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Mesenchymal cells reactivate Snail1 expression to drive three-dimensional invasion programs

¹ Division of Molecular Medicine and Genetics, Department of Internal Medicine, ² the Life Sciences Institute, ³ the Biomedical Research Core Facilities, ⁴ Department of Human Genetics, ⁵ Department of Pathology, and ⁶ Program in Cell and Molecular Biology, University of Michigan, Ann Arbor, MI 48109
⁷ Institute of Biomedicine/Anatomy, University of Helsinki, FIN-00014 Helsinki, Finland
⁸ Programa de Recerca en Cancer, Institut Municipal d'Investigació Mèdica Hospital del Mar Universitat Pompeu Fabra, 08003 Barcelona, Spain
⁹ Institute of Pathology, Technical University of Munich, D-81675 Munich, Germany
¹⁰ The Finsen Laboratory, Department 3735, Rigshospitalet, DK-2200 Copenhagen N, Denmark

Correspondence to Stephen J. Weiss: sjweiss{at}umich.edu

Epithelial–mesenchymal transition (EMT) is required for mesodermal differentiation during development. The zinc-finger transcription factor, Snail1, can trigger EMT and is sufficient to transcriptionally reprogram epithelial cells toward a mesenchymal phenotype during neoplasia and fibrosis. Whether Snail1 also regulates the behavior of terminally differentiated mesenchymal cells remains unexplored. Using a Snai1 conditional knockout model, we now identify Snail1 as a regulator of normal mesenchymal cell function. Snail1 expression in normal fibroblasts can be induced by agonists known to promote proliferation and invasion in vivo. When challenged within a tissue-like, three-dimensional extracellular matrix, Snail1-deficient fibroblasts exhibit global alterations in gene expression, which include defects in membrane type-1 matrix metalloproteinase (MT1-MMP)-dependent invasive activity. Snail1-deficient fibroblasts explanted atop the live chick chorioallantoic membrane lack tissue-invasive potential and fail to induce angiogenesis. These findings establish key functions for the EMT regulator Snail1 after terminal differentiation of mesenchymal cells.

© 2009 Rowe et al.
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	Introduction

Top Abstract Introduction Results and discussion Materials and methods References

 
Snail1, a zinc finger–type transcriptional repressor, initiates an epithelial–mesenchymal transition (EMT) that is critical for the morphogenetic events that characterize developmental programs such as gastrulation (Carver et al., 2001; Nieto, 2002; Murray et al., 2007). Snail1 triggers this transdifferentiation program, in part, by repressing epithelial markers and related cell–cell junction proteins while coordinately acting as a major cytoskeletal regulator (Batlle et al., 2000; Cano et al., 2000; Moreno-Bueno et al., 2006; Peinado et al., 2007). The aberrant postnatal expression of Snail1 is sufficient to confer a mesenchymal, fibroblast-like phenotype in differentiated epithelial cells during pathological states associated with cancer and fibrosis (Yook et al., 2005, 2006; Boutet et al., 2006; Moreno-Bueno et al., 2006; Olmeda et al., 2007a,b; Peinado et al., 2007).

At sites of active tissue remodeling, changes in vascular permeability disperse serum-derived soluble growth factors within the interstitial compartment, which serve to activate signal transduction cascades in resident fibroblasts (Martin, 1997; Bhowmick et al., 2004; Dong et al., 2004; Orimo et al., 2005; Klapholz-Brown et al., 2007). Accordingly, these agonists trigger changes in gene expression programs that shift the fibroblast phenotype from a quiescent status to an "activated" state characterized by increased proliferation, tissue-invasive activity, and the induction of angiogenesis (Martin, 1997; Iyer et al., 1999; Bhowmick et al., 2004; Sabeh et al., 2004; Klapholz-Brown et al., 2007). Growth factors capable of promoting the activated fibroblast phenotype, such as PDGF-BB, have been identified (Dong et al., 2004; Gao et al., 2005), but key transcription factors that regulate downstream gene programs remain largely uncharacterized. Herein, we identify Snail1 as a critical regulator of both fibroblast gene expression programs and fibroblast function in vitro as well as in vivo. The results demonstrate that Snail1, a master EMT inducer, continues to subserve vital cellular functions following mesenchymal cell terminal differentiation.

	Results and discussion

Top Abstract Introduction Results and discussion Materials and methods References

 
Under serum-free conditions, fibroblasts do not express detectable levels of Snail1 mRNA or protein (Fig. 1, A and B). In contrast, in the presence of 10% serum or PDGF-BB, both Snail1 mRNA and intranuclear protein levels are strongly induced in mouse as well as human fibroblasts (Fig. 1, A–C). In epithelial cells, Snail1 protein half-life is controlled by GSK3-β–dependent and –independent ubiquitination pathways that lead to proteasome-mediated Snail1 destruction (Zhou et al., 2004; Yook et al., 2005, 2006; Vernon and LaBonne, 2006). As expected, blockade of fibroblast proteasome activity with the inhibitor, MG132, results in a marked accumulation of the Snail1 protein (Fig. 1 B). In the GSK3-β–dependent pathway, Snail1 is marked for ubiquitination after phosphorylation of its N-terminal domain (Zhou et al., 2004; Vernon and LaBonne, 2006; Yook et al., 2006). As PDGF-BB signaling can inhibit GSK3-β activity via the phosphatidylinositol 3-kinase (PI3K)/Akt-dependent phosphorylation of GSK3-β serine 9 (Ser⁹; Julien et al., 2007), Akt phosphorylation, Ser⁹ phosphorylation, and Snail1 protein levels were monitored in fibroblasts in the absence or presence of the PI3K inhibitor, LY 294002. As predicted, treatment of serum-starved fibroblasts with PDGF-BB induces an increase in phospho-Akt and Ser⁹ GSK3-β levels in tandem with an increase in Snail1 protein (Fig. 1, D and E). In the presence of LY 294002, however, both Akt and Ser⁹ GSK3-β phosphorylation are blocked, and Snail1 levels fall to undetectable levels (Fig. 1, D and E).

View larger version (55K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Expression and regulation of Snail1 in activated fibroblasts. (A) Mouse dermal fibroblasts were cultured in the presence or absence of 10% serum for 24 h, and Snail1 mRNA was assessed by RT-PCR. (B) Mouse dermal fibroblasts were cultured serum-free, or in the presence of 10% serum, serum plus 10 ng/ml PDGF-BB, or serum plus 10 µM MG132 for 24 h, and Snail1 protein was monitored by Western blotting. (C) Mouse fibroblasts (top) or human foreskin fibroblasts (bottom) were cultured serum-free or in the presence of 10% serum for 24 h, and Snail1 protein were localized by immunocytochemistry with the anti-Snail1 173EC2 monoclonal antibody (mouse fibroblasts) or the Sn9H2 monoclonal antibody (human fibroblasts). Nuclei were stained with DAPI (blue). Bar, 50 µm. (D) Mouse dermal fibroblasts were cultured serum-free for 48 h, followed by stimulation with 10 ng/ml PDGF-BB in the presence or absence of 10 µM LY294002 for 10 min. Levels of phospho-Ser473 Akt, phospho-Ser9 GSK3-β, and β-actin were assessed by Western blotting. (E) Mouse dermal fibroblasts were cultured serum-free for 24 h, followed by stimulation with 10 ng/ml PDGF-BB for 12 h in the presence or absence of LY294002, and Snail1 protein levels were determined by Western blotting.

View larger version (34K): [in this window] [in a new window] [Download PPT slide]   Figure 2. A model of Snail1 deficiency in mouse fibroblasts. (A) Schematic of targeting strategy used to generate a mouse Snai1 conditional knockout allele. B, BglII; H, NcoI. (B) Embryonic stem cell clones were screened for recombination of the targeting vector at the 5' (NcoI) or 3' (BglII) ends. (C) Example of genotyping results demonstrating amplification of the Snai1wt and Snai1fl alleles with P1 and P2. (D) Snai1wt/fl dermal fibroblasts were infected either with a control adenovirus (β-gal) or adeno-Cre, and recombination of the Snai1fl allele was assessed by the loss of the 420–base pair amplicon when genomic DNA was amplified with P1 and P2 (top) and the appearance of a single amplicon with P3 and P4 (bottom). (E–G) Snai1fl/fl dermal fibroblasts were infected with either a control adenovirus or a Cre adenovirus, and Snai1 recombination was assessed by quantitative PCR (E), Western blotting (F), or immunocytochemistry for Snail1 with mAb 173EC2 (red) with propidium iodide (PI) counterstaining (blue; G). Error bars indicate ± 1 SEM. Bar, 30 µm.

View larger version (37K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Snail1 is a master regulator of fibroblast gene expression programs. (A) Heat map of microarray data for three biological replicates of Snail1fl/fl and Snail1–/– fibroblasts. The 20 most highly up-regulated and down-regulated transcripts in Snail1–/– fibroblasts are presented. The key on the bottom assigns heat map colors to the absolute gene expression value on a log2 scale. (B) GO terms identifying biological processes differentially expressed in Snai1fl/fl and deficient fibroblasts. (C) List of motility- and invasion-associated genes regulated by Snail1 in fibroblasts.

View larger version (73K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Snail1 regulates the type I collagenolytic and 3D invasive activities of fibroblasts. (A and B) Snai1fl/fl or Snai1–/– fibroblasts were embedded in a 100-µl plug of cross-linked, fibrillar type I collagen (2.2 mg/ml), which was embedded within a larger, cell-free collagen matrix in the presence of 10 ng/ml PDGF-BB and 10% serum. Migration was monitored over a 6-d culture period by phase-contrast microscopy, with arrowheads marking the advance of the invading front and the dotted line indicating the boundary between the inner and outer collagen gels (top) or after phalloidin staining in the bottom panels (red). (B) 3D invasion depth and the number of invading cells were measured after a 6-d culture period (n = 3; mean ± SEM). *, P < 0.01. (C) Snail1fl/fl or Snail1–/– fibroblasts were cultured in 3D collagen for 48 h and stained for cortactin (green), F-actin (red), and nuclei (DAPI, blue), and images were captured by confocal laser microscopy. Cortactin-rich cellular processes are indicated by arrowheads. (D and E) Fibroblasts were cultured atop a 3D bed of Alexa 594–labeled type I collagen (left, gray; right, blue) for 5 d, collagen degradation was monitored by confocal laser microscopy in sections costained for cortactin (green) and F-actin (D, middle and right panels, red), and photomicrographs were quantified using ImageQuant software. Degraded areas in left panels are demarcated by broken lines in the middle and right panels. (E) The degraded area and relative cortactin signal are presented as representative results of three experiments with the mean ± SEM (*, P < 0.05). (F and G) Snail1fl/fl or Snail1–/– fibroblasts were cultured on a bed of type I collagen and costained for MT1-MMP (red) and either cortactin (top, green) or F-actin (bottom, green; 60x magnification). The broken lines in F demarcate the invadopodial clusters used for colocalization analysis. (G) MT1-MMP signal colocalizing with cortactin and actin within invadopodia clusters was quantified using MetaMorph software. *, P < 0.005. Error bars indicate ± 1 SEM. Bars: (A, top) 200 µm; (A, bottom) 100 µm; (C and D) 10 µm; (F) 5 µm.

Though Snail1-deficient cells display defects in the pericellular proteolysis and invasion of homogeneous collagenous barriers in vitro, connective tissue barriers in vivo are more complex, multimolecular composites of ECM macromolecules (Grinnell, 2003; Hotary et al., 2003; Yamada and Cukierman, 2007; Zhou et al., 2008). As such, wild-type and Snail1-deleted fibroblasts were cultured atop the chorioallantoic membrane (CAM) of live chick embryos (Sabeh et al., 2004), a tissue characterized by a type IV collagen-rich basement membrane and an underlying interstitium containing both type I and type III collagens (the stroma also contains blood vessels circumscribed by type IV collagen-positive basement membranes; Fig. 5 A). Although wild-type fibroblasts efficiently breach the CAM basement membrane and invade into the underlying stroma, Snail1-deficient fibroblasts exhibit a complete defect in invasion and fail to penetrate the CAM surface (Fig. 5, B and C), a phenotype identical to that described previously for MT1-MMP–null fibroblasts (Sabeh et al., 2004). In vivo, fibroblasts can initiate neovascularization during wound healing (Martin, 1997), but Snail1-deficient fibroblasts also demonstrate a significantly attenuated ability to induce neovessel formation (Fig. 5, B and D). Neither proliferative nor apoptotic indices of the fibroblasts are affected in the CAM model (Fig. 5, E and F). Collectively, the data identify Snail1 as a master regulator of activated fibroblast function in vivo by controlling tissue-invasive as well as proangiogenic functions.

View larger version (77K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Snail1 and the fibroblast wound response in vivo. (A) CAM sections were stained with (left to right) hematoxylin and eosin (H/E) or antibodies against type IV, I, and III collagens, and photographed using light or fluorescence microscopy. (B) Snai1fl/fl or Snai1–/– fibroblasts were labeled with green fluorescent nanobeads, and cultured atop the live CAM. After 24 h, CAMs were sectioned and assessed for invasion by fluorescence microscopy for labeled cells (green), type IV collagen (red), cell nuclei (left, blue), or H/E staining (right). The broken line marks the upper surface of the CAM. Bar, 100 µm. (C) CAM invasion was quantified by measuring the invasive area demarcated by distribution of fluorescent beads in the CAM stroma or the depth of the invasive front (n = 5; mean ± SEM). *, P = 0.02 for invasion area; *, P = 0.01 for invasion depth. (D) Using type IV collagen signal in the CAM interstitium as an index of neovessel formation, CAM stromal angiogenesis was quantified (n = 11 for wild-type, n = 9 for null; mean ± SEM, *P < 0.01). (E) CAM sections were stained for the apoptosis marker TUNEL (red) and counterstained with DAPI (blue), and the percentage of TUNEL-positive cells was quantified (n = 3; mean ± SEM). (F) CAM sections were stained for the proliferation marker Ki67 (red), nuclei were counterstained with DAPI (blue), and the percentage of Ki67-positive nuclei was quantified (n = 3; mean ± SEM). Bars: (A and B) 100 µm; (E and F) 50 µm.

To date, analyses of Snail1 function in mammalian cells have focused on the ability of the transcription factor to initiate the transdifferentiation of normal or neoplastic epithelial cells. The findings presented herein, coupled with the fact that Snail1 protein is expressed in fibroblasts localized at damaged or carcinomatous tissues in vivo (Franci et al., 2006; Rosivatz et al., 2006), demonstrate that Snail1 activity is not confined to epithelial cells alone. Although our studies have focused on the role of Snail1 in regulating fibroblast function, it is intriguing to note that Snail1 may also be expressed in the neoplastic mesenchyme (Franci et al., 2006). Indeed, large T antigen/Ras-transformed fibroblasts are similarly reliant on Snail1 for the expression of a tissue-invasive phenotype (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200810113/DC1). Hence, in addition to its essential roles in EMT, we propose that Snail1 now be considered as a transcription factor capable of exerting key regulatory effects in the mesenchyme during development as well as disease.

	Materials and methods

Top Abstract Introduction Results and discussion Materials and methods References

 
Mice
To generate the Snai1 conditional knockout mouse, a targeting vector was constructed consisting of a flippase recognition target (FRT)-flanked phosphoglycerine kinase (PGK)-neo cassette 3' to the loxp-flanked exon 3 of mouse Snai1, predicted to encode the two C-terminal zinc finger domains as well as the polyadenylation sequence for the Snai1 mRNA. Approximately 4 kb of flanking genomic sequence was then inserted at the 5' and 3' ends of the loxp-flanked exon and FRT-flanked neomycin cassette to promote homologous recombination. The linearized targeting vector was electroporated into W4 embryonic stem cells (Auerbach et al., 2000), and stable transfected clones were selected with G418. Clones were screened for targeting of the Snai1 by Southern blotting, and recombination was verified at both the 5' and 3' ends of the construct. Of 100 clones screened, 3 were identified with correct targeting and used for injection into C57BL/6NCrl x (C57BL/6J x DBA/2J)F1 blastocysts to produce chimeric mice. Out of three chimeric lines produced, two lines transmitted the targeted Snai1 allele (Snai1^tm1Stjw) through the germ line. β-actin FLPe mice (stock No. 003800; Jackson ImmunoResearch Laboratories) were backcrossed to C57/6J mice to generate a congenic strain before mating with chimeras for excision of the FRT-flanked PGK-neo cassette to generate the Snai1^fl (Snai1^tm2Stjw) allele. Snai1^fl/fl homozygous conditional knockout mice were born in the expected Mendelian ratios, which indicates that the Snai1^fl allele functions equivalently to the wild-type Snai1 allele.

Antibodies and reagents
The 173EC2, 173EC3, and Sn9H2 anti-Snail1, and anti–MT1-MMP mAb1 antibodies were prepared and characterized as described previously (Franci et al., 2006; Rosivatz et al., 2006; Ingvarsen et al., 2008). The anti–GSK3-β phospho-serine 9 and anti-Akt phospho-serine 473 antibodies were obtained from Cell Signaling Technology. The anti-cortactin, anti-actin, and anti-Ki67 antibodies were obtained from Santa Cruz Biotechnology, Inc., Sigma-Aldrich, and Abcam, respectively. Adeno–β-gal and Adeno-Cre (transgenes driven by a cytomegalovirus promoter) were obtained from the University of Michigan Vector Core. LY-294002 and MG132 were obtained from EMD and Sigma-Aldrich, respectively. Apoptotic cell death was measured with an in situ apoptosis detection kit (ApopTag Red) according to the manufacturer's instructions (Millipore).

Western blotting
For Western blotting, the following primary antibody dilutions were used: 173EC2 hybridoma supernatant (1:40), 173EC3 affinity-purified antibody (1:10,000), anti–GSK3-β phospho-serine 9 and anti-Akt phospho-serine 473 antibodies (1:1,000), and anti-actin (1:4,000).

Quantitative PCR
Quantitative PCR was performed using the SYBR green PCR master mix (Applied Biosystems) according to the manufacturer's instructions. Primers for mouse cortactin were: forward, 5'-GCAGCCATCCCAGGTGTTTTAGTT-3', and reverse, 5'-CTTTGGTCCCCTTTCCTCCTCTTC-3'; mouse MT1-MMP primers were: forward, 5'-TGATTCTGCCGAGCCCTGGACTGT-3', and reverse, 5'-TGAGGGGGCATCTTTGTGGGTGAC-3'; mouse Snail primers were: forward, 5'-CTGCTTCGAGCCATAGAACTAAAG-3', and reverse, 5'-GAGGGGAACTATTGCATAGTCTGT-3'; and glyceraldehyde 3-phosphate dehydrogenase primers were: forward, 5'-CCAAGGTCATCCATGACAACT-3', and reverse, 5'-GTCATACCAGGAAATGAGCTTGACA-3'.

Immunofluorescence
For Snail1 immunocytochemistry, cells were fixed in 4% paraformaldehyde, permeabilized with 1% sodium dodecyl sulfate, denatured with 6M urea and 0.1% glycine, pH 3.5, blocked with 3% goat serum, and incubated with either 173EC2 (1:5) or 173EC3 (1:1,000) overnight, followed by detection with Alexa 488–labeled anti–mouse secondary antibody (Invitrogen). The Alexa 532–labeled anti–MT1-MMP mAb1 was used at 5 µg/ml, and the anti-cortactin antibody was used at a dilution of 1:40 after paraformaldehyde fixation and permeabilization with Triton X-100. The anti-cortactin antibody was detected with an Alexa 488–labeled, anti–rabbit secondary antibody (Invitrogen). Cells were counterstained with either 4',6-diamidino-2-phenylindole or propidium iodide (Invitrogen). Confocal images of cells were acquired on a confocal microscope (FV500) using a 60x water immersion lens with a 1.20 numerical aperture using FluoView software (all from Olympus). All images comparing Snail1 wild-type and deficient cells were acquired with equal photomultiplier tube intensity and gain settings. Phase contrast images were acquired with a inverted microscope (DM-ILB; Leica) with a 20x objective and 0.40 numerical aperture, and CAM images were acquired on a microscope (DM-LB; Leica) with a 20x objective and 0.50 numerical aperture (Leica). Phase contrast and CAM images were acquired and analyzed with SPOT cameras and software (Diagnostic Instruments, Inc.).

Image analysis
To analyze MT1-MMP in invadopodia, confocal cross sections of invadopodia costained for MT1-MMP and cortactin were analyzed with MetaMorph software (MDS Analytical Technology). Collagen degradation was analyzed using ImageQuant 5.2 software (GE Healthcare). Invadopodial clusters were traced, and the areas containing MT1-MMP and cortactin colocalization were quantified.

Cell culture and invasion assays
To analyze 3D invasion, 50,000 fibroblasts were embedded in 100 µl of type I collagen gel (2.2 mg/ml) isolated from rat tail (Sabeh et al., 2004). After gelling, the plug was embedded in a cell-free, 500 µl collagen gel (2.2 mg/ml) cultured within a 24-well plate. After allowing the surrounding collagen matrix to gel (1 h at 37°C), fibroblast invasion was stimulated with serum and 10 ng/ml PDGF-BB (Millipore). Invasion distance from the inner collagen plug into the outer collagen gel was quantified. CAM invasion assays were conducted using 11-d-old chick embryos where fibroblasts labeled with Fluoresbrite-carboxylated nanospheres (Polysciences, Inc.) were cultured atop the CAM for 24 h. Invasion depth was defined as the leading front of at least three invading cells in 10 fields in frozen sections (Sabeh et al., 2004). The invasion area was defined as the area occupied by invading cells in at least 10 fields (Sabeh et al., 2004), whereas angiogenesis was quantified by type IV collagen staining (Bajou et al., 2001). Snail1 conditional knockout transformed fibroblasts were generated by isolating embryonic day 13.5 mouse embryonic fibroblasts followed by serial retroviral introduction of the polyoma large T and activated Ras oncogenes (Land et al., 1983).

Transcriptional profiling
Total RNA was isolated from fibroblast cultures in 3D collagen, then labeled and hybridized to mouse 430 2.0 cDNA microarrays (Affymetrix). Three replicates each of Snail1 wild-type and deficient cultures were analyzed by the University of Michigan Microarray Core. Differentially expressed probe sets were determined using a minimum fold change of 1.5 and a maximum p-value of 0.005. GO analysis was performed to identify biological processes transcriptionally regulated by Snail. GO coefficients were calculated as –log(p-value).

Online supplemental material
Fig. S1 shows analysis of fibroblast function under 2D culture conditions. Fig. S2 shows transcript analysis by quantitative PCR and rescue of the Snail1-deficient phenotype by reconstitution with Snail1. Fig. S3 characterizes and analyzes the tissue-invasive potential of wild-type and Snail1-deficient transformed mouse embryonic fibroblasts. Table S1 shows a list of genes differentially expressed between Snail1 wild-type and deficient fibroblasts. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200810113/DC1.

	Acknowledgments

 
This work was supported by the National Institutes of Health (grant CA116516 to S.J. Weiss) and funds from the Cancer Biology Training Program (grant T32-CA009676) and the Breast Cancer Research Foundation. We thank E. Hughes (Transgenics Core) for technical assistance and Steve Lentz of the Morphology and Image Analysis Core (DK20572 and DK34933) for valuable assistance with confocal microscopy.

Submitted: 17 October 2008

Accepted: 24 December 2008

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