Gene Profiles in the Early Stage of Neuronal Differentiation of Mouse Bone Marrow Stromal Cells Induced by Basic Fibroblast Growth Factor.

A stably established population of mouse bone marrow stromal cells (BMSCs) with self-renewal and multilineage differentiation potential was expanded in vitro for more than 50 passages. These cells express high levels of mesenchymal stem cell markers and can be differentiated into adipogenic, chondrogenic, and osteogenic lineages in vitro. Subjected to basic fibroblast growth factor (bFGF) treatment, a typical neuronal phenotype was induced in these cells, as supported by neuronal morphology, induction of neuronal markers, and relevant electrophysiological excitability. To identify the genes regulating neuronal differentiation, cDNA microarray analysis was conducted using mRNAs isolated from cells differentiated for different time periods (0, 4, 24, and 72 h) after bFGF treatment. Various expression patterns of neuronal genes were stimulated by bFGF. These gene profiles were shown to be involved in developmental, functional, and structural integration of the nervous system. The expression of representative genes stimulated by bFGF in each group was verified by RT-PCR. Amongst proneural genes, the mammalian achate-schute homolog 1 (Mash-1), a basic helix-loop-helix transcriptional factor, was further demonstrated to be significantly upregulated. Overexpression of Mash-1 in mouse BMSCs was shown to induce the expression of neuronal specific enolase (NSE) and terminal neuronal morphology, suggesting that Mash-1 plays an important role in the induction of neuronal differentiation of mouse BMSCs.

Neural cell adhesion molecule modulates mesenchymal stromal cell migration via activation of MAPK/ERK signaling.

Mesenchymal Stromal Cells (MSCs) represent promising tools for cellular therapy owing to their multipotentiality and ability to localize to injured, inflamed sites and tumor. Various approaches to manipulate expression of MSC surface markers, including adhesion molecules and chemokine receptors, have been explored to enhance homing of MSCs. Recently, Neural Cell Adhesion Molecule (NCAM) has been found to be expressed on MSCs yet its function remains largely elusive. Herein, we show that bone marrow-derived MSCs from NCAM deficient mice exhibit defective migratory ability and significantly impaired adipogenic and osteogenic differentiation potential. We further explore the mechanism governing NCAM mediated migration of MSCs by showing the interplay between NCAM and Fibroblast Growth Factor Receptor (FGFR) induces activation of MAPK/ERK signaling, thereby the migration of MSCs. In addition, re-expression of NCAM180, but not NCAM140, could restore the defective MAPK/ERK signaling thereby the migration of NCAM deficient MSCs. Finally, we demonstrate that NCAM180 expression level could be manipulated by pro-inflammatory cytokine Tumor Necrosis Factor (TNF)-α treatment. Overall, our data reveal the vital function of NCAM in MSCs migration and differentiation thus raising the possibility of manipulating NCAM expression to enhance homing and therapeutic potential of MSCs in cellular therapy.

Clonal analysis for elucidating the lineage potential of embryonic NG2+ cells.

BACKGROUND AIMS: The widespread NG2-expressing neural progenitors in the central nervous system (CNS) are considered to be multifunctional cells with lineage plasticity, thereby possessing the potential for treating CNS diseases. Their lineages and functional characteristics have not been completely unraveled. The present study aimed to disclose the lineage potential of clonal NG2(+) populations in vitro and in vivo. METHODS: Twenty-four clones from embryonic cerebral cortex-derived NG2(+) cells were induced for oligodendrocyte, astrocyte, neuronal and chondrocyte differentiation. The expression profiles of neural progenitor markers chondroitin sulfate proteoglycan 4 (NG2), platelet-derived growth factor-α receptor (PDGFαR); nestin and neuronal cell surface antigen (A2B5) were subsequently sorted on cells with distinct differentiation capacity. Transplantation of these NG2(+) clones into the spinal cord was used to examine their lineage potential in vivo. RESULTS: In vitro differentiation analysis revealed that all the clones could differentiate into oligodendrocytes, and seven of them were bipotent (oligodendrocytes and astrocytes). Amazingly, one clone exhibited a multipotent capacity of differentiating into not only neuronal-glial lineages but also chondrocytes. These distinct subtypes were further found to exhibit phenotypic heterogeneity based on the examination of a spectrum of neural progenitor markers. Transplanted clones survived, migrated extensively and differentiated into oligodendrocytes, astrocytes or even neurons to integrate with the host spinal cord environment. CONCLUSIONS: These results suggest that NG2(+) cells contain heterogeneous progenitors with distinct differentiation capacities, and the immortalized clonal NG2(+) cell lines might provide a cell source for treating spinal cord disorders.

A novel role for neural cell adhesion molecule in modulating insulin signaling and adipocyte differentiation of mouse mesenchymal stem cells.

Neural cell adhesion molecule (NCAM) has recently been found on adult stem cells, but its biological significance remains largely unknown. In this study, we used bone-marrow-derived mesenchymal stem cells (MSCs) from wild-type and NCAM knockout mice to investigate the role of NCAM in adipocyte differentiation. It was demonstrated that NCAM isoforms 180 and 140 but not NCAM-120 are expressed on almost all wild-type MSCs. Upon adipogenic induction, Ncam(-/-) MSCs exhibited a marked decrease in adipocyte differentiation compared with wild-type cells. The role of NCAM in adipocyte differentiation was also confirmed in NCAM-silenced preadipocyte 3T3-L1 cells, which also had a phenotype with reduced adipogenic potential. In addition, we found that Ncam(-/-) MSCs appeared to be insulin resistant, as shown by their impaired insulin signaling cascade, such as the activation of the insulin-IGF-1 receptor, PI3K-Akt and CREB pathways. The PI3K-Akt inhibitor, LY294002, completely blocked adipocyte differentiation of MSCs, unveiling that the reduced adipogenic potential of Ncam(-/-) MSCs is due to insulin resistance as a result of loss of NCAM function. Furthermore, insulin resistance of Ncam(-/-) MSCs was shown to be associated with induction of tumor necrosis factor α (TNF-α), a key mediator of insulin resistance. Finally, we demonstrated that re-expression of NCAM-180, but not NCAM-140, inhibits induction of TNF-α and thereby improves insulin resistance and adipogenic potential of Ncam(-/-) MSCs. Our results suggest a novel role of NCAM in promoting insulin signaling and adipocyte differentiation of adult stem cells. These findings raise the possibility of using NCAM intervention to improve insulin resistance.

Neural cell adhesion molecule potentiates the growth of murine melanoma via ß-catenin signaling by association with fibroblast growth factor receptor and glycogen synthase kinase-3ß.

The neural cell adhesion molecule (NCAM) was recently shown to be involved in the progression of various tumors with diverse effects. We previously demonstrated that NCAM potentiates the cellular invasion and metastasis of melanoma. Here we further report that the growth of melanoma is obviously retarded when the expression of NCAM is silenced. We found that the proliferation of murine B16F0 melanoma cells, their colony formation on soft agar, and growth of transplanted melanoma in vivo are clearly inhibited by the introduction of NCAM siRNA. Interestingly, change of NCAM expression level is shown to regulate the activity of Wnt signaling molecule, ß-catenin, markedly. This novel machinery requires the function of FGF receptor and glycogen synthase kinase-3ß but is independent of the Wnt receptors, MAPK-Erk and PI3K/Akt pathways. In addition, NCAM is found to form a functional complex with ß-catenin, FGF receptor, and glycogen synthase kinase-3ß. Moreover, up-regulation of NCAM140 and NCAM180 appears more potent than NCAM120 in activation of ß-catenin, suggesting that the intracellular domain of NCAM is required for facilitating the ß-catenin signaling. Furthermore, the melanoma cells also exhibit distinct differentiation phenotypes with the NCAM silencing. Our findings reveal a novel regulatory role of NCAM in the progression of melanoma that might serve as a new therapeutic target for the treatment of melanoma.

Neural cell adhesion molecule potentiates invasion and metastasis of melanoma cells through CAMP-dependent protein kinase and phosphatidylinositol 3-kinase pathways.

Neural cell adhesion molecule (NCAM) has been implicated in tumor metastasis yet its function in melanoma progression remains unclear. Here, we demonstrate that stably silencing NCAM expression in mouse melanoma B16F0 cells perturbs their cellular invasion and metastatic dissemination in vivo. The pro-invasive function of NCAM is exerted via dual mechanisms involving both cAMP-dependent protein kinase (PKA) and phosphatidylinositol 3-kinase (PI3K) pathways. Pharmacologic inhibition of PKA and PI3K leads to impaired cellular invasion. In contrast, forced expression of constitutively activated Akt, the major downstream target of PI3K, restores the defective cellular invasiveness of NCAM knock-down (KD) B16F0 cells. Furthermore, attenuation of either PKA or Akt activity in NCAM KD cells is shown to affect their common downstream target, transcription factor cAMP response element binding protein (CREB), which in turn down-regulates mRNA expression of matrix metalloproteinase-2 (MMP-2), thus contributes to impaired cellular invasion and metastasis of melanoma cells. Together, these findings indicate that NCAM potentiates cellular invasion and metastasis of melanoma cells through stimulation of PKA and PI3K signaling pathways thus suggesting the potential implication of anti-NCAM strategy in melanoma treatment.

Insulin-like growth factor 1 protects human neuroblastoma cells SH-EP1 against MPP+-induced apoptosis by AKT/GSK-3ß/JNK signaling.

Parkinson's disease (PD) is primarily caused by severe degeneration and loss of dopamine neurons in the substantia nigra pars compacta. Thus, preventing the death of dopaminergic neurons is thought to be a potential strategy to interfere with the development of PD. In the present work, we studied the effect of insulin-like growth factor-1 (IGF-1) on 1-methyl-4-phenylpyridinium (MPP+)-induced apoptosis in human neuroblastoma SH-EP1 cells. We found that the PI3K/AKT pathway plays a central role in IGF-mediated cell survival against MPP+ neurotoxicity. Furthermore, we demonstrated that the protective effect of AKT is largely dependent on the inactivation of GSK-3ß, since inhibition of GSK-3ß by its inhibitor, BIO, could mimic the protective effect of IGF-1 on MPP+-induced cell death in SH-EP1 cells. Interestingly, the IGF-1 potentiated PI3K/AKT activity is found to negatively regulate the JNK related apoptotic pathway and this negative regulation is further shown to be mediated by AKT-dependent GSK-3ß inactivation. Thus, our results demonstrated that IGF-1 protects SH-EP1 cells from MPP+-induced apoptotic cell death via PI3K/AKT/GSK-3ß pathway, which in turn inhibits MPP+-induced JNK activation.

NCAM-mediated locomotor recovery from spinal cord contusion injury involves neuroprotection, axon regeneration, and synaptogenesis.

The expression level of neural cell adhesion molecule (NCAM), which plays a critical role in pathways involving development and plasticity of the nervous system, changes markedly after spinal cord injury (SCI). However, the significance of NCAM-involved mechanisms in SCI remains elusive. The present study demonstrates that NCAM-deficient (ND) mice exhibited significantly poorer locomotor activity than wildtype (WT) littermates with the same injury intensity by the contusion model. To determine detailed contribution of NCAM, quantitative immunohistochemistry examination was performed on the injured spinal cord of 6mm along the rostrocaudal axis in the animals for up to 5 weeks after SCI. Overall level of NCAM decreased initially in the lesion site but increased around the center of the injury thereafter. At acute stage, more apoptotic cells were found in the gray and white matter in ND mice. Between the two animal groups, no obvious difference in expression levels of GFAP (astrocytosis marker) and MBP (remyelination marker) was detected. However, diverse expression trends of NF200 (axon marker), GAP-43 (synaptogenesis indicator) and phosphorylated ERK (active signal molecule) were observed in the area encompassing the lesion site, and remarkable differences were illustrated between WT mice and ND littermates. Detailed analysis indicates that NCAM-mediated pathways may be involved in the activation of ERK at acute stages and bi-phasic upregulation of GAP-43 expression at acute and sub-acute stages after SCI to promote cell survival, outgrowth of regenerated axons, synaptogenesis, and function recovery.

NF-kappaB mediates MPP+-induced apoptotic cell death in neuroblastoma cells SH-EP1 through JNK and c-Jun/AP-1.

Parkinson's disease (PD) is characterized by a progressive loss of dopaminergic neurons in substantia nigra with unknown etiology. Neuropathology seen in the brains of PD patients can be closely mimicked by MPP(+)-induced neurotoxicity in vitro. In this study, we used an S-type human neuroblastoma cell line (SH-EP1) as a model to investigate the involvement of NF-kappaB and JNK pathways in MPP(+)-induced neurotoxicity. We show that NF-kappaB was activated by MPP(+) as evidenced by NF-kappaB p65 nuclear translocation, the increased DNA binding activity and a rapid phosphorylation of NF-kappaB inhibitor (IkappaBalpha). NF-kappaB partially mediated the neurotoxicity of MPP(+), as suggested by the reduction of MPP(+)-induced cell death by both a specific IkappaB kinase (IKK) inhibitor and a dominant negative form of IkappaBalpha (IkappaBalpha-M). Besides NF-kappaB, JNK and c-Jun/AP-1 were also activated upon MPP(+) stimulation. Inhibition of JNK activation with a specific JNK inhibitor partially reduced the MPP(+)-mediated cell death. Similarly, inhibition of c-Jun/AP-1 activation, either by a dominant negative c-Jun or c-Jun/AP-1 inhibitor, significantly attenuated MPP(+)-mediated cell death. These results suggest that both JNK and c-Jun/AP-1 activation are pro-apoptotic. Furthermore, we provide clear evidence for the existence of a crosstalk between the NF-kappaB and JNK signaling as MPP(+)-induced activation of JNK and c-Jun/AP-1 was strongly down-regulated in IkappaBalpha-M cells. In conclusion, we demonstrate that in SH-EP1 cells MPP(+)-induced neurotoxicity is partially mediated by NF-kappaB which in turn acts on the activation of JNK and c-Jun/AP-1. These results may point to a combined inhibition of NF-kappaB and JNK as a new approach to PD therapy.