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Rho guanine nucleotide dissociation inhibitor 1 (RhoGDI1) plays a critical role in regulating the activity of Rho guanosine triphosphatases (GTPases). Phosphorylation of RhoGDI1 dynamically modulates the activation of Rho GTPases, influencing cell proliferation and migration. This study explored the involvement of Never In Mitosis A (NIMA)-related serine/threonine protein kinase 2 (NEK2) in phosphorylating RhoGDI1 and its implications in cancer cell behavior associated with tumor progression. We employed GST pull-down assays and immunoprecipitation to investigate the interaction between NEK2 and RhoGDI1. Truncation fragments identified the region of RhoGDI1 responsible for binding with NEK2. Phosphorylation assays determined the site of NEK2-mediated phosphorylation on RhoGDI1. Functional assays were conducted using overexpression of the RhoGDI1 substitution mutant to assess their impact on cancer cell behavior. NEK2 directly bound to RhoGDI1 and phosphorylated it at Ser174. This phosphorylation event facilitated cancer cell proliferation and motility by activating RhoA and Rac1. The RhoGDI1 aa 112-134 region was critical for the binding to NEK2. Disruption of the NEK2-RhoGDI1 interaction through overexpression of a RhoGDI1 truncated fragment (aa 112-134) led to diminished RhoGDI1 phosphorylation and RhoA/Rac1 activation induced by NEK2, resulting in reduced cancer cell proliferation and migration. Moreover, in vivo studies showed reduced tumor growth and lung metastasis when the NEK2-RhoGDI1 interaction was disrupted. This study indicates that NEK2 promotes the metastatic behaviors of cancer cells by activating RhoA and Rac1 by phosphorylating RhoGDI1.

Purpose: CKS2 was identified as an upregulated gene in gastric cancer via our DNA microarray. This study was to verify the upregulation of CKS2 in many gastric cancer patients and to examine the CKS2-mediated cellular response., Methods: CKS2 upregulation was analyzed using reverse transcriptase PCR, real-time PCR, and immunohistochemical and clinicopathological analyses. GFP-CKS2 or CKS2-siRNA was used to analyze the cellular localization and proliferation., Results: The strong upregulation of mRNA and protein levels of CKS2 was identified. In CKS2-overexpressing cells, tumor suppressor p53 and p21(cip1) were downregulated and cell growth was increased. In contrast, CKS2-siRNA-transfected cells showed an increased tumor suppressor expression and decreased cell growth., Conclusions: We showed that CKS2 was significantly upregulated in gastric cancers and a high level of CKS2 was highly correlated with histologic tumor differentiation and pathological grade of the tumor size, lymph node, and metastasis stage. We suggest that the cell cycle regulator CKS2 might be deeply involved in gastric cancer progression.

Aims: The Wnt/beta-catenin pathway plays a critical part in several cell physiology events associated with embryonic development and adult homeostasis, including determination, proliferation, migration, and differentiation. However, the role of Wnt signaling in osteoblastogenesis from mesenchymal stem cells (MSC) remains a controversial matter. Therefore, in the present study, we investigated how ICAT (inhibitor of beta-catenin and TCF-4), a negative regulator of the Wnt signaling pathway, influenced differentiation and proliferation of human adipose tissue-derived stromal cells (hASC)., Main Methods: To mediate ICAT overexpression in hASC, we used a lentiviral gene transfer technique. We further determined the role of ICAT by RNAi technique., Key Findings: ICAT-transduced hASC exhibited lower TCF promoter activity and cellular growth capacity than control cells, but ICAT overexpression did not affect hASC attachment efficiency. ICAT overexpression also increased osteogenic differentiation. Conversely, introduction of an ICAT siRNA oligonucleotide increased TCF promoter activity and cellular proliferation, but it inhibited osteogenic differentiation., Significance: Taken together, these findings indicated that ICAT participated in regulating hASC proliferation and differentiation by modulating Wnt signaling.

Cardiovascular complications in diabetic patients have been reported to be related to the impaired proangiogenic actions of endothelial progenitor cells. In this study, we investigated the functions of adipose tissue-derived stromal cells (ASCs) in diabetes. We induced type I diabetes in rats by a intraperitoneal injection of 60 mg/kg of streptozotocin (STZ) and type II diabetes by the combined treatment of high fat diet and 45 mg/kg of STZ. Rat ASCs (rASCs) isolated from the adipose tissues in the interscapular and abdominal region of type I or type II diabetic rats showed lower proliferating ability than those of control rats. Diabetic rASCs showed lower blood flow recovery than those of control rats in a hindlimb ischemia model of nude mouse. When ASCs isolated from rat and human were exposed to high glucose concentrations, their proliferating abilities and improved blood flow in a hindlimb ischemia model were compromised, compared with ASCs that were maintained at control glucose concentrations. However, the same concentrations of mannitol did not affect these characteristics. Exposure of human ASCs (hASCs) to high glucose concentrations increased reactive oxygen species (ROS) production, and the addition of ROS scavengers [N-acetylcysteine (NAC) or catalase] to high glucose media partially decreased the high glucose-induced inhibitory effect on proliferating ability in hASCs. However, hASCs treated with high glucose medium for 6 days showed lower proliferation in control culture medium, which was not recovered by the addition of NAC or catalase. These data indicate that ASCs isolated from diabetic rats and exposed at high concentration of glucose have an impaired proangiogenic action and that the functional impairment is partly due to ROS generated by chronic exposure to high glucose concentrations.

Abstract: NDRG2 is a member of the N-myc downstream regulated gene (NDRG) family, implicated in cell growth and differentiation. Investigation of NDRG2 molecular interactions by yeast two-hybrid screening identified prenylated Rab acceptor-1 (PRA1), involved in vesicle trafficking and protein transport, as binding partner. Binding of NDRG2 (and NDRG1-4) with PRA1 in vitro was confirmed by GST pull-down assay and immunoprecipitation, and colocalization was verified by confocal microscopy in HCT116 cells. Intracellular coexpression showed that NDRG2 and PRA1 synergistically downregulate T-cell factor (TCF) promoter activity and GSK3β phosphorylation. Results suggest that NDRG2 and PRA1 might act synergistically to prevent signaling of TCF/β-catenin. Structured summary of protein interactions: NDRG2a binds to PRA1 by pull down (View interaction) NDRG2a physically interacts with PRA1 by two hybrid (View Interaction: 1, 2) PRA1 physically interacts with NDRG2a by anti tag coimmunoprecipitation (View interaction) NDRG2a , PRA1 and Catenin beta colocalize by cosedimentation (View interaction) NDRG2a and PRA1 colocalize by fluorescence microscopy (View Interaction: 1, 2, 3) [Copyright &y& Elsevier]

Abstract: Background: Cysteine proteases like cathepsins are widely distributed proteolytic enzymes and form tight equimolar complexes with cystatins at their active sites. Among cystatins, CST1, encoding cystatin SN, is a member of the type 2 salivary cystatin family found in a variety of fluids and secretions, including plasma, tears, and saliva. CST1 was identified as an upregulated gene in gastric cancer tissues compared to noncancerous regions using our Affymetrix GeneChip microarray. Methods: The upregulation of CST1 in gastric cancer was analyzed using RT-PCR (n =15), immnohistochemistry, and clinicopathological (n =77) analysis. CST1-siRNA was used for the suppression of CST1 gene expression and cathepsin proteolytic activity was assayed. Results: CST1 was upregulated in cancerous lesions of gastric cancer tissues compared to noncancerous regions and clinicopathological analysis showed a significant correlation between high expression of CST1 and pTNM stage (p =0.044). In CST1-siRNA transfected cells, cell proliferation was reduced and the proteolytic activity of cathepsins was increased. Conclusions: CST1 might be highly involved in gastric tumorigenesis and regulate the proteolytic activity of cysteine proteases. [Copyright &y& Elsevier]

Abstract: The prenylated Rab acceptor 1 (PRA1) is a ubiquitously expressed 21kDa protein containing two transmembrane domains that possibly induce its localization to the Golgi complex. It binds to prenylated Rab GTPases and VAMP2. In this study, we report that PRA1-overexpressing cells exhibited a significantly retarded growth rate as compared to that of the mock-transfected cells, and the transcriptional activity of TCF, as evaluated by TOPflash luciferase reporter assay, was profoundly reduced in the PRA1-overexpressed cells. These intracellular functions of PRA1 were verified by introducing deletion mutant or site-directed mutants, or small interfering RNA of PRA1. In addition, the translocation of β-catenin from the cytosol to the nucleus was blocked to a significant degree in the PRA1-cells, and the interaction of PRA1 and β-catenin was identified by confocal microscopy and immunoprecipitation analysis. Finally, we observed that the inhibition of TCF/β-catenin signaling by PRA1 is associated with ERK1/2 dephosphorylation. Therefore, our data suggest that the in vivo modulation of PRA1 may be involved in TCF/β-catenin signaling, as well as cellular proliferation and tumorigenesis. [Copyright &y& Elsevier]

Abstract: Mesenchymal stem cells (MSCs) have been discovered in a multitude of organs, but their distribution and identity are still uncertain. Furthermore, loose connective tissue (LCT) is dispersed throughout virtually all organs, but its biological role in tissue homeostasis is unclear. Here, we describe a unique organ culture system to explore the omnipresence and in situ identity of MSCs among the LCTs. This culture system included the use of the fibrin hydrogel coupled with dynamic culture conditions, using native LCTs obtained from various organs as starting materials. This culture allowed MSC outgrowth into the hydrogel to be robustly supported, while maintaining the structural integrity of LCTs during in vitro culture. Subcultured outgrown cells fulfilled the minimal requirements for defining MSCs on the basis of clonogenicity, multipotency, and immunophenotypic characteristics. In vitro label-retaining assay demonstrated that the numbers of mobilized and proliferated cells in situ increased in the pericapillary region and expressed both MSCs and pericytes markers, indicating that the in situ identity of MSCs represents a certain population of pericapillary pericytes. Our results indicate that this culture system affords a unique strategy for both isolating MSCs and recapitulating their niche in LCTs. [Copyright &y& Elsevier]