Gli Transcription Factors Research Articles

Hedgehog signaling activates a mammalian heterochronic gene regulatory network controlling differentiation timing across lineages.

Many developmental signaling pathways have been implicated in lineage-specific differentiation; however, mechanisms that explicitly control differentiation timing remain poorly defined in mammals. We report that murine Hedgehog signaling is a heterochronic pathway that determines the timing of progenitor differentiation. Hedgehog activity was necessary to prevent premature differentiation of second heart field (SHF) cardiacprogenitors in mouse embryos, and the Hedgehog transcription factor GLI1 was sufficient to delay differentiation of cardiac progenitors invitro. GLI1 directly activated a de novo progenitor-specific network invitro, akin to that of SHF progenitors invivo, which prevented the onset of the cardiac differentiation program. A Hedgehog signaling-dependent active-to-repressive GLI transition functioned as a differentiation timer, restricting the progenitor network to the SHF. GLI1 expression was associated with progenitor status across germ layers, and it delayed the differentiation of neural progenitors invitro, suggesting a broad role for Hedgehog signaling as a heterochronic pathway.

Canonical Hedgehog Pathway and Noncanonical GLI Transcription Factor Activation in Cancer.

The Hedgehog signaling pathway is one of the fundamental pathways required for development and regulation of postnatal regeneration in a variety of tissues. The pathway has also been associated with cancers since the identification of a mutation in one of its components, PTCH, as the cause of Basal Cell Nevus Syndrome, which is associated with several cancers. Our understanding of the pathway in tumorigenesis has expanded greatly since that initial discovery over two decades ago. The pathway has tumor-suppressive and oncogenic functions depending on the context of the cancer. Furthermore, noncanonical activation of GLI transcription factors has been reported in a number of tumor types. Here, we review the roles of canonical Hedgehog signaling pathway and noncanonical GLI activation in cancers, particularly epithelial cancers, and discuss an emerging concept of the distinct outcomes that these modes have on cancer initiation and progression.

A novel mechanism of regulation of the oncogenic transcription factor GLI3 by toll-like receptor signaling.

The transcription factor GLI3 is a member of the GLI family and has been shown to be regulated by canonical hedgehog (HH) signaling through smoothened (SMO). Little is known about SMO-independent regulation of GLI3. Here, we identify TLR signaling as a novel pathway regulating GLI3 expression. We show that GLI3 expression is induced by LPS/TLR4 in human monocyte cell lines and peripheral blood CD14+ cells. Further analysis identified TRIF, but not MyD88, signaling as the adapter used by TLR4 to regulate GLI3. Using pharmacological and genetic tools, we identified IRF3 as the transcription factor regulating GLI3 downstream of TRIF. Furthermore, using additional TLR ligands that signal through TRIF such as the TLR4 ligand, MPLA and the TLR3 ligand, Poly(I:C), we confirm the role of TRIF-IRF3 in the regulation of GLI3. We found that IRF3 directly binds to the GLI3 promoter region and this binding was increased upon stimulation of TRIF-IRF3 with Poly(I:C). Furthermore, using Irf3−/− MEFs, we found that Poly(I:C) stimulation no longer induced GLI3 expression. Finally, using macrophages from mice lacking Gli3 expression in myeloid cells (M-Gli3−/−), we found that in the absence of Gli3, LPS stimulated macrophages secrete less CCL2 and TNF-α compared with macrophages from wild-type (WT) mice. Taken together, these results identify a novel TLR-TRIF-IRF3 pathway that regulates the expression of GLI3 that regulates inflammatory cytokines and expands our understanding of the non-canonical signaling pathways involved in the regulation of GLI transcription factors.

100 Novel chromatin-associated activity and substrates for the polarity kinase aPKC in BCC drug resistance

Basal cell carcinomas (BCCs) are GLI transcription factor dependent and one of the most common and accessible human tumors making them ideal for studying the molecular and cellular basis of tumor evolution and resistance. We previously identified a GLI nuclear resistance mechanism involving the interaction between polarity kinase atypical PKC (aPKC) and the deacetylase HDAC1. While we showed that HDAC1-dependent GLI deacetylation drives GLI away from the nuclear envelope to chromatin through the LAP2 isoform-dependent Gli1 nuclear chaperoning system, how nuclear aPKC accumulates and functions remains mysterious.

115 The impact of aging on murine basal cell carcinoma development

Basal cell carcinoma (BCC) is the most common skin malignancy and most frequently occurs in patients over 70 years of age. Dysregulated Hedgehog (Hh) signaling and consequent activation of GLI transcription factors is a molecular hallmark of human BCC and drives BCC-like tumor development in genetically engineered mice, which are typically studied at a young age. We examined the impact of aging on BCC tumorigenesis in young versus aged cohorts of Lgr6-CreER;R26-LSL-rtTA;tetO-GLI2A mice, in which topical 4-OHT treatment activates Cre function and rtTA expression in Lgr6+ cells and their progeny, and treatment with doxycycline leads to expression of a tetO-driven activator form of GLI2.

ERK2 MAP kinase regulates SUFU binding by multisite phosphorylation of GLI1.

Crosstalk between the Hedgehog and MAPK signaling pathways occurs in several types of cancer and contributes to clinical resistance to Hedgehog pathway inhibitors. Here we show that MAP kinase-mediated phosphorylation weakens the binding of the GLI1 transcription factor to its negative regulator SUFU. ERK2 phosphorylates GLI1 on three evolutionarily conserved target sites (S102, S116, and S130) located near the high-affinity binding site for SUFU; these phosphorylations cooperate to weaken the affinity of GLI1-SUFU binding by over 25-fold. Phosphorylation of any one, or even any two, of the three sites does not result in the level of SUFU release seen when all three sites are phosphorylated. Tumor-derived mutations in R100 and S105, residues bordering S102, also diminish SUFU binding, collectively defining a novel evolutionarily conserved SUFU affinity-modulating region. In cultured mammalian cells, GLI1 variants containing phosphomimetic substitutions of S102, S116, and S130 displayed an increased ability to drive transcription. We conclude that multisite phosphorylation of GLI1 by ERK2 or other MAP kinases weakens GLI1-SUFU binding, thereby facilitating GLI1 activation and contributing to both physiological and pathological crosstalk.

Cytoskeletal regulation of a transcription factor by DNA mimicry via coiled-coil interactions.

A long-established strategy for transcription regulation is the tethering of transcription factors to cellular membranes. By contrast, the principal effectors of Hedgehog signalling, the GLI transcription factors, are regulated by microtubules in the primary cilium and the cytoplasm. How GLI is tethered to microtubules remains unclear. Here, we uncover DNA mimicry by the ciliary kinesin KIF7 as a mechanism for the recruitment of GLI to microtubules, wherein the coiled-coil dimerization domain of KIF7, characterized by its striking shape, size and charge similarity to DNA, forms a complex with the DNA-binding zinc fingers in GLI, thus revealing a mode of tethering a DNA-binding protein to the cytoskeleton. GLI increases KIF7 microtubule affinity and consequently modulates the localization of both proteins to microtubules and the cilium tip. Thus, the kinesin-microtubule system is not a passive GLI tether but a regulatable platform tuned by the kinesin-transcription factor interaction. We retooled this coiled-coil-based GLI-KIF7 interaction to inhibit the nuclear and cilium localization of GLI. This strategy can potentially be exploited to downregulate erroneously activated GLI in human cancers.

Sex-specific phenotypes in the aging mouse heart and consequences for chronic fibrosis.

The incidence of diastolic dysfunction increases with age in both humans and mice. This is characterized by increased passive stiffness and slower relaxation of the left ventricle. The stiffness arises at least partially from progressively increased interstitial collagen deposition because of highly secretory fibroblasts. In the past, we demonstrated that AMPK activation via the drug 5-aminoimidazole-4-carboxamide riboside (AICAR) in middle-aged mice reduced adverse remodeling after myocardial infarction. Therefore, as an attempt to normalize the fibroblast phenotype, we used 21-mo-old male and female mice and treated them with AICAR (0.166 mg/g body wt) where each mouse was followed in a functional study over a 3-mo period. We found sex-related differences in extracellular matrix (ECM) composition as well as heart function indices at baseline, which were further accentuated by AICAR treatment. AICAR attenuated the age-related increase in left atrial volume (LAV, an indicator of diastolic dysfunction) in female but not in male hearts, which was associated with reduced collagen deposition in the old female heart, and reduced the transcription factor Gli1 expression in cardiac fibroblasts. We further demonstrated that collagen synthesis was dependent on Gli1, which is a target of AMPK-mediated degradation. By contrast, AICAR had a minor impact on cardiac fibroblasts in the old male heart because of blunted AMPK phosphorylation. Hence, it did not significantly improve old male heart function indices. In conclusion, we demonstrated that male and female hearts are phenotypically different, and sex-specific differences need to be considered when analyzing the response to pharmacological intervention.NEW & NOTEWORTHY The aging heart develops diastolic dysfunction because of increased collagen deposition. We attempted to reduce collagen expression in the old heart by activating AMPK using AICAR. An improvement of diastolic function and reduction of cardiac fibrosis was found only in the female heart and correlated with decreased procollagen expression and increased degradation of the transcription factor Gli1. Male hearts display blunted AICAR-dependent AMPK activation and therefore this treatment had no benefits for the male mice.

PRKCI Mediates Radiosensitivity via the Hedgehog/GLI1 Pathway in Cervical Cancer.

ObjectiveInsensitivity to radiotherapy accounts for the majority of therapeutic failures in cervical cancer (CC) patients who undergo radical radiotherapy. We aimed to elucidate the molecular mechanisms underlying radiosensitivity to identify methods to improve the overall 5-year survival rate. The atypical protein kinase C iota (aPKCι) gene PRKCI exhibits tumor-specific copy number amplification (CNA) in CC. We investigated how PRKCI decreases radiosensitivity in CC and assessed the interplay between PRKCI and the Hedgehog (Hh)/GLI1 pathway in the present research.MethodsThe biological functions of PRKCI in CC radiosensitivity were explored through immunohistochemistry, colony formation, Cell Counting Kit-8 (CCK-8), cell cycle, apoptosis assays, and xenograft models. qRT-PCR, Western blotting analysis, and immunofluorescence assays were utilized to evaluate the interplay between PRKCI and the Hh/GLI1 pathway and its mechanism in PRKCI-decreased radiosensitivity in CC. Furthermore, the effect of auranofin (AF), a selective inhibitor of PKCι, on CC cells was explored through biochemical assays in vitro and in vivo.ResultsWe found that high PRKCI expression was responsible for decreased survival in CC. PRKCI was intimately associated with radiation-triggered alterations in proliferation, the cell cycle, apoptosis, and xenograft growth. The Hh/GLI1 pathway was activated when PRKCI expression was altered. PRKCI functions downstream of the Hh/GLI1 pathway to phosphorylate and activate the transcription factor GLI1. AF acts as a radiosensitizer and showed biological effects in vitro and in vivo.ConclusionsPRKCI is a therapeutic target for regulating radiosensitivity in CC. This molecule regulates radiosensitivity by modulating GLI1 relocalization and phosphorylation in CC via the Hh/GLI1 pathway.

Abstract 2354: Astrocytes activated by tGLI1-expressing breast cancer brain metastases upregulate CNTF to activate astrocytes and promote breast cancer stem cells

Abstract Breast cancer is the most frequently diagnosed cancer, and second most common source of brain metastasis in women. Despite recent advances in detection and therapeutics, patients with breast cancer brain metastases (BCBM) survive only 6-18 months after diagnosis. HER2-enriched breast cancer and triple-negative breast cancer (TNBC) subtypes have the highest propensity to metastasize to the brain. Mechanisms that drive BCBM remain unclear, contributing to limited effective treatments and poor prognoses for HER2-enriched breast cancer and TNBC patients. Truncated glioma-associated oncogene homolog 1 (tGLI1), a gain-of-function GLI1 transcription factor discovered in our lab, promotes BCBM of circulating tumor cells in vivo. We previously reported that tGLI1 enriches the breast cancer stem cell (BCSC) subpopulation, and that tGLI1-positive BCSCs strongly activate astrocytes, the most abundant glial cell type in the brain. The mechanisms by which tGLI1-activated astrocytes promote BCBM have not been investigated. Since tumor-associated astrocytes have been reported to secrete specific cytokines, such as ciliary neurotrophic factor (CNTF), to activate astrocytes and cancer cells by binding to IL-6R and CNTF receptor-alpha (CNTFRα), we determined whether astrocytes activated by tGLI1-positive breast cancer cells secrete elevated levels of CNTF. Results of CNTF ELISA revealed that conditioned media from tGLI1-positive breast cancer cells increases the ability of astrocytes to secrete CNTF compared to GLI1-positive or control cells. Furthermore, we found that exogenous CNTF significantly activates astrocytes and promotes BCSCs, important mediators of tumor progression and metastasis in breast cancer. Analysis of patient datasets reveals CNTFRα is more highly expressed in brain metastases compared to breast cancer and normal breast tissues, suggesting that CNTF secreted by activated astrocytes can promote the growth of BCSCs in the brain. We further found that CNTF and CNTFRα gene signatures are significantly associated with worse brain metastasis-free survival, suggesting an important role for the tGLI1-CNTF-CNTFRα pathway in BCBM development. In summary, our study demonstrates for the first time that astrocytes activated by tGLI1-expressing BCBM secrete CNTF, CNTF further activates astrocytes and promotes BCSCs, and that this tumor-astrocyte interaction could be a novel mechanism for BCBM development and progression. Citation Format: Grace L. Wong, Sherona R. Sirkisoon, Noah R. Aguayo, Daniel L. Doheny, Dongqin Zhu, Angelina T. Regua, Austin Arrigo, Sara G. Manore, Calvin J. Wagner, Alexandra Thomas, Ravi Singh, Fei Xing, Guangxu Jin, Kounosuke Watabe, Hui-Wen Lo. Astrocytes activated by tGLI1-expressing breast cancer brain metastases upregulate CNTF to activate astrocytes and promote breast cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2354.

Expression of tumour transcription factor GLI1 in canine mammary tumours tissue.

BackgroundMammary tumor is one of the most common diseases of canine in pet clinics.ObjectivesThis study investigates the distribution and expression of the tumor transcription factor GLI1 and the downstream proteins, Bmi1 and Sox2, in canine mammary tumors and paracancerous tissues.MethodsCancerous and paracancerous normal mammary tissues were detected using western blotting (WB), and immunohistochemistry.ResultsThe results showed that the histopathology of different types in mammary tumors by microscopic observation. GLI1/Bmi1/Sox2 expression was significantly higher in canine mammary invasive carcinoma than in ductal carcinoma and adjacent normal mammary tissues (p < 0.01). The expression of GLI1 in invasive carcinoma tissues was significantly higher than Bmi1 and Sox2, while Sox2 expression in ductal carcinoma tissues was significantly higher than GLI1 and Bmi1 (p < 0.01). GLI1/Bmi1/Sox2 all showed positive reactions in both mammary tumor and adjacent normal mammary tissues with immunohistochemistry. GLI1 and Sox2 showed strong positive staining in the cytoplasm of invasive mammary carcinoma and ductal carcinoma cells, and weak positive staining in the nuclei. The positive Bmi1 reaction was mainly concentrated in the cytoplasm of invasive carcinoma and ductal carcinoma cells, while the positive reaction on the cell membrane was weak.ConclusionsWe speculate that GLI1 and related proteins play an important role in regulating the proliferation and differentiation of tumors. Therefore, it provides important reference for the pathogenesis and pathogenicity of canine mammary tumor.

Indolactam Dipeptides as Nanomolar Gli Inhibitors.

The Gli transcription factors within the Hedgehog (Hh) signaling pathway play essential roles in human development. However, the reactivation of Gli proteins in adult tissue is tumorigenic and drives the progression of several cancers, including the majority of basal cell carcinomas. Here we describe a novel set of indolactam dipeptides that target protein kinase C (PKC), exploiting the unique capacity of PKC isozymes to act as regulators of Gli. We devised an efficient synthetic route for the indolactam-based natural product (-)-pendolmycin and a series of analogues, and we evaluated these analogues in mechanistically distinct Gli reporter assays. The lead compound from these studies, N-hexylindolactam V, exhibits superior Gli suppression relative to clinical inhibitors and blocks the growth of Gli-dependent basal cell carcinoma cells. More broadly, our structure-activity studies provide inroads for the development of novel Gli antagonists and new avenues for combating Gli-driven cancers.

Acro-callous syndrome: A case report.

A malformative syndrome of unknown prevalence, the diagnosis is based on morphological ultrasound and magnetic resonance imaging of the fetus. The transmission is genetic and autosomal recessive. Courtnes has established 4 diagnostic criteria to affirm the acrocal syndrome and to eliminate the amalgam with other polymalformative syndrome.

Hepatic Hedgehog Signaling Participates in the Crosstalk between Liver and Adipose Tissue in Mice by Regulating FGF21.

The Hedgehog signaling pathway regulates many processes during embryogenesis and the homeostasis of adult organs. Recent data suggest that central metabolic processes and signaling cascades in the liver are controlled by the Hedgehog pathway and that changes in hepatic Hedgehog activity also affect peripheral tissues, such as the reproductive organs in females. Here, we show that hepatocyte-specific deletion of the Hedgehog pathway is associated with the dramatic expansion of adipose tissue in mice, the overall phenotype of which does not correspond to the classical outcome of insulin resistance-associated diabetes type 2 obesity. Rather, we show that alterations in the Hedgehog signaling pathway in the liver lead to a metabolic phenotype that is resembling metabolically healthy obesity. Mechanistically, we identified an indirect influence on the hepatic secretion of the fibroblast growth factor 21, which is regulated by a series of signaling cascades that are directly transcriptionally linked to the activity of the Hedgehog transcription factor GLI1. The results of this study impressively show that the metabolic balance of the entire organism is maintained via the activity of morphogenic signaling pathways, such as the Hedgehog cascade. Obviously, several pathways are orchestrated to facilitate liver metabolic status to peripheral organs, such as adipose tissue.

Ehrlichia SLiM ligand mimetic activates Hedgehog signaling to engage a BCL-2 anti-apoptotic cellular program.

Ehrlichia chaffeensis (E. chaffeensis) has evolved eukaryotic ligand mimicry to repurpose multiple cellular signaling pathways for immune evasion. In this investigation, we demonstrate that TRP120 has a novel repetitive short linear motif (SLiM) that activates the evolutionarily conserved Hedgehog (Hh) signaling pathway to inhibit apoptosis. In silico analysis revealed that TRP120 has sequence and functional similarity with Hh ligands and a candidate Hh ligand SLiM was identified. siRNA knockdown of Hh signaling and transcriptional components significantly reduced infection. Co-immunoprecipitation and surface plasmon resonance demonstrated that rTRP120-TR interacted directly with Hh receptor Patched-2 (PTCH2). E. chaffeensis infection resulted in early upregulation of Hh transcription factor GLI-1 and regulation of Hh target genes. Moreover, soluble recombinant TRP120 (rTRP120) activated Hh and induced gene expression consistent with the eukaryotic Hh ligand. The TRP120-Hh-SLiM (NPEVLIKD) induced nuclear translocation of GLI-1 in THP-1 cells and primary human monocytes and induced a rapid and expansive activation of Hh pathway target genes. Furthermore, Hh activation was blocked by an α-TRP120-Hh-SLiM antibody. TRP120-Hh-SLiM significantly increased levels of Hh target, anti-apoptotic protein B-cell lymphoma 2 (BCL-2), and siRNA knockdown of BCL-2 dramatically inhibited infection. Blocking Hh signaling with the inhibitor Vismodegib, induced a pro-apoptotic cellular program defined by decreased mitochondria membrane potential, significant reductions in BCL-2, activation of caspase 3 and 9, and increased apoptotic cells. This study reveals a novel E. chaffeensis SLiM ligand mimetic that activates Hh signaling to maintain E. chaffeensis infection by engaging a BCL-2 anti-apoptotic cellular program.

Unconventional GPCR‐PKA Signaling in the Hedgehog Pathway

The Hedgehog pathway controls tissue and organ development throughout animal evolution and drives several widespread skin and brain cancers. A pivotal step in Hh signal transduction is the activation of GLI transcription factors by the atypical G protein‐coupled receptor (GPCR) Smoothened (SMO). How SMO activates GLI has remained unclear for decades. The principal issue is that SMO appears to defy standard signaling paradigms utilized by nearly all other GPCRs.Here we show that SMO employs a decoy substrate sequence to physically block the active site of the PKA catalytic subunit (PKA‐C) and extinguish its enzymatic activity. As a result, GLI is released from phosphorylation‐induced inhibition. Using a combination of in vitro, cellular, and organismal models, we demonstrate that interfering with SMO / PKA pseudosubstrate interactions prevents Hh signal transduction. The mechanism we uncovered echoes one utilized by the Wnt cascade, revealing an unexpected similarity in how these two essential developmental and cancer pathways signal intracellularly.Our findings help to resolve a longstanding mystery in developmental and cancer signaling, and reveal a new mechanism of receptor‐kinase communication that may apply broadly throughout the GPCR superfamily. Our work also suggests new mechanism‐inspired therapeutic strategies to manage cancers driven by ectopic Hedgehog pathway activity.

GSK-3\u03b2 suppression upregulates Gli1 to alleviate osteogenesis inhibition in titanium nanoparticle-induced osteolysis

Wear particle-induced periprosthetic osteolysis (PPO) have become a major reason of joint arthroplasty failure and secondary surgery following joint arthroplasty and thus pose a severe threat to global public health. Therefore, determining how to effectively suppress particle-induced PPO has become an urgent problem. The pathological mechanism involved in the PPO signaling cascade is still unclear. Recently, the interaction between osteogenic inhibition and wear particles at the implant biological interface, which has received increasing attention, has been revealed as an important factor in pathological process. Additionally, Hedgehog (Hh)-Gli1 is a crucial signaling cascade which was regulated by multiple factors in numerous physiological and pathological process. It was revealed to exert a crucial part during embryonic bone development and metabolism. However, whether Hh-Gli1 is involved in wear particle-induced osteogenic inhibition in PPO remains unknown. Our present study explored the mechanism by which the Hh-Gli1 signaling cascade regulates titanium (Ti) nanoparticle-induced osteolysis. We found that Hh-Gli1 signaling was dramatically downregulated upon Ti particle treatment. Mechanistically, glycogen synthesis kinase 3β (GSK-3β) activation was significantly increased in Ti particle-induced osteogenic inhibition via changes in GSK-3β phosphorylation level and was found to participate in the posttranslational modification and degradation of the key transcription factor Gli1, thus decreasing the accumulation of Gli1 and its translocation from the cytoplasm to the nucleus. Collectively, these findings suggest that the Hh-Gli1 signaling cascade utilizes a GSK3β-mediated mechanism and may serve as a rational new therapeutic target against nanoparticle-induced PPO.Graphical

IL25 Enhanced Colitis-Associated Tumorigenesis in Mice by Upregulating Transcription Factor GLI1.

Interleukin-25 (IL17E/IL25) plays a critical role in colitis and intestinal homeostasis. However, the expression and biological role of IL25 in colorectal cancer is not properly understood. In this study, we show that IL25 is mainly expressed by cancer stem cells in the colorectal cancer microenvironment. Genetic deletion of IL25 inhibited tumor formation and growth and prolonged survival in AOM/DSS-treated mice. IL25 stimulated cancer organoid and cancer cells sphere formation and prevented the tumor from chemotherapy-induced apoptosis. Mechanistically, IL25 upregulated stem cell genes LGR5, CD133, and ABC transporters via activating the Hedgehog signaling pathway. IL25 inhibited phosphorylation of AMPK and promoted GLI1 accumulation to maintain cancer stem cells. Moreover, IL25 expression was associated with poor survival in patients with metastatic colorectal cancer. Taken together, our work reveals an immune-associated mechanism that intrinsically confers cancer cell stemness properties. Our results first demonstrated that IL25, as a new potent endogenous Hedgehog pathway agonist, could be an important prognostic factor and therapeutic target for CRC.

A Critical Function for the Transcription Factors GLI1 and GLI2 in the Proliferation and Survival of Human Mast Cells

Mast cell hyperactivity and accumulation in tissues are associated with allergy and other mast cell-related disorders. However, the molecular pathways regulating mast cell survival in homeostasis and disease are not completely understood. As glioma-associated oncogene (GLI) proteins are involved in both tissue homeostasis and in the hematopoietic system by regulating cell fate decisions, we sought to investigate the role for GLI proteins in the control of proliferation and survival of human mast cells. GLI1 transcripts were present in primary human mast cells and mast cell lines harboring or not activating mutations in the tyrosine kinase receptor KIT (HMC-1.1 and HMC-1.2, and LAD2 cells, respectively), while GLI2 transcripts were only present in HMC-1.1 and HMC-1.2 cells, suggesting a role for oncogenic KIT signaling in the regulation of GLI2. Reduction in GLI activity by small molecule inhibitors, or by shRNA-mediated knockdown of GLI1 or GLI2, led to increases in apoptotic cell death in both cultured human and murine mast cells, and reduced the number of peritoneal mast cells in mice. Although GLI proteins are typically activated via the hedgehog pathway, steady-state activation of GLI in mast cells occurred primarily via non-canonical pathways. Apoptosis induced by GLI silencing was associated with a downregulation in the expression of KIT and of genes that influence p53 stability and function including USP48, which promotes p53 degradation; and iASPP, which inhibits p53-induced transcription, thus leading to the induction of p53-regulated apoptotic genes. Furthermore, we found that GLI silencing inhibited the proliferation of neoplastic mast cell lines, an effect that was more pronounced in rapidly growing cells. Our findings support the conclusion that GLI1/2 transcription factors are critical regulators of mast cell survival and that their inhibition leads to a significant reduction in the number of mast cells in vitro and in vivo, even in cells with constitutively active KIT variants. This knowledge can potentially be applicable to reducing mast cell burden in mast cell-related diseases.

Real time imaging of the trafficking of a Hedgehog pathway kinesin in the primary cilium

The primary cilium is a hub of cellular signaling. In vertebrates, signaling through the Hedgehog (Hh) pathway, which is essential for embryonic development, critically depends on the cilium. Pathway activation results in a precisely choreographed sequence of core proteins entering and exiting the primary cilium, which leads to the activation of Gli transcription factors, major effectors of Hh pathway. However, the dynamics of only a few membrane-associated pathway proteins in the cilium have been imaged at high spatial-temporal resolution.