Pleiotropy in FOXC1-attributable phenotypes involves altered ciliation and cilia-dependent signaling.

Alterations to cilia are responsible for a wide range of severe disease; however, understanding of the transcriptional control of ciliogenesis remains incomplete. In this study we investigated whether altered cilia-mediated signaling contributes to the pleiotropic phenotypes caused by the Forkhead transcription factor FOXC1. Here, we show that patients with FOXC1-attributable Axenfeld-Rieger Syndrome (ARS) have a prevalence of ciliopathy-associated phenotypes comparable to syndromic ciliopathies. We demonstrate that altering the level of Foxc1 protein, via shRNA mediated inhibition, CRISPR/Cas9 mutagenesis and overexpression, modifies cilia length in vitro. These structural changes were associated with substantially perturbed cilia-dependent signaling [Hedgehog (Hh) and PDGFRα], and altered ciliary compartmentalization of the Hh pathway transcription factor, Gli2. Consistent with these data, in primary cultures of murine embryonic meninges, cilia length was significantly reduced in heterozygous and homozygous Foxc1 mutants compared to controls. Meningeal expression of the core Hh signaling components Gli1, Gli3 and Sufu was dysregulated, with comparable dysregulation of Pdgfrα signaling evident from significantly altered Pdgfrα and phosphorylated Pdgfrα expression. On the basis of these clinical and experimental findings, we propose a model that altered cilia-mediated signaling contributes to some FOXC1-induced phenotypes.

mTORC1 hampers Hedgehog signaling in Tsc2 deficient cells.

The mTORC1-complex is negatively regulated by TSC1 and TSC2. Activation of Hedgehog signaling is strictly dependent on communication between Smoothened and the Hedgehog-signaling effector and transcription factor, GLI2, in the primary cilium. Details about this communication are not known, and we wanted to explore this further. Here we report that in Tsc2 -/- MEFs constitutively activated mTORC1 led to mis-localization of Smoothened to the plasma membrane, combined with increased concentration of GLI2 in the cilia and reduced Hedgehog signaling, measured by reduced expression of the Hedgehog target gene, Gli1 Inhibition of mTORC1 rescued the cellular localization of Smoothened to the cilia, reduced the cilia concentration of GLI2, and restored Hedgehog signaling. Our results reveal evidence for a two-step activation process of GLI2. The first step includes GLI2 stabilization and cilium localization, whereas the second step includes communication with cilia-localized Smoothened. We found that mTORC1 inhibits the second step. This is the first demonstration that mTORC1 is involved in the regulation of Hedgehog signaling.

PP2A phosphatase regulatory subunit PPP2R3C is a new positive regulator of the hedgehog signaling pathway.

Cellular signaling pathways rely on posttranslational modifications (PTMs) to finely regulate protein functions, particularly transcription factors. The Hedgehog (Hh) signaling cascade, crucial for embryonic development and tissue homeostasis, is susceptible to aberrations that lead to developmental anomalies and various cancers. At the core of Hh signaling are Gli proteins, whose dynamic balance between activator (GliA) and repressor (GliR) states shapes cellular outcomes. Phosphorylation, orchestrated by multiple kinases, is pivotal in regulating Gli activity. While kinases in this context have been extensively studied, the role of protein phosphatases, particularly Protein Phosphatase 2A (PP2A), remains less explored. This study unveils a novel role for the B″gamma subunit of PP2A, PPP2R3C, in Hh signaling regulation. PPP2R3C interacts with Gli proteins, and its disruption reduces Hedgehog pathway activity as measured by reduced expression of Gli1/2 and Hh target genes upon Hh signaling activation, and reduced growth of a Hh signaling-dependent medulloblastoma cell line. Moreover, we establish an antagonistic connection between PPP2R3C and MEKK1 kinase in Gli protein phosphorylation, underscoring the intricate interplay between kinases and phosphatases in Hh signaling pathway. This study sheds light on the previously understudied role of protein phosphatases in Hh signaling and provides insights into their significance in cellular regulation.

Tumor-Associated Macrophage-Derived TGF-ß1 Activates GLI2 via the Smad2/3 Signaling Pathway to Affect Cisplatin Resistance in Lung Adenocarcinoma.

BACKGROUND: Transforming growth factor-ß1 (TGF-ß1) is an immunosuppressive cytokine that is highly expressed in the tumor microenvironment (TME) of lung adenocarcinoma (LUAD). TGF-ß1 plays important roles in regulating tumor metastasis and chemotherapy resistance. However, the specific molecular mechanisms by which TGF-ß1 regulates cisplatin resistance in the TAM of LUAD remain unclear. MATERIALS AND METHODS: THP-1 induced macrophages were co-cultured with A549 and H1975 cells, and subsequently transfected with silencing TGF-ß1 (siTGF-ß1), GLI2 (siGLI2), a GLI2 overexpression plasmid, and their negative controls. Cellular activity was measured by CCK-8 and colony formation assays. Cell apoptosis was evaluated by flow cytometry and TUNEL staining. Transwell assays were performed to assess cell migration and invasion capabilities. The levels of Smad2/3, GLI2, cyclin D, and cyclin E expression were evaluated by qPCR, western blotting, and immunofluorescence methods. TGF-ß1 levels were determined by ELISA. RESULTS: Macrophages suppressed the apoptosis and promoted the migration and invasion of LUAD cells. TAM siTGF-ß1 downregulated the Smad2/3 signaling pathways and GLI2 expression, deceased cell proliferation, and promoted apoptosis. SiGLI2 increased apoptosis and decreased the proliferation of LUAD cell lines. GLI2 decreased cisplatin resistance in LUAD cells. CONCLUSION: High expression of TGF-ß1 in the TAM positively activates GLI2 expression via the Smad2/3 pathway, which subsequently regulates cyclin D and cyclin E expression, and promotes the cisplatin resistance of LUAD.

Hedgehog components are overexpressed in a series of liver cancer cases.

Liver cancers, including hepatocellular carcinoma (HCC), are the sixth most common cancer and the third leading cause of cancer-related death worldwide, representing a global public health problem. This study evaluated nine patients with HCC. Six of the cases involved hepatic explants, and three involved hepatic segmentectomy for tumor resection. Eight out of nine tumors were HCC, with one being a combined hepatocellular-cholangiocarcinoma tumor. Conventional markers of hepatocellular differentiation (Hep Par-1, arginase, pCEA, and glutamine synthetase) were positive in all patients, while markers of hepatic precursor cells (CK19, CK7, EpCAM, and CD56) were negative in most patients, and when positive, they were detected in small, isolated foci. Based on in silico analysis of HCC tumors from The Cancer Genome Atlas database, we found that Hedgehog (HH) pathway components (GLI1, GLI2, GLI3 and GAS1) have high connectivity values (module membership > 0.7) and are strongly correlated with each other and with other genes in biologically relevant modules for HCC. We further validated this finding by analyzing the gene expression of HH components (PTCH1, GLI1, GLI2 and GLI3) in our samples through qPCR, as well as by immunohistochemical analysis. Additionally, we conducted a chemosensitivity analysis using primary HCC cultures treated with a panel of 18 drugs that affect the HH pathway and/or HCC. Most HCC samples were sensitive to sunitinib. Our results offer a comprehensive view of the molecular landscape of HCC, highlighting the significance of the HH pathway and providing insight into focused treatments for HCC.

Positive regulation of Hedgehog signaling via phosphorylation of GLI2/GLI3 by DYRK2 kinase.

Hedgehog (Hh) signaling, an evolutionarily conserved pathway, plays an essential role in development and tumorigenesis, making it a promising drug target. Multiple negative regulators are known to govern Hh signaling; however, how activated Smoothened (SMO) participates in the activation of downstream GLI2 and GLI3 remains unclear. Herein, we identified the ciliary kinase DYRK2 as a positive regulator of the GLI2 and GLI3 transcription factors for Hh signaling. Transcriptome and interactome analyses demonstrated that DYRK2 phosphorylates GLI2 and GLI3 on evolutionarily conserved serine residues at the ciliary base, in response to activation of the Hh pathway. This phosphorylation induces the dissociation of GLI2/GLI3 from suppressor, SUFU, and their translocation into the nucleus. Loss of Dyrk2 in mice causes skeletal malformation, but neural tube development remains normal. Notably, DYRK2-mediated phosphorylation orchestrates limb development by controlling cell proliferation. Taken together, the ciliary kinase DYRK2 governs the activation of Hh signaling through the regulation of two processes: phosphorylation of GLI2 and GLI3 downstream of SMO and cilia formation. Thus, our findings of a unique regulatory mechanism of Hh signaling expand understanding of the control of Hh-associated diseases.

CPLANE protein INTU regulates growth and patterning of the mouse lungs through cilia-dependent Hh signaling.

Congenital lung malformations are fatal at birth in their severe forms. Prevention and early intervention of these birth defects require a comprehensive understanding of the molecular mechanisms of lung development. We find that the loss of inturned (Intu), a cilia and planar polarity effector gene, severely disrupts growth and branching morphogenesis of the mouse embryonic lungs. Consistent with our previous results indicating an important role for Intu in ciliogenesis and hedgehog (Hh) signaling, we find greatly reduced number of primary cilia in both the epithelial and mesenchymal tissues of the lungs. We also find significantly reduced expression of Gli1 and Ptch1, direct targets of Hh signaling, suggesting disruption of cilia-dependent Hh signaling in Intu mutant lungs. An agonist of the Hh pathway activator, smoothened, increases Hh target gene expression and tubulogenesis in explanted wild type, but not Intu mutant, lungs, suggesting impaired Hh signaling response underlying lung morphogenetic defects in Intu mutants. Furthermore, removing both Gli2 and Intu completely abolishes branching morphogenesis of the lung, strongly supporting a mechanism by which Intu regulates lung growth and patterning through cilia-dependent Hh signaling. Moreover, a transcriptomics analysis identifies around 200 differentially expressed genes (DEGs) in Intu mutant lungs, including known Hh target genes Gli1, Ptch1/2 and Hhip. Genes involved in muscle differentiation and function are highly enriched among the DEGs, consistent with an important role of Hh signaling in airway smooth muscle differentiation. In addition, we find that the difference in gene expression between the left and right lungs diminishes in Intu mutants, suggesting an important role of Intu in asymmetrical growth and patterning of the mouse lungs.

The anti-tumor activity of tangeretin in esophageal squamous cell carcinoma by inhibiting GLI2-mediated transcription of GPNMB.

Tangeretin (Tan), a citrus flavonoid, possesses a strong anti-tumor efficacy in various human cancers. However, the precise role of Tan in the development of esophageal squamous cell carcinoma (ESCC) remains unclear. RNA sequencing (RNA-seq) analysis was performed to observe the Tan-related genes in Tan-treated TE-1 cells. The direct relationship between GLI family zinc finger 2 (GLI2) and the promoter of glycoprotein non-metastatic melanoma protein B (GPNMB) was predicted by bioinformatics analysis and validated by luciferase reporter and chromatin immunoprecipitation (ChIP) assays. Cell survival after Tan treatment was assessed by CCK8 assay. Gene expression levels were evaluated by a qRT-PCR, western blot, or immunofluorescence method. Cell migration and invasion were detected by wound-healing and transwell assays. The function of Tan in vivo was examined using xenograft studies. Our data indicated anti-migration and anti-invasion functions of Tan in ESCC cells in vitro. Tan also diminished tumor growth in vivo. Mechanistically, Tan diminished the expression and transcriptional activity of GLI2 in ESCC cells. Silencing of GLI2 resulted in decreased expression of GPNMB by inhibiting GPNMB transcription via the binding site at the GPNMB promoter at position +(1539-1550). Moreover, Tan down-regulated GPNMB expression in ESCC cells, and re-expression of GPNMB reversed anti-migration and anti-invasion functions of Tan in ESCC cells. Our findings uncover anti-migration and anti-invasion effects of Tan in ESCC cells by down-regulating GPNMB by suppressing GLI2-mediated GPNMB transcription, providing new evidence that Tan can function as a therapeutic agent against ESCC.

KRAS Promotes GLI2-Dependent Transcription during Pancreatic Carcinogenesis.

Aberrant activation of GLI transcription factors has been implicated in the pathogenesis of different tumor types including pancreatic ductal adenocarcinoma. However, the mechanistic link with established drivers of this disease remains in part elusive. In this study, using a new genetically engineered mouse model overexpressing constitutively active mouse form of GLI2 and a combination of genome-wide assays, we provide evidence of a novel mechanism underlying the interplay between KRAS, a major driver of pancreatic ductal adenocarcinoma development, and GLI2 to control oncogenic gene expression. These mice, also expressing KrasG12D, show significantly reduced median survival rate and accelerated tumorigenesis compared with the KrasG12D only expressing mice. Analysis of the mechanism using RNA sequencing demonstrate higher levels of GLI2 targets, particularly tumor growth-promoting genes, including Ccnd1, N-Myc, and Bcl2, in KrasG12D mutant cells. Furthermore, chromatin immunoprecipitation sequencing studies showed that in these cells KrasG12D increases the levels of trimethylation of lysine 4 of the histone 3 (H3K4me3) at the promoter of GLI2 targets without affecting significantly the levels of other major active chromatin marks. Importantly, Gli2 knockdown reduces H3K4me3 enrichment and gene expression induced by mutant Kras. In summary, we demonstrate that Gli2 plays a significant role in pancreatic carcinogenesis by acting as a downstream effector of KrasG12D to control gene expression.

Distinct expression patterns of Hedgehog signaling components in mouse gustatory system during postnatal tongue development and adult homeostasis.

The Hedgehog (HH) pathway regulates embryonic development of anterior tongue taste fungiform papilla (FP) and the posterior circumvallate (CVP) and foliate (FOP) taste papillae. HH signaling also mediates taste organ maintenance and regeneration in adults. However, there are knowledge gaps in HH pathway component expression during postnatal taste organ differentiation and maturation. Importantly, the HH transcriptional effectors GLI1, GLI2 and GLI3 have not been investigated in early postnatal stages; the HH receptors PTCH1, GAS1, CDON and HHIP, required to either drive HH pathway activation or antagonism, also remain unexplored. Using lacZ reporter mouse models, we mapped expression of the HH ligand SHH, HH receptors, and GLI transcription factors in FP, CVP and FOP in early and late postnatal and adult stages. In adults we also studied the soft palate, and the geniculate and trigeminal ganglia, which extend afferent fibers to the anterior tongue. Shh and Gas1 are the only components that were consistently expressed within taste buds of all three papillae and the soft palate. In the first postnatal week, we observed broad expression of HH signaling components in FP and adjacent, non-taste filiform (FILIF) papillae in epithelium or stroma and tongue muscles. Notably, we observed elimination of Gli1 in FILIF and Gas1 in muscles, and downregulation of Ptch1 in lingual epithelium and of Cdon, Gas1 and Hhip in stroma from late postnatal stages. Further, HH receptor expression patterns in CVP and FOP epithelium differed from anterior FP. Among all the components, only known positive regulators of HH signaling, SHH, Ptch1, Gli1 and Gli2, were expressed in the ganglia. Our studies emphasize differential regulation of HH signaling in distinct postnatal developmental periods and in anterior versus posterior taste organs, and lay the foundation for functional studies to understand the roles of numerous HH signaling components in postnatal tongue development.

The HH-GLI2-CKS1B network regulates the proliferation-to-maturation transition of cardiomyocytes.

Cardiomyocyte (CM) proliferation and maturation are highly linked processes, however, the extent to which these processes are controlled by a single signaling axis is unclear. Here, we show the previously undescribed role of Hedgehog (HH)-GLI2-CKS1B cascade in regulation of the toggle between CM proliferation and maturation. Here we show downregulation of GLI-signaling in adult human CM, adult murine CM, and in late-stage hiPSC-CM leading to their maturation. In early-stage hiPSC-CM, inhibition of HH- or GLI-proteins enhanced CM maturation with increased maturation indices, increased calcium handling, and transcriptome. Mechanistically, we identified CKS1B, as a new effector of GLI2 in CMs. GLI2 binds the CKS1B promoter to regulate its expression. CKS1B overexpression in late-stage hiPSC-CMs led to increased proliferation with loss of maturation in CMs. Next, analysis of datasets of patients with heart disease showed a significant enrichment of GLI2-signaling in patients with ischemic heart failure (HF) or dilated-cardiomyopathy (DCM) disease, indicating operational GLI2-signaling in the stressed heart. Thus, the Hh-GLI2-CKS1B axis regulates the proliferation-maturation transition and provides targets to enhance cardiac tissue engineering and regenerative therapies.

A Pilot Immunohistochemical Study Identifies Hedgehog Pathway Expression in Sinonasal Adenocarcinoma.

Tumors of the head and neck, more specifically the squamous cell carcinoma, often show upregulation of the Hedgehog signaling pathway. However, almost nothing is known about its role in the sinonasal adenocarcinoma, either in intestinal or non-intestinal subtypes. In this work, we have analyzed immunohistochemical staining of six Hedgehog pathway proteins, sonic Hedgehog (SHH), Indian Hedgehog (IHH), Patched1 (PTCH1), Gli family zinc finger 1 (GLI1), Gli family zinc finger 2 (GLI2), and Gli family zinc finger 3 (GLI3), on 21 samples of sinonasal adenocarcinoma and compared them with six colon adenocarcinoma and three salivary gland tumors, as well as with matching healthy tissue, where available. We have detected GLI2 and PTCH1 in the majority of samples and also GLI1 in a subset of samples, while GLI3 and the ligands SHH and IHH were generally not detected. PTCH1 pattern of staining shows an interesting pattern, where healthy samples are mostly positive in the stromal compartment, while the signal shifts to the tumor compartment in tumors. This, taken together with a stronger signal of GLI2 in tumors compared to non-tumor tissues, suggests that the Hedgehog pathway is indeed activated in sinonasal adenocarcinoma. As Hedgehog pathway inhibitors are being tested in combination with other therapies for head and neck squamous cell carcinoma, this could provide a therapeutic option for patients with sinonasal adenocarcinoma as well.

Ptch1 is essential for cochlear marginal cell differentiation and stria vascularis formation.

A common cause of deafness in humans is dysregulation of the endocochlear potential generated by the stria vascularis (SV). Thus, proper formation of the SV is critical for hearing. Using single-cell transcriptomics and a series of Shh signaling mutants, we discovered that the Shh receptor Patched1 (Ptch1) is essential for marginal cell (MC) differentiation and SV formation. Single-cell RNA sequencing analyses revealed that the cochlear roof epithelium is already specified into discrete domains with distinctive gene expression profiles at embryonic day 14, with Gsc as a marker gene of the MC lineage. Ptch1 deficiency leads to defective specification of MC precursors along the cochlear basal-apical regions. We demonstrated that elevated Gli2 levels impede MC differentiation through sustaining Otx2 expression and maintaining the progenitor state of MC precursors. Our results uncover an early specification of cochlear non-sensory epithelial cells and establish a crucial role of the Ptch1-Gli2 axis in regulating the development of SV.

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.

Hedgehog pathway negatively regulated depleted uranium-induced nephrotoxicity.

Depleted uranium (DU) retains the radiological toxicities, which accumulates preferentially in the kidneys. Hedgehog (Hh) pathway plays a critical role in tissue injury. However, the role of Hh in DU-induced nephrotoxicity was still unclear. This study was carried out to investigate the effect of Gli2, which was an important transcription effector of Hh signaling, on DU induced nephrotoxicity. To clarify it, CK19 positive tubular epithelial cells specific Gli2 conditional knockout (KO) mice model was exposed to DU, and then histopathological damage and Hh signaling pathway activation was analyzed. Moreover, HEK-293 T cells were exposed to DU with Gant61 or Gli2 overexpression, and cytotoxicity of DU as analyzed. Results showed that DU caused nephrotoxicity accompanied by activation of Hh signaling pathway. Meanwhile, genetic KO of Gli2 reduced DU-induced nephrotoxicity by normalizing biochemical indicators and reducing Hh pathway activation. Pharmacologic inhibition of Gli1/2 by Gant61 reduced DU induced cytotoxicity by inhibiting apoptosis, ROS formation and Hh pathway activation. However, overexpression of Gli2 aggravated DU-induced cytotoxicity by increasing the levels of apoptosis and ROS formation. Taken together, these results revealed that Hh signaling negatively regulated DU-inducted nephrotoxicity, and that inhibition of Gli2 might serve as a promising nephroprotective target for DU-induced kidney injury.

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.

Upregulation of apoptotic genes and downregulation of target genes of Sonic Hedgehog signaling pathway in DAOY medulloblastoma cell line treated with arsenic trioxide.

Sonic hedgehog (SHH) medulloblastoma etiology is associated with the SHH molecular pathway activation at different levels. We investigated the effect of arsenic trioxide as a downstream-level inhibitor of the SHH signaling pathway on morphology, cytotoxicity, migration, and SHH-related and apoptotic gene expression of DAOY cells. Cells were treated at various arsenic trioxide (ATO)concentrations (1, 2, 3, 5, and 10 µM) for different times (24 and 48 hr). Following treatments, the morphology of the cells was investigated at ×20 and ×40 magnification by an inverted microscope. Then, cytotoxicity was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue assays. Cell migration was analyzed through the wound-healing assay. Furthermore, the expression of SHH-related (GLI1, GLI2, SMO, and MYCN) and apoptotic genes (BAX, BCL2, and TP53) was assessed by real-time quantitative polymerase chain reaction (qPCR). Finally, GLI1, SMO, and MYCN markers were analyzed through immunocytochemistry. Data were analyzed by SPSS (version 16) and P≤0.05 was considered significant. Morphological changes were seen at 3 and 2 µM in 24 and 48 hr of treatment, respectively. The MTT assay showed a dose-dependent cytotoxicity indicating an IC50 value of 3.39±0.35 and 2.05±0.64 µM in 24 and 48hr treatment, respectively. In addition, the trypan blue assay showed higher IC50 values of 4.29±0.25 and 3.92±0.22 µM in 24 and 48 hr treatment, respectively. The wound-healing assay indicated a dose-dependent reduction of cell migration speed showing a 50% reduction at 2.89±0.26 µM. Significant downregulation of GLI1 and GLI2, as well as the upregulation of BAX, BAX/BCL2 ratio, and TP53 were evident. Significant increases in GLI1 and MYCN markers were also evident in immunocytochemistry. ATO, as a downstream effective inhibitor of the SHH pathway, substantially leads to cell death, cell migration inhibition, apoptosis upregulation, and downregulation of SHH target genes in DAOY medulloblastoma. Since ATO is a toxic chemotherapeutic agent, it must be used at low concentrations (2 µM) in order not to damage healthy cells.

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.