Kuan Min Fang

Microglial phagocytosis attenuated by short-term exposure to exogenous ATP through P2X7 receptor action

Microglia, the CNS resident macrophages responsible for the clearance of degenerating cellular fragments, are essential to tissue remodeling and repair after CNS injury. ATP can be released in large amounts after CNS injury and may mediate microglial activity through the ionotropic P2X and the metabotropic P2Y receptors. This study indicates that exposure to a high concentration of ATP for 30 min rapidly induces changes of the microglial cytoskeleton, and significantly attenuates microglial phagocytosis. A pharmacological approach showed that ATP‐induced inhibition of microglial phagocytotic activity was due to P2X7R activation, rather than that of P2YR. Activation of P2X7R by its agonist, 2′‐3′‐O‐(4‐benzoyl)benzoyl‐ATP (BzATP), produced a Ca2+‐independent reduction in microglial phagocytotic activity. In addition, the knockdown of P2X7R expression by lentiviral‐mediated shRNA interference or the blockade of P2X7R activation by the specific antagonists, oxidized ATP (oxATP) and brilliant blue G, has efficiently restored the phagocytotic activity of ATP and BzATP‐treated microglia. Our results reveal that P2X7R activation may induce the formation of a Ca2+‐independent signaling complex, which results in the reduction of microglial phagocytosis. This suggests that exposure to ATP for a short‐term period may cause insufficient clearance of tissue debris by microglia through P2X7R activation after CNS injury, and that blockade of this receptor may preserve the phagocytosis of microglia and facilitate CNS tissue repair.

Effects of Combinatorial Treatment with Pituitary Adenylate Cyclase Activating Peptide and Human Mesenchymal Stem Cells on Spinal Cord Tissue Repair

The aim of this study is to understand if human mesenchymal stem cells (hMSCs) and neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) have synergistic protective effect that promotes functional recovery in rats with severe spinal cord injury (SCI). To evaluate the effect of delayed combinatorial therapy of PACAP and hMSCs on spinal cord tissue repair, we used the immortalized hMSCs that retain their potential of neuronal differentiation under the stimulation of neurogenic factors and possess the properties for the production of several growth factors beneficial for neural cell survival. The results indicated that delayed treatment with PACAP and hMSCs at day 7 post SCI increased the remaining neuronal fibers in the injured spinal cord, leading to better locomotor functional recovery in SCI rats when compared to treatment only with PACAP or hMSCs. Western blotting also showed that the levels of antioxidant enzymes, Mn-superoxide dismutase (MnSOD) and peroxiredoxin-1/6 (Prx-1 and Prx-6), were increased at the lesion center 1 week after the delayed treatment with the combinatorial therapy when compared to that observed in the vehicle-treated control. Furthermore, in vitro studies showed that co-culture with hMSCs in the presence of PACAP not only increased a subpopulation of microglia expressing galectin-3, but also enhanced the ability of astrocytes to uptake extracellular glutamate. In summary, our in vivo and in vitro studies reveal that delayed transplantation of hMSCs combined with PACAP provides trophic molecules to promote neuronal cell survival, which also foster beneficial microenvironment for endogenous glia to increase their neuroprotective effect on the repair of injured spinal cord tissue.

Induced interleukin-33 expression enhances the tumorigenic activity of rat glioma cells

BACKGROUND Glioma development is a multistep process associated with progressive genetic alterations but also regulated by cellular and noncellular components in a tumor-associated niche. METHODS Using 2 rat C6 glioma cell clones with different tumorigenesis, named C6-1 and C6-2, this study characterized genes associated with enhanced tumorigenic features of glioma cells by comparative cDNA microarray analysis combined with Q-PCR. Neurospehere formation and clonogenicity were examined to determine the growth of tumorigenic C6 glioma cells. The lentivirus-mediated gene knockdown approach was conducted to determine the role of interleukin-33 (IL-33) in glioma cell proliferation and migration. Transwell cell invasion assay was used to examine microglia migration induced by tumorigenic C6 cells. RESULTS The functional analysis of gene ontology (GO) biological processes shows that the upregulated genes found in tumorigenic C6 (C6-1) cells are closely related to cell proliferation. Tumorigenic C6 cells expressed cytokines and chemokines abundantly. Among these genes, IL-33 was profoundly induced in tumorigenic C6 cells with the expression of IL-33 receptor ST2. Furthermore, the growth rate and colony formation of tumorigenic C6 cells were attenuated by the inhibition of IL-33 and ST2 gene expression. Moreover, IL-33 was involved in tumorigenic glioma cell migration and regulation of the expression of several glioma-associated growth factors and chemokines in tumorigenic C6 cells. CONCLUSION Accordingly, we concluded that glioma cells with abundant production of IL-33 grow rapidly; moreover, the interactions of multiple cytokines/chemokines induced by glioma cells may develop a microenvironment that facilitates microglia/macrophage infiltration and fosters glioma growth in the brain.

Expression of macrophage inflammatory protein-1α and monocyte chemoattractant protein-1 in glioma-infiltrating microglia: Involvement of ATP and P2X7 receptor

Chemokines can be produced by gliomas, which mediate the infiltration of microglia, a characteristic feature of glioma‐associated neuropathogenesis. ATP that is released at a high level from glioma has been reported to play a regulatory role in chemokine production in cultured glioma cells. The objective of this study was to define the potential role of extracellular ATP in the regulation of macrophage inflammatory protein‐1α (MIP‐1α) and monocyte chemoattractant protein‐1(MCP‐1) expression in glioma‐associated microglia/macrophages. The results showed that Iba1+ and ED1+ microglia existed in the tumor at 3 and 7 day after injection of C6 glioma cells into the rat cerebral cortex (dpi). ED1+ microglia/macrophages or Iba1+ microglia in the glioma were also colocalized to MIP‐1α‐ and MCP‐1‐expressing cells. In vitro study indicated that treatment with ATP and BzATP (an agonist for ATP ionotropic receptor P2X7R) caused an increase in the intracellular levels of microglial MIP‐1α and MCP‐1. By using an extracellular Ca2+ chelator (EGTA) and P2X7R antagonists, oxidized ATP (oxATP) and brilliant blue G (BBG), we demonstrated that BzATP‐induced production of MIP‐1α and MCP‐1 levels was due to P2X7R activation and Ca2+‐dependent regulation. Coadministration of C6 glioma cells and oxATP into the rat cerebral cortex resulted in a reduction of MIP‐1α‐ and MCP‐1‐expressing microglia/macrophages. We suggest, based on the results from in vivo and in vitro studies, that a massive amount of ATP molecules released in the glioma tumor site may act as the regulator with P2X7R signaling that increases MIP‐1α and MCP‐1 expression in tumor‐infiltrating microglia/macrophages.

Enhanced cell growth and tumorigenicity of rat glioma cells by stable expression of human CD133 through multiple molecular actions

CD133 (Prominin‐1/AC133) is generally treated as a cell surface marker found on multipotent stem cells and tumor stem‐like cells, and its biological function remains debated. Genetically modified rat glioma cell lines were generated by lentiviral gene delivery of human CD133 into rat C6 glioma cells (hCD133+‐C6) or by infection of C6 cells with control lentivirus (mock‐C6). Stable hCD133 expression promoted the self‐renewal ability of C6‐formed spheres with an increase in the expression of the stemness markers, Bmi‐1 and SOX2. Akt phosphorylation, Notch‐1 activation, and Notch‐1 target gene expression (Hes‐1, Hey1 and Hey2) were increased in hCD133+‐C6 when compared to mock‐C6. The inhibition of Akt phosphorylation, Notch‐1 activation, and Hes‐1 in hCD133+‐C6 cells effectively suppressed their clonogenic ability, indicating that these factors are involved in expanding the growth of hCD133+‐C6. An elevated expression of GTPase‐activating protein 27 (Arhgap27) was detected in hCD133+‐C6. A decline in the invasion of hCD133+‐C6 by knockdown of Arhgap27 expression indicated the critical role of Arhgap27 in promoting cell migration of hCD133+‐C6. In vivo study further showed that hCD133+‐C6 formed aggressive tumors in vivo compared to mock‐C6. Exposure of hCD133+‐C6 to arsenic trioxide not only reduced Akt phosphorylation, Notch‐1 activation and Hes‐1 expression in vitro, but also inhibited their tumorigenicity in vivo. The results show that C6 glioma cells with stable hCD133 expression enhanced their stemness properties with increased Notch‐1/Hes‐1 signaling, Akt activation, and Arhgap27 action, which contribute to increased cell proliferation and migration of hCD133+‐C6 in vitro, as well as progressive tumor formation in vivo.

Mps one binder 2 gene upregulation in the stellation of astrocytes induced by cAMP-dependent pathway

Astrocytes, the major glial population in the central nervous system (CNS), play an important role in neuronal homeostasis, neurogenesis, and synaptogenesis. The cells have a stellate shape with elaborated processes in the developing CNS. Cultured astrocytes become stellate when the cells undergo differentiation in response to stimuli. Nevertheless, the molecular mechanism for astrocytic stellation is poorly understood. Here, we showed that the addition of serum induced a flat polygonal shape in cultured astrocytes with a reduced level of Mps one binder 2 (Mob2) that is involved in neurite growth by forming stable complex with a nuclear Ser/Thr kinase Dbf2‐related protein kinase 1 (NDR1). Furthermore, exposure to a membrane permeable cAMP analogue, dbcAMP, not only induced astrocytic stellation, but also caused an increase in Mob2 expression. Similarly, the upregulation of Mob2 mRNA expression was induced by exposure of astrocytes to pituitary adenylyl cyclase‐activating polypeptide (PACAP). Pretreatment with a cAMP/protein kinase A (PKA) inhibitor, KT‐5720, significantly blocked the effect of dbcAMP and PACAP on induced upregulation of Mob2 mRNA expression in astrocytes. In addition, the process withdrawal of dbcAMP‐treated astrocytes was caused by the inhibition of Mob2 expression using lentivirus‐mediated Mob2 shRNA delivery system. Based on our findings, we suggest that Mob2 is involved in PKA signaling‐mediated astrocytic stellation. J. Cell. Biochem. 113: 3019–3028, 2012.

Functional Role of Matrix gla Protein in Glioma Cell Migration

Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor subtype. Despite that metastasis of GBM beyond the central nervous system (CNS) is rare, its malignancy is attributed to the highly infiltration trait, leading to the difficulty of complete surgical excision. Matrix gla protein (MGP) is a vitamin K-dependent small secretory protein, and functions as a calcification inhibitor. The involvement of MGP function in glioma cell dynamics remains to be clarified. The study showed that a low proliferative rat C6 glioma cell line named as C6-2 exhibited faster migratory and invasive capability compared to that observed in a high tumorigenic rat C6 glioma cell line (called as C6-1). Interestingly, C6-2 cells expressed higher levels of MGP molecules than C6-1 cells did. Lentivirus-mediated short hairpin RNA (shRNA) against MGP gene expression (MGP-KD) in C6-2 cells or lentivirus-mediated overexpression of MGP transcripts in C6-1 cells resulted in the morphological alteration of the two cell lines. Moreover, MGP-KD caused a decline in cell migration and invasion ability of C6-2 cells. In contrast, increased expression of MGP in C6-1 cells promoted their cell migration and invasion. The observations were further verified by the results from the implantation of C6-1 and C6-2 cells into ex vivo brain slice and in vivo rat brain. Thus, our results demonstrate that the manipulation of MGP expression in C6 glioma cells can mediate glioma cell migratory activity. Moreover, our findings indicate the possibility that high proliferative glioma cells expressing a high level of MGP may exist and contribute to tumor infiltration and recurrence.

Reduction of CD200 expression in glioma cells enhances microglia activation and tumor growth.

CD200, a type I transmembrane glycoprotein, can interact with its receptor CD200R, which plays an inhibitory role in the activation of microglia—the resident macrophages of the central nervous system. In this study, the rat C6 glioma cell line (C6‐1) that was previously characterized with high in vivo tumorigenicity was found to generate CD200 mRNA abundantly. However, CD200 expression was barely detected in another C6 glioma cell clone (C6‐2) that was previously found to display low tumorigenic behavior. The results from CD200 immunohistochemistry on human glioma tissue array also showed that tumor cells in Grade I–II astrocytoma expressed a lower level of CD200 immunoreactivity than those detected in Grade III–IV glioblastoma multiforme. C6‐1 transfectants with stable downregulation of CD200 gene expression using lentivirus knockdown approach were generated (C6‐KD). Microglia and iNOS+ cells were increased when microglia were co‐cultured with C6‐KD cells. The colony formation of C6‐KD was also augmented when those cells were co‐cultured with microglia. Yet, increased colony formation of C6‐KD transfectants in the co‐culture with microglia was effectively suppressed by interleukin (IL)‐4 and IL‐10. The in vivo results indicated that the tumor formation of C6‐1 cells in rat brain was promoted after CD200 gene knockdown. Moreover, CD11b+ activated microglia and iNOS+ microglia were highly accumulated in the tumor site formed by C6‐KD. In conclusion, our findings demonstrate that the downregulation of CD200 expression in CD200‐rich glioma cells could foster the formation of an activated microglia–associated tumor microenvironment, leading to glioma progression.

Function of B-cell CLL/lymphoma 11B in glial progenitor proliferation and oligodendrocyte maturation

B-cell CLL/lymphoma 11B (Bcl11b) – a C2H2 zinc finger transcriptional factor – is known to regulate neuronal differentiation and function in the development of the central nervous system (CNS). Although its expression is reduced during oligodendrocyte (OLG) differentiation, its biological role in OLGs remains unknown. In this study, we found that the downregulation of Bcl11b gene expression in glial progenitor cells (GPCs) by lentivirus-mediated gene knockdown (KD) causes a reduction in cell proliferation with inhibited expression of stemness-related genes, while increasing the expression of cell cyclin regulator p21. In contrast, OLG specific transcription factors (Olig1) and OLG cell markers, including myelin proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG), were upregulated in Bcl11b-KD GPCs. Chromatin immunoprecipitation (ChIP) analysis indicated that Bcl11b bound to the promoters of Olig1 and PLP, suggesting that Bcl11b could act as a repressor for Olig1 and PLP, similar to its action on p21. An increase in the number of GC+- or PLP+- OLGs derived from Bcl11b-KD GPCs or OLG precursor cells was also observed. Moreover, myelin basic protein (MBP) expression in OLGs derived from Bcl11b-KD GPCs was enhanced in hippocampal neuron co-cultures and in cerebellar brain-slice cultures. The in vivo study using a lysolecithin-induced demyelinating animal model also indicated that larger amounts of MBP+-OLGs and PLP+-OLGs derived from implanted Bcl11b-KD GPCs were present at the lesioned site of the white matter than in the scramble group. Taken together, our results provide insight into the functional role of Bcl11b in the negative regulation of GPC differentiation through the repression of OLG differentiation-associated genes.