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. 2010 Jul;177(1):404-14.
doi: 10.2353/ajpath.2010.091079. Epub 2010 May 27.

Sonic hedgehog acts as a negative regulator of {beta}-catenin signaling in the adult tongue epithelium

Fabian T Schneider  1 , Anne Schänzer, Cathrin J Czupalla, Sonja Thom, Knut Engels, Mirko H H Schmidt, Karl H Plate, Stefan Liebner
Affiliations

Sonic hedgehog acts as a negative regulator of {beta}-catenin signaling in the adult tongue epithelium

Fabian T Schneider et al. Am J Pathol. 2010 Jul.

Abstract

Wnt/beta-catenin signaling has been implicated in taste papilla development; however, its role in epithelial maintenance and tumor progression in the adult tongue remains elusive. We show Wnt/beta-catenin pathway activation in reporter mice and by nuclear beta-catenin staining in the epithelium and taste papilla of adult mouse and human tongues. beta-Catenin activation in APC(min/+) mice, which carry a mutation in adenomatous poliposis coli (APC), up-regulates Sonic hedgehog (Shh) and Jagged-2 (JAG2) in the tongue epithelium without formation of squamous cell carcinoma (SCC). We demonstrate that Shh suppresses beta-catenin transcriptional activity in a signaling-dependent manner in vitro and in vivo. A similar regulation and function was observed for JAG2, suggesting that both pathways negatively regulate beta-catenin, thereby preventing SCC formation in the tongue. This was supported by reduced nuclear beta-catenin in the tongue epithelium of Patched(+/-) mice, exhibiting dominant active Shh signaling. At the invasive front of human tongue cancer, nuclear beta-catenin and Shh were increased, suggesting their participation in tumor progression. Interestingly, Shh but not JAG2 was able to reduce beta-catenin signaling in SCC cells, arguing for a partial loss of negative feedback on beta-catenin transcription in tongue cancer. We show for the first time that the putative Wnt/beta-catenin targets Shh and JAG2 control beta-catenin signaling in the adult tongue epithelium, a function that is partially lost in lingual SCC.

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Figures

Figure 1
Figure 1
Increased nuclear β-catenin in the tongue epithelium of APCmin/+ mice. A, D, G, and J: The tongue epithelium of APCmin/+ mice (G and J) shows increased nuclear localization of β-catenin compared with controls (A and D). Note that positive nuclei are located in the epithelium and in taste buds; arrowheads point to nuclear β-catenin. M: Quantification of β-catenin positive nuclei in APCmin/+ compared with wild-type (WT) mice (**P < 0.005). B, E, H, and K: Cell proliferation was not altered in APCmin/+ mice (H and K) compared with controls (B and E), determined by Ki-67 antigen immunofluorescence. C, F, I, and L: apoptosis was not significantly altered in the tongue epithelium of APCmin/+ mice (I and L) compared with wild-type (C and F) as evaluated by TUNEL staining. N: Combined graph showing quantification of Ki-67 and TUNEL positive cells in APCmin/+ mice compared with wild-type. For statistical analysis, three animals of each genotype were analyzed for nuclear β-catenin, Ki-67, and TUNEL staining.
Figure 2
Figure 2
Shh and the Hh target gene Ptch1 are up-regulated in APCmin/+ tongue epithelium. B, D, and F: Shh is augmented in papillae of APCmin/+ mice compared with controls (A, C, and E) as revealed by immunohistochemistry (A–D) and in situ hybridization (E and F), respectively. Arrowheads indicate Shh positive cells in the fungiform papilla. G: Quantitative real-time PCR (n = 3) for Shh, Ptch1, and Gli1 revealed significant up-regulation of Shh and Ptch1 in APCmin/+ mice, whereas Gli1 was not altered (n = 3 tongue epithelia of APCmin/+ and wild-type) (**P < 0.005). Data are normalized against G6PDX and polr2a. Control values were set to 100% (dashed line).
Figure 3
Figure 3
In vitro up-regulation of Shh inhibits sTOP/sFOP-FLASH in HEK293 cells. A: HEK293 cells were transiently transfected with the super(8×)TOP-FLASH and the super(8×)FOP-FLASH reporter plasmid, respectively, and stimulated with 20 mmol/L LiCl. After transfection with increasing amounts of pCMV-Sport6.1-Shh plasmid, activation of the sTOP-FLASH reporter was measured after 24 hours. B: To check the mShh biological activity in HEK293 cells transfected with the 8 × 3′Gli-BSδ51LucII or the 8 × 3′GliM3-BSδ51LucII luciferase reporter, after stimulation of HEK293 cells either by mGli1 transfection as a positive control or by Shh-CM, luciferase activity was measured after 24 hours (**P < 0.0081; *P < 0.0157). Validation of mShh protein expression in HEK293 cells transfected with the pCMV-Sport6.1-Shh or vector control (VC) by Western blot analysis of cell lysate and CM on a 12% SDS-polyacrylamide gel electrophoresis. C and D: Shh effect on sTOP/sFOP-FLASH reporter in HEK293 cells. Stimulation of canonical Wnt signaling either by 20 mmol/L LiCl or LEFΔN-βCTA transfection. C: 500 ng pCMV-Sport6.1-Shh-plasmid (*P < 0.0147; **P < 0.0015) or (D) Shh-CM were used (**P < 0.002; *P < 0.0241). Assays were repeated in three individual experiments; SD is shown as SEM.
Figure 4
Figure 4
Expression of Shh and nuclear β-catenin is down-regulated in Ptch+/− animals compared with wild-type. A–D: Ptch+/− mice showed reduced levels of nuclear β-catenin (B and D) compared with control animals (A and C; n = 3); arrowheads point to nuclear β-catenin; insets show higher magnification. Quantification of nuclear β-catenin staining (I) showed a highly significant reduction in Ptch+/− mice tongue epithelium (***P < 0.0001; SD is shown as SEM). E–H: Shh was reduced evidenced by IHC in the tongue epithelium of Ptch+/− mice (F and H) compared with wild-type (E and G). J: qRT-PCR for Shh, Ptch1, and Gli1 in Ptch+/− versus wild-type mice (***P < 0.0001; *P < 0.0187). Data are normalized against G6PDX and polr2a. Control values were set to 100% (dashed line); three tongue epithelia of Ptch+/− and control animals were analyzed for qPCR; SD is shown as SEM.
Figure 5
Figure 5
Shh and β-catenin in SCC of grades I to III. Invasive zone of human SCC section of grade I, grade II, and grade III tumors. A, E, and I: CK 5/6 staining reveals the epithelial nature of the tumor. Note the increased staining of single cells with increased tumor malignancy. B, F, and J: β-catenin staining is predominantly junctional in low-grade tumors and shows increased frequency of nuclear localization in higher grades at the invasive front. Insets show higher magnification. C, G, and K: Increase in Shh staining along with increasing tumor grade. Note Shh positive cells at the invasive front of the tumor tissue. D, H, and L: Increase in MIB1 staining for proliferating cells along with increasing tumor malignancy parallels with tumor progression. Nuclear counter staining in blue; insets show higher magnification of MIB1 positive cells (World Health Organization grade I, n = 3; II, n = 13; III, n = 3). M: Human SCC25 cells transiently transfected with sTOP-FLASH or sFOP-FLASH reporter plasmids, stimulated with LEFΔN-βCTA co-transfected with or without the pCMV-Sport6.1-Shh plasmid (*P < 0.0362). Experiments were performed in three individual experiments by using FuGene-HD (Roche); SD is shown as SEM.
Figure 6
Figure 6
Jagged-2 and Notch2 are up-regulated in the tongue epithelium of APCmin/+ mice, suppressing β-catenin signaling. A: qRT-PCR of APCmin/+ versus wild-type mice tongue epithelium showed an up-regulation of the Notch pathway genes Jagged2 (**P < 0.0051) and Notch2 (*P < 0.0393). In situ hybridization for Jagged2 verified increased mRNA in APCmin/+ mice versus wild-type. B: sTOP/sFOP-FLASH reporter transiently transfected in HEK293; stimulation with 50 ng LEFΔN-βCTA, co-transfected with 300 ng Notch2 or 600 ng Jagged2 (***P < 0.0001) or both (***P < 0.0001). Reduction of the sTOP-FLASH compared with LEFΔN-βCTA unstimulated as control (dashed line) is not influenced by the Presenellin inhibitor DAPT (10 μmol/L; ***P < 0.0006). The assay was performed in five individual experiments; SD is shown as SEM. Western blot analysis of Jagged2 and Notch2 over expression in HEK293 cells. Note the two forms of Notch2; upper band shows full length (FL), and the lower band shows Notch intracellular domain on a 7% SDS-polyacrylamide gel electrophoresis. C: Transient transfected SCC25 cells with the sTOP/sFOP-FLASH reporter and the same amounts of LEFΔN-βCTA, Jagged2, and Notch2 as indicated in B. The assay was performed in three individual experiments; SD is shown as SEM.
Figure 7
Figure 7
Interaction scheme of Shh and activated β-catenin signaling. Schematic illustration of the hypothesized inhibition of β-catenin by canonical Shh signaling together with Jagged2-Notch2. Left side: If Shh binds to Ptch, it looses its inhibitory effect on SMO. This activates the Gli proteins for target gene translation (left side). The APC mutation in APCmin/+ leads to accumulation of cytosolic and nuclear β-catenin and leads to Wnt-specific target gene translation (middle). The activation of the Shh signaling is able to inhibit β-catenin downstream of the degradation complex. It is unknown whether this inhibition mechanism works through a novel pathway initiated by the receptor complex Ptch/SMO or takes place further downstream. Right side: JAG2 might be a second control mechanism for β-catenin via an unknown mechanism, as canonical Notch signaling is not required.
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