Integral Membrane Protein 2A Is a Negative Regulator of Canonical and Non-Canonical Hedgehog Signalling.

The Hedgehog (Hh) receptor PTCH1 and the integral membrane protein 2A (ITM2A) inhibit autophagy by reducing autolysosome formation. In this study, we demonstrate that ITM2A physically interacts with PTCH1; however, the two proteins inhibit autophagic flux independently, since silencing of ITM2A did not prevent the accumulation of LC3BII and p62 in PTCH1-overexpressing cells, suggesting that they provide alternative modes to limit autophagy. Knockdown of ITM2A potentiated PTCH1-induced autophagic flux blockade and increased PTCH1 expression, while ITM2A overexpression reduced PTCH1 protein levels, indicating that it is a negative regulator of PTCH1 non-canonical signalling. Our study also revealed that endogenous ITM2A is necessary for timely induction of myogenic differentiation markers in C2C12 cells since partial knockdown delays the timing of differentiation. We also found that basal autophagic flux decreases during myogenic differentiation at the same time that ITM2A expression increases. Given that canonical Hh signalling prevents myogenic differentiation, we investigated the effect of ITM2A on canonical Hh signalling using GLI-luciferase assays. Our findings demonstrate that ITM2A is a strong negative regulator of GLI transcriptional activity and of GLI1 stability. In summary, ITM2A negatively regulates canonical and non-canonical Hh signalling.

Ubiquitin-protein ligase Ubr5 cooperates with hedgehog signalling to promote skeletal tissue homeostasis.

Mammalian Hedgehog (HH) signalling pathway plays an essential role in tissue homeostasis and its deregulation is linked to rheumatological disorders. UBR5 is the mammalian homologue of the E3 ubiquitin-protein ligase Hyd, a negative regulator of the Hh-pathway in Drosophila. To investigate a possible role of UBR5 in regulation of the musculoskeletal system through modulation of mammalian HH signaling, we created a mouse model for specific loss of Ubr5 function in limb bud mesenchyme. Our findings revealed a role for UBR5 in maintaining cartilage homeostasis and suppressing metaplasia. Ubr5 loss of function resulted in progressive and dramatic articular cartilage degradation, enlarged, abnormally shaped sesamoid bones and extensive heterotopic tissue metaplasia linked to calcification of tendons and ossification of synovium. Genetic suppression of smoothened (Smo), a key mediator of HH signalling, dramatically enhanced the Ubr5 mutant phenotype. Analysis of HH signalling in both mouse and cell model systems revealed that loss of Ubr5 stimulated canonical HH-signalling while also increasing PKA activity. In addition, human osteoarthritic samples revealed similar correlations between UBR5 expression, canonical HH signalling and PKA activity markers. Our studies identified a crucial function for the Ubr5 gene in the maintenance of skeletal tissue homeostasis and an unexpected mode of regulation of the HH signalling pathway.

Long non-coding RNA HOTAIR induces GLI2 expression through Notch signalling in systemic sclerosis dermal fibroblasts.

OBJECTIVES: Systemic sclerosis (SSc) is characterised by tissue fibrosis of the major organs of the body including the skin, lungs and heart. We have previously reported that the lncRNA HOTAIR plays a central role in the activation of SSc myofibroblasts, the key cellular elements of fibrosis. HOTAIR induces fibroblast activation through H3K27me3-mediated activation of the Notch signalling pathway. Here we aimed to identify the signalling events downstream of Notch that drive SSc myofibroblast activation. METHODS: Patient fibroblasts were obtained from full-thickness forearm skin biopsies of 3 adult patients with SSc of recent onset. The lncRNA HOTAIR was expressed in healthy dermal fibroblasts by lentiviral transduction. Hedgehog signalling pathway was inhibited with GANT61 and GLI2 siRNA. Gamma secretase inhibitors RO4929097 and DAPT were used to block Notch signalling. GSK126 was used to inhibit Enhancer of Zeste 2 (EZH2). RESULTS: Overexpression of HOTAIR in dermal fibroblasts induced the expression of the Hedgehog pathway transcription factor GLI2. This is mediated by activation of Notch signalling following epigenetic downregulation of miRNA-34a expression. Inhibition of H3K27 methylation and Notch signalling reduced expression of GLI2 in HOTAIR-expressing fibroblasts as well as in SSc dermal fibroblasts. Importantly, the inhibition of GLI2 function using GANT61 or siRNA mitigates the pro-fibrotic phenotype induced by HOTAIR. CONCLUSIONS: Our data indicates that GLI2 expression is stably upregulated in SSc myofibroblasts through HOTAIR and that GLI2 mediates the expression of pro-fibrotic markers downstream of Notch.

Overexpression of Desmoglein 2 in a Mouse Model of Gorlin Syndrome Enhances Spontaneous Basal Cell Carcinoma Formation through STAT3-Mediated Gli1 Expression.

Activation of the hedgehog pathway is causative of virtually all sporadic and Gorlin syndrome-related basal cell carcinomas (BCCs), with loss of function of Ptc1 being the most common genomic lesion. Sporadic BCCs also overexpress Dsg2, a desmosomal cadherin normally found in the basal layer. Using a mouse model of Gorlin syndrome (Ptc1+/lacZ mice), we found that overexpressing Dsg2 in the basal layer (K14-Dsg2/Ptc1+/lacZ mice) or the superficial epidermis (Inv-Dsg2/Ptc1+/lacZ mice) resulted in increased spontaneous BCC formation at 3 and 6 months, respectively. The tumors did not show loss of heterozygosity of Ptc1, despite high levels of Gli1 and phosphorylated Stat3. A panel of sporadic human BCCs showed increased staining of both Dsg2 and phosphorylated Stat3 in all nine samples. Overexpression of Dsg2 in ASZ001 cells, a Ptc1-/- BCC cell line, induced Stat3 phosphorylation and further increased Gli1 levels, in both an autocrine and paracrine manner. Three different Stat3 inhibitors reduced viability and Gli1 expression in ASZ001 cells but not in HaCaT cells. Conversely, stimulation of Stat3 in ASZ001 cells with IL-6 increased Gli1 expression. Our results indicate that Dsg2 enhances canonical hedgehog signaling downstream of Ptc1 to promote BCC development through the activation of phosphorylated Stat3 and regulation of Gli1 expression.