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Liver Int. 2005 Jun;25(3):667-76.Click here to read Links

Expression of p28GANK and its correlation with RB in human hepatocellular carcinoma.

Tan L, Fu XY, Liu SQ, Li HH, Hong Y, Wu MC, Wang HY.

International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute, Second Military Medical University, Shanghai, China.

BACKGROUND: Aberrance of retinoblastoma protein (RB) signal pathway is known to play an important role in the carcinogenesis of human hepatocellular carcinoma (HCC). p28GANK, originally purified from human 26S proteasome as a non-ATPase subunit, was recently found in HCC and shown to interact with RB. The aim of this study was to investigate the expression profile of p28GANK and its correlation with RB in HCC. METHODS: The expression of p28GANK was evaluated in 55 surgically resected HCCs by immunohistochemistry (IHC), and the associations were explored between p28GANK level and clinicopathologic features as well as tumor suppressor RB. Western blotting was performed to determine p28GANK expression level in 12 HCCs. Immunofluorescence stainings of p28GANK and RB in U2-OS cells were examined by confocal microscopy. RESULTS: Positive p28GANK cytoplasmic staining was recognized in 55 HCCs. Nuclear positive occurrence of p28(GANK) in HCCs was more frequent than paracancerous hepatic tissues (P < 0.05). The overexpression probability of p28GANK was inversely associated with Edmonson's grade: overexpression occurred in nine out of 11 (81.8%), 22 out of 35 (62.9%) and two out of nine (22.2%) in I-II, III and IV graded cases, respectively (P = 0.004). Total cellular expression of p28GANK had curvilinear correlation with the nuclear expression of RB (r = 0.475, P = 0.019), while the nuclear expression of p28GANK had not. Western blot analysis showed that up-regulation of p28GANK expression was found in nine out of 12 HCCs compared with paracancerous liver tissues. Exogenously expressed p28GANK colocalized with RB in cytoplasm of U2-OS cells. CONCLUSIONS: These results confirm the role of p28GANK as a highly expressed oncoprotein in HCC by in situ examination. Its overexpression correlates with the differentiation status of HCC. The whole cellular p28GANK activation, not nuclear portion only, influences the alteration of RB. Underlying nuclear translocation of p28GANK may contribute to the counteraction against RB through a feed back loop. These data provide new evidence for p28GANK to be used as a promising drug target of a therapeutic agent against HCC.

PMID: 15910504 [PubMed - indexed for MEDLINE]

 

 

 

 

 

 

J Cell Biol. 003 Apr 14;161(1):67-77. Epub 2003 Apr 7.Click here to read Click here to readLinks

Arrest of mammalian fibroblasts in G1 in response to actin inhibition is dependent on retinoblastoma pocket proteins but not on p53.

Lohez OD, Reynaud C, Borel F, Andreassen PR, Margolis RL.

Institut de Biologie Structurale Jean Ebel (Commissariat ŕ l'Energie Atomique-Centre National de la Recherche Scientifique-Université Joseph Fourier), Grenoble cedex 1, France.

 

p53 and the retinoblastoma (RB) pocket proteins are central to the control of progression through the G1 phase of the cell cycle. The RB pocket protein family is downstream of p53 and controls S-phase entry. Disruption of actin assembly arrests nontransformed mammalian fibroblasts in G1. We show that this arrest requires intact RB pocket protein function, but surprisingly does not require p53. Thus, mammalian fibroblasts with normal pocket protein function reversibly arrest in G1 on exposure to actin inhibitors regardless of their p53 status. By contrast, pocket protein triple knockout mouse embryo fibroblasts and T antigen-transformed rat embryo fibroblasts lacking both p53 and RB pocket protein function do not arrest in G1. Fibroblasts are very sensitive to actin inhibition in G1 and arrest at drug concentrations that do not affect cell adhesion or cell cleavage. Interestingly, G1 arrest is accompanied by inhibition of surface ruffling and by induction of NF2/merlin. The combination of failure of G1 control and of tetraploid checkpoint control can cause RB pocket protein-suppressed cells to rapidly become aneuploid and die after exposure to actin inhibitors, whereas pocket protein-competent cells are spared. Our results thus establish that RB pocket proteins can be uniquely targeted for tumor chemotherapy.

PMID: 12682090 [PubMed - indexed for MEDLINE]

PMCID: PMC2172876

 

 

 

 

 

J Allergy Clin Immunol. 2005 Dec;116(6):1256-63.

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Chronic exposure to TNF-alpha increases airway mucus gene expression in vivo.

Busse PJ, Zhang TF, Srivastava K, Lin BP, Schofield B, Sealfon SC, Li XM.

Division of Clinical Immunology, Mount Sinai School of Medicine, New York, NY 10029, USA. paula.busse@mssm.edu

BACKGROUND: Hypersecretion of mucus plays an important role in the pathogenesis and severity of asthma. The primary proteins in mucus are mucin glycoproteins; MUC-5AC is the primary airway mucin gene. The calcium chloride-activated channel gene hCLCA1 (gob-5 in the mouse) has been suggested to increase MUC-5AC gene expression, and both are increased in asthmatic patients and murine models. TNF-alpha increases the expression of these genes in vitro but has not been investigated in vivo. OBJECTIVE: We sought to determine whether TNF-alpha increases gene expression of gob-5 and MUC-5AC and induces mucus cell metaplasia in vivo. METHODS: Naive BALB/c mice received 50 ng of recombinant murine TNF-alpha (rmTNF-alpha) intratracheally daily for 1, 2, or 3 weeks; another group received the same dose of intratracheal rmTNF-alpha daily for 3 weeks and then alternate-day treatment for 3 additional weeks (total of 6 weeks). AKR mice received 50 ng of rmTNF-alpha intratracheally for 3 or 6 weeks daily. Naive nontreated mice were used as control animals. Airway gene products for gob-5 and MUC-5AC were determined by means of real-time PCR. Lung tissue sections were stained with periodic acid-Schiff/Alcian blue to assess mucus cell metaplasia. RESULTS: rmTNF-alpha significantly increased gene expression of airway gob-5 and MUC-5AC after 2 weeks in the BALB/c mice. There was noticeable mucus staining in all mice treated for at least 3 weeks with TNF-alpha and in 80% of the mice receiving 2 weeks of treatment. After 3 weeks of treatment, the AKR mice also showed increased gob-5 expression. CONCLUSIONS: This study demonstrates for the first time that TNF-alpha alone in vivo is sufficient to increase airway mucus gene expression in 2 murine strains.

PMID: 16337454 [PubMed - indexed for MEDLINE]

Am J Respir Crit Care Med. 2005 Feb 15;171(4):305-14. Epub 2004 Nov 5.

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Metalloproteinases mediate mucin 5AC expression by epidermal growth factor receptor activation.

Deshmukh HS, Case LM, Wesselkamper SC, Borchers MT, Martin LD, Shertzer HG, Nadel JA, Leikauf GD.

University of Cincinnati, P.O. Box 670056, Cincinnati, OH 45267-0056, USA.

Chronic obstructive pulmonary disease is marked by alveolar enlargement and excess production of airway mucus. Acrolein, a component of cigarette smoke, increases mucin 5AC (MUC5AC), a prevalent airway mucin in NCI-H292 cells by transcriptional activation, but the signal transduction pathways involved in acrolein-induced MUC5AC expression are unknown. Acrolein depleted cellular glutathione at doses of 10 muM or greater, higher than those sufficient (0.03 muM) to increase MUC5AC mRNA, suggesting that MUC5AC expression was independent of oxidative stress. In contrast, acrolein increased MUC5AC mRNA levels by phosphorylating epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase 3/2, or MAPK 3/2(ERK1/2). Pretreating the cells with an EGFR-neutralizing antibody, or a metalloproteinase inhibitor, decreased the acrolein-induced MUC5AC mRNA increase. Small, interfering RNA directed against ADAM17 or MMP9 inhibited the acrolein-induced MUC5AC mRNA increase. Acrolein increased the release and subsequent activation of pro-MMP9. Acrolein increased MMP9 and decreased tissue inhibitor of metalloproteinase 3 (TIMP3), an endogenous inhibitor of ADAM17, transcripts. Together, these data suggest that acrolein induces MUC5AC expression via an initial ligand-dependent activation of EGFR mediated by ADAM17 and MMP9. In addition, a prolonged effect of acrolein may be mediated by altering MMP9 and TIMP3 transcription.

PMID: 15531749 [PubMed - indexed for MEDLINE]

 

Zhonghua Jie He He Hu Xi Za Zhi. 2004 Sep;27(9):585-8.

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[Expression of epidermal growth factor receptor and MUC5AC on human airway with chronic obstructive pulmonary disease]

[Article in Chinese]

Mao L, Bai CX, Zhang M, Wang YH, Chen J.

Department of Pulmonary Diseases, Zhongshan Hospital, Fudan University, Shanghai 200032, China.

OBJECTIVE: To determine the relationship between epidermal growth factor receptor (EGFR) expression and mucin 5AC (MUC5AC) synthesis in human airways with chronic obstructive pulmonary disease (COPD). METHODS: Lung specimens were obtained from 38 patients who were undergoing lobectomy, and the samples were taken from areas remote to the lesion. EGFR and MUC5AC protein expression were examined using immunohistochemical analysis and Western blot in peripheral airways (less than 2 mm in diameter) from 16 subjects with COPD, 10 subjects with a history of more than 30 pack-year smoking and 12 nonsmokers or exsmokers (0 - 10 pack-year smoker). RESULTS: Weak EGFR protein signals were detected in the lungs of the controls (2.01 +/- 1.02) in comparison with stronger signal in the COPD patients (4.62 +/- 1.65, P < 0.01) and the smokers (4.89 +/- 1.89, P < 0.01). EGFR immunoreactivity was observed mainly in goblet cells in the controls, the percentage of positive cell being 71.1% +/- 14.3%, which was higher than those in COPD (21.1% +/- 8.6%) or in the smoker (21.9% +/- 9.7%) airways. In contrast, COPD or smoker airways showed more expression of EGFR which expressed mainly in basal cells (42.9% +/- 14.2%, 52.1% +/- 13.5%, respectively) than that in the control airways (23.7% +/- 9.5%, P < 0.01). However there was no significant differences in EGFR expression and location in peripheral airways between COPD patients and smokers (P > 0.05). There was significant positive correlation between EGFR immunoreactivity and the area of the MUC5AC positive staining in all subjects (r = 0.877 4, P < 0.001). CONCLUSION: It is suggested that EGFR activation is involved in mucin expression in COPD airways.

PMID: 15498267 [PubMed - indexed for MEDLINE]

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Differential Regulation of CD40-Mediated TNF Receptor-Associated Factor Degradation in B Lymphocytes.

Moore CR, Bishop GA.

Interdisciplinary Graduate Program in Immunology.

Engagement of CD40 on murine B cells by its ligand CD154 induces the binding of TNFR-associated factors (TRAFs) 1, 2, 3, and 6, followed by the rapid degradation of TRAFs 2 and 3. TRAF degradation occurs in response to signaling by other TNFR superfamily members, and is likely to be a normal regulatory component of signaling by this receptor family. In this study, we found that receptor-induced TRAF degradation limits TRAF2-dependent CD40 signals to murine B cells. However, TRAFs 1 and 6 are not degraded in response to CD40 engagement, despite their association with CD40. To better understand the mechanisms underlying differential TRAF degradation, mixed protein domain TRAF chimeras were analyzed in murine B cells. Chimeras containing the TRAF2 zinc (Zn) domains induced effective degradation, if attached to a TRAF domain that binds to the PXQXT motif of CD40. However, the Zn domains of TRAF3 and TRAF6 could not induce degradation in response to CD40, regardless of the TRAF domains to which they were attached. Our data indicate that TRAF2 serves as the master regulator of TRAF degradation in response to CD40 signaling, and this function is dependent upon both the TRAF Zn domains and receptor binding position.

PMID: 16148124 [PubMed - in process]

 

 

B Cell Maturation Antigen, the Receptor for a Proliferation-Inducing Ligand and B Cell-Activating Factor of the TNF Family, Induces Antigen Presentation in B Cells.

Yang M, Hase H, Legarda-Addison D, Varughese L, Seed B, Ting AT.

Immunobiology Center, Mount Sinai School of Medicine, New York, NY 10029.

B cell maturation Ag (BCMA), a member of the TNFR superfamily expressed on B cells, binds to a proliferation-inducing ligand (APRIL) and B cell-activating factor of the TNF family (BAFF) but the specific B cell responses regulated by BCMA remain unclear. This study demonstrates that ligation of A20 B cells transfected with BCMA induces the expression of CD40, CD80/B7-1, CD86/B7-2, MHC class II, and CD54/ICAM-1, which subsequently enhances the presentation of OVA peptide Ag to DO11.10 T cells. BCMA expression in murine splenic B cells can be induced with IL-4 and IL-6, allowing subsequent treatment with APRIL or agonist anti-BCMA to similarly induce Ag presentation. A comparative analysis of hybrid receptors of TNFR2 fused to the cytoplasmic domains of APRIL/BAFF receptors found that only BCMA, but not transmembrane activator and calcium-modulator and cyclophilin ligand interactor or BAFF-R, is capable of activating Ag presentation. Although all three receptors can trigger NF-kappaB signaling, only BCMA activates the JNK pathway conferring on BCMA the specific ability to activate this Ag presentation response.

PMID: 16116167 [PubMed - in process]

 

 

 

 

 

ICAM-1 gene expression in endothelial cells: Effects on the inhibition of STAT3 phosphorylation.
Resveratrol suppresses IL-6-induced
Wung BS, Hsu MC, Wu CC, Hsieh CW.

Department of Applied Microbiology, National Chiayi University, Chiayi, Taiwan.

Resveratrol, a polyphenolic phytoaxelin present in red wine, has been suggested to protect against atherosclerosis and cardiovascular disease because of its antioxidant effects. Intercellular adhesion molecule (ICAM-1), induced by cytokines, has been hypothesized to play a role in the early events during atherosclerosis. In this study we tested the effects of resveratrol upon both IL-6-induced ICAM-1 gene expression and its underlying signaling pathways in endothelial cells (ECs). Resveratrol was found to inhibit both TNFalpha- and IL-6-induced ICAM-1 gene expression at the promoter, transcriptional and protein levels. Resveratrol also abrogates the tyr705 phosphorylation of STAT3 in IL-6-treated ECs, in a dose- and time-dependent manner. Although quercetin had similar effects, resveratrol showed higher inhibitory properties following 2-4 h pretreatments. Resveratrol has been shown to induce the activity of endothelial nitric oxide synthase (eNOS) and increase NO production. Consistent with this, the treatment of ECs with a NO donor (SNAP) reduces IL-6-induced STAT3 phosphorylation. Conversely, exposure of ECs to a NOS inhibitor reversed the effects of resveratrol upon IL-6-induced STAT3 phosphorylation. Furthermore, ECs transfected with constitutively active Rac1 (RacV12) showed increases in ICAM-1 promoter activity, intracellular reactive oxygen species (ROS) levels and STAT3 phosphorylation, and these increases were attenuated by resveratrol treatment. In summary, we demonstrate for the first time that resveratrol inhibits IL-6-induced ICAM-1 gene expression, in part, by interfering with Rac-mediated pathways via the attenuation of STAT3 phosphorylation. This study therefore provides important new insights that may contribute to the proposed beneficial effects of resveratrol in endothelial responses to cytokines during inflammation.

PMID: 16150460 [PubMed - as supplied by publisher]