Role of Sonic Hedgehog Signaling in Oligodendrocyte Differentiation

During development, the secreted molecule Sonic Hedgehog (Shh) is required for lineage specification and proliferation of oligodendrocyte progenitors (OLPs), which are the glia cells responsible for the myelination of axons in the central nervous system (CNS). Shh signaling has been implicated in controlling both the generation of oligodendrocytes (OLGs) during embryonic development and their production in adulthood. Although, some evidence points to a role of Shh signaling in OLG development, its involvement in OLG differentiation remains to be fully determined. The objective of this study was to assess whether Shh signaling is involved in OLG differentiation after neural stem cell commitment to the OLG lineage. To address these questions, we manipulated Shh signaling using cyclopamine, a potent inhibitor of Shh signaling activator Smoothened (Smo), alone or combined with the agonist SAG in OLG primary cultures and assessed expression of myelin-specific markers. We found that inactivation of Shh signaling caused a dose-dependent decrease in myelin basic protein (MBP) and myelin associated glycoprotein (MAG) in differentiating OLGs. Co-treatment of the cells with SAG reversed the inhibitory effect of cyclopamine on both myelin-specific protein levels and morphological changes associated with it. Further experiments are required to elucidate the molecular mechanism by which Shh signaling regulates OLG differentiation.     

The Neuroprotective Roles of Sonic Hedgehog Signaling Pathway in Ischemic Stroke

Ischemic stroke is characterized by high morbidity, mortality and disability rate worldwide. Because of its complexity in pathogenesis and lack of effective therapeutic strategies and drugs, great breakthrough has not yet been made in the treatment of cerebral ischemic stroke. Therefore, to explore a more effective and safer therapeutic strategy for cerebral ischemic stroke has been the focus of numerous researchers. Neuroprotective effects of sonic hedgehog (Shh) signaling pathway in ischemic stroke have been reported in recent studies, but have not been fully elucidated. In our review, we elaborate the roles of Shh signaling in ischemic stroke from different aspects, including oxidative stress, excitotoxicity, neuroinflammation, apoptosis, angiogenesis, neuroplasticity, neurogenesis, astrogliosis and oligodendrogenesis. Meanwhile, Shh signaling based therapeutic approaches for cerebral ischemic stroke are also included in our review. We hope it will benefit the readers to better understand the roles of Shh signaling pathway in cerebral ischemic stroke and provide more comprehensive insights for basic research and novel strategies for the clinical treatment of cerebral ischemic stroke.     

Sonic Hedgehog Signaling Pathway Supports Cancer Cell Growth during Cancer Radiotherapy

Tumor growth after radiotherapy is a commonly recognized cause of therapeutic failure. In this way, we examined tumor cell growth after radiotherapy by establishing a cancer cell growth model in vitro. We accomplished this model by seeding non-irradiated firefly luciferase2 and green fluorescent protein fusion gene (Fluc) labeled living cancer reporter cells onto a feeder layer of irradiated cancer cells. The living tumor cell growth was monitored by bioluminescence imaging. The living reporter cells grew faster when seeded onto lethally irradiated feeder cells than when seeded onto non-irradiated feeder cells or when seeded in the absence of feeder cells. We found that the expression levels of the Shh and Gli1 proteins, both of which are critical proteins in Sonic hedgehog (SHH) signaling, were increased after irradiation and that this expression was positively correlated with reporter cell growth. Moreover, the dying cell stimulation of living tumor cell growth was enhanced by the addition of SHH signaling agonists and inhibited by SHH signaling antagonists. SHH agonists also enhanced reporter cell growth in the absence of irradiated feeder cells, suggesting this mechanism plays a role in feeder cell growth stimulation. Given these results, we conclude that SHH signaling activation plays an important role during tumor repopulation after radiotherapy.     

Sonic Hedgehog as a mediator of long-range signaling

The ability of Hedgehog (Hh) proteins to exert their biological effects is regulated by a series of post-translational processes. These processes include an intramolecular cleavage, covalent addition of cholesterol and/or palmitate, and conversion into a multimeric freely diffusible form. The processing of Hh proteins affects their trafficking, potency, and ability to signal over many cell diameters. Accordingly, the loss of gene products required for these processes abrogates the Hh proteins' abilities to exert their effects, which can be long range, short range, or both. We review here recent evidence demonstrating that Hh proteins are directly responsible for their long-range biological effects. Additionally, we integrate both genetic and biochemical data to delineate a model illustrating how the unusual biochemistry of Hh family members may allow them to act as morphogens, signaling over both short and long distances.     

The Role of Sonic Hedgehog Signaling in Osteoclastogenesis and Jaw Bone Destruction

Sonic hedgehog (SHH) and its signaling have been identified in several human cancers, and increased levels of its expression appear to correlate with disease progression and metastasis. However, the role of SHH in bone destruction associated with oral squamous cell carcinomas is still unclear. In this study we analyzed SHH expression and the role played by SHH signaling in gingival carcinoma-induced jawbone destruction. From an analysis of surgically resected lower gingival squamous cell carcinoma mandible samples, we found that SHH was highly expressed in tumor cells that had invaded the bone matrix. On the other hand, the hedgehog receptor Patched and the signaling molecule Gli-2 were highly expressed in the osteoclasts and the progenitor cells. SHH stimulated osteoclast formation and pit formation in the presence of the receptor activator for nuclear factor-κB ligand (RANKL) in CD11b⁺ mouse bone marrow cells. SHH upregulated phosphorylation of ERK1/2 and p38 MAPK, NFATc1, tartrate-resistant acid phosphatase (TRAP), and Cathepsin K expression in RAW264.7 cells. Our results suggest that tumor-derived SHH stimulated the osteoclast formation and bone resorption in the tumor jawbone microenvironment.     

Analysis of the Sonic Hedgehog Signaling Pathway in Normal and Abnormal Bladder Development

In this study, we examined the expression of Sonic Hedgehog, Patched, Gli1, Gli2, Gli3 and Myocardin in the developing bladders of male and female normal and megabladder (mgb−/−) mutant mice at embryonic days 12 through 16 by in situ hybridization. This analysis indicated that each member of the Sonic Hedgehog signaling pathway as well as Myocardin displayed distinct temporal and spatial patterns of expression during normal bladder development. In contrast, mgb−/− bladders showed both temporal and spatial changes in the expression of Patched, Gli1 and Gli3 as well as a complete lack of Myocardin expression. These changes occurred primarily in the outer mesenchyme of developing mgb−/− bladders consistent with the development of an amuscular bladder phenotype in these animals. These results provide the first comprehensive analysis of the Sonic Hedgehog signaling pathway during normal bladder development and provide strong evidence that this key signaling cascade is critical in establishing radial patterning in the developing bladder. In addition, the lack of detrusor smooth muscle development observed in mgb−/− mice is associated with bladder-specific temporospatial changes in Sonic Hedgehog signaling coupled with a lack of Myocardin expression that appears to result in altered patterning of the outer mesenchyme and poor initiation and differentiation of smooth muscle cells within this region of the developing bladder.     

Cortical Neural Stem Cell Lineage Progression Is Regulated by Extrinsic Signaling Molecule Sonic Hedgehog

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Exploring Sonic Hedgehog Cell Signaling in Neurogenesis: Its Potential Role in Depressive Behavior

Neurochemical Research - Depression is the most prevalent form of neuropsychiatric disorder affecting all age groups globally. As per the estimation of the World Health Organization (WHO),...     

Retinoic Acid Signaling Regulates Sonic Hedgehog and Bone Morphogenetic Protein Signalings During Genital Tubercle Development

Retinoic acid (RA) plays pivotal roles in organogenesis, and both excessive and reduced amounts of RA cause developmental abnormalities. Reproductive organs are susceptible to teratogen toxigenicity, and the genital tubercle (GT) is one such representative organ. The physiological function of endogenous RA signaling and the mechanisms of RA‐induced teratogenicity are poorly understood during the GT development. The objective of this study is to understand the developmental and teratogenic roles of RA during GT development by analyzing genetically modified mouse models. We found dynamic patterns of gene expression for the RA‐synthesizing enzyme, Raldh2, and for the RA‐catabolizing enzyme, Cyp26b1, during GT development. Rarb, an indicator gene for RA signaling, starts its expression in the prospective corpus cavernosum penis and in the urethral plate epithelium (UE), which plays central roles during GT development. Excessive RA signaling in Cyp26b1^?/? mutants leads to abnormal extents of cell proliferation and differentiation during GT development, and also upregulates expression of growth factor signalings. They include Sonic hedgehog (Shh) signaling and Bone morphogenetic protein (Bmp) signaling, which are expressed in the UE and its bilateral mesenchyme. RA signaling positively regulatesShh and Bmp4 expression during GT development as testified also by the experiment of RA administration and analyses of loss–of‐function of RA signaling mutants. Thus, RA signaling is involved in the developmental cascade necessary for UE formation and GT development.     

The Ciliary G-Protein-Coupled Receptor Gpr161 Negatively Regulates the Sonic Hedgehog Pathway via cAMP Signaling

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Highlights? The GPCR Gpr161 localizes to primary cilia in a Tulp3/IFT-A-dependent manner ? Complete loss of Gpr161 increases Shh signaling in the mouse neural tube ? Gpr161 couples protein kinase A activation to Shh signaling via cAMP signaling ? Shh signaling internalizes Gpr161 from the cilia, preventing its activity     

Sonic Hedgehog Regulates Osteoblast Function by Focal Adhesion Kinase Signaling in the Process of Fracture Healing

Several biological studies have indicated that hedgehog signaling plays an important role in osteoblast proliferation and differentiation, and sonic hedgehog (SHH) expression is positively correlated with phosphorylated focal adhesion kinase (FAK) Tyr³⁹⁷. However, the relationship between them and their role in the process of normal fracture repair has not been clarified yet. Immunohistochemical analysis revealed that SHH and pFAK Tyr³⁹⁷ were expressed in bone marrow cells and that pFAK Tyr³⁹⁷ was also detected in ALP-positive osteoblasts near the TRAP-positive osteoclasts in the fracture site in the ribs of mice on day 5 after fracture. SHH and pFAK Tyr³⁹⁷ were detectable in osteoblasts near the hypertrophic chondrocytes on day 14. In vitro analysis showed that SHH up-regulated the expression of FAK mRNA and pFAK Tyr³⁹⁷ time dependently in osteoblastic MC3T3-E1 cells. Functional analysis revealed that 5 lentivirus encoding short hairpin FAK RNAs (shFAK)-infected MC3T3-E1 cell groups displayed a round morphology and decreased proliferation, adhesion, migration, and differentiation. SHH stimulated the proliferation and differentiation of MC3T3-E1 cells, but had no effect on the shFAK-infected cells. SHH also stimulated osteoclast formation in a co-culture system containing MC3T3-E1 and murine CD11b⁺ bone marrow cells, but did not affect the shFAK-infected MC3T3-E1 co-culture group. These data suggest that SHH signaling was activated in osteoblasts at the dynamic remodeling site of a bone fracture and regulated their proliferation and differentiation, as well as osteoclast formation, via FAK signaling.     

Methoprene Photolytic Compounds Disrupt Zebrafish Development,Producing Phenocopies of Mutants in the Sonic Hedgehog Signaling Pathway

Environmental chemicals have been proposed to impact endocrine or retinoid pathways, causing developmental abnormalities in humans and other vertebrates. Presented evidence shows that exposure of zebrafish embryos to sunlight-induced photolytic products of the pesticide methoprene results in developmental defects in the head, heart, pectoral fins, and somites, and in spinal motor and optic nerve axons. Exposed embryos are phenocopies of zebrafish you-type mutants and, as in the mutant sonic-you, show underexpression of the signaling protein sonic hedgehog. Reduced expression of sonic hedgehog is also displayed in embryos treated with the retinoic acid synthesis inhibitor citral. This study identifies citral-related compounds as embryonic signaling disruptors of potential environmental concern.     

重组人SHH蛋白N端在HEK293T细胞中的分泌表达与鉴定

目的:获得有活性的Sonic Hedgehog(SHH)蛋白N端结构域蛋白纯品,该结构域是SHH蛋白与受体结合结构域,可用作抗原,用于研制抗SHH的中和抗体。方法:应用PCR技术从商业化人Shh基因中分别扩增该基因5'端591和600 bp的片段,并插入真核表达载体pL293,分别在HEK293T细胞中进行瞬时分泌表达,通过His标签纯化后获得SHH-591和SHH-600蛋白纯品,SDS-PAGE和Western印迹对表达产物进行分析,并通过ELISA进行结合活性鉴定。结果:构建了重组表达载体pL293-Shh-N591和pL293-Shh-N600,酶切鉴定和测序证实含有目的基因片段,真核瞬时表达产物均在相对分子质量约20×103处可见与预期相符的条带,该条带可被His标签抗体所识别;纯化获得了SHH-591-His和SHH-600-His蛋白纯品;ELISA结合实验结果显示SHH-591-His和SHH-600-His均能与抗His标签抗体结合,而SHH-591-His与SHH中和抗体的结合能力更强。结论:获得了真核表达的SHH-N蛋白SHH-591-His,可用于下一步中和抗体药物的筛选和后续研究。     

Identification of N-terminal Residues of Sonic Hedgehog Important for Palmitoylation by Hedgehog Acyltransferase

Sonic Hedgehog (Shh) is a secreted morphogen that regulates embryonic development. After removal of the signal peptide, Shh is processed to the mature, active form through autocleavage and a series of lipid modifications, including the attachment of palmitate. Covalent attachment of palmitate to the N-terminal cysteine of Shh is catalyzed by Hedgehog acyltransferase (Hhat) and is critical for proper signaling. The sequences within Shh that are responsible for palmitoylation by Hhat are not known. Here we show that the first six amino acids of mature Shh (CGPGRG) are sufficient for Hhat-mediated palmitoylation. Alanine scanning mutagenesis was used to determine the role of each amino acid and the positional sequence requirement in a cell-based Shh palmitoylation assay. Mutation of residues in the GPGR sequence to Ala had no effect on palmitoylation, provided that a positively charged residue was present within the first seven residues. The N-terminal position exhibited a strong but not exclusive requirement for Cys. Constructs with an N-terminal Ala were not palmitoylated. However, an N-terminal Ser served as a substrate for Hhat, but not the Drosophila melanogaster ortholog Rasp, highlighting a critical difference between the mammalian and fly enzymes. These findings define residues and regions within Shh that are necessary for its recognition as a substrate for Hhat-mediated palmitoylation. Finally, we report the results of a bioinformatics screen to identify other potential Hhat substrates encoded in the human genome.     

Resveratrol Pretreatment Decreases Ischemic Injury and Improves Neurological Function Via Sonic Hedgehog Signaling After Stroke in Rats

Resveratrol has neuroprotective effects for ischemic cerebral stroke. However, its neuroprotective mechanism for stroke is less well understood. Beneficial actions of the activated Sonic hedgehog (Shh) signaling pathway in stroke, such as improving neurological function, promoting neurogenesis, anti-oxidative, anti-apoptotic, and pro-angiogenic effects, have been noted, but relatively little is known about the role of Shh signaling in resveratrol-reduced cerebral ischemic injury after stroke. The present study tests whether the Shh pathway mediates resveratrol to decrease cerebral ischemic injury and improve neurological function after stroke. We observed that resveratrol pretreatment significantly improved neurological function, decreased infarct volume, enhanced vitality, and reduced apoptosis of neurons in vivo and vitro after stroke. Meanwhile, expression levels of Shh, Ptc-1, Smo, and Gli-1 mRNAs were significantly upregulated and Gli-1 was relocated to the nucleus. Intriguingly, in vivo and in vitro inhibition of the Shh signaling pathway with cyclopamine, a Smo inhibitor, completely reversed the above effects of resveratrol. These results suggest that decreased cerebral ischemic injury and improved neurological function by resveratrol may be mediated by the Shh signaling pathway.     

Dual Function of UNC-51-like Kinase 3 (Ulk3) in the Sonic Hedgehog Signaling Pathway

The Sonic hedgehog (Shh) signaling pathway controls a variety of developmental processes and is implicated in tissue homeostasis maintenance and neurogenesis in adults. Recently, we identified Ulk3 as an active kinase able to positively regulate Gli proteins, mediators of the Shh signaling in mammals. Here, we provide several lines of evidence that Ulk3 participates in the transduction of the Shh signal also independently of its kinase activity. We demonstrate that Ulk3 through its kinase domain interacts with Suppressor of Fused (Sufu), a protein required for negative regulation of Gli proteins. Sufu blocks Ulk3 autophosphorylation and abolishes its ability to phosphorylate and positively regulate Gli proteins. We show that Shh signaling destabilizes the Sufu-Ulk3 complex and induces the release of Ulk3. We demonstrate that the Sufu-Ulk3 complex, when co-expressed with Gli2, promotes generation of the Gli2 repressor form, and that reduction of the Ulk3 mRNA level in Shh-responsive cells results in higher potency of the cells to transmit the Shh signal. Our data suggests a dual function of Ulk3 in the Shh signal transduction pathway and propose an additional way of regulating Gli proteins by Sufu, through binding to and suppression of Ulk3.