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author:("Si, yiliang")
1.  Progastrin Stimulates Colonic Cell Proliferation via CCK2R- and β-Arrestin–Dependent Suppression of BMP2 
Gastroenterology  2013;145(4):10.1053/j.gastro.2013.07.034.
Background & Aims
Progastrin stimulates colonic mucosal proliferation and carcinogenesis through the cholecystokinin 2 receptor (CCK2R)—partly by increasing numbers of colonic progenitor cells. However, little is known about the mechanisms by which progastrin stimulates colonic cell proliferation. We investigated the role of bone morphogenetic proteins (BMPs) in progastrin induction of colonic cell proliferation via CCK2R.
Methods
We performed microarray analysis to compare changes in gene expression in the colonic mucosa of mice that express a human progastrin transgene (hGAS), gastrin knockout (GAS−/−) mice, and C57BL/6 mice (controls); the effects of progastrin were also determined on in vitro colonic crypt cultures from cholecystokinin 2 receptor knockout (CCK2R−/−) and wild-type mice. Human colorectal and gastric cancer cells that expressed CCK2R were incubated with progastrin or Bmp2 protein; levels of β-arrestin-1 and -2 (ARRB1 and ARRB2) were knocked down using small interfering RNAs. Cells were analyzed for progastrin binding, proliferation, changes in gene expression, and symmetric cell division.
Results
The BMP pathway was downregulated in the colons of hGAS mice, compared with controls. Progastrin suppressed transcription of Bmp2 through a pathway that required CCK2R and was mediated by ARRB1 and ARRB2. In mouse colonic epithelial cells, downregulation of Bmp2 led to decreased phosphorylation of Smads1/5/8 and suppression of Id4. In human gastric and colorectal cancer cell lines, CCK2R was necessary and sufficient for progastrin binding and induction of proliferation; these effects were blocked when cells were incubated with recombinant Bmp2. Incubation with progastrin increased the number of CD44+, bromodeoxyuridine+, and NUMB+ cells, indicating an increase in symmetric divisions of putative cancer stem cells.
Conclusions
Progastrin stimulates proliferation in colons of mice and cultured human cells via CCK2R- and ARRB1- and 2-dependent suppression of Bmp2 signaling. This process promotes symmetric cell division.
doi:10.1053/j.gastro.2013.07.034
PMCID: PMC3829714  PMID: 23891976
Progastrin; CCK2R; BMP
2.  Bone marrow cells as precursors of the tumor stroma 
Experimental cell research  2013;319(11):1650-1656.
Cancer is a systemic disease. Local and distant factors conspire to promote or inhibit tumorigenesis. The bone marrow is one important source of tumor promoting cells. These include the important mature and immature hematopoietic cells as well as circulating mesenchymal progenitors. Recruited bone marrow cells influence carcinogenesis at the primary site, within the lymphoreticular system and even presage metastasis through their recruitment to distant organs. In this review we focus on the origins and contribution of cancer-associated fibroblasts in tumorigenesis. Mesenchymal cells present an important opportunity for targeted cancer prevention and therapy.
doi:10.1016/j.yexcr.2013.03.006
PMCID: PMC4097986  PMID: 23499739
Cancer; Fibroblasts; Tumor microenvironment; Mesenchyme; Carcinogenesis
3.  Infusion of Mesenchymal Stem Cells Ameliorates Hyperglycemia in Type 2 Diabetic Rats 
Diabetes  2012;61(6):1616-1625.
Infusion of mesenchymal stem cells (MSCs) has been shown to effectively lower blood glucose in diabetic individuals, but the mechanism involved could not be adequately explained by their potential role in promoting islet regeneration. We therefore hypothesized that infused MSCs might also contribute to amelioration of the insulin resistance of peripheral insulin target tissues. To test the hypothesis, we induced a diabetic rat model by high-fat diet/streptozotocin (STZ) administration, performed MSC infusion during the early phase (7 days) or late phase (21 days) after STZ injection, and then evaluated the therapeutic effects of MSC infusion and explored the possible mechanisms involved. MSC infusion ameliorated hyperglycemia in rats with type 2 diabetes (T2D). Infusion of MSCs during the early phase not only promoted β-cell function but also ameliorated insulin resistance, whereas infusion in the late phase merely ameliorated insulin resistance. Infusion of MSCs resulted in an increase of GLUT4 expression and an elevation of phosphorylated insulin receptor substrate 1 (IRS-1) and Akt (protein kinase B) in insulin target tissues. This is the first report of MSC treatment improving insulin sensitivity in T2D. These data indicate that multiple roles and mechanisms are involved in the efficacy of MSCs in ameliorating hyperglycemia in T2D.
doi:10.2337/db11-1141
PMCID: PMC3357293  PMID: 22618776
4.  LRP16 Integrates into NF-κB Transcriptional Complex and Is Required for Its Functional Activation 
PLoS ONE  2011;6(3):e18157.
Background
Nuclear factor κB (NF-κB)-mediated pathways have been widely implicated in cell survival, development and tumor progression. Although the molecular events of determining NF-κB translocation from cytoplasm to nucleus have been extensively documented, the regulatory mechanisms of NF-κB activity inside the nucleus are still poorly understood. Being a special member of macro domain proteins, LRP16 was previously identified as a coactivator of both estrogen receptor and androgen receptor, and as an interactor of NF-κB coactivator UXT. Here, we investigated the regulatory role of LRP16 on NF-κB activation.
Methodology
GST pull-down and coimmunoprecipitation (CoIP) assays assessed protein-protein interactions. The functional activity of NF-κB was assessed by luciferase assays, changes in expression of its target genes, and its DNA binding ability. Annexin V staining and flow cytometry analysis were used to evaluate cell apoptosis. Immunohistochemical staining of LRP16 and enzyme-linked immunosorbent assay-based evaluation of active NF-κB were performed on primary human gastric carcinoma samples.
Results
We demonstrate that LRP16 integrates into NF-κB transcriptional complex through associating with its p65 component. RNA interference knockdown of the endogenous LRP16 in cells leads to impaired NF-κB activity and significantly attenuated NF-κB-dependent gene expression. Mechanistic analysis revealed that knockdown of LRP16 did not affect tumor necrosis factor α (TNF-α)-induced nuclear translocation of NF-κB, but blunted the formation or stabilization of functional NF-κB/p300/CREB-binding protein transcription complex in the nucleus. In addition, knockdown of LRP16 also sensitizes cells to apoptosis induced by TNF-α. Finally, a positive link between LRP16 expression intensity in nuclei of tumor cells and NF-κB activity was preliminarily established in human gastric carcinoma specimens.
Conclusions
Our findings not only indicate that LRP16 is a crucial regulator for NF-κB activation inside the nucleus, but also suggest that LRP16 may be an important contributor to the aberrant activation of NF-κB in tumors.
doi:10.1371/journal.pone.0018157
PMCID: PMC3069058  PMID: 21483817
5.  Keratin 18 attenuates estrogen receptor α-mediated signaling by sequestering LRP16 in cytoplasm 
BMC Cell Biology  2009;10:96.
Background
Oncogenesis in breast cancer is often associated with excess estrogen receptor α(ERα) activation and overexpression of its coactivators. LRP16 is both an ERα target gene and an ERα coactivator, and plays a crucial role in ERα activation and proliferation of MCF-7 breast cancer cells. However, the regulation of the functional availability of this coactivator protein is not yet clear.
Results
Yeast two-hybrid screening, GST pulldown and coimmunoprecipitation (CoIP) identified the cytoplasmic intermediate filament protein keratin 18 (K18) as a novel LRP16-interacting protein. Fluorescence analysis revealed that GFP-tagged LRP16 was primarily localized in the nuclei of mock-transfected MCF-7 cells but was predominantly present in the cytoplasm of K18-transfected cells. Immunoblotting analysis demonstrated that the amount of cytoplasmic LRP16 was markedly increased in cells overexpressing K18 whereas nuclear levels were depressed. Conversely, knockdown of endogenous K18 expression in MCF-7 cells significantly decreased the cytoplasmic levels of LRP16 and increased levels in the nucleus. CoIP failed to detect any interaction between K18 and ERα, but ectopic expression of K18 in MCF-7 cells significantly blunted the association of LRP16 with ERα, attenuated ERα-activated reporter gene activity, and decreased estrogen-stimulated target gene expression by inhibiting ERα recruitment to DNA. Furthermore, BrdU incorporation assays revealed that K18 overexpression blunted the estrogen-stimulated increase of S-phase entry of MCF-7 cells. By contrast, knockdown of K18 in MCF-7 cells significantly increased ERα-mediated signaling and promoted cell cycle progression.
Conclusions
K18 can effectively associate with and sequester LRP16 in the cytoplasm, thus attenuating the final output of ERα-mediated signaling and estrogen-stimulated cell cycle progression of MCF-7 breast cancer cells. Loss of K18 increases the functional availability of LRP16 to ERα and promotes the proliferation of ERα-positive breast tumor cells. K18 plays an important functional role in regulating the ERα signaling pathway.
doi:10.1186/1471-2121-10-96
PMCID: PMC2804594  PMID: 20035625
6.  FHL2 interacts with and acts as a functional repressor of Id2 in human neuroblastoma cells 
Nucleic Acids Research  2009;37(12):3996-4009.
Inhibitor of differentiation 2 (Id2) is a natural inhibitor of the basic helix–loop–helix transcription factors. Although Id2 is well known to prevent differentiation and promote cell-cycle progression and tumorigenesis, the molecular events that regulate Id2 activity remain to be investigated. Here, we identified that Four-and-a-half LIM-only protein 2 (FHL2) is a novel functional repressor of Id2. Moreover, we demonstrated that FHL2 can directly interact with all members of the Id family (Id1–4) via an N-terminal loop–helix structure conserved in Id proteins. FHL2 antagonizes the inhibitory effect of Id proteins on basic helix–loop–helix protein E47-mediated transcription, which was abrogated by the deletion mutation of Ids that disrupted their interaction with FHL2. We also showed a competitive nature between FHL2 and E47 for binding Id2, whereby FHL2 prevents the formation of the Id2–E47 heterodimer, thus releasing E47 to DNA and restoring its transcriptional activity. FHL2 expression was remarkably up-regulated during retinoic acid-induced differentiation of neuroblastoma cells, during which the expression of Id2 was opposite to that. Ectopic FHL2 expression in neuroblastoma cells markedly reduces the transcriptional and cell-cycle promoting functions of Id2. Altogether, these results indicate that FHL2 is an important repressor of the oncogenic activity of Id2 in neuroblastoma cells.
doi:10.1093/nar/gkp332
PMCID: PMC2709579  PMID: 19417068
7.  Id2 promotes the invasive growth of MCF-7 and SKOV-3 cells by a novel mechanism independent of dimerization to basic helix-loop-helix factors 
BMC Cancer  2009;9:75.
Background
Inhibitor of differentiation 2 (Id2) is a critical factor for cell proliferation and differentiation in normal vertebrate development. Most of the biological function of Id2 has been ascribed to its helix-loop-helix motif. Overexpression of Id2 is frequently observed in various human tumors, but its role for invasion potential in tumor cells is dispute. We aimed to reveal the role of Id2 in invasion potential in poorly invasive and estrogen receptor α (ERα)-positive MCF-7 and SKOV-3 cancer cells.
Methods
MCF-7 and SKOV-3 cells were stably transfected with the wild-type, degradation-resistant full-length or helix-loop-helix (HLH)-deleted Id2, respectively. Protein levels of Id2 and its mutants and E-cadherin were determined by western blot analysis and mRNA levels of Id2 and its mutants were determined by RT-PCR. The effects of Id2 and its mutants on cell proliferation were determined by [3H]-thymidine incorporation assay and the 3- [4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) dye method. The in vitro invasion potential of cells was evaluated by Transwell assay. Cell motility was assessed by scratch wound assay. The promoter activity of E-cadherin was determined by cotransfection and luciferase assays.
Results
Ectopic transfection of the wild-type Id2 markedly increased the protein and mRNA expression of Id2 in MCF-7 and SKOV-3 cells; the protein level but not mRNA level was further increased by transfection with the degradation-resistant Id2 form. The ectopic expression of Id2 or its mutants did not alter proliferation of either MCF-7 or SKOV-3 cells. Transfection of the wild-type Id2 significantly induced the invasion potential and migratory capacity of cells, which was further augmented by transfection with the degradation-resistant full-length or HLH-deleted Id2. E-cadherin protein expression and transactivation of the proximal E-cadherin promoter were markedly suppressed by the degradation-resistant full-length or HLH-deleted Id2 but not wild-type Id2. Ectopic expression of E-cadherin in MCF-7 and SKOV-3 cells only partially blunted the invasion potential induced by the degradation-resistant HLH-deleted Id2.
Conclusion
Overexpression of Id2 in ERα-positive epithelial tumor cells indeed increases the cells' invasive potential through a novel mechanism independent of dimerization to basic helix-loop-helix factors. E-cadherin contributes only in part to Id2-induced cell invasion when Id2 is accumulated to a higher level in some specific cell types.
doi:10.1186/1471-2407-9-75
PMCID: PMC2654660  PMID: 19257909

Results 1-7 (7)