Examination of piperonyl butoxide developmental toxicity as a Sonic hedgehog pathway inhibitor targeting limb and palate morphogenesis.

Piperonyl butoxide (PBO) is a pesticide synergist with widespread use and human exposure that was discovered to inhibit Sonic hedgehog (Shh) signaling, a pathway required for numerous developmental processes. Previous examinations of PBO's potential for developmental toxicity have generated seemingly conflicting results. We investigated the impact of acute PBO exposure targeting Shh pathway activity during palate and limb morphogenesis. Timed-pregnant C57BL/6 J mice were exposed to a single PBO dose (67-1800 mg/kg) at gestational day (GD) 9.75, and litters were collected at GD10.25 and GD10.75 to examine Shh pathway activity or GD17 for phenotypic assessment. PBO exposure induced dose-dependent limb malformations and cleft palate in the highest dose group. Following PBO exposure, reduced expression of the Shh pathway activity markers Gli1 and Ptch1 was observed in the embryonic limb buds and craniofacial processes. These findings provide additional evidence that prenatal PBO exposure targeting Shh pathway activity can result in malformations in mice that parallel common etiologically complex human birth defects.

Cdc14B/Cyclin B1 signaling modulates the pathogenesis of sonic hedgehog subtype medulloblastoma.

Medulloblastoma (MB) is a severe malignancy of the central nervous system that predominantly occurs in the cerebellum of children. Overactivation of the sonic hedgehog (Shh) signaling pathway is the primary cause of the development and progression of Shh subtype MB, although the detailed mechanisms underlying this process remain largely elusive. In this study, we discovered that Shh can promote proliferation in MB cells through non-canonical Hedgehog signaling. This involves Shh binding to Patched 1, disrupting its interaction with Cyclin B1, allowing for nuclear translocation of Cyclin B1, and inducing the activation of genes involved in cell division. Furthermore, we observed that deregulation of Cdc14B leads to the stabilization of the Cyclin B1/CDK1 complex in MB cells through activating Cdc25C, a phosphatase known to help maintain Cyclin B1 stability. Our findings highlight the role of Cdc14B/Cdc25C/CDK1/Cyclin B1 in mediating Hedgehog signaling-driven pathogenesis in MB and have implications for identifying potential therapeutic targets.

Non-canonical non-genomic morphogen signaling in anucleate platelets: a critical determinant of prothrombotic function in circulation.

Circulating platelets derived from bone marrow megakaryocytes play a central role in thrombosis and hemostasis. Despite being anucleate, platelets express several proteins known to have nuclear niche. These include transcription factors and steroid receptors whose non-genomic functions are being elucidated in platelets. Quite remarkably, components of some of the best-studied morphogen pathways, namely Notch, Sonic Hedgehog (Shh), and Wnt have also been described in recent years in platelets, which regulate platelet function in the context of thrombosis as well as influence their survival. Shh and Notch pathways in stimulated platelets establish feed-forward loops of autocrine/juxtacrine/paracrine non-canonical signaling that helps perpetuate thrombosis. On the other hand, non-canonical Wnt signaling is part of a negative feedback loop for restricting platelet activation and possibly limiting thrombus growth. The present review will provide an overview of these signaling pathways in general. We will then briefly discuss the non-genomic roles of transcription factors and steroid receptors in platelet activation. This will be followed by an elaborate description of morphogen signaling in platelets with a focus on their bearing on platelet activation leading to hemostasis and thrombosis as well as their potential for therapeutic targeting in thrombotic disorders.

Folate deficiency reduced aberrant level of DOT1L-mediated histone H3K79 methylation causes disruptive SHH gene expression involved in neural tube defects.

BACKGROUND: Neural tube defects (NTDs) are one of the most severe congenital abnormalities characterized by failures of the neural tube to close during early embryogenesis. Maternal folate deficiency could impact the occurrence of NTDs, however, the mechanisms involved in the cause of NTDs are poorly defined. RESULTS: Here, we report that histone H3 methyltransferase disruptor of telomeric silencing 1-like (DOT1L) expression was significantly downregulated, and low levels of H3K79me2 were found in the corresponding NTDs samples with their maternal serum folate under low levels. Using ChIP-seq assays, we found that a decrease of H3K79me2 downregulates the expression of Shh and Sufu in mouse embryonic stem cells (mESC) under folate deficiency. Interestingly, folate antagonist methotrexate treatment led to attenuation of H3K79me2 due to Dot1l, affecting Shh and Sufu genes regulation. Upon further analysis, we find that the genes Shh and Sufu are both downregulated in the brain tissues of mice and humans with NTDs. There was a positive correlation between the transcription levels of Shh, Sufu and the protein levels of DOT1L by Pearson correlation analysis. CONCLUSION: Our results indicate that abnormal Shh and Sufu genes expression reduced by aberrant Dot1l-mediated H3K79me2 levels could be the cause of NTDs occurrence.

Hedgehog signalling in allograft vasculopathy: a new therapeutic target?

Allograft vasculopathy (AV) leads to chronic rejection of organ transplants, but its causes are obscure. New research from the Jane-Wit laboratory showed that Sonic Hedgehog (SHH) signalling from damaged graft endothelium drives vasculopathy by promoting proinflammatory cytokine production and NLRP3-inflammasome activation in alloreactive CD4+PTCH1hiPD-1hiT memory cells, offering new diagnostic and therapeutic strategies.

Interference of sympathetic overactivation restores limbal stem/progenitor cells function and accelerates corneal epithelial wound healing in diabetic mice.

Diabetic keratopathy (DK), the diabetic complication in the cornea, is characterized by the delayed epithelial regeneration and sensory nerve degeneration. The involvement of limbal stem/progenitor cells (LSPCs) dysfunction has been reported, however the pathogenic mechanisms remain unclear. Here, we confirmed the dysfunction of LSPCs in diabetic mouse and human corneas. The sympathetic nerve in the cornea was adjacent to LSPCs, and the sympathetic overactivation was found in diabetic mice. Surgical and pharmacological ablation of sympathetic nerves rescued the LSPCs function and promoted corneal epithelial regeneration in diabetic mice. In contrast, both topical norepinephrine (NE) application and chemogenetic sympathetic overactivation directly impaired the stemness and proliferation characteristics of LSPCs, as well as the normal epithelial regeneration. Moreover, we identified that ß2-adrenoceptor (Adrb2) was the predominant adrenergic receptor expressed in LSPCs by corneal limbal single-cell sequencing and real time PCR (RT-PCR) analysis of sorted LSPCs. The Adrb2 knockout mice exhibited the enhancement of epithelial regeneration and LSPCs function, compared with the wild-type mice. Similarly, topical application of the Adrb2 specific antagonist ICI 118, 551 effectively accelerated diabetic corneal epithelial regeneration with the restored LSPCs function. Mechanistically, sonic hedgehog (Shh) activity mediated the downstream effects of NE-Adrb2 signaling pathway in regulating LSPCs and epithelial regeneration. Taken together, our data revealed the involvement of sympathetic overactivation in the impairment of diabetic LSPCs function and corneal epithelial regeneration through the NE-Adrb2-Shh signaling pathway. The interference of sympathetic overactivation may provide novel treatment strategies for diabetic keratopathy.

Adult Medulloblastoma: Updates on Current Management and Future Perspectives.

Medulloblastoma (MB) is a malignant embryonal tumor of the posterior fossa belonging to the family of primitive neuro-ectodermic tumors (PNET). MB generally occurs in pediatric age, but in 14-30% of cases, it affects the adults, mostly below the age of 40, with an incidence of 0.6 per million per year, representing about 0.4-1% of tumors of the nervous system in adults. Unlike pediatric MB, robust prospective trials are scarce for the post-puberal population, due to the low incidence of MB in adolescent and young adults. Thus, current MB treatments for older patients are largely extrapolated from the pediatric experience, but the transferability and applicability of these paradigms to adults remain an open question. Adult MB is distinct from MB in children from a molecular and clinical perspective. Here, we review the management of adult MB, reporting the recent published literature focusing on the effectiveness of upfront chemotherapy, the development of targeted therapies, and the potential role of a reduced dose of radiotherapy in treating this disease.

Shh and Olig2 sequentially regulate oligodendrocyte differentiation from hiPSCs for the treatment of ischemic stroke.

Rationale: Demyelination is a major component of white matter injury, characterized by oligodendrocyte (OL) death and myelin sheath loss, which result in memory loss and cognitive impairment in the context of ischemic stroke. Accumulating evidence has shown that OLs can be generated by the direct activation of defined transcription factors (TFs) in human induced pluripotent stem cells (hiPSCs); however, the rapid acquisition of single TF-induced OL progenitor cells (OPCs) as cell therapy for ischemic stroke remains to be thoroughly explored. Methods: A stable, chemically defined protocol was used to generate a substantial number of transplantable and functional OLs through the partial inhibition of sonic hedgehog (Shh) activity by GANT61 during neural induction from hiPSCs and sequential induction of TF Olig2 overexpression. Transcriptome and metabolome analyses further revealed a novel molecular event in which Olig2 regulates OL differentiation from hiPSC-derived neural progenitor cells (NPCs). Olig2-induced NG2+ OPCs (Olig2-OPCs) were then evaluated for their therapeutic potential in cell-based therapy for ischemic stroke. Results: GANT61 treatment resulted in a motor neuron (MN)-OL fate switch during neural induction, and induced overexpression of Olig2 accelerated oligodendroglial lineage cell specification. Olig2-OPCs expressed typical oligodendroglial lineage marker genes, including NKX2.2, CSPG4, and ST8SIA1, and displayed superior ability to differentiate into mature OLs in vitro. Mechanistically, Olig2-OPCs showed increased gene expression of the peroxisome proliferator-activated receptor γ (PPARγ) signaling pathway, and activated CEPT1-mediated phospholipogenesis. Functionally, inhibiting PPARγ and knocking down CEPT1 further compromised the terminal differentiation of Olig2-OPCs. Most importantly, when transplanted into a rat model of transient middle cerebral artery occlusion (tMCAO), Olig2-OPCs efficiently promoted neurological functional recovery by reducing neuronal death, promoting remyelination, and rescuing spatial memory decline. Conclusions: We developed a stable, chemically defined protocol to generate OPCs/OLs with partial inhibition of Shh activity by GANT61 from hiPSCs and sequentially induced the expression of the single TF Olig2. Olig2-OPC transplantation may be an ideal alternative approach for ischemic stroke rehabilitation therapy.

The Cross-Talks Among Bone Morphogenetic Protein (BMP) Signaling and Other Prominent Pathways Involved in Neural Differentiation.

The bone morphogenetic proteins (BMPs) are a group of potent morphogens which are critical for the patterning, development, and function of the central nervous system. The appropriate function of the BMP pathway depends on its interaction with other signaling pathways involved in neural differentiation, leading to synergistic or antagonistic effects and ultimately favorable biological outcomes. These opposite or cooperative effects are observed when BMP interacts with fibroblast growth factor (FGF), cytokines, Notch, Sonic Hedgehog (Shh), and Wnt pathways to regulate the impact of BMP-induced signaling in neural differentiation. Herein, we review the cross-talk between BMP signaling and the prominent signaling pathways involved in neural differentiation, emphasizing the underlying basic molecular mechanisms regarding the process of neural differentiation. Knowing these cross-talks can help us to develop new approaches in regenerative medicine and stem cell based therapy. Recently, cell therapy has received significant attention as a promising treatment for traumatic or neurodegenerative diseases. Therefore, it is important to know the signaling pathways involved in stem cell differentiation toward neural cells. Our better insight into the cross-talk of signaling pathways during neural development would improve neural differentiation within in vitro tissue engineering approaches and pre-clinical practices and develop futuristic therapeutic strategies for patients with neurological disease.

Deleterious Rare Mutations of GLI1 Dysregulate Sonic Hedgehog Signaling in Human Congenital Heart Disease.

The Glioma-associated oncogene (Gli) family members of zinc finger DNA-binding proteins are core effectors of Sonic hedgehog (SHH) signaling pathway. Studies in model organisms have identified that the Gli genes play critical roles during organ development, including the heart, brain, kidneys, etc. Deleterious mutations in GLI genes have previously been revealed in several human developmental disorders, but few in congenital heart disease (CHD). In this study, the mutations in GLI1-3 genes were captured by next generation sequencing in human cohorts composed of 412 individuals with CHD and 213 ethnically matched normal controls. A total of 20 patient-specific nonsynonymous rare mutations in coding regions of human GLI1-3 genes were identified. Functional analyses showed that GLI1 c.820G> T (p.G274C) is a gain-of-function mutation, while GLI1 c.878G>A (p.R293H) and c.1442T>A (p.L481X) are loss-of-function mutations. Our findings suggested that deleterious rare mutations in GLI1 gene broke the balance of the SHH signaling pathway regulation and may constitute a great contribution to human CHD, which shed new light on understanding genetic mechanism of embryo cardiogenesis regulated by SHH signaling.

Bone-targeted nanoplatform enables efficient modulation of bone tumor microenvironment for prostate cancer bone metastasis treatment.

As there is currently no effective therapy for patients with prostate cancer (PCa) bone metastasis, it was stringent to explore the relevant treatment strategies. Actually, the interaction between cancer cells and bone microenvironment plays important role in prostate cancer bone metastasis, especially the Sonic hedgehog protein (SHH) signaling in the bone microenvironment. The SHH promotes osteoblast maturation and osteoblast then secretes RANKL to induce osteoclastogenesis. Herein, this study develops bone-targeting calcium phosphate lipid hybrid nanoparticles (NPs) loaded with docetaxel (DTXL) and SHH siRNA for PCa bone metastasis treatment. For bone targeting purposes, the nanoplatform was modified with alendronate (ALN). (DTXL + siRNA)@NPs-ALN NPs effectively change the bone microenvironment by inhibiting the SHH paracrine and autocrine signaling, enhancing the anti-tumor effects of DTXL. Besides showing good in vitro cellular uptake, the NPs-ALN also inhibited tumor growth both in vitro and in vivo by inducing apoptosis, cell cycle arrest, and autophagy. This DDS comprised of (DTXL + siRNA)-loaded NPs provides an excellent strategy to treat PCa bone metastasis.

Corrigendum: High Circulating Sonic Hedgehog Protein Is Associated With Poor Outcome in EGFR-Mutated Advanced NSCLC Treated With Tyrosine Kinase Inhibitors.

[This corrects the article DOI: 10.3389/fonc.2021.747692.].

GLI3 Is Required for OLIG2+ Progeny Production in Adult Dorsal Neural Stem Cells.

The ventricular-subventricular zone (V-SVZ) is a postnatal germinal niche. It holds a large population of neural stem cells (NSCs) that generate neurons and oligodendrocytes for the olfactory bulb and (primarily) the corpus callosum, respectively. These NSCs are heterogeneous and generate different types of neurons depending on their location. Positional identity among NSCs is thought to be controlled in part by intrinsic pathways. However, extrinsic cell signaling through the secreted ligand Sonic hedgehog (Shh) is essential for neurogenesis in both the dorsal and ventral V-SVZ. Here we used a genetic approach to investigate the role of the transcription factors GLI2 and GLI3 in the proliferation and cell fate of dorsal and ventral V-SVZ NSCs. We find that while GLI3 is expressed in stem cell cultures from both dorsal and ventral V-SVZ, the repressor form of GLI3 is more abundant in dorsal V-SVZ. Despite this high dorsal expression and the requirement for other Shh pathway members, GLI3 loss affects the generation of ventrally-, but not dorsally-derived olfactory interneurons in vivo and does not affect trilineage differentiation in vitro. However, loss of GLI3 in the adult dorsal V-SVZ in vivo results in decreased numbers of OLIG2-expressing progeny, indicating a role in gliogenesis.

Molecular Targeted Therapies: Time for a Paradigm Shift in Medulloblastoma Treatment?

Medulloblastoma is a rare malignancy of the posterior cranial fossa. Although until now considered a single disease, according to the current WHO classification, it is a heterogeneous tumor that comprises multiple molecularly defined subgroups, with distinct gene expression profiles, pathogenetic driver alterations, clinical behaviors and age at onset. Adult medulloblastoma, in particular, is considered a rarer "orphan" entity in neuro-oncology practice because while treatments have progressively evolved for the pediatric population, no practice-changing prospective, randomized clinical trials have been performed in adults. In this scenario, the toughest challenge is to transfer the advances in cancer genomics into new molecularly targeted therapeutics, to improve the prognosis of this neoplasm and the treatment-related toxicities. Herein, we focus on the recent advances in targeted therapy of medulloblastoma based on the new and deeper knowledge of disease biology.

A Direct in vitro Fatty Acylation Assay for Hedgehog Acyltransferase.

Several assays have been developed to monitor the in vitro catalytic activity of Hedgehog acyltransferase (Hhat), an enzyme critical to the Hedgehog signaling pathway in cells. However, the majority of these previously reported assays involve radioactive fatty acyl donor substrates, multiple steps to achieve product readout, or specialized equipment. To increase safety, efficiency, and convenience, we developed a direct, fluorescent in vitro assay to monitor Hhat activity. Our assay utilizes purified Hhat, a fluorescently labeled fatty acyl-CoA donor substrate, and a Sonic hedgehog (Shh) peptide recipient substrate sufficient for fatty acylation. The protocol is a straightforward process that yields direct readout of fatty acylated Shh peptide via fluorescence detection of the transferred fatty acyl group. This protocol was validated in: J Biol Chem (2022), DOI: 10.1016/j.jbc.2022.102422 Graphical abstract Graphical abstract adapted from Schonbrun and Resh (2022).

Silencing GS Homeobox 2 Alleviates Gemcitabine Resistance in Pancreatic Cancer Cells by Activating SHH/GLI1 Signaling Pathway.

Sonic hedgehog (SHH) signaling pathway and glioma-associated oncogene homolog 1 (GLI1) play important roles in the initiation and progression of pancreatic ductal adenocarcinoma (PDAC). GS homeobox 2 (GSX2, formerly GSH2) is a downstream target of SHH signaling, but its role in pancreatic cancer remains unclear. This study evaluates the role of GSH2 in the development and drug resistance of pancreatic cancer. Both cell culture and xenograft mouse model were used. Immunohistochemistry, Western blotting and quantitative RT-PCR were used to examine the expression of GSH2 and other related molecules. CCK8 assay was used to test the cell proliferation, and flow cytometry used to examine cell apoptosis upon gemcitabine treatment. It was found that GSH2 is overexpressed in human pancreatic cancer tissues and cells. The expression of SHH and GLI1 was reversely correlated with GSH2 in pancreatic cancer cells. SHH and GLI1 have protein-protein interactions with GSH2. GSH2 silencing in pancreatic cancer cells inhibited cell proliferation, migration and invasion, increased cell apoptosis and sensitized pancreatic cancer cells to gemcitabine treatment. Furthermore, in vivo study demonstrated that silencing GSH2 increased the efficacy of gemcitabine-based treatment. Our results indicate that GSH2 is overexpressed in pancreatic cancer. GSH2 silencing in pancreatic cancer alleviates gemcitabine resistance by activating SHH/GLI1 pathway. Thus, targeting GSH2 in PDAC could be a novel cancer therapeutic strategy.

High Circulating Sonic Hedgehog Protein Is Associated With Poor Outcome in EGFR-Mutated Advanced NSCLC Treated With Tyrosine Kinase Inhibitors.

INTRODUCTION: Growing preclinical evidence has suggested that the Sonic hedgehog (Shh) pathway is involved in resistance to tyrosine kinase inhibitor (TKI) therapy for EGFR-mutated (EGFRm) non-small cell lung cancer (NSCLC). However, little is known concerning the prognostic value of this pathway in this context. MATERIALS AND METHODS: We investigated the relationship between plasma levels of Shh and EGFRm NSCLC patients' outcome with EGFR TKIs. We included 74 consecutive patients from two institutions with EGFRm advanced NSCLC treated by EGFR TKI as first-line therapy. Plasma samples were collected longitudinally for each patient and were analyzed for the expression of Shh using an ELISA assay. The activation of the Shh-Gli1 pathway was assessed through immunohistochemistry (IHC) of Gli1 and RT-qPCR analysis of the transcripts of Gli1 target genes in 14 available tumor biopsies collected at diagnosis (baseline). RESULTS: Among the 74 patients, only 61 had baseline (diagnosis) plasma samples, while only 49 patients had plasma samples at the first evaluation. Shh protein was detectable in all samples at diagnosis (n = 61, mean = 1,041.2 ± 252.5 pg/ml). Among the 14 available tumor biopsies, nuclear expression of Gli1 was observed in 57.1% (8/14) of patients' biopsies. Shh was significantly (p < 0.05) enriched in youth (age < 68), male, nonsmokers, patients with a PS > 1, and patients presenting more than 2 metastatic sites and L858R mutation. Higher levels of Shh correlated with poor objective response to TKI, shorter progression-free survival (PFS), and T790M-independent mechanism of resistance. In addition, the rise of plasma Shh levels along the treatment was associated with the emergence of drug resistance in patients presenting an initial good therapy response. CONCLUSION: These data support that higher levels of plasma Shh at diagnosis and increased levels of Shh along the course of the disease are related to the emergence of TKI resistance and poor outcome for EGFR-TKI therapy, suggesting that Shh levels could stand both as a prognostic and as a resistance biomarker for the management of EGFR-mutated NSCLC patients treated with EGFR-TKI.

Intertwined Signaling Pathways Governing Tooth Development: A Give-and-Take Between Canonical Wnt and Shh.

Teeth play essential roles in life. Their development relies on reciprocal interactions between the ectoderm-derived dental epithelium and the underlying neural crest-originated mesenchyme. This odontogenic process serves as a prototype model for the development of ectodermal appendages. In the mouse, developing teeth go through distinct morphological phases that are tightly controlled by epithelial signaling centers. Crucial molecular regulators of odontogenesis include the evolutionarily conserved Wnt, BMP, FGF and sonic hedgehog (Shh) pathways. These signaling modules do not act on their own, but are closely intertwined during tooth development, thereby outlining the path to be taken by specific cell populations including the resident dental stem cells. Recently, pivotal Wnt-Shh interaction and feedback loops have been uncovered during odontogenesis, showing conservation in other developing ectodermal appendages. This review provides an integrated overview of the interplay between canonical Wnt and Shh throughout mouse tooth formation stages, extending from the initiation of dental placode to the fully formed adult tooth.

Sonic Hedgehog Induces Mesenchymal Stromal Cell Senescence-Associated Secretory Phenotype and Chondrocyte Apoptosis in Human Osteoarthritic Cartilage.

Hedgehog (HH) signaling plays a critical role in osteoarthritis (OA) pathogenesis, but the molecular mechanism remains to be elucidated. We show here that Sonic Hedgehog (SHH) gene expression is initiated in human normal cartilage stromal cells (NCSC) and increased in OA cartilage mesenchymal stromal cells (OA-MSCs) during aging. Manifesting a reciprocal cellular distribution pattern, the SHH receptors PTCH1 and SMO and transcription factors GLI2 and GLI3 are expressed by chondrocytes (OAC) in OA cartilage. SHH autocrine treatment of osteoarthritis MSC stimulates proliferation, chondrogenesis, hypertrophy, and replicative senescence with elevated SASP gene expression including IL1B, IL6, CXCL1, and CXCL8. SHH paracrine treatment of OAC suppresses COL2A1, stimulates MMP13, and induces chondrocyte apoptosis. The OA-MSC conditioned medium recapitulates the stimulatory effects of SHH on OAC catabolism and apoptosis. SHH knock-down in OA-MSC not only inhibits catabolic and senescence marker expression in OA-MSC, but also abolishes the effect of the OA-MSC conditioned medium on OAC catabolism and apoptosis. We propose that SHH is a key mediator between OA-MSC and OA chondrocytes interaction in human OA cartilage via two mechanisms: (1) SHH mediates MSC growth and aging by activating not only its proliferation and chondrogenesis, but also low-grade inflammation and replicative senescence, and (2) SHH mediates OA-MSC-induced OAC catabolism and apoptosis by creating a pro-inflammatory microenvironment favoring tissue degeneration during OA pathogenesis.