Acetylcholine induces mesenchymal stem cell migration via Ca2+ /PKC/ERK1/2 signal pathway

Acetylcholine (ACh) plays an important role in neural and non-neural function, but its role in mesenchymal stem cell (MSC) migration remains to be determined. In the present study, we have found that ACh induces MSC migration via muscarinic acetylcholine receptors (mAChRs). Among several mAChRs, MSCs express mAChR subtype 1 (m1AChR). ACh induces MSC migration via interaction with mAChR1. MEK1/2 inhibitor PD98059 blocks ERK1/2 phosphorylation while partially inhibiting the ACh-induced MSC migration. InsP3Rs inhibitor 2-APB that inhibits MAPK/ERK phosphorylation completely blocks ACh-mediated MSC migration. Interestingly, intracellular Ca(2+) ATPase-specific inhibitor thapsigargin also completely blocks ACh-induced MSC migration through the depletion of intracellular Ca(2+) storage. PKCα or PKCβ inhibitor or their siRNAs only partially inhibit ACh-induced MSC migration, but PKC-ζ siRNA completely inhibits ACh-induced MSC migration via blocking ERK1/2 phosphorylation. These results indicate that ACh induces MSC migration via Ca(2+), PKC, and ERK1/2 signal pathways.     

Inhibition of GPR40 protects MIN6 β cells from palmitate-induced ER stress and apoptosis

Chronic exposure to elevated concentration of free fatty acids (FFA) has been verified to induce endoplasmic reticulum (ER) stress, which leads to pancreatic β-cell apoptosis. As one of the medium and long chain FFA receptors, GPR40 is highly expressed in pancreatic β cells, mediates both acute and chronic effects of FFA on β-cell function, but the role of GPR40 in FFA-induced β-cell apoptosis remains unclear. In this study, we investigated the possible effects of GPR40 in palmitate-induced MIN6 β-cell apoptosis, and found that DC260126, a novel small molecular antagonist of GPR40, could protect MIN6 β cells from palmitate-induced ER stress and apoptosis. Similar results were observed in GPR40-deficient MIN6 cells, indicating that palmitate-induced β-cell apoptosis is at least partially dependent on ER stress pathway via GRP40.     

Hypoxia modulation of peroxisome proliferator-activated receptors (PPARs) in human glioblastoma stem cells. Implications for therapy

Gliobastoma (GB), the most common adult brain tumor, infiltrates normal brain area rendering impossible the complete surgical resection, resulting in a poor median survival (14–15 months), despite the aggressive multimodality treatments post‐surgery, such as radiation and chemo‐therapy. GB is characterized by hypoxic and necrotic regions due to a poorly organized tumor vascularization, leading to inadequate blood supply and consequently to hypoxic and necrotic areas. We have previously shown that, under hypoxia GB primary cells increased the expression of stemness markers as well as the expression of the nuclear receptor peroxisome proliferator‐activated receptor α (PPARα) and also the crucial role played by PPARs in mouse neural stem cells maintenance and differentiation. Due to the importance of lipid signaling in cell proliferation and differentiation, in this work, we analyzed the expression of PPARs in GB neurospheres both in normoxic and hypoxic conditions. The results obtained suggest a differential regulation of the three PPARs by hypoxia, thus indicating a possible therapeutic strategy to counteract GB recurrencies. J. Cell. Biochem. 113: 3342–3352, 2012. © 2012 Wiley Periodicals, Inc.     

Comparative analysis of pre-replication complex proteins in transformed and normal cells

This study examines the abundance of the major protein constituents of the pre-replication complex (pre-RC), both genome-wide and in association with specific replication origins, namely the lamin B2, c-myc, 20mer1, and 20mer2 origins. Several pre-RC protein components, namely ORC1-6, Cdc6, Cdt1, MCM4, MCM7, as well as additional replication proteins, such as Ku70/86, 14-3-3, Cdc45, and PCNA, were comparatively and quantitatively analyzed in both transformed and normal cells. The results show that these proteins are overexpressed and more abundantly bound to chromatin in the transformed compared to normal cells. Interestingly, the 20mer1, 20mer2, and c-myc origins exhibited a two- to threefold greater origin activity and a two- to threefold greater in vivo association of the pre-RC proteins with these origins in the transformed cells, whereas the origin associated with the housekeeping lamin B2 gene exhibited both similar levels of activity and in vivo association of these pre-RC proteins in both cell types. Overall, the results indicate that cellular transformation is associated with an overexpression and increased chromatin association of the pre-RC proteins. This study is significant, because it represents the most systematic comprehensive analysis done to date, using multiple replication proteins and different replication origins in both normal and transformed cell lines.     

Silibinin suppresses the maintenance of colorectal cancer stem-like cells by inhibiting PP2A/AKT/mTOR pathways

Silibinin, an effective chemo-preventive agent in various cancer types, suppresses cancer cell growth, but its effects on cancer stem-like cells (CSLCs) remain unclear. This study aimed to examine whether silibinin inhibited the development of CSLCs and disclose the underlying signaling. The colorectal cancer spheroid culture system was used for enriching CSLCs. The effects of silibinin on CSLCs were evaluated by counting sphere numbers, and calculating the percentage of CD133+ cells by flow cytometry and immunofluorescence both in the absence and presence of different concentrations of silibinin. The results showed the sphere number of CCS was 36 ± 9.6 after 15 days of CSLC enrichment in spheroid culture, and the percentage of CD133+ cells increased to 18 ± 6.4% compared to 3 ± 0.8% before enrichment. Treatment with silibinin reduced the sphere formation to 5 ± 3.3 and decreased the CD133+ percentage to 8 ± 2.3%. Interestingly, treatment of silibinin suppressed the activation of the AKT Ser473/mTOR pathway in spheroid culture through suppressing the activity of protein phosphatase 2Ac subunit (PP2Ac). In a xenograft tumor model, treatment with silibinin also inhibited tumor formation rate and tumor growth. Silibinin, which inhibits colon CSLCs self-renewal and sphere formation by suppressing the PP2Ac/AKT Ser473/mTOR pathway, may be a compound for developing new strategies in modulating CSLCs in cancer therapy.     

Interferon regulatory factor-1 as a positive regulator for high glucose-induced proliferation of vascular smooth muscle cells

High glucose-induced proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the development of diabetic vascular diseases. However, molecular mediators responding for the proliferation of VSMCs remain to be determined. In this study, VSMCs were isolated from the rat thoracic aorta, and two cell models with Irf-1 knockdown and overexpression were established by transfecting cells with pGCsi-FU-Irf-1 and pGC-FU-Irf-1, respectively. Subsequently, high glucose was added to cells to induce proliferation. Proliferation assays were performed to see whether Irf-1 was involved in high glucose-induced proliferation of VSMCs. In addition, the expression of Irf-1 was detected in VSMCs stimulated with high glucose and the thoracic aorta of diabetic rats to confirm the relationship between Irf-1 expression and the proliferation of hyperglycemia-dependent VSMCs. The results showed that Irf-1 expression was significantly higher in the thoracic aorta of diabetic rats and VSMCs stimulated with high glucose than that in nondiabetic rats and untreated cells. Overexpression of Irf-1 accelerated the proliferation of VSMCs, and down-regulation of Irf-1 expression significantly depressed the proliferative ability of VSMCs under high-glucose conditions, indicating that Irf-1 was a positive regulator for high glucose-induced proliferation of VSMCs. It could be presumed that Irf-1 is associated with the accelerated proliferation of VSMCs in diabetic vascular diseases and may prove to be a potential target gene for disease treatment.     

Chromosome dynamic changes in two cultured chinese human embryonic stem cell lines: Single nucleotide polymorphism, copy number variation and loss of heterozygosity

The quality and safety of human embryonic stem cells (hESCs) in clinical application depend on gene stability. Two Chinese hESC lines, Zh1 and Zh21, were incubated over a long period. We observed and compared the gene stability in the passage numbers 20, 17 for Zh1 cell line and passage numbers 27, 60, 68 for Zh21 cell line. Single nucleotide polymorphisis analysis indicated that hESCs in early passages had relative gene stability; and with the increase in passage number, gene instability became strong. We also found that there were copy number variations (CNVs) in both Zh21 and Zh1. We analyzed the CNVs of Chinese Han Beijing man (CHB; normal Chinese people) and found that the all CNV forms were the loss in Zh21, Zh1, and CHB. We also analyzed and compared the related pathways of the mutant genes. We propose three steps to ensure hESC safety. Firstly, besides the conventional methods such as pluripotent genes, chromosome G‐banding and teratoma, high‐resolution DNA chip analysis should also be adopted; secondly, chromosomal properties are monitored every 10 passages in less than passage 50 and every 5 passages in more than passage 50; thirdly, the related pathways of mutant genes should be observed because only the mutant genes with variations of their related pathways may affected cell functions. J. Cell. Biochem. 113: 3520–3527, 2012. © 2012 Wiley Periodicals, Inc.