Ling Mao

Sonic hedgehog protects cortical neurons against oxidative stress.

Oxidative stress is one of the most important pathological mechanisms in neurodegenerative diseases and ischemia. Recent studies have indicated that the sonic hedgehog (SHH) signaling pathway is involved in these diseases, but the underlying mechanisms remains elusive. Here we report that the SHH pathway was activated in primary cultured cortical neurons after exposure to hydrogen peroxide (H2O2). H2O2 treatment decreased the cell viability of neurons, and inhibition of endogenous SHH signaling exacerbated its neurotoxicity. Activation of SHH signaling protected neurons from H2O2-induced apoptosis and increased the cell viability while those effects were partially reversed by blocking SHH signals. Exogenous SHH increased the activities of Superoxide dismutase (SOD) and Glutathione peroxidase (GSH-PX) in H2O2-treated neurons and decreased production of Malondialdehyde (MDA). It also promoted expression of the anti-apoptotic gene Bcl-2 and inhibited expression of pro-apoptotic gene Bax. Activation of SHH signals upregulated both Neurotrophic factors vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF). Pretreatment with SHH inhibited the activation of ERK (extracellular signal-regulated kinases) signals induced by H2O2. Our findings demonstrate that activation of SHH signaling protects cortical neurons against oxidative stress and suggest a potential role of SHH for the clinic treatments of brain ischemia and neurodegenerative disorders.

MYCN Promotes the Expansion of Phox2B-Positive Neuronal Progenitors to Drive Neuroblastoma Development

Amplification of the oncogene MYCN is a tumorigenic event in the development of a subset of neuroblastomas that commonly consist of undifferentiated or poorly differentiated neuroblasts with unfavorable clinical outcome. The cellular origin of these neuroblasts is unknown. Additionally, the cellular functions and target cells of MYCN in neuroblastoma development remain undefined. Here we examine the cell types that drive neuroblastoma development in TH-MYCN transgenic mice, an animal model of the human disease. Neuroblastoma development in these mice begins with hyperplastic lesions in early postnatal sympathetic ganglia. We show that both hyperplasia and primary tumors are composed predominantly of highly proliferative Phox2B(+) neuronal progenitors. MYCN induces the expansion of these progenitors by both promoting their proliferation and preventing their differentiation. We further identify a minor population of undifferentiated nestin(+) cells in both hyperplastic lesions and primary tumors that may serve as precursors of Phox2B(+) neuronal progenitors. These findings establish the identity of neuroblasts that characterize the tumor phenotype and suggest a cellular pathway by which MYCN can promote neuroblastoma development.

Astrocyte-Derived Sonic Hedgehog Contributes to Angiogenesis in Brain Microvascular Endothelial Cells via RhoA/ROCK Pathway After Oxygen–Glucose Deprivation

The human adult brain possesses intriguing plasticity, including neurogenesis and angiogenesis, which may be mediated by the activated sonic hedgehog (Shh). By employing a coculture system, brain microvascular endothelial cells (BMECs) cocultured with astrocytes, which were incubated under oxygen–glucose deprivation (OGD) condition, we tested the hypothesis that Shh secreted by OGD-activated astrocytes promotes cerebral angiogenesis following ischemia. The results of this study demonstrated that Shh was mainly secreted by astrocytes and the secretion was significantly upregulated after OGD. The proliferation, migration, and tube formation of BMECs cocultured with astrocytes after OGD were significantly enhanced, but cyclopamine (a Shh antagonist) or 5E1 (an antibody of Shh) reversed the change. Furthermore, silencing Ras homolog gene family, member A (RhoA) of BMECs by RNAi and blocking Rho-dependent kinase (ROCK) by Y27632, a specific antagonist of ROCK, suppressed the upregulation of proliferation, migration, and tube formation of BMECs after OGD. These findings suggested that Shh derived from activated astrocytes stimulated RhoA/ROCK pathway in BMECs after OGD, which might be involved in angiogenesis in vitro.

A critical role of Sonic Hedgehog signaling in maintaining the tumorigenicity of neuroblastoma cells

Accumulated evidence suggests a major role for the activation of the Sonic Hedgehog (SHH) signaling pathway in the development of neural crest stem cells that give rise to the sympathetic nervous system. We therefore investigated the involvement of SHH signaling in the pathogenesis of neuroblastoma, a common childhood malignant tumor of the sympathetic nervous system. Human neuroblastoma cell lines and a majority of primary neuroblastoma specimens showed high‐level expression of the pathway targets and components, indicating persistent activation of the SHH pathway. All of the neuroblastoma cell lines we examined expressed significant levels of SHH ligand, suggesting an autocrine, ligand‐dependent activation of the SHH pathway in neuroblastoma cells. Inhibition of SHH signaling by cyclopamine induced apoptosis and blocked proliferation in all major types of neuroblastoma cells, and abrogated the tumorigenicity of neuroblastoma cells. Moreover, the knockdown of GLI2 in neuroblastoma BE (2)‐C and SK‐N‐DZ cell lines resulted in the inhibition of colony formation. Our study has revealed a molecular mechanism for the persistent activation of the SHH pathway which promotes the development of neuroblastoma, and suggests a new approach for the treatment of this childhood malignant tumor. (Cancer Sci 2009; 100: 1848–1855)

The protective effect of sonic hedgehog is mediated by the propidium iodide 3-kinase/AKT/Bcl-2 pathway in cultured rat astrocytes under oxidative stress

In our previous study, we found that the sonic hedgehog (Shh) signaling pathway is activated in neurons under oxidative stress and plays a neuro-protective role [Dai RL, et al. (2011) Neurochem Res 36:67-75]; we are led to postulate that the Shh might be released by astrocytes, thereby protecting neurons against oxidant injury. In primary cultured astrocytes of rats, we found that treatment with 100 μM H₂O₂ for 24 h induced a significant increase in the mRNA and protein levels of Shh, Patched1, and Gli-1, and the increase was substantially greater in astrocytes than in neurons. In the coculture systems of astrocytes and neurons under the H₂O₂ treatment, blocking the Shh signaling pathway with 5E1 (an antibody against the N-terminal domain of Shh) could block the neuroprotective activity of astrocytes on cocultured neurons. In this study, we found that treatment with H₂O₂ (100-800 μM) for 24 h caused cell death of astrocytes in a concentration-dependent manner. MTT reduction and Trypan Blue exclusion assay showed that exogenous Shh increased survival rate of the H₂O₂-treated astrocytes, whereas pretreatment with cyclopamine (a specific inhibitor of the Shh signaling pathway) or 5E1 decreased the survival rate of the H₂O₂-treated astrocytes. Shh also inhibited H₂O₂-induced apoptosis of astrocytes, and this effect could be partially reversed by cyclopamine. We also found that Shh promoted the phosphorylation of AKT, but had no significant effect on p38 or extracellular signal regulated kinases 1 and 2 (ERK 1/2) in H₂O₂-treated astrocytes. Blocking Shh or phosphoinositide 3-kinases (PI3-K)/AKT signaling pathway with cyclopamine or LY294002 decreased the survival rate of astrocytes, induced cell apoptosis, upregulated the expression of Bax, and downregulated the expression of Bcl-2. We are led to conclude that the oxidative stress induces astrocytes to secrete endogenous Shh and exogenous administration of Shh might protect the astrocytes from oxidative stress by activating PI3-K/AKT/Bcl-2 pathway.

Recombinant Human Sonic Hedgehog Protein Regulates the Expression of ZO-1 and Occludin by Activating Angiopoietin-1 in Stroke Damage

This study examines the regulating effect of Sonic Hedgehog (Shh) on the permeability of the blood-brain barrier (BBB) in cerebral ischemia. By employing permanent middle cerebral artery occlusion (pMCAO) model, we find that Shh significantly decreases brain edema and preserves BBB permeability. Moreover, Shh increases zonula occludens-1 (ZO-1), occludin and angiopiotetin-1 (Ang-1) expression in the ischemic penumbra. Blockage of Shh with cyclopamine abolishes the effects of Shh on brain edema, BBB permeability and ZO-1, occludin, Ang-1 expression. Primary brain microvessel endothelial cells (BMECs) and astrocytes were pre-treated with Shh, cyclopamine, Ang-1-neutralizing antibody, and subjected to oxygen-glucose deprivation (OGD). Results show that the Ang-1 protein level in the culture medium of Shh-treated astrocytes is significantly higher. Shh also increased ZO-1, occludin and Ang-1 expression in BMECs, while cyclopamine and Ang-1-neutralizing antibody inhibited the effects of Shh on the ZO-1 and occludin expression, respectively. This study suggests that, under ischemic insults, Shh triggers Ang-1 production predominantly in astrocytes, and the secreted Ang-1 acts on BMECs, thereby upregulating ZO-1 and occludin to repair the tight junction and ameliorate the brain edema and BBB leakage.

MicroRNA-107 contributes to post-stroke angiogenesis by targeting Dicer-1.

Previous studies have suggested that microRNA-107 (miR-107) regulates cell migration in tumor and promotes Hypoxia Inducible Factor 1α (HIF1α) regulated angiogenesis under hypoxia. We found that miR-107 was strongly expressed in ischemic boundary zone (IBZ) after permanent middle cerebral artery occlusion (pMCAO) in rats and inhibition of miR-107 could reduce capillary density in the IBZ after stroke. Such finding led us to hypothesize that miR-107 might regulate post-stroke angiogenesis and therefore serve as a therapeutic target. We also found that antagomir-107, a synthetic miR-107 inhibitor, decreased the number of capillaries in IBZ and increased overall infarct volume after pMCAO in rats. We demonstrated that miR-107 could directly down-regulate Dicer-1, a gene that encodes an enzyme essential for processing microRNA (miRNA) precursors. This resulted in translational desupression of VEGF (vascular endothelial growth factor) mRNA, thereby increasing expression of endothelial cell-derived VEGF (VEGF165/VEGF164), leading to angiogenesis after stroke. This process might be a protective mechanism for ischemia-induced cerebral injury and miR-107 might be used as a novel tool in stroke treatment.

Endogenous Endothelial Progenitor Cells Participate in Neovascularization via CXCR4/SDF‐1 axis and Improve Outcome After Stroke

To study whether endogenous endothelial progenitor cells (EPCs) are involved in neovascularization after stroke.

HOXC9 links cell-cycle exit and neuronal differentiation and is a prognostic marker in neuroblastoma.

Differentiation status in neuroblastoma strongly affects clinical outcomes and inducing differentiation is a treatment strategy in this disease. However, the molecular mechanisms that control neuroblastoma differentiation are not well understood. Here, we show that high-level HOXC9 expression is associated with neuroblastoma differentiation and is prognostic for better survival in neuroblastoma patients. HOXC9 induces growth arrest and neuronal differentiation in neuroblastoma cells by directly targeting both cell-cycle-promoting and neuronal differentiation genes. HOXC9 expression is upregulated by retinoic acid (RA), and knockdown of HOXC9 expression confers resistance to RA-induced growth arrest and differentiation. Moreover, HOXC9 expression is epigenetically silenced in RA-resistant neuroblastoma cells, and forced HOXC9 expression is sufficient to inhibit their proliferation and tumorigenecity. These findings identified HOXC9 as a key regulator of neuroblastoma differentiation and suggested a therapeutic strategy for RA-resistant neuroblastomas through epigenetic activation of HOXC9 expression.

Zinc-finger transcription factor Snail accelerates survival, migration and expression of matrix metalloproteinase-2 in human bone mesenchymal stem cells

Although bone mesenchymal stem cells (BMSC) hold promise in gene therapy and tissue engineering, the inefficient migration and the low capability of subsequent survival of BMSC have largely restrained progress in these studies. Characteristics shared between stem cells and tumorigenic cells prompted us to investigate whether mechanisms of tumor progression contribute to stem cell migration. The transcription factor Snail which functions in epithelial‐mesenchymal transitions (EMT) is responsible for the acquisition of motile and invasive properties of tumor cells. It is not yet known whether Snail acts in the mechanisms of stem cell migration. Here it is shown that ectopic Snail expression increased the migration of BMSC in vitro by a mechanism dependent on the phosphoinositide 3‐kinase (PI‐3K) signaling pathway. Snail expression may contribute to the constitutive activation of signaling pathways of PI‐3K and MAPK and the related MMP‐2 secretion in BMSC. Furthermore, the stem cells expressing Snail were protected from the apoptosis triggered by serum deprivation. These results suggested the possibility for us to optimize the migration of BMSC toward infarcted tissues and their subsequent survival in the local microenvironment, by investigating mechanisms associated with the acquisition of invasiveness by tumor cells.