of semaphorins, VEGFs, and other known interactors of neuropilin1 shows that VEGF-B is the neuropilin1 ligand that has the highest expression in C166 cells. In contrary, semaphorin expression in C166 cells is very low (Table 3).


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  		TABLE 3
The expression of potential ligands and participants in the neuropilin pathway in C166 cells

The expression of known ligands and receptors that bind to or interact with neuropilin1 in C166 cells is shown. The expression values are derived from the oligonucleotide microarray signal values. Only genes, which are expressed in C166 cells, are listed. Mean signal values were calculated for the two Shh-N-treated samples in each time point.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
New evidence has emerged supporting a role for Hedgehog pathway in in vivo wound healing by direct pro-migratory effect on fibroblast and endothelial progenitor cells (13). The molecules mediating this effect remain elusive. Here we show in an in vitro wound healing assay that yolk sac-derived endothelial cells and primary embryonic fibroblasts can respond directly to stimulation by Shh by enhanced migration. By combining microarray gene expression analysis with functional assays, we have identified the neuropilin-flavomonooxygenase pathway as a major molecular pathway mediating the pro-migratory response of these cells to Hedgehog signaling.

Among the 54 genes significantly regulated by Hedgehog in the C166 cell line, 4 genes (beside Gli1 and Patched1) are known transcriptional targets of the Hedgehog pathway, a finding that supports the reliability of our assay. One of these genes, missing in metastasis (MIM)/metastasis suppressor 1(Mtss1), regulates actin cytoskeleton by bundling F-actin (34, 35). The three other genes are insulin-like growth factor binding protein 6 (Igfbp6), neural proliferation, differentiation, and control gene 1 (Npdc1) and embigin (Emb) (36, 37).

The search for over-represented biological themes among the various Hedgehog regulated genes using the Ease score program yielded a group of over-represented functional classifications related to cell motility, angiogenesis, and cytoskeletal organization. The two most significantly induced genes (beside Gli1 and Patched1), Nrp1, and Mical2, participate in regulation of cell migration in response to environmental cues. We have confirmed their induction by Shh by independent experiments in C166 as well as in three other embryonic fibroblasts cell lines and primary embryonic fibroblasts.


Figure 5
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  		FIGURE 5.
Shh induces migration and neuropilin1-Mical2 expression in primary murine fibroblastic cells. A, in vitro scratch assay. The experiment was done as described before, only the cells tested were primary mouse fibroblasts. The scratch assay was done in the presence of 10 µM cyclopamine (Cyclop.) or its solvent ethanol (Etoh). Graph shows the percentage of nonrecovered wound area from the initial wound area after 24 h of migration. Error bars indicate S.D. based on two independent experiments. *, p < 0.0005; **, p < 0.05 versus cyclopamine. B, quantitative real time PCR. Graph shows the fold increase in neuropilin1 (Nrp1), Mical2, and Gli11 expression in control and Shh-treated cells relative to cyclopamine-treated cells. y axis is in logarithmic scale.

 
Neuropilin1 serves as a co-receptor for semaphorin3A, mediating its induction of neural growth cone collapse by forming complexes with the Plexin receptors. In addition, neuropilin1 serves as a co-receptor for VEGF-A, enhancing its promigratory effect, mediated by the VEGFR-2 tyrosine kinase receptor, in endothelial cells. A third role for neuropilin1 is its function as a cell surface adhesion molecule (26). Knock-out of neuropilin1 in the mouse results in abnormal blood vessel formation, including insufficient development of the yolk sac vascular network (38). Strikingly, this vascular phenotype is virtually identical to the phenotype produced by elimination of Hedgehog signaling in the Smoothened knock-out mice.

At later stages of development, neuropilin1 is preferentially expressed in arterial endothelial cells (39) and participates in wound angiogenesis, as demonstrated in a murine model of dermal wound repair (40). Neuropilin2 has also been shown recently to induce endothelial cell migration (41). Because the Hedgehog signal accelerates wound healing in diabetes by increased wound vascularity (13), neuropilins may play a role in mediating this signal.


Figure 6
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  		FIGURE 6.
Neuropilin1 knockdown inhibits Shh-induced C166 migration. A, reduced expression of neuropilin1 (Nrp1) by pRETRO-SUPER RNA interference vector for Nrp1 (pRS-Nrp1). Quantitative real time PCR. Graph shows the relative mRNA levels of Mical2, Nrp1, and Nrp2 in a polyclonal population of puromycin-resistant C166 cells infected with pRS-Nrp1 retrovirus (Nrp1 knockdown) relative to a polyclonal population of puromycin-resistant C166 cells infected by Prs-CONTROL retrovirus (Nrp1 CONTROL). B, panel 1, quantitative real time PCR. Graph shows the fold increase in Mical2, Patched1, and Gli1 expression upon treatment with Shh relative to control in both Nrp1 knock-out and Nrp1 CONTROL polyclonal populations of C166. Panel 2, in vitro scratch assay. Experiment was done as described before using Nrp1 knock-out and Nrp1 CONTROL polyclonal populations of C166. Graph shows the percentage of nonrecovered wound area from the initial wound area after 24 h of migration. Error bars indicate S.D. based on two independent experiments. *, p < 0.0005 versus control.

 
Mical2 belongs to a family of multidomain cytosolic proteins with a flavoprotein monooxygenase domain (27, 28). Flavoprotein monooxygenases are oxidoreductases that use FAD as a cofactor to insert one atom of molecular oxygen into their substrates. In some contexts, these enzymes can act as oxidases and generate reactive oxygen species (42). The Mical proteins may act as platforms to recruit downstream effectors to the Plexin activation site. The activity of these effectors could then be selectively modulated by redox modifications (42).

Thus, Mical2 and neuropilin1 participate in pathways that regulate cell migration in response to variety of environmental cues. We have demonstrated the importance of these genes for the pro-migratory effects of Shh by examining the effects of neuropilin short interfering RNA and of the potent inhibitor of flavoprotein monooxygenases, EGCG. Our findings that neuropilin1 and Mical2 are Hedgehog transcriptional targets in other cell types than endothelium suggest a more universal role for these molecules in mediating Hedgehog cellular activity.


Figure 7
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  		FIGURE 7.
EGCG inhibits Shh-induced C166 migration in a dose-dependent manner. A, C166 in vitro scratch assay. The experiment was done as described before, only EGCG or its solvent PBS were added to the cells 30 min before the wound was applied. EGCG was added at the concentrations of 1 and 2.5 µM. Graph shows the percentage of nonrecovered wound area from the initial wound area after 24 h of migration. B, quantitative real time PCR. Graph shows the fold increase in neuropilin1 (Nrp1), Mical2, and Patched1 expression upon treatment with Shh relative to control, in the presence of 2.5 µM EGCG or its solvent PBS. Error bars indicate S.D. based on two independent experiments. *, p < 0.00005; **, p < 0.05 versus control.

 
Neuropilin1 is a co-receptor of semaphorins and VEGFs. VEGFs enhance endothelial cell migration, whereas binding of semaphorins to neuropilin1 causes regression of neural cells (43). Examination of the gene expression data revealed significant expression of VEGF-B in C166 cells and low to absent expression of semaphorins. VEGF-B has an important role in angiogenesis (44) and can bind neuropilin1 (45). The presence of VEGF-B probably explains why the induction of neuropilin1 by Shh resulted in enhanced cell migration.

The Hedgehog pathway has a central role in the pathogenesis of different cancers (4, 5). Several studies have suggested that neuropilin1 also plays a direct role in tumor growth, migration, and angiogenesis (46–49). Furthermore, it was shown that semaphorins and VEGFs suppress or promote tumor cells growth, respectively, through competitive binding to neuropilin1 (50, 51) and that the enhanced angiogenesis in tumors may be mediated by an elevated VEGF:semaphorin ratio. Our observations raise the hypothesis that Hedgehog signaling may enhance angiogenetic response to VEGFs in tumors by up-regulating neuropilin1 expression. This hypothesis needs to be tested experimentally.

Few studies have demonstrated that Shh has a critical role in morphogenesis of the aortic arch the cardiac outflow tract (52–54). Interestingly, in isl1-cre;smo mutants (in which the Hedgehog signal is ablated within endoderm or adjacent cardiogenic mesoderm), the expression of neuropilin2, which also participates in semaphorin signal transduction, is down-regulated in the cardiac outflow tract (55) supporting our finding that neuropilins are targets of Hedgehog signaling.

Recent experiments have shown that specialized "tip cells," located at the extremities of capillary sprouts, regulate blood vessel branching. Tip cells are very similar to growth cones of developing axons, as they seem to regulate extension of the capillary in response to environmental cues in some systems (56). In this study we show that the Hedgehog pro-migratory effect in the endothelial and fibroblastic lineages may be mediated by molecules that have dual, often opposing, roles in axon guidance and endothelial cell migration.


   FOOTNOTES
 
* This work was supported in part by the Israel Science Foundation, The Leukemia Research Foundation, The Israel Cancer Research Foundation, and The Recannati Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1 and 2 and supplemental Figs. 1 and 2. Back

1 Performed this work in partial fulfillment of the requirements for the M.D.-Ph.D. thesis at Sackler Faculty of Medicine, Tel Aviv University, Israel. Back

2 To whom correspondence should be addressed. E-mail: sizraeli{at}sheba.health.gov.il.

3 The abbreviations used are: Shh, Sonic Hedgehog; PBS, phosphate-buffered saline; EGCG, epigallocatechin gallate; VEGF, vascular endothelial growth factor; DTT, dithiothreitol; MEFs, mouse embryonic fibroblasts. Back


   ACKNOWLEDGMENTS
 
We thank Drs. D. R. Gardber, P. A. Beachy, R. Auerbach, G. Neufeld, R. Agami, R. Haffner, and A. P. McMahon for providing cell lines and reagents. We thank the members of the Izraeli and Rechavi laboratories for assistance and discussions. We also thank Inna Muler for technical support.



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Hedgehog signaling plays a cell-autonomous role in maximizing cardiac developmental potential
Development, November 15, 2008; 135(22): 3789 - 3799.
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Y. Cao, L. Wang, D. Nandy, Y. Zhang, A. Basu, D. Radisky, and D. Mukhopadhyay
Neuropilin-1 Upholds Dedifferentiation and Propagation Phenotypes of Renal Cell Carcinoma Cells by Activating Akt and Sonic Hedgehog Axes
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Y. A. Yoo, M. H. Kang, J. S. Kim, and S. C. Oh
Sonic hedgehog signaling promotes motility and invasiveness of gastric cancer cells through TGF-{beta}-mediated activation of the ALK5-Smad 3 pathway
Carcinogenesis, March 1, 2008; 29(3): 480 - 490.
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