Sonic Hedgehog Signaling Pathway Research Articles

Radial Longitudinal Deficiency: New Perspectives on an Enduring Challenge.

Radial longitudinal deficiency (RLD) is a multidimensional congenital hand difference encompassing skeletal, musculotendinous, and joint components. Managing RLD remains challenging, with numerous surgical procedures over the past century failing to achieve a stable, mobile, growing wrist without recurrence of the deformity. This review investigates new therapeutic approaches for RLD, delving into genetic, embryological, and histological aspects, including proximal muscle involvement and causes of recurrence.A notable association between RLD and preaxial polydactyly, linked to aberrations in the Sonic Hedgehog signaling pathway, is highlighted, suggesting a common embryonic origin. Experimental evidence indicates that ectopic Sonic Hedgehog signaling can result in radial aplasia and preaxial polydactyly. Histopathological studies revealed significant muscle abnormalities in RLD, contributing to deformities and recurrences.Techniques such as preoperative soft tissue distraction show promise, but recurrence rates persist. Alternative surgical procedures, such as vascularized second metatarsophalangeal joint transfer and ulna cuff osteotomy, offer potential improvements by minimizing recurrence and optimizing limb length.Despite advancements, effective management of RLD requires further research into the interplay between genetic factors, muscle abnormalities, and surgical outcomes. This review underscores the importance of early detection, genetic counseling, and a multidisciplinary approach to enhance long-term functional and aesthetic results for RLD patients.

Bisphenol S exposure promotes stemness of triple-negative breast cancer cells via regulating Gli1-mediated Sonic hedgehog pathway

Bisphenol S (BPS), one of the most common alternatives for bisphenol A (BPA), has been implied to increase the risk of breast cancer. Triple-negative breast cancer (TNBC) is a highly aggressive type of breast cancer with a poor prognosis. However, the association between BPS and TNBC remains unclear. Cancer stem cells (CSCs) have a crucial role in breast cancer initiation, metastasis, and recurrence. Here, we proposed that BPS, equivalent to the human internal exposure and the environmental concentrations, enhanced CSC-like properties by upregulating sphere formation, self-renewal, the percentage of CD44+/CD24- cells, and the expression of CSC markers. Moreover, BPS promoted the migration, invasion, and epithelial-mesenchymal transition (EMT) in TNBC cells. Mechanistically, BPS activated the Sonic Hedgehog (SHH) signaling pathway in TNBC cells. Molecular docking analysis further showed that BPS upregulated SHH signaling pathway via directly binding Gli1 protein. Furthermore, inhibitor of SHH pathway or Gli1 siRNA attenuated the promoting effects of BPS on stemness, invasion, and migration of TNBC cells. In summary, our data firstly provide evidence that environmentally relevant BPS concentration treatment significantly enhanced TNBC malignant phenotype by activating the Sonic Hedgehog/Gli1 signaling pathway, raising high concerns about the potential population biology hazards of BPS.

Real-world data on the efficacy and safety of hedgehog pathway inhibitors in patients with basal cell carcinoma: Experience of a tertiary Australian centre.

Basal cell carcinomas (BCCs) are the most common cancers worldwide. Although most BCCs are amenable to local treatment, there are limited therapeutic options for surgically unresectable locally advanced and metastatic BCCs. Activation of the sonic hedgehog signalling pathway plays a significant role in the development of most BCCs. Hedgehog pathway inhibitors (HPIs) can be used to inhibit this pathway. Efficacy and safety data on HPI use in Australia is scarce. This study aims to present the effectiveness and safety of HPI at a tertiary dermatology referral centre. We conducted a retrospective analysis of the clinical charts of all patients with BCC treated with an HPI at a tertiary Dermatology referral centre in New South Wales, Australia from 1 January 2016 to 1 July 2023. Twenty-three patients with BCCs were treated with an HPI; 11 locally advanced, 8 multiple, 3 basal cell naevus syndrome and 1 metastatic. All patients were of Caucasian background, with a median age of 56. Across 41 treatment cycles, the median treatment duration was 4 months. The overall response rate (ORR) was 20/23 (87%) and complete response (CR) rate was 9/23 (39%); patients treated with sonidegib achieved an ORR of 11/12 (92%) and CR of 4/12 (33%), and vismodegib-treated patients achieved an ORR of 9/11 (82%) and CR of 5/11 (45%). Patients who responded to HPI treatment also responded to a subsequent HPI rechallenge. Common treatment emergent adverse events (TEAEs) included muscle spasms, dysgeusia and alopecia. Dysgeusia was more frequent with vismodegib than sonidegib (p = 0.0001). There was no evidence to suggest a difference in other TEAEs between the two HPIs. Four treatment cycles were stopped due to grade 3 muscle spasm. In our cohort of 23 patients being treated with HPI, the ORR was 87% and CR was 39%. All patients who experienced TEAEs and had a drug holiday successfully responded to HPI rechallenge.TEAEs, particularly muscle spasms, are common reasons for treatment cessation. Clinicians should implement strategies to mitigate TEAE to improve drug survivability.

Modulating sonic hedgehog (SHH) pathway to create a rapid CNS-TB model: Facilitating drug discovery

Tuberculous meningitis, a severe complication of Mycobacterium tuberculosis (M. tb) infection, involves the dissemination of bacilli in the brain. This study explored the role of the sonic hedgehog (SHH) signaling pathway in regulating blood-brain barrier (BBB) integrity, M. tb invasion into the central nervous system (CNS), and disease progression of Central Nervous System Tuberculosis (CNS-TB) in a Balb/c mouse model. The modulation of the SHH pathway using agonist Purmorphamine (PUR) and antagonist Cyclopamine (CYC) revealed that CYC treatment led to a rapid and extensive invasion of M. tb in the brain, with bacterial loads increasing by 99 % compared to the untreated-infected group. In contrast, PUR reduced M. tb loads by 50 % and delayed disease progression. Histopathological analysis showed that CYC exacerbated inflammation and immune cell infiltration, while PUR mitigated these responses. Immunohistochemistry demonstrated that CYC caused severe BBB breakdown and reactive gliosis, while PUR partially attenuated this response. Further analysis revealed that CYC upregulated Matrix Metalloproteinase-9 (MMP-9) secretion, a key contributor to BBB disruption. These findings highlight the critical role of the SHH pathway in maintaining BBB integrity and regulating the immunopathological response during CNS-TB, opening up future scope for drug discovery. This Cyclopamine-induced model of rapid M. tb invasion and chronic inflammation provides a new tool for studying CNS-TB pathogenesis and evaluating potential therapeutic interventions targeting the SHH signaling axis. Significance statementUnderstanding how tuberculosis (TB) infection can spread to the brain is crucial, as this “central nervous system TB” (CNS-TB) is a serious and potentially life-threatening health complication. However, studying CNS-TB in humans is very difficult. Animal models are needed to better understand how TB gets into the brain and the resulting damage. This study in mice showed that blocking a signaling pathway called Sonic Hedgehog (SHH) allowed TB to rapidly spread to the brain, damaging the blood-brain barrier and causing severe inflammation. In contrast, activating the SHH pathway helped protect the brain from TB. These findings provide important insights that could lead to new ways to prevent or treat this dangerous form of TB.

Genetic Insights Into Hypothalamic Hamartoma: Unraveling Somatic Variants.

Hypothalamic hamartomas (HHs) are rare developmental brain lesions associated with drug-resistant epilepsy and often subjected to epilepsy surgery. Brain somatic variants in genes affecting the Sonic hedgehog (Shh) and primary cilia signaling pathways have been implicated in approximately 50% of nonsyndromic HH cases. This study aims to characterize a new cohort of 9 HH cases and elucidate their genetic etiology. We recruited 9 HH cases including 8 nonsyndromic cases of which 4 were type IV HH. Genomic DNA was extracted from peripheral blood and surgical brain tissues, and somatic variants were investigated using high-depth whole-exome sequencing. Pathogenic somatic variants in known HH genes (GLI3, OFD1, and PRKACA) were identified in 7 of the 9 cases. In addition, a 2-hit mutational event comprising a germline variant (predicted to impair kinase activity) and a somatic loss-of-heterozygosity was identified in TNK2, a gene encoding a brain-expressed tyrosine kinase. Our findings reinforce the role of somatic variants in Shh and cilia genes in HH cases while also shedding light on TNK2 as a potential novel disease-causing gene. This study emphasizes the increasing importance of brain mosaicism in epilepsy disorders and underscores the critical role of genetic diagnosis derived from resected brain tissue.

Sonic hedgehog signaling facilitates pyroptosis in mouse heart following ischemia/reperfusion via enhancing the formation of CARD10-BCL10-MALT1 complex

Pyroptosis has been found to contribute to myocardial ischemia/reperfusion (I/R) injury, but the exact mechanisms that initiate myocardial pyroptosis are not fully elucidated. Sonic hedgehog (SHH) signaling is activated in heart suffered I/R, and intervention of SHH signaling has been demonstrated to protect heart from I/R injury. Caspase recruitment domain-containing protein 10 (CARD10)-B cell lymphoma 10 (BCL10)-mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) (CBM) complex could transduce signals from the membrane and induce inflammatory pathways in non-hematopoietic cells, which could be a downstream effector of SHH signaling pathway. This study aims to explore the role of SHH signaling in I/R-induced myocardial pyroptosis and its relationship with the CBM complex. C57BL/6J mice were subjected to 45 min-ischemia followed by 24 h-reperfusion to establish a myocardial I/R model, and H9c2 cells underwent hypoxia/reoxygenation (H/R) to mimic myocardial I/R model in vitro. Firstly, SHH signaling was significantly activated in heart suffered I/R in an autocrine- or paracrine-dependent manner via its receptor PTCH1, and inhibition of SHH signaling decreased myocardial injury via reducing caspase-11-dependent pyroptosis, concomitant with attenuating CBM complex formation. Secondly, suppression of SHH signaling decreased protein kinase C α (PKCα) level, but inhibition of PKCα attenuated CBM complex formation without impacting the protein levels of SHH and PTCH1. Finally, disruption of the CBM complex prevented MALT1 from recruiting of TRAF6, which was believed to trigger the caspase-11-dependent pyroptosis. Based on these results, we conclude that inhibition of SHH signaling suppresses pyroptosis via attenuating PKCα-mediated CARD10-BCL10-MALT1 complex formation in mouse heart suffered I/R.

Shh regulates M2 microglial polarization and fibrotic scar formation after ischemic stroke

BackgroundFibrotic scar formation is a critical pathological change impacting tissue reconstruction and functional recovery after ischemic stroke. The regulatory mechanisms behind fibrotic scarring in the central nervous system (CNS) remain largely unknown. While macrophages are known to play a role in fibrotic scar formation in peripheral tissues, the involvement of microglia, the resident immune cells of the CNS, in CNS fibrosis requires further exploration. The Sonic Hedgehog (Shh) signaling pathway, pivotal in embryonic development and tissue regeneration, is also crucial in modulating fibrosis in peripheral tissues. However, the impact and regulatory mechanisms of Shh on fibrotic scar formation post-ischemic stroke have not been thoroughly investigated. MethodsThis study explores whether Shh can regulate fibrotic scar formation post-ischemic stroke and its underlying mechanisms through in vivo and in vitro manipulation of Shh expression. ResultsOur results showed that Shh expression was upregulated in the serum of acute ischemic stroke patients, as well as in the serum, CSF, and ischemic regions of MCAO/R mice. Moreover, the upregulation of Shh expression was positively correlated with fibrotic scar formation and M2 microglial polarization. Shh knockdown inhibited fibrotic scar formation and M2 microglial polarization while aggravating neurological deficits in MCAO/R mice. In vitro, adenoviral knockdown or Smoothened Agonist (SAG) activation of Shh expression in BV2 cells following OGD/R regulated their polarization and influenced the expression of TGFβ1 and PDGFA, subsequently affecting fibroblast activation. ConclusionThese results suggest that Shh regulates M2 microglial polarization and fibrotic scar formation after cerebral ischemia.

Two-way Dispatched function in Sonic hedgehog shedding and transfer to high-density lipoproteins.

The Sonic hedgehog (Shh) signaling pathway controls embryonic development and tissue homeostasis after birth. This requires regulated solubilization of dual-lipidated, firmly plasma membrane-associated Shh precursors from producing cells. Although it is firmly established that the resistance-nodulation-division transporter Dispatched (Disp) drives this process, it is less clear how lipidated Shh solubilization from the plasma membrane is achieved. We have previously shown that Disp promotes proteolytic solubilization of Shh from its lipidated terminal peptide anchors. This process, termed shedding, converts tightly membrane-associated hydrophobic Shh precursors into delipidated soluble proteins. We show here that Disp-mediated Shh shedding is modulated by a serum factor that we identify as high-density lipoprotein (HDL). In addition to serving as a soluble sink for free membrane cholesterol, HDLs also accept the cholesterol-modified Shh peptide from Disp. The cholesteroylated Shh peptide is necessary and sufficient for Disp-mediated transfer because artificially cholesteroylated mCherry associates with HDL in a Disp-dependent manner, whereas an N-palmitoylated Shh variant lacking C-cholesterol does not. Disp-mediated Shh transfer to HDL is completed by proteolytic processing of the palmitoylated N-terminal membrane anchor. In contrast to dual-processed soluble Shh with moderate bioactivity, HDL-associated N-processed Shh is highly bioactive. We propose that the purpose of generating different soluble forms of Shh from the dual-lipidated precursor is to tune cellular responses in a tissue-type and time-specific manner.

Two-way Dispatched function in Sonic hedgehog shedding and transfer to high-density lipoproteins

The Sonic hedgehog (Shh) signaling pathway controls embryonic development and tissue homeostasis after birth. This requires regulated solubilization of dual-lipidated, firmly plasma membrane-associated Shh precursors from producing cells. Although it is firmly established that the resistance-nodulation-division transporter Dispatched (Disp) drives this process, it is less clear how lipidated Shh solubilization from the plasma membrane is achieved. We have previously shown that Disp promotes proteolytic solubilization of Shh from its lipidated terminal peptide anchors. This process, termed shedding, converts tightly membrane-associated hydrophobic Shh precursors into delipidated soluble proteins. We show here that Disp-mediated Shh shedding is modulated by a serum factor that we identify as high-density lipoprotein (HDL). In addition to serving as a soluble sink for free membrane cholesterol, HDLs also accept the cholesterol-modified Shh peptide from Disp. The cholesteroylated Shh peptide is necessary and sufficient for Disp-mediated transfer because artificially cholesteroylated mCherry associates with HDL in a Disp-dependent manner, whereas an N-palmitoylated Shh variant lacking C-cholesterol does not. Disp-mediated Shh transfer to HDL is completed by proteolytic processing of the palmitoylated N-terminal membrane anchor. In contrast to dual-processed soluble Shh with moderate bioactivity, HDL-associated N-processed Shh is highly bioactive. We propose that the purpose of generating different soluble forms of Shh from the dual-lipidated precursor is to tune cellular responses in a tissue-type and time-specific manner.

Does the sonic hedgehog signaling pathway play a role in anti-reflux mechanism of bladder in children?

A hedgehog family ligand, namely, sonic hedgehog (SHH), was reported to be important in the development of bladder and ureter smooth muscle. In this prospective study, we aimed to determine protein expression of SHH in resected ureterovesical junction (UVJ) segments of children with vesicoureteral reflux (VUR). The study group included 19 children; 12 (63%) girls, 7 (37%) boys, who had ureteroneocystostomy operation; 3 (15.7%) right sided, 7 (36.8%) left sided, 9 (47.3%) bilateral, due to primary VUR between years 2015 and 2018. Totally, 28 UVJ segments were examined for Western Blot analysis to determine related protein expression levels. The mean Western blot band area of SHH gene pathway related protein was 3880.69 (2059.55-13941.61) while the mean area of β-Actin, the house-keeping gene, was 20180.25 (9530.39-26709.75) (p = 0.001). Correlation analyses between grade of reflux and protein expression of SHH gene pathways revealed no significant relation (p = 0.300). When the UV samples were grouped as low- and high-grade reflux and compared in terms of SHH protein expression levels, no statistically significant difference was found between groups (p = 0.818). We concluded that SHH signaling molecule which is effective in development of bladder and ureter smooth musculature might also be effective in etiopathology of reflux.

Zoledronic acid-treated rats show altered jaw histology and gene expression in non-exposed MRONJ

BackgroundDiagnosis and management of medication-related osteonecrosis of the jaws (MRONJ) prior to clinical exposure induced by trauma may lead to improved patient management. Currently, few studies have examined early histologic and molecular MRONJ-related changes in the jaws. PurposeThis study aimed to identify histological and gene expression changes in the maxilla and mandible of Sprague-Dawley (SD) rats after zoledronic acid (ZA) treatment. Study Design, Setting, and SampleThis was an in-vivo animal study. The experiments were conducted in the laboratory at the Stomatology Hospital of Tianjin Medical University. A total of 12 SD rats were included. Predictor variableThe predictor variable was ZA exposure. Twelve SD rats were divided into two groups: experimental (n = 6) and control (n = 6), and they were intraperitoneally injected with ZA and saline, respectively. Main outcome variableThe outcome variables were histological and molecular changes. The maxilla, mandible, and ilium bone tissue samples were examined using Masson's trichrome and hematoxylin-eosin staining. Gene expression changes were identified using transcriptome sequencing, Kyoto encyclopedia of genes and genomes (KEGG), and gene interactome network analysis. The key changes were validated using the quantitative real-time polymerase chain reaction and immunohistochemistry. CovariatesNone. AnalysesThe t-test, Chi-square test, and Fisher's exact probability method were used for statistical analyses using the Statistical package for the social sciences software (version 26.0). ResultsAll animals remained healthy during the experiments. Histological staining revealed that the percentage of empty bone lacunae in the maxilla and mandible was significantly higher than that in the ilium (P < 0.01). In total, 552 genes were screened using transcriptome sequencing. The sonic hedgehog (Shh) signaling pathway was highly enriched. The key gene for the Shh interaction was distal-less homeobox 5 (Dlx-5). The Shh, Dlx-5, and bone morphogenetic protein 2 genes and protein expression levels in the maxilla and mandible were higher in the experimental group than in the control group (P < 0.05). Conclusions and RelevanceMRONJ-induced osteonecrosis and gene expression changes precede trauma-induced clinical changes in the SD rat model. These findings may provide additional support for timely and clinically early diagnosis and intervention.

Benzaldehyde stimulates autophagy via the sonic hedgehog signaling pathway in mouse brain astrocytes after treatment with Angiostrongylus cantonensis excretory-secretory products

Autophagy is a vital cellular process responsible for digesting various cytoplasmic organelles. This process plays a crucial role in maintaining cell survival and homeostasis, especially under conditions that cause nutrient deficiency, cellular damage, and oxidative stress. Neuroangiostrongyliasis is an infection caused by the parasitic nematode Angiostrongylus cantonensis and is considered as an emerging disease in many parts of the world. However, effective therapeutic strategies for neuroangiostrongyliasis still need to be further developed. In this study, we investigated the effects of benzaldehyde treatment on autophagy and sonic hedgehog (Shh) signaling in A. cantonensis-infected mice and its mechanisms. First, we found autophagosome generation in the central nervous system after A. cantonensis infection. Next, benzaldehyde combined with albendazole treatment reduced eosinophilic meningitis and upregulated the expression of Shh signaling- and autophagy-related molecules in A. cantonensis-infected mouse brains. In vitro experiments demonstrated that benzaldehyde could induce autophagy via the Shh signaling pathway in A. cantonensis excretory-secretory products (ESPs)-treated mouse astrocytes. Finally, benzaldehyde treatment also decreased lipid droplet accumulation and increased cholesterol production by activating the Shh pathway after ESPs treatment. In conclusion, these findings suggested that benzaldehyde treatment could alleviate brain damage by stimulating autophagy generation through the Shh signaling pathway.

The silent information regulator 1 agonist SRT1720 reduces experimental intracerebral hemorrhagic brain injury by regulating the blood-brain barrier integrity.

Intracerebral hemorrhage (ICH) is a significant public health matter that has no effective treatment. ICH-induced destruction of the blood-brain barrier (BBB) leads to neurological deterioration. Astrocytic sonic hedgehog (SHH) alleviates brain injury by maintaining the integrity of the BBB after ICH. Silent information regulator 1 (SIRT1) is neuroprotective in several central nervous system diseases via BBB regulation. It is also a possible influential factor of the SHH signaling pathway. Nevertheless, the role of SIRT1 on BBB and the underlying pathological process associated with the SHH signaling pathway after ICH remain unclear. We established an intracerebral hemorrhagic mouse model by collagenase injection. SRT1720 (a selective agonist of SIRT1) was used to evaluate the effect of SIRT1 on BBB integrity after ICH. SIRT1 expression was reduced in the mouse brain after ICH. SRT1720 attenuated neurobehavioral impairments and brain edema of ICH mouse. After ICH induction, SRT1720 improved BBB integrity and tight junction expressions in the mouse brain. The SHH signaling pathway-related factors smoothened and glioma-associated oncogene homolog-1 were increased with the intervention of SRT1720, while cyclopamine (a specific inhibitor of the SHH signaling pathway) reversed these effects. These findings suggest that SIRT1 protects from ICH by altering BBB permeability and tight junction expression levels. This process is associated with the SHH signaling pathway, suggesting that SIRT1 may be a potential therapeutic target for ICH.

Study of the Immune Infiltration and Sonic Hedgehog Expression Mechanism in Synovial Tissue of Rheumatoid Arthritis-Related Interstitial Lung Disease under Machine Learning CIBERSORT Algorithm.

Rheumatoid arthritis-related interstitial lung disease (RA-ILD) is one of the common complications in patients with RA, which affects their quality of life. The CIBERSORT algorithm is widely employed to determine the proportion of immune cells (ICs) in diseased tissues, while the Sonic Hedgehog (Shh) signaling pathway, as an imperative regulatory factor, has also attracted attention in the pathology of RA-ILD. This work was to explore the mechanisms of RA-ILD immune infiltration and synovial tissue (ST) Shh expression based on the CIBERSORT algorithm. The differential genes of RA-ILD were subjected to pathway enrichment analysis using R language. The content and proportion of 22 types of ICs in RA-ILD lung tissues were analyzed using machine learning-based CIBERSORT algorithm. Meanwhile, immunoblotting was employed to detect and analyze the expression of Shh, Smoothened (Smo), and bone morphogenetic proteins (BMPs) proteins in ST samples from RA-ILD and Ctrl groups (RA patients without ILD). The hub target genes in the protein network associated with RA-ILD include BSG, CCL2, CTLA4, FGFBP1, GLI1, HHIP, HLA-DRB1, IFNAR1, IL17A, IL23A, IL-6, INPP4A, LILRB1, MUC5B, PADI4, PPM1A, PTCH1, PTPN22, RSPO4, Shh, SMO, STAT4, SUFU, TAOK2, TIMP2, and TWSG1, which are involved in multiple pathways, such as B cell regulation, transcription factors of the Shh pathway, and ST immune tolerance-related pathways. In the immunological analysis of RA-ILD using the CIBERSORT algorithm, HLA (r = -0.26), PTPN22 (r = -0.36), STAT4 (r = -0.18), IL-6 (r = -0.17), CTLA4 (r = -0.27), and PADI4 (r = -0.21) were all found to exhibit negative correlations with CD4+T cells (P < 0.05). Monocytes were found to be more abundant in RA-ILD patients' serum versus the Ctrl group. Shh, Smo, and BMP expressions were drastically lower in the RA-ILD group versus Ctrl group (P < 0.05). Significant immune cell infiltration was observed in the lung tissues of RA-ILD patients. Further analysis utilizing the CIBERSORT algorithm revealed alterations in the proportions of different IC subtypes, indicating their association with disease severity and prognosis. Moreover, there was a significant decrease in the expression levels of Shh, Smo, and BMP. These findings underscore the importance of immune cells in the pathophysiology of RA-ILD and suggest a potential involvement of the Shh signaling pathway in the pathogenesis of RA-ILD.

Glioma-associated oncogene (GLI)-specific decoy oligodeoxynucleotide induces apoptosis and attenuates proliferation, colony formation, and migration in liver cancer cells

Aim: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-associated death. The Sonic Hedgehog (SHH) signaling pathway participates in the initiation, progression, migration, and recurrence of HCC cancer stem cells. Furthermore, SHH regulates various cellular behaviors such as proliferation, differentiation, survival, self-renewal, epithelial-mesenchymal transition (EMT), and SHH autoregulation. Glioma-associated oncogene (GLI) family zinc finger are key transcription factors in the development of many organs and are deregulated in cancer. In this study, Huh-7 cells were treated with GLI-specific decoy oligodeoxynucleotide (ODN) to evaluate its anticancer impact. Methods: The transfection efficiency of GLI-specific decoy ODN was measured using fluorescent microscopy. Then, the effects of GLI-specific decoy ODN on apoptosis, viability, proliferation rate, colony formation, and migration capacities of Huh-7 cells were assessed. Furthermore, the expression of genes associated with the alteration of SHH was assessed. Results: Treatment of Huh-7 cells with GLI-specific decoy ODN decreased cell viability (56.36% ± 3%). Expression of certain genes such as c-MYC , SNAI2 , ZEB1 , and PROM1 decreased dramatically, while the expression of CDH1 increased significantly. Furthermore, the treated cells’ proliferation, colony formation, and migration capacity decreased considerably. This treatment induced apoptosis in the Huh-7 cells. Conclusion: Inhibition of the SHH signaling pathway using GLI-specific decoy ODN led to a decline in the growth rate of HCC cells, decreased migration, and attenuated EMT progression.

ARL13B promotes cell cycle through the sonic hedgehog signaling pathway to alleviate nerve damage during cerebral ischemia/reperfusion in rats

Cerebral ischemia/reperfusion (CIRI) is a leading cause of death worldwide. A small GTPase known as ADP-ribosylation factor-like protein 13B (ARL13B) is essential in several illnesses. The role of ARL13B in CIRI remains unknown, though. A middle cerebral artery occlusion/reperfusion (MCAO/R) in rats as well as an oxygen-glucose deprivation/reoxygenation (OGD/R) models in PC12 cells were constructed. The neuroprotective effects of ARL13B against MCAO/R were evaluated using neurological scores, TTC staining, rotarod testing, H&E staining, and Nissl staining. To detect the expression of proteins associated with the SHH pathway and apoptosis, western blotting and immunofluorescence were employed. Apoptosis was detected using TUNEL assays and flow cytometry. There was increased expression of ARL13B in cerebral ischemia/reperfusion models. However, ARL13B knockdown aggravated CIRI nerve injury by inhibiting the sonic hedgehog (SHH) pathway. In addition, the use of SHH pathway agonist (SAG) can increased ARL13B expression, reverse the effects of ARL13B knockdown exacerbating CIRI nerve injury. ARL13B alleviated cerebral infarction and pathological injury and played a protective role against MCAO/R. Furthermore, ARL13B significantly increased the expression of SHH pathway-related proteins and the anti-apoptotic protein BCL-2, while decreased the expression of pro-apoptotic protein BAX, thus reducing apoptosis. The results from the OGD/R model in PC12 cells were consistent with those obtained in vivo. Surprisingly, we demonstrated that ARL13B regulates the cell cycle to protect against CIRI nerve injury. Our findings indicate that ARL13B protects against CIRI by reducing apoptosis through SHH-dependent pathway activation, and suggest that ARL13B plays a crucial role in CIRI pathogenesis.

Lack of Nuclear Localization of the Creb3l1 Transcription Factor Causes Defects in Caudal Fin Bifurcation in Zebrafish Danio rerio.

The formation of normal bone and bone healing requires the cAMP-responsive element binding protein 3-like-1 (Creb3l1) transmembrane transcription factor, as deletion of the murine CREB3L1 results in osteopenic animals with limited capacity to repair bone after a fracture. Creb3l1 undergoes regulated intramembrane proteolysis (RIP) to release the N-terminal transcription activating (TA) fragment that enters the nucleus and regulates the expression of target genes. To expand our understanding of Creb3l1's role in skeletal development and skeletal patterning, we aimed to generate animals expressing only the TA fragment of Creb3l1 lacking the transmembrane domain and thereby not regulated through RIP. However, the CRISPR/Cas9-mediated genome editing in zebrafish Danio rerio caused a frameshift mutation that added 56 random amino acids at the C-terminus of the TA fragment (TA+), making it unable to enter the nucleus. Thus, TA+ does not regulate transcription, and the creb3l1TA+/TA+ fish do not mediate creb3l1-dependent transcription. We document that the creb3l1TA+/TA+ fish exhibit defects in the patterning of caudal fin lepidotrichia, with significantly distalized points of proximal bifurcation and decreased secondary bifurcations. Moreover, using the caudal fin amputation model, we show that creb3l1TA+/TA+ fish have decreased regeneration and that their regenerates replicate the distalization and bifurcation defects observed in intact fins of creb3l1TA+/TA+ animals. These defects correlate with altered expression of the shha and ptch2 components of the Sonic Hedgehog signaling pathway in creb3l1TA+/TA+ regenerates. Together, our results uncover a previously unknown intersection between Creb3l1 and the Sonic Hedgehog pathway and document a novel role of Creb3l1 in tissue patterning.

MDB-57. PROTEOMIC ANALYSIS REVEALS AMBRA1 AS A NEW CRL3REN/KCTD11 SUBSTRATE PROMOTING SONIC HEDGEHOG-DEPENDENT MEDULLOBLASTOMA

Abstract BACKGROUND Sonic Hedgehog (SHH) signaling pathway is fundamental for a proper embryonic development and its uncontrolled activation is responsible for various cancers. One the most relevant SHH-dependent tumors is medulloblastoma (MB), a highly heterogeneous brain malignancy that predominantly occurs in childhood. Understanding the complex networks that govern SHH aberrant activation represents a dramatic challenge for the development of efficient personalized therapies. Here, we identify a new role of the pro-autophagic protein AMBRA1 as interactor of REN/KCTD11 (here REN), the substrate-receptor subunit of the Cullin3-RING ubiquitin ligase complex (CRL3REN) and tumor suppressor lost in ~30% of human SHH-MBs. We show a novel mechanism of action of AMBRA1 in SHH-MB pathogenesis whereby its highly expression, due to loss of REN, drives the activation of GLI1, the final effector of SHH signaling. METHODS Proteomic analysis, co-immunoprecipitation, ubiquitylation and cycloheximide assays have been performed to demonstrate that AMBRA1 is a substrate of ubiquitylation of CRL3REN and to define AMBRA1 mechanism of action in SHH-MB tumorigenesis. Transcriptomic analysis, in vitro and in vivo proliferation assays have been carried out on murine and patient-derived xenograft (PDX) models to examine the effect of AMBRA1 modulation on SHH-MB growth. RESULTS We demonstrate that CRL3REN binds and induces polyubiquitylation and proteasome-mediated degradation of AMBRA1. Interestingly, we found that genetic depletion of AMBRA1 strongly reduces SHH-dependent MB growth both in vitro and in vivo, an effect associated with the decrease in the expression of GLI1 and other SHH target genes. We hypothesize that AMBRA1 could impact on the stability of GLI1 by counteracting the activity of E3 ubiquitin ligases regulating GLI proteins. Remarkably, transcriptomic analysis of SHH PDX upon AMBRA1 knockdown revealed an impairment of SHH activity and an upregulation of cerebellum differentiation. CONCLUSION Our findings identify AMBRA1 as a novel CRL3REN substrate and promising therapeutic target for SHH-dependent cancer.

MDB-79. THE DEUBIQUITINATING ENZYME USP7 COOPERATES WITH SONIC HEDGEHOG SIGNALING TO PROMOTE MEDULLOBLASTOMA

Abstract BACKGROUND Constitutive activation of the Sonic Hedgehog (SHH) signaling pathway during cerebellar development may lead to medulloblastoma (MB), one of the most common malignant pediatric brain tumor. Clinically, SHH-MB with mutations downstream of SMO or TP53-mutated are a real challenge and alternative targeted therapies are sorely needed for this group of patients. In SHH-MB, the transcription factor Atoh1 is constitutively overexpressed and essential for tumor initiation and progression. Previous work demonstrated that proteasomal-mediated degradation of Atoh1 induces tumor regression in vitro and in vivo, therefore deciphering mechanisms controlling Atoh1 stabilization should provide new avenues for drug development in SHH-MB. METHODS Using a proteomic approach, we identified the deubiquitinating enzyme Usp7 as a positive regulator of Atoh1 in MB cells. We then applied a SILAC-based quantitative ubiquitinomics to investigate the Usp7 target landscape in SHH-MB. We also employed several biochemical approaches to demonstrate the functional interaction between Atoh1 and Usp7 in SHH-MB. In parallel, we validated the impact of Usp7 depletion in vitro and in vivo using relevant SHH-MB models. Last, we investigated large transcriptomic and proteomic datasets to unveil the prognostic value of Usp7 in SHH-MB human tumors. RESULTS We demonstrate that Usp7 binds and accumulates Atoh1 protein by counteracting its ubiquitylation. Post-natal electroporation in vivo directly into the developing cerebella also indicates that overexpression of Usp7 promotes proliferation, whereas in the absence of Atoh1 Usp7 was no longer able to spread cell growth, demonstrating a functional link between the Usp7-Atoh1 axis. In vivo, disruption of Usp7 expression in SHH-MB blocks tumor proliferation. Pharmacological inhibition of Usp7 triggers tumor growth in a syngeneic SHH MB mouse model. Lastly, SHH-MB cells with either TP53 or SMO downstream mutations are sensitive to Usp7 inhibition. CONCLUSIONS Overall, our data pinpoint Usp7 as a promising target against Atoh1, a master regulator of SHH-MB.

A novel missense variant located within the zinc finger domain of the GLI3 gene was identified in a Vietnamese pedigree with index finger polydactyly.

Polydactyly, particularly of the index finger, remains an intriguing anomaly for which no specific gene or locus has been definitively linked to this phenotype. In this study, we conducted an investigation of a three-generation family displaying index finger polydactyly. Exome sequencing was conducted on the patient, with a filtration to identify potential causal variation. Validation of the obtained variant was conducted by Sanger sequencing, encompassing all family members. Exome analysis uncovered a novel heterozygous missense variant (c.1482A>T; p.Gln494His) at the zinc finger DNA-binding domain of the GLI3 protein within the proband and all affected family members. Remarkably, the variant was absent in unaffected individuals within the pedigree, underscoring its association with the polydactyly phenotype. Computational analyses revealed that GLI3 p.Gln494His impacts a residue that is highly conserved across species. The GLI3 zinc finger DNA-binding region is an essential part of the Sonic hedgehog signaling pathway, orchestrating crucial aspects of embryonic development through the regulation of target gene expression. This novel finding not only contributes valuable insights into the molecular pathways governing polydactyly during embryonic development but also has the potential to enhance diagnostic and screening capabilities for this condition in clinical settings.