Volume 137, Issue 3, Pages (September 2009)

Slides:



Advertisements
Similar presentations
Volume 114, Issue 5, Pages (May 1998)
Advertisements

Genetic Inactivation of RelA/p65 Sensitizes Adult Mouse Hepatocytes to TNF-induced Apoptosis In Vivo and In Vitro  Fabian Geisler, Hana Algül, Stephan.
Volume 133, Issue 1, Pages (July 2007)
Volume 132, Issue 3, Pages (March 2007)
Volume 132, Issue 2, Pages (February 2007)
Volume 140, Issue 2, Pages e2 (February 2011)
Volume 131, Issue 3, Pages (September 2006)
Foxf2 in Intestinal Fibroblasts Reduces Numbers of Lgr5+ Stem Cells and Adenoma Formation by Inhibiting Wnt Signaling   Ali Moussavi Nik, Azadeh Reyahi,
Volume 135, Issue 4, Pages (October 2008)
Volume 132, Issue 2, Pages (February 2007)
Volume 133, Issue 1, Pages (July 2007)
Volume 136, Issue 1, Pages (January 2009)
Genetic Inactivation of RelA/p65 Sensitizes Adult Mouse Hepatocytes to TNF-induced Apoptosis In Vivo and In Vitro  Fabian Geisler, Hana Algül, Stephan.
Volume 131, Issue 4, Pages (October 2006)
Volume 133, Issue 1, Pages (July 2007)
Volume 135, Issue 2, Pages e3 (August 2008)
Volume 132, Issue 4, Pages (April 2007)
Volume 138, Issue 3, Pages e4 (March 2010)
Growth Hormone Inhibits Signal Transducer and Activator of Transcription 3 Activation and Reduces Disease Activity in Murine Colitis  Xiaonan Han, Danuta.
Volume 68, Issue 2, Pages (August 2005)
Aryl Hydrocarbon Receptor Regulates Pancreatic IL-22 Production and Protects Mice From Acute Pancreatitis  Jing Xue, David T.C. Nguyen, Aida Habtezion 
Volume 134, Issue 4, Pages e2 (April 2008)
Volume 75, Issue 11, Pages (June 2009)
Volume 137, Issue 1, Pages e3 (July 2009)
Volume 143, Issue 5, Pages (November 2012)
Volume 25, Issue 4, Pages (April 2014)
Volume 134, Issue 1, Pages (January 2008)
Volume 132, Issue 5, Pages (May 2007)
Shobhan Gaddameedhi, Christopher P. Selby, Michael G
Proteasome Inhibition of Pathologic Shedding of Enterocytes to Defend Barrier Function Requires X-Linked Inhibitor of Apoptosis Protein and Nuclear Factor.
Volume 142, Issue 2, Pages (February 2012)
Volume 137, Issue 3, Pages (September 2009)
Volume 131, Issue 5, Pages (November 2006)
Volume 141, Issue 4, Pages e2 (October 2011)
Volume 138, Issue 2, Pages e2 (February 2010)
Volume 126, Issue 1, Pages (January 2004)
Volume 136, Issue 4, Pages e3 (April 2009)
Loss of Raf Kinase Inhibitor Protein Promotes Cell Proliferation and Migration of Human Hepatoma Cells  Han Chu Lee, Bo Tian, John M. Sedivy, Jack R.
Volume 138, Issue 4, Pages e3 (April 2010)
Volume 131, Issue 5, Pages (November 2006)
Volume 115, Issue 1, Pages (July 1998)
Volume 132, Issue 7, Pages (June 2007)
Volume 136, Issue 4, Pages (April 2009)
Volume 134, Issue 3, Pages (March 2008)
Volume 127, Issue 1, Pages (July 2004)
Volume 139, Issue 6, Pages (December 2010)
Volume 140, Issue 3, Pages (March 2011)
Volume 134, Issue 2, Pages e3 (February 2008)
Autophagy in proximal tubules protects against acute kidney injury
Suppression of Apoptosis, Crypt Hyperplasia, and Altered Differentiation in the Colonic Epithelia of Bak-Null Mice  Carrie A. Duckworth, D. Mark Pritchard 
Volume 133, Issue 2, Pages (August 2007)
Volume 139, Issue 3, Pages e6 (September 2010)
Anne T. Funding, Claus Johansen, Matthias Gaestel, Bo M
Volume 130, Issue 5, Pages (September 2007)
Volume 45, Issue 6, Pages (March 2012)
Volume 138, Issue 3, Pages e5 (March 2010)
Julien Colombani, Cédric Polesello, Filipe Josué, Nicolas Tapon 
Volume 141, Issue 3, Pages (September 2011)
Hus1 Acts Upstream of Chk1 in a Mammalian DNA Damage Response Pathway
Volume 2, Issue 6, Pages (June 2008)
EVA1A/TMEM166 Regulates Embryonic Neurogenesis by Autophagy
Volume 25, Issue 5, Pages (March 2007)
Volume 131, Issue 2, Pages (August 2006)
Yap1 Phosphorylation by c-Abl Is a Critical Step in Selective Activation of Proapoptotic Genes in Response to DNA Damage  Dan Levy, Yaarit Adamovich,
Volume 131, Issue 5, Pages (November 2006)
Galectin-3 Protects Keratinocytes from UVB-Induced Apoptosis by Enhancing AKT Activation and Suppressing ERK Activation  Jun Saegusa, Daniel K. Hsu, Wei.
Proapoptotic Function of the Retinoblastoma Tumor Suppressor Protein
Volume 118, Issue 6, Pages (June 2000)
Volume 1, Issue 2, Pages (August 2007)
Foxf2 in Intestinal Fibroblasts Reduces Numbers of Lgr5+ Stem Cells and Adenoma Formation by Inhibiting Wnt Signaling   Ali Moussavi Nik, Azadeh Reyahi,
Presentation transcript:

Volume 137, Issue 3, Pages 945-954 (September 2009) Induction of Protein Tyrosine Kinase 6 in Mouse Intestinal Crypt Epithelial Cells Promotes DNA Damage–Induced Apoptosis  Andrea Haegebarth, Ansu O. Perekatt, Wenjun Bie, Jessica J. Gierut, Angela L. Tyner  Gastroenterology  Volume 137, Issue 3, Pages 945-954 (September 2009) DOI: 10.1053/j.gastro.2009.05.054 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 PTK6 protein expression is induced by γ-irradiation. (A) Immunoblotting of untreated (0 h) and γ-irradiated (8 Gy) wild-type mouse lysates (6 hours and 72 hours following irradiation) from small intestine with antibodies against PTK6 and β-actin as a loading control. Results obtained using lysates from 3 different animals per time point are shown. (B) Quantitation of PTK6 protein expression. PTK6 protein levels were normalized to β-actin expression. The bars represent the mean ± SD. A significant increase in PTK6 protein expression is detected in irradiated mice (*P = .028; **P = .040). (C) PTK6 is induced in the crypt epithelial cells after irradiation. Immunohistochemistry was performed to examine PTK6 expression in untreated and irradiated wild-type animals. Immunostaining for immunoglobulin G served as a control. While PTK6 protein expression is restricted to the villus epithelium of untreated mice, it is also detected in the crypt of irradiated mice. Bar = 50 μm. (D) Higher magnification showing immunolocalization of PTK6 in crypt cells. At 6 and 72 hours, PTK6-positive cells are evident throughout most of the crypt, with weakest expression in Paneth cells localized at the base. PTK6 immunoreactivity was detected with 3,3′-diaminobenzidine tetrahydrochloride (brown stain). Dashed lines mark a crypt (c) in each panel at the same magnification. V, villi. Gastroenterology 2009 137, 945-954DOI: (10.1053/j.gastro.2009.05.054) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 DNA damage–induced apoptosis is compromised in the absence of PTK6. (A) Radiation-induced apoptosis was examined in the small intestine from untreated and γ-irradiated Ptk6+/+ and Ptk6−/− mice at 0, 6, and 72 hours after irradiation using the TUNEL assay. Labeled apoptotic cells were detected with avidin–fluorescein isothiocyanate (apoptotic cells stain green). Bar = 100 μm. Quantification was performed and is presented as a histogram (B) and frequency histogram (C) of apoptotic cells per crypt-villus unit. (B) At least 50 crypt-villus units per section were scored. Values shown are mean ± SD from 2 sections from at least 3 different mice per group. *P = .026; **P = .032. V, villi; C, crypts. Gastroenterology 2009 137, 945-954DOI: (10.1053/j.gastro.2009.05.054) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 Decreased activation of caspase-3 correlates with reduced apoptosis in Ptk6−/− mice. (A) Activation (cleavage) of caspase-3 was analyzed in sections of small intestine from untreated and γ-irradiated wild-type (Ptk6+/+) and Ptk6−/− mice at 0, 6, and 72 hours after irradiation. Bar = 100 μm. (B) Cleaved caspase-3 levels were determined by immunoblotting. Tissue lysates from small intestine of wild-type (+/+) and knockout (−/−) mice 6 hours after irradiation were subjected to immunoblotting with anti-cleaved caspase-3 antibodies. PTK6 protein and β-actin expression were examined as controls for genotyping and protein loading, respectively. Gastroenterology 2009 137, 945-954DOI: (10.1053/j.gastro.2009.05.054) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 AKT activity is increased in PTK6-deficient mice after irradiation. (A) AKT expression and activation (phosphorylation of Ser 473) were examined by immunoblotting using total tissue lysates from small intestines of Ptk6+/+ and Ptk6−/− mice at 0 and 6 hours after whole-body γ-irradiation (γ-IR). (B) AKT activity was determined using in vitro kinase assays. Endogenous AKT was immunoprecipitated from lysates analyzed in (A) with immobilized AKT antibody and incubated with purified recombinant GSK-3 and ATP in kinase reaction buffer. Reactions were stopped at 0 (control [c]) and 30 minutes and subjected to immunoblotting with phospho-GSK-3β and total GSK-3β antibodies. Increased AKT kinase activity was detected in lysates prepared from mice deficient for PTK6 (*P = .036). Gastroenterology 2009 137, 945-954DOI: (10.1053/j.gastro.2009.05.054) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 Increased MAPK signaling is detected in Ptk6−/− mice following irradiation. (A) Intestinal protein lysates were harvested from 3 untreated wild-type (Ptk6+/+) and 3 Ptk6−/− mice and from 4 Ptk6+/+ and 4 Ptk6−/− mice at 6 hours post γ-irradiation (γ-IR). Immunoblotting was performed to examine ERK1/2 expression and phosphorylation. Increased phosphorylation (activation) of ERK1/2 was detected with phospho-specific antibodies. (B) The relative ratios of phospho-ERK1/2 to total ERK are shown. There is a significant increase in phospho-ERK1/2 levels in the Ptk6-null intestine after γ-irradiation and levels of phospho-ERK1/2 are higher in Ptk6−/− than Ptk6+/+ mice (*P = .031, **P = .016). Gastroenterology 2009 137, 945-954DOI: (10.1053/j.gastro.2009.05.054) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 Inhibitory phosphorylation of the proapoptotic protein BAD is enhanced in the Ptk6−/− intestine after irradiation. (A) Immunoblotting was performed using lysates from Ptk6+/+ and Ptk6−/− intestines at 0 hours and 6 hours post γ-irradiation (γ-IR) with BAD phospho-Ser-112 specific antibodies and antibodies against total BAD. Each lane represents lysate from an independent animal. β-actin expression was examined as a control. (B) There was a significant increase in phospho-BAD levels in the Ptk6−/− intestine at 6 hours following γ-irradiation (*), at which time higher levels of phospho-BAD were detected in Ptk6−/− than Ptk6+/+ intestines (**). *P = .034, **P = .023. Gastroenterology 2009 137, 945-954DOI: (10.1053/j.gastro.2009.05.054) Copyright © 2009 AGA Institute Terms and Conditions

Figure 7 Increased numbers of intestinal crypt epithelial cells proliferate in wild-type mice than Ptk6-null mice in response to DNA damage. (A) Proliferation was analyzed by examining BrdU incorporation in sections of small intestine from untreated and γ-irradiated Ptk6+/+ and Ptk6−/− mice at 0, 6, and 72 hours after irradiation. S-phase cells were pulse labeled with BrdU for 1 hour before animals were killed. BrdU incorporation was detected with antibodies against BrdU and 3,3′-diaminobenzidine tetrahydrochloride (brown). Positive nuclei are detected in the crypts, while some diffuse background staining is seen in the upper villus. v, villi; c, crypts. Untreated Ptk6−/− mice exhibit an extended zone of proliferation at 0 hours, but proliferation levels are higher in wild-type mice at 6 and 72 hours after irradiation. (B) The percent of BrdU-positive cells per crypt in wild-type (+/+) and Ptk6 null (−/−) mice at 0, 6, and 72 hours after irradiation is shown. The bars represent the mean ± SD. *P = .015, **P = .023, ***P = .047. Gastroenterology 2009 137, 945-954DOI: (10.1053/j.gastro.2009.05.054) Copyright © 2009 AGA Institute Terms and Conditions