Crosstalk between NF-κB and β-catenin pathways in bacterial-colonized intestinal epithelial cells

2005 ◽  
Vol 289 (1) ◽  
pp. G129-G137 ◽  
Author(s):  
Jun Sun ◽  
Michael E. Hobert ◽  
Yingli Duan ◽  
Anjali S. Rao ◽  
Tong-Chuan He ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Signaling Pathways ◽  
Signaling Pathway ◽  
Intestinal Inflammation ◽  
Direct Interaction ◽  
Wild Type ◽  
Mediated Effects ◽  
Cell Proliferation And Differentiation ◽  
Important Regulator ◽  
Constitutively Active

Salmonella-epithelial cell interactions are known to activate the proinflammatory NF-κB signaling pathway and have recently been found to also influence the β-catenin signaling pathway, an important regulator of epithelial cell proliferation and differentiation. Here, using polarized epithelial cell models, we demonstrate that these same bacteria-mediated effects also direct the molecular crosstalk between the NF-κB and β-catenin signaling pathways. Convergence of these two pathways is a result of the direct interaction between the NF-κB p50 subunit and β-catenin. We show that PhoPc, the avirulent derivative of a wild-type Salmonella strain, attenuates NF-κB activity by stabilizing the association of β-catenin with NF-κB. In cell lines expressing constitutively active β-catenin, IκBα protein was indirectly stabilized and NF-κB activity was repressed after wild-type Salmonella colonization. Accordingly, constitutively active β-catenin was found to inhibit the secretion of IL-8. Thus our findings strongly suggest that the crosstalk between the β-catenin and NF-κB signaling pathways is an important regulator of intestinal inflammation.

H-Ras Is Involved in the Inside-out Signaling Pathway of Interleukin-3–Induced Integrin Activation

Blood ◽  
1999 ◽  
Vol 93 (5) ◽  
pp. 1540-1548 ◽  
Author(s):  
Hirohiko Shibayama ◽  
Naoyuki Anzai ◽  
Stephen E. Braun ◽  
Seiji Fukuda ◽  
Charlie Mantel ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Dominant Negative ◽  
The Other ◽  
Wild Type ◽  
Integrin Activation ◽  
Mapk Kinase ◽  
Interleukin 3 ◽  
Other Hand ◽  
Inside Out ◽  
Constitutively Active

Abstract The proto-oncogene product, p21ras, has been implicated in the cellular mechanism of adhesion, although its precise role has been controversial. Numerous cytokines and growth-factors activate Ras, which is an important component of their growth-promoting signaling pathways. On the other hand, the role of Ras in cytokine-induced adhesion has not been elucidated. We therefore investigated the function of H-Ras in the inside-out signaling pathway of interleukin-3 (IL-3)–induced integrin activation in the murine Baf3 cell line after transfection of cells with either constitutively active, dominant-negative, or wild-type H-Ras cDNAs. Adhesion of Baf3 cells to fibronectin was induced by IL-3 in a dose-dependent manner via very late antigen-4 (VLA-4; 4β1 integrins) and VLA-5 (5β1 integrins) activation. On the other hand, IL-4 did not induce the adhesion of Baf3 cells to fibronectin, although IL-4 did stimulate the cell proliferation of Baf3 cells. Constitutively active H-Ras–transfected Baf3 cells adhered to fibronectin without IL-3 stimulation through VLA-4 and VLA-5, whereas dominant-negative H-Ras–transfected Baf3 cells showed significantly less adhesion induced by IL-3 compared with wild-type and constitutively active H-Ras–transfected Baf3 cells. Anti-β1 integrin antibody (clone; 9EG7), which is known to change integrin conformation and activate integrins, induced the adhesion of dominant-negative H-Ras–transfected Baf3 cells as much as the other types of H-Ras–transfected Baf3 cells. 8-Br-cAMP, Dibutyryl-cAMP, Ras-Raf-1 pathway inhibitors, and PD98059, a MAPK kinase inhibitor, suppressed proliferation and phosphorylation of MAPK detected by Western blotting with anti–phospho-MAPK antibody, but not adhesion of any type of H-Ras–transfected Baf3 cells, whereas U-73122, a phospholipase C (PLC) inhibitor, suppressed adhesion of these cells completely. These data indicate that H-Ras and PLC, but not Raf-1, MAPK kinase, or the MAPK pathway, are involved in the inside-out signaling pathway of IL-3–induced VLA-4 and VLA-5 activation in Baf3 cells.

scholarly journals Proline Reverses the Abnormal Phenotypes of Colletotrichum trifolii Associated with Expression of Endogenous Constitutively Active Ras

2002 ◽  
Vol 68 (4) ◽  
pp. 1647-1651 ◽  
Author(s):  
Stephen D. Memmott ◽  
Young-sil Ha ◽  
Martin B. Dickman
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Signaling Pathways ◽  
G Protein ◽  
Growth Medium ◽  
Causative Organism ◽  
Wild Type ◽  
Colletotrichum Trifolii ◽  
Rich Media ◽  
Constitutively Active

ABSTRACT Colletotrichum trifolii is the causative organism of alfalfa anthracnose. We previously cloned and characterized the small prototypical G protein, Ras, of C. trifolii, which is involved in the signaling pathways that mediate interaction between the pathogen and its host. Transformants expressing constitutively active forms of Ras have growth medium-dependent phenotypes. In nutrient-rich media (e.g., yeast extract and peptone), the phenotype of the transformants was indistinguishable from that of the wild type. However, during nutrient starvation, the transformants lose polarity, have distended hyphae, and fail to sporulate and produce appressoria. Since peptone caused the phenotype to revert, amino acids were tested singly and in combination to identify the responsible amino acid(s). We found that 1.6 mM proline in the medium reverses the constitutively active Ras phenotype.

scholarly journals Lipopolysaccharide Stimulates p62-Dependent Autophagy-Like Aggregate Clearance in Hepatocytes

2014 ◽  
Vol 2014 ◽  
pp. 1-13 ◽  
Author(s):  
Christine Chen ◽  
Meihong Deng ◽  
Qian Sun ◽  
Patricia Loughran ◽  
Timothy R. Billiar ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Liver Injury ◽  
Signaling Pathways ◽  
Signaling Pathway ◽  
Energy Homeostasis ◽  
Misfolded Proteins ◽  
Wild Type ◽  
Cell Lysates ◽  
Lc3 Puncta ◽  
Tlr4 Activation

Impairment of autophagy has been associated with liver injury. TLR4-stimulation by LPS upregulates autophagy in hepatocytes, although the signaling pathways involved remain elusive. The objective of this study was to determine the signaling pathway leading to LPS-stimulated autophagy in hepatocytes. Cell lysates from livers of wild type (WT; C57BL/6) mice given LPS (5 mg/kg-IP) and hepatocytes from WT, TLR4ko, and MyD88ko mice treated with LPS (100 ng/mL) up to 24 h were collected. LC3II, p62/SQSTM1, Nrf2, and beclin1 levels were determined by immunoblot, immunofluorescence, and qPCR. Autophagy-like activation was measured by GFP-LC3-puncta formation and LC3II-expression. Beclin1, Nrf2, p62, MyD88, and TIRAP were knocked-down using siRNA. LC3II-expression increased in both liver and hepatocytes after LPS and was dependent on TLR4. Beclin1 expression did not increase after LPS in hepatocytes and beclin1-knockdown did not affect LC3II levels. In hepatocytes given LPS, expression of p62 increased and p62 colocalized with LC3. p62-knockdown prevented LC3II puncta formation. LPS-induced LC3II/p62-puncta also required MyD88/TIRAP signaling and localization of both Nrf2 and NFκB transcription factors to the nucleus to upregulate p62-expression. Therefore, TLR4-activation by LPS in hepatocytes induces a p62-mediated, not beclin1-mediated, autophagy-like clearance pathway that is hepatoprotective by clearing aggregate-prone or misfolded proteins from the cytosol and preserving energy homeostasis under stress.

H-Ras Is Involved in the Inside-out Signaling Pathway of Interleukin-3–Induced Integrin Activation

Blood ◽  
1999 ◽  
Vol 93 (5) ◽  
pp. 1540-1548 ◽  
Author(s):  
Hirohiko Shibayama ◽  
Naoyuki Anzai ◽  
Stephen E. Braun ◽  
Seiji Fukuda ◽  
Charlie Mantel ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Dominant Negative ◽  
The Other ◽  
Wild Type ◽  
Integrin Activation ◽  
Mapk Kinase ◽  
Interleukin 3 ◽  
Other Hand ◽  
Inside Out ◽  
Constitutively Active

The proto-oncogene product, p21ras, has been implicated in the cellular mechanism of adhesion, although its precise role has been controversial. Numerous cytokines and growth-factors activate Ras, which is an important component of their growth-promoting signaling pathways. On the other hand, the role of Ras in cytokine-induced adhesion has not been elucidated. We therefore investigated the function of H-Ras in the inside-out signaling pathway of interleukin-3 (IL-3)–induced integrin activation in the murine Baf3 cell line after transfection of cells with either constitutively active, dominant-negative, or wild-type H-Ras cDNAs. Adhesion of Baf3 cells to fibronectin was induced by IL-3 in a dose-dependent manner via very late antigen-4 (VLA-4; 4β1 integrins) and VLA-5 (5β1 integrins) activation. On the other hand, IL-4 did not induce the adhesion of Baf3 cells to fibronectin, although IL-4 did stimulate the cell proliferation of Baf3 cells. Constitutively active H-Ras–transfected Baf3 cells adhered to fibronectin without IL-3 stimulation through VLA-4 and VLA-5, whereas dominant-negative H-Ras–transfected Baf3 cells showed significantly less adhesion induced by IL-3 compared with wild-type and constitutively active H-Ras–transfected Baf3 cells. Anti-β1 integrin antibody (clone; 9EG7), which is known to change integrin conformation and activate integrins, induced the adhesion of dominant-negative H-Ras–transfected Baf3 cells as much as the other types of H-Ras–transfected Baf3 cells. 8-Br-cAMP, Dibutyryl-cAMP, Ras-Raf-1 pathway inhibitors, and PD98059, a MAPK kinase inhibitor, suppressed proliferation and phosphorylation of MAPK detected by Western blotting with anti–phospho-MAPK antibody, but not adhesion of any type of H-Ras–transfected Baf3 cells, whereas U-73122, a phospholipase C (PLC) inhibitor, suppressed adhesion of these cells completely. These data indicate that H-Ras and PLC, but not Raf-1, MAPK kinase, or the MAPK pathway, are involved in the inside-out signaling pathway of IL-3–induced VLA-4 and VLA-5 activation in Baf3 cells.

scholarly journals Role of Phosphoinositide 3-Kinase in the Aggressive Tumor Growth of HT1080 Human Fibrosarcoma Cells

2001 ◽  
Vol 21 (17) ◽  
pp. 5846-5856 ◽  
Author(s):  
Swati Gupta ◽  
Selma Stuffrein ◽  
Rina Plattner ◽  
Michael Tencati ◽  
Christa Gray ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Cell Lines ◽  
Cross Talk ◽  
Mutant Allele ◽  
Ras Signaling ◽  
Wild Type ◽  
Ht1080 Cells ◽  
Phosphoinositide 3 Kinase ◽  
Constitutively Active

ABSTRACT We have developed a model system of human fibrosarcoma cell lines that do or do not possess and express an oncogenic mutant allele of N-ras. HT1080 cells contain an endogenous mutant allele of N-ras, whereas the derivative MCH603 cell line contains only wild-type N-ras. In an earlier study (S. Gupta et al., Mol. Cell. Biol. 20:9294–9306, 2000), we had shown that HT1080 cells produce rapidly growing, aggressive tumors in athymic nude mice, whereas MCH603 cells produced more slowly growing tumors and was termed weakly tumorigenic. An extensive analysis of the Ras signaling pathways (Raf, Rac1, and RhoA) provided evidence for a potential novel pathway that was critical for the aggressive tumorigenic phenotype and could be activated by elevated levels of constitutively active MEK. In this study we examined the role of phosphoinositide 3-kinase (PI 3-kinase) in the regulation of the transformed and aggressive tumorigenic phenotypes expressed in HT1080 cells. Both HT1080 (mutant N-ras) and MCH603 (wild-type N-ras) have similar levels of constitutively active Akt, a downstream target of activated PI 3-kinase. We find that both cell lines constitutively express platelet-derived growth factor (PDGF) and PDGF receptors. Transfection with tumor suppressor PTEN cDNA into HT1080 and constitutively active PI 3-kinase–CAAX cDNA into MCH603 cells, respectively, resulted in several interesting and novel observations. Activation of the PI 3-kinase/Akt pathway, including NF-κB, is not required for the aggressive tumorigenic phenotype in HT1080 cells. Activation of NF-κB is complex: in MCH603 cells it is mediated by Akt, whereas in HT1080 cells activation also involves other pathway(s) that are activated by mutant Ras. A threshold level of activation of PI 3-kinase is required in MCH603 cells before stimulatory cross talk to the RhoA, Rac1, and Raf pathways occurs, without a corresponding activation of Ras. The increased levels of activation seen were similar to those observed in HT1080 cells, except for Raf and MEK, which were more active than HT1080 levels. This cross talk results in conversion to the aggressive tumorigenic phenotype. This latter observation is consistent with our previous observation that overstimulation of the activity of endogenous members of Ras signaling pathways, activated MEK in particular, is a prerequisite for aggressive tumorigenic growth.

Adaptor Protein Lnk Negatively Regulates Bcr-Abl-Induced Cell Proliferation through Inhibition of the Stat5 Signaling Pathway.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3406-3406
Author(s):  
Takayuki Tabayashi ◽  
Sigal Gery ◽  
Maya Koren-Michowitz ◽  
H.Phillip Koeffler
Keyword(s):  
Cell Proliferation ◽  
Growth Inhibition ◽  
Signaling Pathways ◽  
Signaling Pathway ◽  
Myeloproliferative Neoplasms ◽  
Lymphoblastic Leukemia ◽  
Adaptor Protein ◽  
Sh2 Domain ◽  
Dependent Manner ◽  
Wild Type

Abstract Abstract 3406 Adaptor protein Lnk negatively regulates not only several hematopoietic cytokine receptors including MPL, EpoR and c-Kit, but also non-receptor tyrosine kinases such as JAK2 and Src. Our previous studies demonstrated that Lnk, when expressed in hematopoietic cell lines, binds and regulates the mutant proteins, JAK2V617F and MPLW515L. Recent in vivo studies have shown that Lnk has an important role in the development of myeloproliferative neoplasms. These data suggest that Lnk may have the ability to inhibit constitutively activated signaling pathways in hematopoietic malignancies. However, how Lnk can attenuate the activity of Bcr-Abl is unclear. In the present study, we tested the hypothesis that Lnk might play a role in regulating Bcr-Abl function. In order to assess if Lnk can inhibit the proliferation of Bcr-Abl-positive hematopoietic cells, Bcr-Abl-expressing BaF3 cells were stably transfected with either Lnk (BaF3/Bcr-Abl/Lnk) or vector only (BaF3/ Bcr-Abl). Colony-formation assays revealed that Lnk significantly inhibited the proliferation of Bcr-Abl-expressing BaF3 cells. Similarly, overexpression of Lnk inhibited growth in the human CML cell line, K562. To determine the cause of growth inhibition by Lnk, assays for apoptosis were performed. Annexin V staining demonstrated that Lnk overexpression induced apoptosis in Bcr-Abl-expressing BaF3 cells. Western blotting analysis of protein lysates from BaF3/ Bcr-Abl /Lnk cells and BaF3/ Bcr-Abl cells found that Lnk-mediated growth inhibition was associated with downregulation of the Stat5 signaling pathway, but not associated with MAPK and PI3K signaling pathways. In addition, experiments in 293T cells expressing Bcr-Abl and Stat5 with either wild-type Lnk or SH2 mutant Lnk revealed that wild-type Lnk, but not SH2 mutant Lnk, inhibited phosphorylation of Stat5. Interestingly, Lnk inhibited Bcr-Abl-induced Stat5 phosphorylation in a dose-dependent manner. These data suggest that the SH2 domain of Lnk is essential for Lnk–mediated downregulation of the Stat5 signaling pathway in Bcr-Abl-positive cells. Taken together, our data suggest that Lnk inhibits Bcr-Abl-induced cell proliferation by attenuating the Stat5 signal transduction and may become a therapeutic target for Bcr-Abl-positive leukemias such as chronic myeloid leukemia and Philadelphia chromosome positive acute lymphoblastic leukemia. Disclosures: No relevant conflicts of interest to declare.

Novel anti-inflammatory functions for endothelial and myeloid cyclooxygenase-2 in a new mouse model of Crohn's disease

2010 ◽  
Vol 298 (6) ◽  
pp. G842-G850 ◽  
Author(s):  
Junji Watanabe ◽  
James A. Lin ◽  
Ajay J. Narasimha ◽  
Ani Shahbazian ◽  
Tomo-o Ishikawa ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Intestinal Inflammation ◽  
Myeloid Cell ◽  
Cyclooxygenase 2 ◽  
Wild Type ◽  
Tnf Α ◽  
Important Regulator ◽  
Established Cell ◽  
Cox 2

Cyclooxygenase-2 (COX-2) is an important regulator of inflammation implicated in the development of a variety of diseases, including inflammatory bowel disease (IBD). However, the regulation of intestinal inflammation by COX-2 is poorly understood. We previously reported that COX-2−/− mice fed a cholate-containing high-fat (CCHF) diet had high mortality of unknown mechanisms attributable to severe intestinal inflammation in the ileo-ceco-colic junction that presented characteristics similar to Crohn's disease (CD). To further characterize the role of COX-2 in intestinal inflammation, we established cell-specific conditional COX-2−/− mice. Endothelial cell-specific (COX-2−E/−E) and myeloid cell-specific (COX-2−M/−M) COX-2−/− mice, but not wild-type mice, on the CCHF diet developed localized CD-like pathology at the ileo-ceco-colic junction that was associated with cellular infiltration, increased expression of myeloperoxidase and IL-5, and decreased IL-10 expression. The CD-like pathology in COX-2−E/−E mice was also accompanied by increased expression of cytokines (IL-6, TNF-α, and INF-γ), compared with wild-type mice and COX-2−M/−M mice. In contrast, the ileo-ceco-colic inflammation in COX-2−M/−M mice was associated with more pronounced infiltration of granulocytes and macrophages than COX-2−E/−E mice. COX-2−ME/−ME (COX-2−M/−M × COX-2−E/−E) mice on the CCHF diet developed CD-like pathology in the ileo-ceco-colic junction reminiscent of total COX-2−/− mice on CCHF diet and wild-type mice on CCHF diet treated with COX-2 inhibitor, celecoxib. The pathology of diet-mediated ileo-ceco-colic inflammation in COX-2−/− mice offers an excellent model system to elucidate the protective roles of endothelial and myeloid COX-2 and the molecular pathogenesis of CD.

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