An In Silico Study on Withania somnifera Bioactives and Curcumin Analogs as Potential Inducers of Smoothened (Smo) Receptor of Sonic Hedgehog (SHH) Pathway to Promote Oligodendrogenesis.

Demyelinating disorder is a subset of neurodegenerative conditions wherein factors such as aging and/or auto-immune attack cause damage and degradation of myelin sheath which enwraps the neuronal axons. Lowered axonal integrity and sub-par conduction of nerve impulses due to impaired action potentials make neurodegeneration imminent as the neurons do not have mitotic ability to replenish their numbers. Oligodendrocytes (OLs) myelinate the axonal segments of neurons and perform neuronal maintenance. Neuroregenerative stem cell therapy exploits this property for remyelination by targeting OL replenishment using in vitro stem cell differentiation protocols for inducing OL lineage cells. But some shortcomings of such protocols are over-reliance on synthetic inducers, lengthy differentiation process, low differentiation efficiency besides being financially expensive. This in silico study sought to identify herbal substitutes of currently available OL-lineage-specific synthetic inducers from a virtual library of curcumin analogs and Withania somnifera bioactives. Smoothened (Smo) receptor belonging to the canonical sonic hedgehog (SHH) signaling pathway promotes in vivo differentiation of OLs as well as their subsequent lineage progression to myelinating OLs. Therefore, we performed pharmacokinetics prediction for the bioactives followed by their molecular docking and molecular dynamics simulation with Smo. From a pool of 1289 curcumin analogs and 80 Withania somnifera-derived bioactives, the best docked ligands were identified as the compounds with PubChem IDs 68815167 and 25880, respectively. Molecular dynamics simulation of these ligands further concluded the Withania somnifera bioactive 25880 to have the best activity with Smo. This compound may be deemed as a potential lead molecule for an agonistic interaction with and activation of Smo to initialize its downstream signaling cascade for enriching OL differentiation.

Surgical Debulking Modifies Notch Signaling and May Improve Vismodegib Effectiveness for Locally Advanced Basal Cell Carcinoma.

Smoothened inhibitors, such as vismodegib, exhibit remarkable success in treating patients with locally advanced basal cell carcinoma (LaBCC). Yet, vismodegib efficacy is hindered by notable side effects, which often lead to treatment discontinuation and subsequent relapse in patients with LaBCC. Prolonged remission was previously reported in patients with LaBCCs who underwent surgical debulking before starting vismodegib. In this study, we enrolled 4 patients with LaBCC who underwent debulking followed by vismodegib therapy to assess their clinical outcomes and analyze the cutaneous molecular changes occurring as a result of surgical intervention. After LaBCC debulking, patients underwent a punch biopsy of residual basal cell carcinoma tissue 1 week later. RT-qPCR analysis of 24 Notch and Wnt signaling-associated genes revealed elevated PTCH1, HEY2, LGR6, FZD2, LEF1, ALCAM, and RUNX1 expressions in follow-up biopsies compared with those in patient-matched debulked tissue. Immunoblot and immunostaining further confirmed elevated Notch signaling in follow-up biopsy tissue compared with that in patient-matched debulked tumor tissue. Patients 1, 3, and 4 displayed a clinical response to debulking followed by vismodegib, whereas patient 2 was lost to follow-up after debulking. These findings suggest that surgical manipulation of LaBCCs is correlated with molecular alterations in signaling pathways associated with cellular reprogramming.

Identification of Meibomian gland stem cell populations and mechanisms of aging.

Meibomian glands secrete lipid-rich meibum, which prevents tear evaporation. Aging-related Meibomian gland shrinkage may result in part from stem cell exhaustion and is associated with evaporative dry eye disease, a common condition lacking effective treatment. The identities and niche of Meibomian gland stem cells and the signals controlling their activity are poorly defined. Using snRNA-seq, in vivo lineage tracing, ex vivo live imaging, and genetic studies in mice, we identified markers for stem cell populations that maintain distinct regions of the gland and uncovered Hh signaling as a key regulator of stem cell proliferation. Consistent with this, human Meibomian gland carcinoma exhibited increased Hh signaling. Aged glands displayed decreased Hh and EGF signaling, deficient innervation, and loss of collagen I in niche fibroblasts, indicating that alterations in both glandular epithelial cells and their surrounding microenvironment contribute to age-related degeneration. These findings suggest new approaches to treat aging-associated Meibomian gland loss.

Oral smoothened inhibitors for Gorlin syndrome: A clinical review.

Although smoothened inhibitors (SMOi) have demonstrated efficacy in the management of basal cell carcinoma, no guidelines are available on how to utilize SMOi in the treatment of Gorlin syndrome (GS). This review's objective is to assess the clinical response to SMOi in GS, provide practical guidance for clinicians, and identify areas for future research. Through comprehensive searches of previous publications and expert opinion, this review demonstrates that intermittent dosing of SMOi and daily dosing have similar efficacy. While the adverse events of SMOi may result in their discontinuation during treatment of GS, intermittent dosing may improve compliance.

Roles of astrocytic sonic hedgehog production and its signal for regulation of the blood-brain barrier permeability.

Sonic hedgehog (Shh) is a secreted glycopeptide belonging to the hedgehog family that is essential for morphogenesis during embryonic development. The Shh signal is mediated by two membrane proteins, Patched-1 (Ptch-1) and Smoothened (Smo), following the activation of transcription factors such as Gli. Shh decreases the permeability of the blood-brain barrier (BBB) and plays a key role in its function. In the damaged brain, BBB function is remarkably disrupted. The BBB disruption causes brain edema and neuroinflammation resulting from the extravasation of serum components and the infiltration of inflammatory cells into the cerebral parenchyma. Multiple studies have suggested that astrocyte is a source of Shh and that astrocytic Shh production is increased in the damaged brain. In various experimental animal models of acute brain injury, Shh or Shh signal activators alleviate BBB disruption by increasing tight junction proteins in endothelial cells. Furthermore, activation of astrocytic Shh signaling reduces reactive astrogliosis, neuroinflammation, and increases the production of vascular protective factors, which alleviates BBB disruption in the damaged brain. These findings suggest that astrocytic Shh and Shh signaling protect BBB function in the damaged brain and that target drugs for Shh signaling are expected to be novel therapeutic drugs for acute brain injuries.

Activation of Shh/Smo is sufficient to maintain oligodendrocyte precursor cells in an undifferentiated state and is not necessary for myelin formation and (re)myelination.

Myelination is the terminal step in a complex and precisely timed program that orchestrates the proliferation, migration and differentiation of oligodendroglial cells. It is thought that Sonic Hedgehog (Shh) acting on Smoothened (Smo) participates in regulating this process, but that these effects are highly context dependent. Here, we investigate oligodendroglial development and remyelination from three specific transgenic lines: NG2-CreERT2 (control), Smofl/fl/NG2-CreERT2 (loss of function), and SmoM2/NG2-CreERT2 (gain of function), as well as pharmacological manipulation that enhance or inhibit the Smo pathway (Smoothened Agonist (SAG) or cyclopamine treatment, respectively). To explore the effects of Shh/Smo on differentiation and myelination in vivo, we developed a highly quantifiable model by transplanting oligodendrocyte precursor cells (OPCs) in the retina. We find that myelination is greatly enhanced upon cyclopamine treatment and hypothesize that Shh/Smo could promote OPC proliferation to subsequently inhibit differentiation. Consistent with this hypothesis, we find that the genetic activation of Smo significantly increased numbers of OPCs and decreased oligodendrocyte differentiation when we examined the corpus callosum during development and after cuprizone demyelination and remyelination. However, upon loss of function with the conditional ablation of Smo, myelination in the same scenarios are unchanged. Taken together, our present findings suggest that the Shh pathway is sufficient to maintain OPCs in an undifferentiated state, but is not necessary for myelination and remyelination.

A sterol analog inhibits hedgehog pathway by blocking cholesterylation of smoothened.

The hedgehog (Hh) signaling pathway has long been a hotspot for anti-cancer drug development due to its important role in cell proliferation and tumorigenesis. However, most clinically available Hh pathway inhibitors target the seven-transmembrane region (7TM) of smoothened (SMO), and the acquired drug resistance is an urgent problem in SMO inhibitory therapy. Here, we identify a sterol analog Q29 and show that it can inhibit the Hh pathway through binding to the cysteine-rich domain (CRD) of SMO and blocking its cholesterylation. Q29 suppresses Hh signaling-dependent cell proliferation and arrests Hh-dependent medulloblastoma growth. Q29 exhibits an additive inhibitory effect on medulloblastoma with vismodegib, a clinically used SMO-7TM inhibitor for treating basal cell carcinoma (BCC). Importantly, Q29 overcomes resistance caused by SMO mutants against SMO-7TM inhibitors and inhibits the activity of SMO oncogenic variants. Our work demonstrates that the SMO-CRD inhibitor can be a new way to treat Hh pathway-driven cancers.

Characterization of Sonic Hedgehog transcripts in the adult mouse brain: co-expression with neuronal and oligodendroglial markers.

In the adult mammalian brain, astrocytes are proposed to be the major Sonic Hedgehog (Shh)-responsive cells. However, the sources of the Shh molecule mediating activation of the pathway are still poorly characterized. The present work investigates the distribution and phenotype of cells expressing Shh mRNA in the adult mouse brain. Using single-molecule fluorescent in situ hybridization (smfISH), we report much broader expression of Shh transcripts in almost all brain regions than originally reported. We identify Shh mRNA in HuC/D+ neuronal populations, including GABAergic (glutamic acid decarboxylase 67, Gad67), cholinergic (choline acetyltransferase, ChAT), dopaminergic (tyrosine hydroxylase, TH), nitrergic (neuronal nitric oxide synthase, nNOS), and in a small population of oligodendroglial cells expressing Sox10 and Olig2 mRNA transcription factors. Further analysis of Shh mRNA in cerebral cortical and hypothalamic neurons suggests that Shh is also expressed by glutamatergic neurons. Interestingly, we did not observe substantial Desert Hedgehog and Indian Hedgehog mRNA signals, nor Shh signals in S100ß+ astrocytes and Iba1+ microglial cells. Collectively, the present work provides the most robust central map of Shh-expressing cells to date and underscores the importance of nitrergic neurons in regulating Shh availability to brain cells. Thus, our study provides a framework for future experiments aimed at better understanding of the functions of Shh signaling in the brain in normal and pathological states, and the characterization of novel regulatory mechanisms of the signaling pathway.

Association of the combined parameters including the frequency of primary cilia, PD-L1, Smoothened protein, membranous ß-catenin and cytoplasmic ß-catenin expression with the outcome of patients with clear cell renal cell carcinoma.

AIMS: The objective of this study was to investigate the association and combined prognostic significance of the PD-L1, Smoothened protein and ß-catenin expressions in patients with clear cell renal cell carcinoma (ccRCC). METHODS: The PD-L1, Smoothened protein and ß-catenin expression were evaluated in 104 ccRCC patients. All studied tumor samples were acquired from nephrectomy specimens of primary tumors and not from biopsies or metastases. An indirect immunohistochemistry using polyclonal rabbit anti-Smoothened antibody, monoclonal mouse anti-human ß-catenin-1 antibody, immunohistochemical assay PD-L1 28-8 pharmDx using monoclonal rabbit anti-PD-L1 antibody and anti-VHL (C- terminal) rabbit antibody was used. Immunohistochemistry was scored semiquantitavely. RESULTS: Median overall survival (OS) was significantly better in patients with lower PD-L1 expression (≤5%), Smoothened protein (SMO) expression (<5%) or cytoplasmic ß-catenin expression (≤75%) than in patients with higher expressions of these biomarkers (P<0.001, P=0.047, and P<0.001, respectively). Membranous ß-catenin showed an opposite effect with its lower expression (≤75%) being associated with longer OS (P=0.020). There was significant association between PD-1 and PD-L1 expression (P=0.007) and significant association of tumor grade (WHO 2016) with membranous ß-catenin (P<0.001), cytoplasmic ß-catenin (P=0.005), pVHL (P=0.042), PD-L1 (P=0.049) and PD-1 (P=0.028) expression. CONCLUSION: The present study provides the first data on the potential association and combined prognostic significance of frequency of primary cilia, PD-L1, Smoothened protein and ß-catenin expression with the outcome in clear cell renal cell carcinoma.

Downregulation of Rab23 inhibits hepatocellular carcinoma by repressing SHH signaling pathway.

Hepatocellular carcinoma (HCC) is the sixth most common malignant tumors and the third leading cause of cancer-related death worldwide. As an oncogene, Rab23 has been shown to be significantly related to the growth and migration of hepatocellular carcinoma in both in vitro and in vivo studies, but its underlying mechanism remains obscure. In the present study, we examined the effect of inhibiting Rab23 expression on the pathological progression of HCC. The correlation between liver Rab23 gene expression and survival probability in human HCC patients was analyzed using the TCGA database and CPTAC database. Rab23 knockdown hepatocellular carcinoma cell line was generated through lentiviral transduction, then we established a nude HCC xenograft model by subcutaneously implanting the transfected cells. The analysis of gene and protein expression was carried out using Western blot or RT-qPCR, respectively. Flow cytometry analysis was used to detect the level of apoptosis. The expression levels of key proteins involved in the Sonic Hedgehog (SHH) signaling pathway were assessed. The results showed that HCC patients with low levels of hepatic Rab23 mRNA and protein had a better survival tendency than those with higher levels of Rab23. Cell proliferations were reduced and apoptosis levels were increased after Knocking down Rab23 in HCC cell lines. Furthermore, in vivo studies have demonstrated that suppression of the Rab23 gene results in decreased tumor size, proliferation rate, and reduced levels of SHH-related proteins Smoothened and GLI-1. The above results suggest that Rab23 is involved in the pathological progression of HCC as an important regulator of the SHH signaling pathway, which also provides an important research basis for new therapeutic strategies for HCC.

Fully activated structure of the sterol-bound Smoothened GPCR-Gi protein complex.

Smoothened (SMO) is an oncoprotein and signal transducer in the Hedgehog signaling pathway that regulates cellular differentiation and embryogenesis. As a member of the Frizzled (Class F) family of G protein-coupled receptors (GPCRs), SMO biochemically and functionally interacts with Gi family proteins. However, key molecular features of fully activated, G protein-coupled SMO remain elusive. We present the atomistic structure of activated human SMO complexed with the heterotrimeric Gi protein and two sterol ligands, equilibrated at 310 K in a full lipid bilayer at physiological salt concentration and pH. In contrast to previous experimental structures, our equilibrated SMO complex exhibits complete breaking of the pi-cation interaction between R4516.32 and W5357.55, a hallmark of Class F receptor activation. The Gi protein couples to SMO at seven strong anchor points similar to those in Class A GPCRs: intracellular loop 1, intracellular loop 2, transmembrane helix 6, and helix 8. On the path to full activation, we find that the extracellular cysteine-rich domain (CRD) undergoes a dramatic tilt, following a trajectory suggested by positions of the CRD in active and inactive experimental SMO structures. Strikingly, a sterol ligand bound to a shallow transmembrane domain (TMD) site in the initial structure migrates to a deep TMD pocket found exclusively in activator-bound SMO complexes. Thus, our results indicate that SMO interacts with Gi prior to full activation to break the molecular lock, form anchors with Gi subunits, tilt the CRD, and facilitate migration of a sterol ligand in the TMD to an activated position.

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

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

Smoothened mediates medaka spermatogonia proliferation via Gli1-Rgcc-Cdk1 axis†.

The proliferation of spermatogonia directly affects spermatogenesis and male fertility, but its underlying molecular mechanisms are poorly understood. In this study, Smoothened (Smo), the central transducer of Hedgehog signaling pathway, was characterized in medaka (Oryzias latipes), and its role and underlying mechanisms in the proliferation of spermatogonia were investigated. Smo was highly expressed in spermatogonia. In ex vivo testicular organ culture and a spermatogonial cell line (SG3) derived from medaka mature testis, Smo activation promoted spermatogonia proliferation, while its inhibition induced apoptosis. The expression of glioma-associated oncogene homolog 1 (gli1) and regulator of cell cycle (rgcc) was significantly upregulated in SG3 after Smo activation. Furthermore, Gli1 transcriptionally upregulated the expression of rgcc, and Rgcc overexpression rescued cell apoptosis caused by Smo or Gli1 inhibition. Co-immunoprecipitation assay indicated that Rgcc could interact with cyclin-dependent kinase 1 (Cdk1) to regulate the cell cycle of spermatogonia. Collectively, our study firstly reveals that Smo mediates the proliferation of spermatogonia through Gli1-Rgcc-Cdk1 axis. In addition, Smo and Gli1 are necessary of the survival of spermatogonia. This study deepens our understanding of spermatogonia proliferation and survival at the molecular level, and provides insights into male fertility control and reproductive disease treatment.

Smoothened antagonist sonidegib affects the development of D. melanogaster larvae via suppression of epidermis formation.

Hedgehog (Hh) signaling is essential for the regulation of embryonic growth and development, the maintenance of stem cell autostasis, and tissue formation, whether in vertebrates or invertebrates. However, exploration into the Hh pathway antagonists in Drosophila or other pests of agricultural importance has been scant. In order to gain a better understanding of the potential utility of the antagonists in insect investigations, a conventional Hh antagonist, sonidegib, was used to evaluate the effects on the development of Drosophila larvae. The results showed that early instar larvae exposed to sonidegib exhibited new epidermal abnormalities and decreased motility after molting. Transcriptome analysis revealed that Sonidegib had a profound effect on chitin-based cuticle development throughout all stages of larvae. Physiological experiments revealed that sonidegib suppressed the epidermis formation and decreased the chitin content. The results of this study shed new light on the potential use of Hh antagonists in agricultural pest management.

Expression Pattern of Sonic Hedgehog, Patched and Smoothened in Clear Cell Renal Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the deadliest neoplasm of the urinary tract, and we are still far from completely understanding ccRCC development and treatment. The renal tissue paraffin blocks (20) of patients with ccRCC were collected at the University Hospital in Split from 2019 to 2020, and tissue sections were stained with patched (PTCH), anti-smoothened (SMO) and anti-Sonic Hedgehog (SHH) antibodies. SHH was highly expressed (31.9%) in grade 1 tumour, it being higher than all other grades and the control (p < 0.001-p < 0.0001). The trend of a linear decrease in the expression of SHH was observed with the progression of the tumour grade (p < 0.0001). PTCH expression was significantly lower in grades 1 and 2 in comparison to the control (p < 0.01) and grade 4 (p < 0.0001). A significant increase in the expression of SMO was found in grade 4 compared to all other grades (p < 0.0001) and the control (p < 0.001). The strong expression of SHH was observed in carcinoma cells of the G1 stage with a diffuse staining pattern (>50% of neoplastic cells). Stroma and/or inflammatory infiltrate display no staining and no expression of SHH in G1 and G2, while mild focal staining (10-50% of neoplastic cells) was observed in G3 and G4. Patients with high PTCH and low SMO expression had significant time survival differences (p = 0.0005 and p = 0.029, respectively). Therefore, high levels of PTCH and low levels of SMO expression are important markers of better survival rates in ccRCC patients.

Role of smoothened and sfrp genes in Eupentacta fraudatrix regeneration.

The secreted frizzled-related proteins (sfrp) and smoothened (smo) genes and their possible role in the regeneration of internal organs in the holothurian Eupentacta fraudatrix were studied. In this species, two sfrp genes were identified: sfrp1/2/5, sfrp3/4 and one smo gene. Their expression was analysed during regeneration of the aquapharyngeal bulb (AB) and intestine, and these genes were knock down by RNA interference. It has been shown that the expression of these genes is extremely important for the formation of AB. In all animals subjected to knockdown, at 7 days after evisceration, a full-sized AB rudiment was not formed. As a result of sfrp1/2/5 knockdown, the process of extracellular matrix remodelling in AB is interrupted, that leading to clusters of dense connective tissue formation, which slows down cell migration. When sfrp3/4 is knockdown, the connective tissue of AB anlage is completely disrupted and its symmetry is broken. The effect of smo knockdown was expressed in a significant impairment of AB regeneration, when connections between ambulacras were not formed after evisceration. However, despite severe disturbances in AB regeneration, a normal-sized gut anlage developed in all cases, which suggests that the regeneration of the digestive tube and AB occur independently of each other.

Ligand-based 3D pharmacophore modeling, virtual screening, and molecular dynamic simulation of potential smoothened inhibitors.

CONTEXT: The Hedgehog (Hh) signaling pathway is a crucial regulator of various cellular processes. Dysregulated activation of the Smoothened (SMO) oncoprotein, a key component of the Hh pathway, has been implicated in several types of cancer. Although SMO inhibitors are important anti-cancer therapeutics, drug-resistant SMO mutants have emerged, limiting their efficacy. This study aimed to discover stable SMO inhibitors for both wild-type and mutant SMOs, using a 12-feature pharmacophore model validated for virtual screening. One lead compound, LCT10312, was identified with high affinity to SMO and showed a significant conformational change in the SMO structure upon binding. Molecular dynamic simulation revealed stable interaction of LCT10312 with SMO and large atom motions, indicating SMO structural fluctuation. The lead compound showed high predicted binding scores to several clinically relevant SMO mutants. METHODS: A ligand-based pharmacophore model was developed from 25 structurally clustered SMO inhibitors using LigandScout v3.12 software and virtually screened for hit identification from a library of 511,878 chemicals. Molecular docking was employed to identify potential leads based on SMO affinities. Molecular dynamic simulation (MDS) with GROMACS v5.1.4 was performed to analyze the structural changes of SMO oncoprotein upon binding lead compound(s) and cyclopamine as the control for 100 ns. The binding affinity of lead compound(s) was predicted on clinical and laboratory SMO mutants.

The role of Smo-Shh/Gli signaling activation in the prevention of neurological and ageing disorders.

Sonic hedgehog (Shh) signaling is an essential central nervous system (CNS) pathway involved during embryonic development and later life stages. Further, it regulates cell division, cellular differentiation, and neuronal integrity. During CNS development, Smo-Shh signaling is significant in the proliferation of neuronal cells such as oligodendrocytes and glial cells. The initiation of the downstream signalling cascade through the 7-transmembrane protein Smoothened (Smo) promotes neuroprotection and restoration during neurological disorders. The dysregulation of Smo-Shh is linked to the proteolytic cleavage of GLI (glioma-associated homolog) into GLI3 (repressor), which suppresses target gene expression, leading to the disruption of cell growth processes. Smo-Shh aberrant signalling is responsible for several neurological complications contributing to physiological alterations like increased oxidative stress, neuronal excitotoxicity, neuroinflammation, and apoptosis. Moreover, activating Shh receptors in the brain promotes axonal elongation and increases neurotransmitters released from presynaptic terminals, thereby exerting neurogenesis, anti-oxidation, anti-inflammatory, and autophagy responses. Smo-Shh activators have been shown in preclinical and clinical studies to help prevent various neurodegenerative and neuropsychiatric disorders. Redox signalling has been found to play a critical role in regulating the activity of the Smo-Shh pathway and influencing downstream signalling events. In the current study ROS, a signalling molecule, was also essential in modulating the SMO-SHH gli signaling pathway in neurodegeneration. As a result of this investigation, dysregulation of the pathway contributes to the pathogenesis of various neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD).Thus, Smo-Shh signalling activators could be a potential therapeutic intervention to treat neurocomplications of brain disorders.

B13, a well-tolerated inhibitor of hedgehog pathway, exhibited potent anti-tumor effects against colorectal carcinoma in vitro and in vivo.

Abnormal activation of Hedgehog (Hh) signaling pathway mediates the genesis and progression of various tumors [1]. Currently, three drugs targeting the Hh signaling component Smoothened (Smo) have been marketed for the clinical treatment of basal cell tumors or acute myeloid leukemia. However, drug resistance is a common problem in those drugs, so the study of Smo inhibitors that can overcome drug resistance has important guiding significance for clinical adjuvant drugs. MTT assay, clone formation assay and EdU assay were used to detect the proliferation inhibitory activity of the drugs on tumor cells. The effect of B13 on cell cycle and apoptosis were detected by flow cytometry. An acute toxicity test was used to detect the toxicity of B13 in vivo, and xenograft tumor model was used to detect the efficacy of B13 in vivo. The binding of B13 to Smo was studied by BODIPY-cyclopamine competitive binding assay and molecular docking. The effect of B13 on the expression and localization of downstream target gene Gli1/2 of Smo was investigated by Western Blot and immunofluorescence assay. SmoD473H mutant cell line was constructed to study the effect of B13 against drug resistance. (1) B13 had the strongest inhibitory activity against colorectal cancer cells. (2) B13 can effectively inhibit the clone formation and EdU positive rate of colon cancer cells. (3) B13 can block the cell cycle in the G2/M phase and cell apoptosis. (4) B13 has low toxicity in vivo, and its efficacy in vivo is better than that of the Vismodegib. (5) Molecular docking and BODIPY-cyclopamine experiments showed that B13 could bind to Smo protein. (6) B13 can inhibit the protein expression of Gli1, the downstream of Smo, and inhibit its entry into the nucleus. (7) B13 could inhibit the expression of Gli1 in the HEK293 cells with SmoD473H, and the molecular docking results showed that B13 could bind SmoD473H protein. B13 with the best anti-tumor activity was screened out by MTT assay. In vitro, pharmacodynamics experiments showed that B13 could effectively inhibit the proliferation and metastasis of colorectal cancer cells, induce cell cycle arrest, and induce cell apoptosis. In vivo pharmacodynamics experiments showed that B13 was superior to Vismodegib in antitumor activity and had low toxicity in vivo. Mechanism studies have shown that B13 can bind Smo protein, inhibit the expression of downstream Gli1 and its entry into the nucleus. Notably, B13 overcomes resistance caused by SmoD473H mutations.

Activation of the Hedgehog signaling pathway leads to fibrosis in aortic valves.

BACKGROUND: Fibrosis is a pathological wound healing process characterized by excessive extracellular matrix deposition, which interferes with normal organ function and contributes to ~ 45% of human mortality. Fibrosis develops in response to chronic injury in nearly all organs, but the a cascade of events leading to fibrosis remains unclear. While hedgehog (Hh) signaling activation has been associated with fibrosis in the lung, kidney, and skin, it is unknown whether hedgehog signaling activation is the cause or the consequence of fibrosis. We hypothesize that activation of hedgehog signaling is sufficient to drive fibrosis in mouse models. RESULTS: In this study, we provide direct evidence to show that activation of Hh signaling via expression of activated smoothened, SmoM2, is sufficient to induce fibrosis in the vasculature and aortic valves. We showed that activated SmoM2 -induced fibrosis is associated with abnormal function of aortic valves and heart. The relevance of this mouse model to human health is reflected in our findings that elevated GLI expression is detected in 6 out of 11 aortic valves from patients with fibrotic aortic valves. CONCLUSIONS: Our data show that activating hedgehog signaling is sufficient to drive fibrosis in mice, and this mouse model is relevant to human aortic valve stenosis.