Hh Signaling Pathway Research Articles

Pharmacology of Veratrum californicum Alkaloids as Hedgehog Pathway Antagonists.

Veratrum californicum contains steroidal alkaloids that function as inhibitors of hedgehog (Hh) signaling, a pathway involved in the growth and differentiation of cells and normal tissue development. This same Hh pathway is abnormally active for cell proliferation in more than 20 types of cancer. In this current study, alkaloids have been extracted from the root and rhizome of V. californicum, followed by their separation into five fractions using high performance liquid chromatography. Mass spectrometry was used to identify the presence of twenty-five alkaloids, nine more than are commonly cited in literature reports, and the Bruker Compass Data Analysis software was used to predict the molecular formula for every detected alkaloid. The Gli activity of the raw extract and each fraction were compared to 0.1 µM cyclopamine, and fractions 1, 2, and 4 showed increased bioactivity through suppression of the Hh signaling pathway. Fractions 2 and 4 had enhanced bioactivity, but fraction 1 was most effective in inhibiting Hh signaling. The composition of fraction 1 consisted of veratrosine, cycloposine, and potential isomers of each.

The H2Valdien derivatives regulate the epithelial-mesenchymal transition of hepatoma carcinoma cells through the Hedgehog signaling pathway.

This research delves into the influence of H2Valdien derivatives on the proliferation, migration, and apoptosis induction in hepatoma carcinoma cells (HepG2, Huh-7, and SMMC-7721), with a specific emphasis on inhibiting epithelial-mesenchymal transition (EMT) through modulation of the Hedgehog (Hh) signaling pathway. Utilizing the cell counting kit-8 method, flow cytometry, TUNEL assay, wound healing, and transwell assays, we observed a dose-dependent growth arrest and apoptosis induction in HepG2, Huh-7, and SMMC-7721 cells. Notably, H2Valdien derivatives exhibited a capacity to reduce migration and invasion, impacting the expression of EMT-associated proteins such as N-cadherin, vimentin, and E-cadherin. Mechanistically, these derivatives demonstrated the inhibition of the Hh signaling pathway by inactivating Sonic Hh (Shh) and smoothened proteins. This study underscores the robust antiproliferative and apoptosis-inducing effects of H2Valdien derivatives on hepatoma carcinoma cells and elucidates their regulatory role in EMT through modulation of the Hh signaling pathway, providing valuable insights for potential therapeutic interventions.

USP5 promotes tumorigenesis by activating Hedgehog/Gli1 signaling pathway in osteosarcoma.

Changes in protein ubiquitination have been linked to cancer. Deubiquitinating enzymes (DUBs) counteract E3 ligase activities and have emerged as promising targets for cancer treatment. Ubiquitin-specific peptidase 5 (USP5) is a member of the DUBs family and has been implicated in promoting tumorigenesis in numerous cancers. However, the clinical significance and biological function of USP5 in osteosarcoma (OS) remains unclear. Here, we found elevated USP5 expression in OS tissues compared with normal bone tissues. Furthermore, we observed significant associations of elevated USP5 levels with increased mortality and more malignant phenotypes in OS patients. Moreover, our results revealed that USP5 could facilitate metastasis and cell progression in OS by activating the hedgehog (Hh) signaling pathway using cultured cells and animal tumor models. Mechanistically, USP5 appeared to stabilize and deubiquitinate Gli1, a key mediator of the Hh signaling pathway. Additionally, the oncogenic effect of USP5 in OS was dependent on Gli1 stability. Our findings support the model where USP5 contributes to OS pathogenesis by activating the Hh/Gli1 signaling pathway, making USP5 a potential diagnostic and therapeutic target for OS.

Current Status of Hedgehog Signaling Inhibitors.

The Hedgehog (Hh) signaling pathway plays a crucial role in diverse biological processes such as cell differentiation, proliferation, senescence, tumorigenesis, malignant transformation, and drug resistance. Aberrant Hh signaling, resulting from mutations and excessive activation, can contribute to the development of various diseases during different stages of biogenesis and development. Moreover, it has been linked to unfavorable outcomes in several human cancers, including basal cell carcinoma (BCC), multiple myeloma (MM), melanoma, and breast cancer. Hence, the presence of mutations and excessive activation of the Hh pathway presents obstacles and constraints in the realm of cancer treatment. Extant research has demonstrated that small molecule inhibitors are regarded as the most effective therapeutic approaches for targeting the Hh pathway in contrast to traditional chemotherapy and radiotherapy. Consequently, this review focuses on the present repertoire of small molecule inhibitors that target various components of the Hh pathway, including Hh ligands, Ptch receptors, Smo transmembrane proteins, and Gli nuclear transcription factors. This study provides a comprehensive analysis of small molecules' structural and functional aspects in the preclinical and clinical management of cancer. Additionally, it elucidates the obstacles encountered in targeting the Hh pathway for human cancer therapy and proposes potential therapeutic approaches.

Role of Hedgehog Signaling Pathways in Multipotent Mesenchymal Stem Cells Differentiation.

Multipotent mesenchymal stem cells (MSCs) have high self-renewal and multi-lineage differentiation potentials and low immunogenicity, so they have attracted much attention in the field of regenerative medicine and have a promising clinical application. MSCs originate from the mesoderm and can differentiate not only into osteoblasts, cartilage, adipocytes, and muscle cells but also into ectodermal and endodermal cell lineages across embryonic layers. To design cell therapy for replacement of damaged tissues, it is essential to understand the signaling pathways, which have a major impact on MSC differentiation, as this will help to integrate the signaling inputs to initiate a specific lineage. Hedgehog (Hh) signaling plays a vital role in the development of various tissues and organs in the embryo. As a morphogen, Hh not only regulates the survival and proliferation of tissue progenitor and stem populations but also is a critical moderator of MSC differentiation, involving tri-lineage and across embryonic layer differentiation of MSCs. This review summarizes the role of Hh signaling pathway in the differentiation of MSCs to mesodermal, endodermal, and ectodermal cells.

Identification of ancestral gnathostome Gli3 enhancers with activity in mammals.

Abnormal expression of the transcriptional regulator and hedgehog (Hh) signaling pathway effector Gli3 is known to trigger congenital disease, most frequently affecting the central nervous system (CNS) and the limbs. Accurate delineation of the genomic cis-regulatory landscape controlling Gli3 transcription during embryonic development is critical for the interpretation of noncoding variants associated with congenital defects. Here, we employed a comparative genomic analysis on fish species with a slow rate of molecular evolution to identify seven previously unknown conserved noncoding elements (CNEs) in Gli3 intronic intervals (CNE15-21). Transgenic assays in zebrafish revealed that most of these elements drive activities in Gli3 expressing tissues, predominantly the fins, CNS, and the heart. Intersection of these CNEs with human disease associated SNPs identified CNE15 as a putative mammalian craniofacial enhancer, with conserved activity in vertebrates and potentially affected by mutation associated with human craniofacial morphology. Finally, comparative functional dissection of an appendage-specific CNE conserved in slowly evolving fish (elephant shark), but not in teleost (CNE14/hs1586) indicates co-option of limb specificity from other tissues prior to the divergence of amniotes and lobe-finned fish. These results uncover a novel subset of intronic Gli3 enhancers that arose in the common ancestor of gnathostomes and whose sequence components were likely gradually modified in other species during the process of evolutionary diversification.

The deubiquitinating enzyme USP44 suppresses hepatocellular carcinoma progression by inhibiting Hedgehog signaling and PDL1 expression

Hepatocellular carcinoma (HCC) is one of the deadliest malignancies in the world. Research into the key genes that maintain the malignant behavior of cancer cells is crucial for the treatment of HCC. Here, we identified ubiquitin‐specific peptidase 44 (USP44), a member of the deubiquitinase family, as a novel regulator of HCC progression. The tumor suppressive function of USP44 was evaluated in a series of in vitro and in vivo experiments. Through quantitative proteomics examination, we demonstrated that USP44 inhibits HCC PDL1 expression by downregulating the Hedgehog (Hh) signaling pathway. Mechanistically, we found that USP44 directly interacts with Itch, an E3 ligase involved in Hh signaling, and promotes the deubiquitination and stabilization of Itch. These events result in the proteasomal degradation of Gli1 and subsequent inactivation of Hh signaling, which ultimately suppresses PDL1 expression and the progression of HCC. Furthermore, the HCC tissue microarray was analyzed by immunohistochemistry to evaluate the pathological relevance of the USP44/Itch/Gli1/PDL1 axis. Finally, the Gli1 inhibitor GANT61 was found to act in synergy with anti-PDL1 therapy. Overall, USP44 can act as a suppressive gene in HCC by modulating Hh signaling, and co-inhibition of Gli1 and PDL1 might be an effective novel combination strategy for treating HCC patients.

Colorectal Cancer Stem Cells and Targeted Agents.

Since their discovery, cancer stem cells have become a hot topic in cancer therapy research. These cells possess stem cell-like self-renewal and differentiation capacities and are important factors that dominate cancer metastasis, therapy-resistance and recurrence. Worse, their inherent characteristics make them difficult to eliminate. Colorectal cancer is the third-most common cancer and the second leading cause of cancer death worldwide. Targeting colorectal cancer stem cells (CR-CSCs) can inhibit colorectal cancer metastasis, enhance therapeutic efficacy and reduce recurrence. Here, we introduced the origin, biomarker proteins, identification, cultivation and research techniques of CR-CSCs, and we summarized the signaling pathways that regulate the stemness of CR-CSCs, such as Wnt, JAK/STAT3, Notch and Hh signaling pathway. In addition to these, we also reviewed recent anti-CR-CSC drugs targeting signaling pathways, biomarkers and other regulators. These will help researchers gain insight into the current agents targeting to CR-CSCs, explore new cancer drugs and propose potential therapies.

Quercetin ameliorates liver fibrosis in Wilson disease and EMT involving suppression of the Hedgehog signaling pathway

Liver fibrosis is a fundamental pathological alteration observed in individuals with Wilson disease. Quercetin (Que) has shown potential in inhibiting fibrosis in various diseases. However, the precise mechanism by which Que alleviates liver fibrosis in Wilson disease remains unclear. The present study aimed to investigate the potential role of Que in liver fibrosis of Wilson disease using male Toxic Milk (TX) mice and Cu2+-induced hepatoblastoma cells (HepG2 cells). In TX mice, Analyses using Histopathology, Immunohistochemical staining, Enzyme-linked immunosorbent assay (ELISA), and Western blot, revealed that Que treatment facilitated the efflux of Cu2+ from the liver, improvement of liver function indicators and pathological damage of liver tissue, and significantly inhibited Hedghog (Hh) signaling pathway, leading to significant downregulation of key proteins and genes involved in this pathway, including Gli1, Ptch1, Smo, and Shh. Moreover, Que can reverse epithelial-mesenchymal transition (EMT), and we have also investigated key proteins and genes of EMT and the specific transcription factors controlling E-cadherin expression at the transcriptional level (Slug, Snail, ZEB-1, and Twist) and found that Que can regulatory their expression levels. In Cu2+-induced HepG2 cells, we using Hh Signaling pathway Inhibitor: GDC-0449, found blocking Hh Signaling pathway can reverse the EMT and consistent with in vivo results. Therefore, Que may have the potential to ameliorate liver fibrosis in Wilson disease and EMT by suppressing the Hh signaling pathway.

Soluble vascular endothelial glycocalyx proteoglycans as potential therapeutic targets in inflammatory diseases.

Reducing the activity of cytokines and leukocyte extravasation is an emerging therapeutic strategy to limit tissue-damaging inflammatory responses and restore immune homeostasis in inflammatory diseases. Proteoglycans embedded in the vascular endothelial glycocalyx, which regulate the activity of cytokines to restrict the inflammatory response in physiological conditions, are proteolytically cleaved in inflammatory diseases. Here we critically review the potential of proteolytically shed, soluble vascular endothelial glycocalyx proteoglycans to modulate pathological inflammatory responses. Soluble forms of the proteoglycans syndecan-1, syndecan-3 and biglycan exert beneficial anti-inflammatory effects by the removal of chemokines, suppression of proinflammatory cytokine expression and leukocyte migration, and induction of autophagy of proinflammatory M1 macrophages. By contrast, soluble versikine and decorin enhance proinflammatory responses by increasing inflammatory cytokine synthesis and leukocyte migration. Endogenous syndecan-2 and mimecan exert proinflammatory effects, syndecan-4 and perlecan mediate beneficial anti-inflammatory effects and glypican regulates Hh and Wnt signaling pathways involved in systemic inflammatory responses. Taken together, targeting the vascular endothelial glycocalyx-derived, soluble syndecan-1, syndecan-2, syndecan-3, syndecan-4, biglycan, versikine, mimecan, perlecan, glypican and decorin might be a potential therapeutic strategy to suppress overstimulated cytokine and leukocyte responses in inflammatory diseases.

Inhibition of Hedgehog signaling ameliorates foam cell formation by promoting autophagy in early atherosclerosis

Macrophages are the origin of most foam cells in the early stage of atherosclerotic plaques. However, the mechanism involved in the formation of macrophage-derived foam cell formation remains unclear. Here, we revealed that the hedgehog (Hh) signaling is critical in autophagy-lysosome pathway regulation and macrophage-derived foam cell formation. Inhibition of Hh signaling by vismodegib ameliorated lipid deposition and oxidative stress level in atherosclerotic plaques in high-fat diet-fed apoE−/− mice. For mechanistic study, how the Hh signaling modulate the process of foam cell formation were accessed afterward. Unexpectedly, we found that suppression of Hh signaling in apoE−/− mice had no significant impact on circulating cholesterol levels, indicating that Hh pathway modulate the procession of atherosclerotic plaque not through a traditional lipid-lowing mechanism. Instead, vismodegib was found to accelerate autophagosomes maturation as well as cholesterol efflux in macrophage-derived foam cell and in turn improve foam cell formation, while autophagy inhibitors (LY294002 or CQ) administration significantly attenuated vismodegib-induced cholesterol efflux and reversed the effect on foam cell formation. Therefore, our result demonstrated that inhibition of the Hh signaling pathway increases cholesterol efflux and ameliorates macrophage-derived foam cell formation by promoting autophagy in vitro. Our data thus suggested a novel therapeutic target of atherosclerosis and indicated the potential of vismodegib to treat atherosclerosis.

Asymmetric Synthesis of Cyclopamine, a Hedgehog (Hh) Signaling Pathway Inhibitor.

Cyclopamine is a teratogenic steroidal alkaloid, which inhibits the Hedgehog (Hh) signaling pathway by targeting the Smoothened (Smo) receptor. Suppression of Hh signaling with synthetic small molecules has been pursued as a therapeutic approach for the treatment of cancer. We report herein the asymmetric synthesis of cyclopamine based on a two-stage relay strategy. Stage-I: total synthesis of veratramine through a convergent approach, wherein a crucial photoinduced excited-state Nazarov reaction was applied to construct the basic [6-6-5-6] skeleton of C-nor-D-homo-steroid. Stage-II: conversion of veratramine to cyclopamine was achieved through a sequence of chemo-selective redox manipulations.

Signaling Pathways and Therapeutic Strategies in Advanced Basal Cell Carcinoma.

Non-melanoma skin cancers (NMSCs) are the most common human neoplasms world-wide. In detail, basal cell carcinoma (BCC) is the most frequent malignancy in the fair-skinned population. The incidence of BCC remains difficult to assess due to the poor registration practice; however, it has been increasing in the last few years. Approximately, 85% of sporadic BCCs carry mutations in Hedgehog pathway genes, especially in PTCH, SUFU and SMO genes, which lead to the aberrant activation of GLI transcriptional factors, typically silent in cells of adult individuals. The management of advanced BCC (aBCC), both metastatic (mBCC) and locally advanced BCC (laBCC), not candidates for surgical excision or radiotherapy, remains challenging. The discovery of mutations in the Hh signaling pathway has paved the way for the development of Hh pathway inhibiting agents, such as vismodegib and sonidegib, which have represented a breakthrough in the aBCC management. However, the use of these agents is limited by the frequent occurrence of adverse events or the development of drug resistance. In this review, we thoroughly describe the current knowledge regarding the available options for the pharmacological management of aBCCs and provide a forward-looking update on novel therapeutic strategies that could enrich the therapeutic armamentarium of BCC in the near future.

Targeting hedgehog-driven mechanisms of drug-resistant cancers.

Due to the cellular plasticity that is inherent to cancer, the acquisition of resistance to therapy remains one of the biggest obstacles to patient care. In many patients, the surviving cancer cell subpopulation goes on to proliferate or metastasize, often as the result of dramatically altered cell signaling and transcriptional pathways. A notable example is the Hedgehog (Hh) signaling pathway, which is a driver of several cancer subtypes and aberrantly activated in a wide range of malignancies in response to therapy. This review will summarize the field's current understanding of the many roles played by Hh signaling in drug resistance and will include topics such as non-canonical activation of Gli proteins, amplification of genes which promote tolerance to chemotherapy, the use of hedgehog-targeted drugs and tool compounds, and remaining gaps in our knowledge of the transcriptional mechanisms at play.

Triptolide inhibits epithelial ovarian tumor growth by blocking the hedgehog/Gli pathway.

Epithelial ovarian cancer (EOC), the most predominant subtype of ovarian cancer (OC), involves poor prognosis and exhibits high aggression. Triptolide (TPL), like other Chinese herbs, has historically played a significant role in modern medicine. The screening system based on Gli-dependent luciferase reporter activity assessed the effects of over 800 natural medicinal materials on hedgehog (Hh) signaling pathway activity and discovered that TPL had an excellent inhibitory effect on Hh signaling pathway activity. However, the significance and mechanism of TPL involvement in regulating the Hh pathway have not been well explored. Thus, this work aimed to understand better how TPL affects the Hh pathway activity, which, in turn, influences the biological behavior of EOC. Our findings observed that Smo agonist SAG-induced EOC cell proliferation, migration, and invasion were drastically reversed by TPL in a concentration-dependent pattern. Further evidence suggested that TPL promotes the degradation of Gli1 and Gli2 to inhibit the activity of the Hh signaling pathway by relying on Gli1 and Gli2 ubiquitination. Our in vivo studies also confirmed that TPL could significantly inhibit the tumor growth of EOC. Taken together, our results revealed that one of the antitumor mechanisms of TPL was the targeted inhibition of the Hh/Gli pathway.

THU403 Fluoxetine Administration During The Peripartal Period And The Effect On Calcium Metabolism And Serotonin Signaling In The Mammary Gland

Abstract Disclosure: H. Fricke: None. C.J. Krajco: None. M.J. Perry: None. L.L. Hernandez: None. Postpartum depression affects an estimated 10 to 15% of the population. Selective serotonin reuptake inhibitors (SSRIs) are the most commonly prescribed class of antidepressants during pregnancy and lactation and are considered safe to use during pregnancy and lactation. During lactation, bone resorption is increased to mobilize calcium via a serotonin-dependent hormonal cascade. In the mammary gland, serotonin acts via the Hedgehog (Hh) signaling pathway to liberate calcium from bone for milk. Previously, our lab has determined that a high dose of the SSRI fluoxetine (FLX) during the peripartal period impacts calcium and serotonin metabolism during lactation. We hypothesize that a low dose of FLX during the peripartal period will upregulate Hh signaling and subsequently modulate calcium and serotonin metabolism during lactation, and that this impact will vary depending on whether FLX was administered during lactation only or throughout gestation and lactation. Female C57BL/6J mice were randomized to receive FLX (2 mg/kg) or saline daily via i.p. injection from parturition to the end of lactation (D21; lactation only), or from P0 to D21 (gestation + lactation), creating the following treatments: lactation saline (LS; n=10), lactation FLX (n=16), gestation + lactation saline (G+LS; n=5), and gestation + lactation FLX (G+LF; n=15). Circulating serotonin and calcium levels were measured in the serum at P0, the beginning of lactation (D2), and D21, and mammary glands were harvested at D21 for gene expression analysis. When normalized to P0 measurements, there were no differences in circulating calcium or serotonin levels between control and FLX groups at D2 or D21 when treated during lactation only, or gestation and lactation. However, G+LF mice tended to have a lower circulating serotonin level than G+LS mice on D21 (p=0.0770). At D21, gene expression of glioblastoma transcription factor 1 (GLI1) and sonic hedgehog (SHH), both members of the Hh signaling pathway, were altered in the G+LF group. There was a tendency for GLI1 expression to be increased in the G+LF dams (p=0.0634) and SHH was significantly increased in the G+LF dams (p=0.0382) when compared to the G+LS dams. These data suggest that a low dose of FLX during lactation only may not have a significant impact on calcium and serotonin metabolism via alterations in Hh signaling in the mammary gland but may be impacted when administered throughout the entire peripartal period. Presentation: Thursday, June 15, 2023

CRB3 navigates Rab11 trafficking vesicles to promote γTuRC assembly during ciliogenesis.

The primary cilium plays important roles in regulating cell differentiation, signal transduction, and tissue organization. Dysfunction of the primary cilium can lead to ciliopathies and cancer. The formation and organization of the primary cilium are highly associated with cell polarity proteins, such as the apical polarity protein CRB3. However, the molecular mechanisms by which CRB3 regulates ciliogenesis and the location of CRB3 remain unknown. Here, we show that CRB3, as a navigator, regulates vesicle trafficking in γ-tubulin ring complex (γTuRC) assembly during ciliogenesis and cilium-related Hh and Wnt signaling pathways in tumorigenesis. Crb3 knockout mice display severe defects of the primary cilium in the mammary ductal lumen and renal tubule, while mammary epithelial-specific Crb3 knockout mice exhibit the promotion of ductal epithelial hyperplasia and tumorigenesis. CRB3 is essential for lumen formation and ciliary assembly in the mammary epithelium. We demonstrate that CRB3 localizes to the basal body and that CRB3 trafficking is mediated by Rab11-positive endosomes. Significantly, CRB3 interacts with Rab11 to navigate GCP6/Rab11 trafficking vesicles to CEP290, resulting in intact γTuRC assembly. In addition, CRB3-depleted cells are unresponsive to the activation of the Hh signaling pathway, while CRB3 regulates the Wnt signaling pathway. Therefore, our studies reveal the molecular mechanisms by which CRB3 recognizes Rab11-positive endosomes to facilitate ciliogenesis and regulates cilium-related signaling pathways in tumorigenesis.

CRB3 navigates Rab11 trafficking vesicles to promote γTuRC assembly during ciliogenesis

The primary cilium plays important roles in regulating cell differentiation, signal transduction, and tissue organization. Dysfunction of the primary cilium can lead to ciliopathies and cancer. The formation and organization of the primary cilium are highly associated with cell polarity proteins, such as the apical polarity protein CRB3. However, the molecular mechanisms by which CRB3 regulates ciliogenesis and the location of CRB3 remain unknown. Here, we show that CRB3, as a navigator, regulates vesicle trafficking in γ-tubulin ring complex (γTuRC) assembly during ciliogenesis and cilium-related Hh and Wnt signaling pathways in tumorigenesis. Crb3 knockout mice display severe defects of the primary cilium in the mammary ductal lumen and renal tubule, while mammary epithelial-specific Crb3 knockout mice exhibit the promotion of ductal epithelial hyperplasia and tumorigenesis. CRB3 is essential for lumen formation and ciliary assembly in the mammary epithelium. We demonstrate that CRB3 localizes to the basal body and that CRB3 trafficking is mediated by Rab11-positive endosomes. Significantly, CRB3 interacts with Rab11 to navigate GCP6/Rab11 trafficking vesicles to CEP290, resulting in intact γTuRC assembly. In addition, CRB3-depleted cells are unresponsive to the activation of the Hh signaling pathway, while CRB3 regulates the Wnt signaling pathway. Therefore, our studies reveal the molecular mechanisms by which CRB3 recognizes Rab11-positive endosomes to facilitate ciliogenesis and regulates cilium-related signaling pathways in tumorigenesis.

A clinicopathological exploration of Hedgehog signaling: implications in oral carcinogenesis.

Hedgehog Signaling, a basic cancer stem cell pathway, plays a major role during the embryonic development, is known to play a quiescent role in adults. However, aberrant activation of Hedgehog signaling in adults is known to play a role in cancer development. Hence, the aim of the study was to identify the role of Hedgehog signaling pathway in the Oral cancers. The expression of Hedgehog signaling pathway was evaluated in 124 patients through the quantitative real-time PCR. The association between the gene expression and clinico-pathological parameters were analyzed using the Pearson chi-square test and survival analysis was carried out using Kaplan-Meier analysis. SHH and GLI1 was found to be significantly associated with the Lymph Node Status and SUFU was significantly associated with the Age. SMO and SUFU were found to have a worse prognosis in oral cancer patients. According to our findings, IHH plays a critical role in the activation of the HH signaling pathway in oral cancer. These findings back up the use of the Hedgehog signaling pathway as a biomarker for early disease prediction in oral cancer, as well as its role in tumor aggressiveness and invasiveness.

Inhibition of Ezh2 redistributes bivalent domains within transcriptional regulators associated with WNT and Hedgehog pathways in osteoblasts

Bivalent epigenomic regulatory domains containing both activating histone 3 lysine 4 (H3K4me3) and repressive lysine 27 (H3K27me3) trimethylation are associated with key developmental genes. These bivalent domains repress transcription in the absence of differentiation signals but maintain regulatory genes in a poised state to allow for timely activation. Previous studies demonstrated that enhancer of zeste homolog 2 (Ezh2), a histone 3 lysine 27 (H3K27) methyltransferase, suppresses osteogenic differentiation and that inhibition of Ezh2 enhances commitment of osteoblast progenitors invitro and bone formation invivo. Here, we examined the mechanistic effects of Tazemetostat (EPZ6438), an Food and Drug Administration approved Ezh2 inhibitor for epithelioid sarcoma treatment, because this drug could potentially be repurposed to stimulate osteogenesis for clinical indications. We find that Tazemetostat reduces H3K27me3 marks in bivalent domains in enhancers required for bone formation and stimulates maturation of MC3T3 preosteoblasts. Furthermore, Tazemetostat activates bivalent genes associated with the Wingless/integrated (WNT), adenylyl cyclase (cAMP), and Hedgehog (Hh) signaling pathways based on transcriptomic (RNA-seq) and epigenomic (chromatin immunoprecipitation [ChIP]-seq) data. Functional analyses using selective pathway inhibitors and silencing RNAs demonstrate that the WNT and Hh pathways modulate osteogenic differentiation after Ezh2 inhibition. Strikingly, we show that loss of the Hh-responsive transcriptional regulator Gli1, but not Gli2, synergizes with Tazemetostat to accelerate osteoblast differentiation. These studies establish epigenetic cooperativity of Ezh2, Hh-Gli1 signaling, and bivalent regulatory genes in suppressing osteogenesis. Our findings may have important translational ramifications for anabolic applications requiring bone mass accrual and/or reversal of bone loss.