Heterozygous missense variant in GLI2 impairs human endocrine pancreas development.

Missense variants are the most common type of coding genetic variants. Their functional assessment is fundamental for defining any implication in human diseases and may also uncover genes that are essential for human organ development. Here, we apply CRISPR-Cas9 gene editing on human iPSCs to study a heterozygous missense variant in GLI2 identified in two siblings with early-onset and insulin-dependent diabetes of unknown cause. GLI2 is a primary mediator of the Hedgehog pathway, which regulates pancreatic ß-cell development in mice. However, neither mutations in GLI2 nor Hedgehog dysregulation have been reported as cause or predisposition to diabetes. We establish and study a set of isogenic iPSC lines harbouring the missense variant for their ability to differentiate into pancreatic ß-like cells. Interestingly, iPSCs carrying the missense variant show altered GLI2 transcriptional activity and impaired differentiation of pancreatic progenitors into endocrine cells. RNASeq and network analyses unveil a crosstalk between Hedgehog and WNT pathways, with the dysregulation of non-canonical WNT signaling in pancreatic progenitors carrying the GLI2 missense variant. Collectively, our findings underscore an essential role for GLI2 in human endocrine development and identify a gene variant that may lead to diabetes.

TGF-ß1/SMAD3-driven GLI2 isoform expression contributes to aggressive phenotypes of hepatocellular carcinoma.

Hedgehog signaling is activated in response to liver injury, and modulates organogenesis. However, the role of non-canonical hedgehog activation via TGF-ß1/SMAD3 in hepatic carcinogenesis is poorly understood. TGF-ß1/SMAD3-mediated non-canonical activation was found in approximately half of GLI2-positive hepatocellular carcinoma (HCC), and two new GLI2 isoforms with transactivating activity were identified. Phospho-SMAD3 interacted with active GLI2 isoforms to transactivate downstream genes in modulation of stemness, epithelial-mesenchymal transition, chemo-resistance and metastasis in poorly-differentiated hepatoma cells. Non-canonical activation of hedgehog signaling was confirmed in a transgenic HBV-associated HCC mouse model. Inhibition of TGF-ß/SMAD3 signaling reduced lung metastasis in a mouse in situ hepatic xenograft model. In another cohort of 55 HCC patients, subjects with high GLI2 expression had a shorter disease-free survival than those with low expression. Moreover, co-positivity of GLI2 with SMAD3 was observed in 87.5% of relapsed HCC patients with high GLI2 expression, indicating an increased risk of post-resection recurrence of HCC. The findings underscore that suppressing the non-canonical hedgehog signaling pathway may confer a potential strategy in the treatment of HCC.

Identification of Fasudil as a collaborator to promote the anti-tumor effect of lenvatinib in hepatocellular carcinoma by inhibiting GLI2-mediated hedgehog signaling pathway.

Lenvatinib is a frontline tyrosine kinase inhibitor for patients with advanced hepatocellular carcinoma (HCC). However, just 25% of patients benefit from the treatment, and acquired resistance always develops. To date, there are neither effective medications to combat lenvatinib resistance nor accurate markers that might predict how well a patient would respond to the lenvatinib treatment. Thus, novel strategies to recognize and deal with lenvatinib resistance are desperately needed. In the current study, a robust Lenvatinib Resistance index (LRi) model to predict lenvatinib response status in HCC was first established. Subsequently, five candidate drugs (Mercaptopurine, AACOCF3, NU1025, Fasudil, and Exisulind) that were capable of reversing lenvatinib resistance signature were initially selected by performing the connectivity map (CMap) analysis, and fasudil finally stood out by conducting a series of cellular functional assays in vitro and xenograft mouse model. Transcriptomics revealed that the co-administration of lenvatinib and fasudil overcame lenvatinib resistance by remodeling the hedgehog signaling pathway. Mechanistically, the feedback activation of EGFR by lenvatinib led to the activation of the GLI2-ABCC1 pathway, which supported the HCC cell's survival and proliferation. Notably, co-administration of lenvatinib and fasudil significantly inhibited IHH, the upstream switch of the hedgehog pathway, to counteract GLI2 activation and finally enhance the effectiveness of lenvatinib. These findings elucidated a novel EGFR-mediated mechanism of lenvatinib resistance and provided a practical approach to overcoming drug resistance in HCC through meaningful drug repurposing strategies.

Ulk4 promotes Shh signaling by regulating Stk36 ciliary localization and Gli2 phosphorylation.

The Hedgehog (Hh) family of secreted proteins governs embryonic development and adult tissue homeostasis through the Gli family of transcription factors. Gli is thought to be activated at the tip of primary cilium, but the underlying mechanism has remained poorly understood. Here, we show that Unc-51-like kinase 4 (Ulk4), a pseudokinase and a member of the Ulk kinase family, acts in conjunction with another Ulk family member Stk36 to promote Gli2 phosphorylation and Hh pathway activation. Ulk4 interacts with Stk36 through its N-terminal region containing the pseudokinase domain and with Gli2 via its regulatory domain to bridge the kinase and substrate. Although dispensable for Hh-induced Stk36 kinase activation, Ulk4 is essential for Stk36 ciliary tip localization, Gli2 phosphorylation, and activation. In response to Hh, both Ulk4 and Stk36 colocalize with Gli2 at ciliary tip, and Ulk4 and Stk36 depend on each other for their ciliary tip accumulation. We further show that ciliary localization of Ulk4 depends on Stk36 kinase activity and phosphorylation of Ulk4 on Thr1023, and that ciliary tip accumulation of Ulk4 is essential for its function in the Hh pathway. Taken together, our results suggest that Ulk4 regulates Hh signaling by promoting Stk36-mediated Gli2 phosphorylation and activation at ciliary tip.

Anoikis regulator GLI2 promotes NC cell immunity escape by TGF-ß-mediated non-classic hedgehog signaling in colorectal cancer: based on artificial intelligence and big data analysis.

BACKGROUND: Anoikis is a speed-limited procedure to inhibit tumor metastasis during epithelial-mesenchymal transition (EMT). Previous studies have explored anoikis-related genes (ARG) in predicting prognosis and distinguishing tumoral immunity in many types of cancer. However, the role of ARGs in regulating NK cell exhaustion (NKE) and in predicting chemotherapy sensitivity is not clear. Therefore, it is necessary to work on it. METHODS: Gene expression profiles and clinical features are collected from TCGA and GEO, and data analysis is performed in R4.2.0. RESULTS: The ARGs-based no-supervised learning algorithm identifies three ARG subgroups, amongst which the prognosis is different. WCGNA and Artificial intelligence (AI) are applied to construct an NKE-related drug sensitivity stratification and prognosis identification model in digestive system cancer. Pathways association analysis screens out GLI2 is a key gene in regulating NKE by non-classic Hedgehog signaling (GLI2/TGF-ß/IL6). In vitro experiments show that down-regulation of GLI2 enhances the CAPE-mediated cell toxicity and accompanies with down-regulation of PD-L1, tumor-derive IL6, and snial1 whereas the expression of cleaved caspas3, cleaved caspase4, cleaved PARP, and E-cadherin are up-regulated in colorectal cancer. Co-culture experiments show that GLI2- decreased colorectal tumor cells lead to down-regulation of TIM-3 and PD1 in NK cells, which are restored by TGF-bate active protein powder. Besides, the Elisa assay shows that GLI2-decreased colorectal tumor cells lead to up-regulation of IFN-gamma in NK cells.

Genetic diagnosis of congenital hypopituitarism in Turkish patients by a target gene panel: novel pathogenic variants in GHRHR, GLI2, LHX4 and POU1F1 genes.

Objective: Congenital hypopituitarism (CH) is a rare disease characterized by one or more hormone deficiencies of the pituitary gland. To date, many genes have been associated with CH. In this study, we identified the allelic variant spectrum of 11 causative genes in Turkish patients with CH. Materials and methods: This study included 47 patients [21 girls (44.6%) and 26 boys (55.4%)] from 45 families. To identify the genetic etiology, we screened 11 candidate genes associated with CH using next-generation sequencing. To confirm and detect the status of the specific familial variant in relatives, Sanger sequencing was also performed. Results: We identified 12 possible pathogenic variants in GHRHR, GH1, GLI2, PROP-1, POU1F1, and LHX4 in 11 patients (23.4%), of which six were novel variants: two in GHRHR, two in POU1F1, one in GLI2, and one in LHX4. In all patients, these variants were most frequently found in GLI2, followed by PROP-1 and GHRHR. Conclusion: Genetic causes were determined in only 23.4% of all patients with CH and 63% of molecularly diagnosed patients (7/11) from consanguineous families. Despite advances in genetics, we were unable to identify the genetic etiology of most patients with CH, suggesting the effect of unknown genes or environmental factors. More genetic studies are necessary to understand the etiology of CH.

Increasing Ciliary ARL13B Expression Drives Active and Inhibitor-Resistant Smoothened and GLI into Glioma Primary Cilia.

ADP-ribosylation factor-like protein 13B (ARL13B), a regulatory GTPase and guanine exchange factor (GEF), enriches in primary cilia and promotes tumorigenesis in part by regulating Smoothened (SMO), GLI, and Sonic Hedgehog (SHH) signaling. Gliomas with increased ARL13B, SMO, and GLI2 expression are more aggressive, but the relationship to cilia is unclear. Previous studies have showed that increasing ARL13B in glioblastoma cells promoted ciliary SMO accumulation, independent of exogenous SHH addition. Here, we show that SMO accumulation is due to increased ciliary, but not extraciliary, ARL13B. Increasing ARL13B expression promotes the accumulation of both activated SMO and GLI2 in glioma cilia. ARL13B-driven increases in ciliary SMO and GLI2 are resistant to SMO inhibitors, GDC-0449, and cyclopamine. Surprisingly, ARL13B-induced changes in ciliary SMO/GLI2 did not correlate with canonical changes in downstream SHH pathway genes. However, glioma cell lines whose cilia overexpress WT but not guanine exchange factor-deficient ARL13B, display reduced INPP5e, a ciliary membrane component whose depletion may favor SMO/GLI2 enrichment. Glioma cells overexpressing ARL13B also display reduced ciliary intraflagellar transport 88 (IFT88), suggesting that altered retrograde transport could further promote SMO/GLI accumulation. Collectively, our data suggest that factors increasing ARL13B expression in glioma cells may promote both changes in ciliary membrane characteristics and IFT proteins, leading to the accumulation of drug-resistant SMO and GLI. The downstream targets and consequences of these ciliary changes require further investigation.

The GLI2/CDH6 axis enhances migration, invasion and mitochondrial fission of stomach adenocarcinoma cells.

It has been reported that cadherin 6 (CDH6) upregulation is associated with enhanced epithelial-to-mesenchymal transition (EMT) in several types of solid tumor cells. The current study aimed to explore the effect of CDH6 on the migration and invasion of stomach adenocarcinoma (STAD) cells, the transcription factors involved in CDH6 dysregulation and their effect on mitochondrial fission. Bioinformatics analysis was performed using data extracted from the Genotype-Tissue Expression Project, the Cancer Genome Atlas and Kaplan-Meier plotter. AGS and HGC27 cells were used to establish an in vitro STAD cell model. The results showed that higher CDH6 expression was associated with significantly shorter overall survival in patients with STAD. In addition, CDH6 overexpression promoted wound healing, enhanced the invasion ability of tumor cells and increased mitochondrial fission. Glioma-associated oncogene family zinc finger 2 (GLI2) could bind to the CDH6 promoter and activate its transcription. Fluorescent labeling also showed that GLI2 overexpression promoted mitochondrial fission. However, CDH6 silencing significantly reduced mitochondrial fragmentation. Besides, GLI2 overexpression notably upregulated phosphorylated-focal adhesion kinase and dynamin-related protein 1. However, the above effects were largely abrogated by CDH6 knockdown. In conclusion, the present study suggested that the novel GLI2/CDH6 axis could enhance the migration, invasion and mitochondrial fission of STAD cells.

Circular RNA circ-FIRRE interacts with HNRNPC to promote esophageal squamous cell carcinoma progression by stabilizing GLI2 mRNA.

Increasing evidence has shown that circular RNAs (circRNAs) interact with RNA-binding proteins (RBPs) and promote cancer progression. However, the function and mechanism of the circRNA/RBP complex in esophageal squamous cell carcinoma (ESCC) are still largely unknown. Herein, we first characterized a novel oncogenic circRNA, circ-FIRRE, by RNA sequencing (Ribo-free) profiling of ESCC samples. Furthermore, we observed marked circ-FIRRE overexpression in ESCC patients with high TNM stage and poor overall survival. Mechanistic studies indicated that circ-FIRRE, as a platform, interacts with the heterogeneous nuclear ribonucleoprotein C (HNRNPC) protein to stabilize GLI2 mRNA by directly binding to its 3'-UTR in the cytoplasm, thereby resulting in elevated GLI2 protein expression and subsequent transcription of its target genes MYC, CCNE1, and CCNE2, ultimately contributing to ESCC progression. Moreover, HNRNPC overexpression in circ-FIRRE knockdown cells notably abolished circ-FIRRE knockdown-mediated Hedgehog pathway inhibition and ESCC progression impairment in vitro and in vivo. Clinical specimen results showed that circ-FIRRE and HNRNPC expression was positively correlated with GLI2 expression, which reveals the clear significance of the circ-FIRRE/HNRNPC-GLI2 axis in ESCC. In summary, our results indicate that circ-FIRRE could serve as a valuable biomarker and potential therapeutic target for ESCC and highlight a novel mechanism of the circ-FIRRE/HNRNPC complex in ESCC progression regulation.

The effects of Gli1 and Gli2 on BMP9-induced osteogenic differentiation of mesenchymal stem cells.

Diseases, such as bone nonunion with bone defects, osteoporosis, etc, seriously endanger people's quality of life, and bone tissue engineering based on mesenchymal stem cells is an effective method to solve such problems. Several studies have shown that BMP9 can effectively promote osteogenic differentiation of MSCs, but the underlying molecular mechanisms are still unclear. Gli1 and Gli2 were important transcription factors and play an important role in the Hedgehog signaling pathway. In this study, we investigated the role of Gli1 and Gli2 in BMP9-induced osteogenic differentiation of MSCs. We found that inhibition of Gli1 and Gli2 weakened BMP9-induced osteogenic differentiation of MSCs, and early osteogenic markers (alkaline phosphatase, ALP), late osteogenic markers (calcium salt deposition), the expression of pivotal osteogenic markers were attenuated, and inhibition of Gli1 and Gli2 weakened the expression of p-Smad1/5/8 and p-p38 induced by BMP9. In conclusion, our study shows that Gli1 and Gli2 play an important role in BMP9-induced osteogenic differentiation.

Targeting GLI1 and GLI2 with small molecule inhibitors to suppress GLI-dependent transcription and tumor growth.

Aberrant activation of Hedgehog (HH) signaling in cancer is the result of genetic alterations of upstream pathway components (canonical) or other oncogenic mechanisms (noncanonical), that ultimately concur to activate the zinc-finger transcription factors GLI1 and GLI2. Therefore, inhibition of GLI activity is a good therapeutic option to suppress both canonical and noncanonical activation of the HH pathway. However, only a few GLI inhibitors are available, and none of them have the profile required for clinical development due to poor metabolic stability and aqueous solubility, and high hydrophobicity. Two promising quinoline inhibitors of GLI were selected by virtual screening and subjected to hit-to-lead optimization, thus leading to the identification of the 4-methoxy-8-hydroxyquinoline derivative JC19. This molecule impaired GLI1 and GLI2 activities in several cellular models interfering with the binding of GLI1 and GLI2 to DNA. JC19 suppressed cancer cell proliferation by enhancing apoptosis, inducing a strong anti-tumor response in several cancer cell lines in vitro. Specificity towards GLI1 and GLI2 was demonstrated by lower activity of JC19 in GLI1- or GLI2-depleted cancer cells. JC19 showed excellent metabolic stability and high passive permeability. Notably, JC19 inhibited GLI1-dependent melanoma xenograft growth in vivo, with no evidence of toxic effects in mice. These results highlight the potential of JC19 as a novel anti-cancer agent targeting GLI1 and GLI2.

CEP164-GLI2 association ensures the hedgehog signaling in pancreatic cancer cells.

Hedgehog (Hh) signaling is involved in multiple biological events including development and cancers. It is processed through primary cilia, which are assembled from the mother centriole in most mammalian cells. Pancreatic ductal adenocarcinoma (PDAC) cells generally lose their primary cilia; thus, the Hh signaling pathway is postulated to be independent of the organelle in PDAC. We previously reported that the mother centriole-specific protein, centrosomal protein 164 (CEP164), is required for centriolar localization of the GLI2 transcription factor in Hh signaling and for suppressing the expression of Hh-target genes. In this study, we demonstrated the physical interaction between CEP164 and GLI2, and delineated their binding modes at the mother centriole. The ectopically expressed GLI2-binding region of CEP164 reduced the centriolar GLI2 localization and enhanced the expression of Hh-target genes in PDAC cells. Furthermore, similar phenotypes were observed in PDAC cells lacking primary cilia. These results suggest that the CEP164-GLI2 association at the mother centriole is responsible for controlling Hh signaling, independent of primary cilia in PDAC cells.

The Role of Hedgehog Signaling in the Melanoma Tumor Bone Microenvironment.

A crucial regulator in melanoma progression and treatment resistance is tumor microenvironments, and Hedgehog (Hh) signals activated in a tumor bone microenvironment are a potential new therapeutic target. The mechanism of bone destruction by melanomas involving Hh/Gli signaling in such a tumor microenvironment is unknown. Here, we analyzed surgically resected oral malignant melanoma specimens and observed that Sonic Hedgehog, Gli1, and Gli2 were highly expressed in tumor cells, vasculatures, and osteoclasts. We established a tumor bone destruction mouse model by inoculating B16 cells into the bone marrow space of the right tibial metaphysis of 5-week-old female C57BL mice. An intraperitoneal administration of GANT61 (40 mg/kg), a small-molecule inhibitor of Gli1 and Gli2, resulted in significant inhibition of cortical bone destruction, TRAP-positive osteoclasts within the cortical bone, and endomucin-positive tumor vessels. The gene set enrichment analysis suggested that genes involved in apoptosis, angiogenesis, and the PD-L1 expression pathway in cancer were significantly altered by the GANT61 treatment. A flow cytometry analysis revealed that PD-L1 expression was significantly decreased in cells in which late apoptosis was induced by the GANT61 treatment. These results suggest that molecular targeting of Gli1 and Gli2 may release immunosuppression of the tumor bone microenvironment through normalization of abnormal angiogenesis and bone remodeling in advanced melanoma with jaw bone invasion.

MRCKα/ß positively regulates Gli protein activity.

Posttranslational modifications (PTMs) are key regulatory events for the majority of signaling pathways. Transcription factors are often phosphorylated on multiple residues, which regulates their trafficking, stability, or transcriptional activity. Gli proteins, transcription factors that respond to the Hedgehog pathway, are regulated by phosphorylation, but the sites and the kinases involved have been only partially described. We identified three novel kinases: MRCKα, MRCKß, and MAP4K5 which physically interact with Gli proteins and directly phosphorylate Gli2 on multiple sites. We established that MRCKα/ß kinases regulate Gli proteins, which impacts the transcriptional output of the Hedgehog pathway. We showed that double knockout of MRCKα/ß affects Gli2 ciliary and nuclear localization and reduces Gli2 binding to the Gli1 promoter. Our research fills a critical gap in our understanding of the regulation of Gli proteins by describing their activation mechanisms through phosphorylation.

Case report: A case of Culler-Jones syndrome caused by a novel mutation of GLI2 gene and literature review.

Culler-Jones syndrome is a rare clinical phenomenon with diverse manifestations and is prone to misdiagnosis. We report one patient who presented with a 10-year history of anosmia and a 1-year history of epididymal pain. Kallmann syndrome was suspected initially. The results of his laboratory tests, imaging, and genetic testing, however, combined to provide a conclusive diagnosis of Culler-Jones syndrome. With the aid of high-throughput sequencing technology, the GLI2 gene c.527A>G (p.Tyr176Cys) heterozygous mutation in the child was identified. No published works have yet described this mutation site. We described Culler-Jones syndrome in a child at length. We recommend that Culler-Jones syndrome be taken into account when considering the spectrum of disorders associated with abnormal growth and development in children. Once diagnosed, individualized hormone replacement treatment is required for each patient.

Glioma-Associated Oncogene Family Zinc Finger 2 (GLI2) Activates Wnt Signaling through Transcriptional Inhibition of Neuronal Precursor Cell-Expressed Developmentally Downregulated 4 (NEDD4L) to Promote Androgen-Induced Granulosa Cell Damage.

OBJECTIVE: Being a prevalent endocrine and metabolic disease, polycystic ovary syndrome (PCOS) severely threatens women's physical and mental health. Glioma-associated oncogene family zinc finger 2 (GLI2) expression is up-regulated in granulosa cells of PCOS patients, but its specific role in PCOS remains unclear. METHODS: Following the treatment of human ovarian granulosa cells (KGN) with dihydrotestosterone (DHT), RT-qPCR and western blot were utilized to check GLI2 expression. After GLI2 expression was silenced, cell activity was detected through CCK8 and apoptosis was examined via TUNEL and western blot. Inflammation and oxidative stress were tested utilizing ELISA and western blot. The binding between GLI2 and neuronal precursor cell-expressed developmentally downregulated 4 (NEDD4L) promoter was predicted by JASPAR database and verified by luciferase reporter and ChIP assay. In addition, RT-qPCR and western blot were applied to check the mRNA and protein expressions of NEDD4L. Following the knockdown of NEDD4L in GLI2-silencing cells, CCK8 assay, TUNEL assay, western blot, ELISA and other methods were performed again. Finally, western blot detected the expressions of Wnt pathway-related proteins. RESULTS: GLI2 was up-regulated in DHT-treated KGN cells. Interference with GLI2 increased the viability, decreased the apoptosis, and inhibited the inflammatory response and oxidative stress of DHT-induced KGN cells. GLI2 could bind to NEDD4L promoter and transcriptionally suppress NEDD4L expression. Further experiments testified that NEDD4L depletion reversed the impacts of GLI2 deficiency on the viability, apoptosis, inflammation, oxidative stress and Wnt signaling pathway in DHT-challenged KGN cells. CONCLUSION: GLI2 activated Wnt signaling to promote androgen-induced granulosa cell damage through transcriptional inhibition of NEDD4L.

[Clinical and genetic analysis of a child with Culler-Jones syndrome due to variant of GLI2 gene].

OBJECTIVE: To explore the genetic basis for a child featuring short stature and postaxial polydactyly. METHODS: A child who presented at Ningbo Women & Children's Hospital in May 2021 due to the"discovery of growth retardation for more than two years" was selected as the subject. Peripheral blood samples of the child and his parents were collected for the extraction of genomic DNA. Whole exome sequencing was carried out for the child, and candidate variant was verified by Sanger sequencing of his family members. RESULTS: The child was found to harbor a heterozygous c.3670C>T (p.Q1224) variant of the GLI2 gene, which may lead to premature termination of protein translation. The variant was not detected in either parent. CONCLUSION: The child was diagnosed with Culler-Jones syndrome. The c.3670C>T (p.Q1224*) variant of the GLI2 gene probably underlay the disease in this child.

Reciprocal FGF19-GLI2 signaling induces epithelial-to-mesenchymal transition to promote lung squamous cell carcinoma metastasis.

PURPOSE: Metastatic lung squamous cell carcinoma (LUSC) is one of the most common causes of cancer death worldwide. As yet, however, the molecular mechanism underlying LUSC metastasis remains elusive. In this study, we report a novel mechanism involving signaling interactions between FGF19 and GLI2 that could drive the progression of LUSC. METHODS: The expression of FGF19 in human LUSC samples was assessed by immunohistochemistry. The concentration of FGF19 in serum samples was assessed by ELISA. RNA sequencing, scratch wound-healing, trans-well, GO analysis, GSEA, luciferase reporter, Western blotting, immunofluorescence and immunohistochemistry assays, as well as an animal model were used to investigate the molecular mechanism underlying FGF19 driven LUSC progression. The therapeutic effect of a GLI2 inhibitor was determined using both in vitro cellular and in vivo animal experiments. RESULTS: We found that FGF19, a member of the fibroblast growth factor family, plays a crucial role in the invasion and metastasis of LUSC, and identified GLI2 as an important downstream effector of FGF19 involved in metastasis. Surprisingly, we found that FGF19 and GLI2 could reciprocally induce the expression of each other, and form a positive feedback loop to promote LUSC cell invasion and metastasis. These findings were corroborated by an association between a poor prognosis of LUSC patients and FGF19/GLI2 co-expression. In addition, we found that the GLI inhibitor GANT61 could effectively reduce FGF19-mediated LUSC invasion and metastasis. CONCLUSION: Our data suggest that FGF19 may serve as a novel biomarker for predicting metastatic LUSC. Intervening with the FGF19-GLI2 feedback loop may be a strategy for the treatment of FGF19-driven LUSC metastasis.

A study on etiology of incontinence in double knockout mouse model.

INTRODUCTION AND OBJECTIVE: Stress urinary incontinence is of concern in both pediatric and adult population. Double mutant GLI family zinc finger Gli2+/-; Gli3Δ699/+ murine model of stress incontinence has been recently developed as a reliable model which does not require surgical manipulation to create incontinence and is shown to survive to adulthood. The aim of this study was to establish the etiology of incontinence in the double mutant Gli2+/-; Gli3Δ699/+ mice. STUDY DESIGN: We used 13 cluster of differentiation 1 (CD-1) mice (7-9 weeks) for demonstration of histology of the bladder and urethra. There were 3 Wild Gli2+/- females, 2 Wild Gli2+/- males, 4 Gli2+/-;Gli3Δ699/+ females and 4 Gli2+/-;Gli3Δ699/+ males. The Wild Gli2+/- mice served as the control group and Gli2+/-;Gli3Δ699/+ mice served as the test group. Additionally, eight 16.5 days mice (2 each of Wild Gli2+/- females, Wild Gli2+/- males, double knockout (DKO) Gli2+/-;Gli3Δ699/+ females and Gli2+/-;Gli3Δ699/+ males) were used to assess the histology of the spinal cord. The gross appearance of bladder and urethra was studied using ink injection assays. Immunohistochemistry was done for smooth muscle actin and cytokeratin. RESULTS: Gross and histologic appearance confirmed the previously reported widening of bladder outlet and hypoplasia of smooth muscles in female urethra and also established them in the male urethra of Gli2+/-;Gli3Δ699/+ mice compared to Gli2+/- mice. The double knockout mice were smaller than the Gli2 mice (5.2 vs 6.1 cm, p = 0.002). Immunohistochemistry demonstrated epithelial hyperplasia and smooth muscle hypoplasia. Additionally, there was prostatic hypoplasia in the Gli2+/-;Gli3Δ699/+ male mice. The spinal cord length for body size appeared comparable between the Gli2+/- and Gli2+/-;Gli3Δ699/+ mice but histological evaluation revealed abnormal development of the caudal end of the vertebral body with premature termination of the spinal cord (Figure). DISCUSSION: The histological changes in the bladder neck and urethra were consistent to those previously reported. While previous report described the findings in female mice only, we confirmed that these findings are also present in males as well as prostatic hypoplasia, a possible additional factor leading to stress incontinence. The most important finding in the present study however, was the detection of premature termination of spinal cord in the DKO Gli2+/-; Gli3Δ699/+ mice which has not been reported previously and is likely a major contributor to incontinence in this model. CONCLUSION: The incontinence in male as well as female Gli2+/-; Gli3Δ699/+ mice is due to both myogenic and neurogenic involvement. These double knockout mice are a valuable model of stress incontinence related to neurogenic bladder due to low outlet resistance.