[Frontiers in Bioscience E3, 341-354, January 1, 2011]

TNF-alpha mediated NF-kappaB activation is constantly extended by transglutaminase 2

Kang-Seo Park1,4, Dae-Seok Kim1, Chunkyu Ko1, Sang-Jin Lee2, Seung Hyun Oh3, Soo-Youl Kim1

1Cancer Cell and Molecular Biology Branch, Goyang, Gyeonggi-do 410-769, Republic of Korea, 2Genitourinary Cancer Branch, Division of Translational and Clinical Research II, National Cancer Center, Goyang, Gyeonggi-do 410-769, Republic of Korea, 3Carcinogenesis Branch, Division of Cancer Biology, Goyang, Gyeonggi-do 410-769, Republic of Korea, 4Department of Biomedical Science, Graduate School, Kyung Hee University, Seoul 130-701, Republic of Korea TA

TABLE OF CONTENTS

1. Abstract
2. Introduction
3...Materials and methods
3.1. Western blotting
3.2. Plasmid constructs and transient transfection
3.3. Reporter assay: SEAP assay, b-galactosidase assay and b-lactamase assay
3.4. Immunocytochemical analysis
3.5. Adenovirus construction expressing TGase 2
3.6. In vivo animal experiments
3.7. Electrophoretic mobility shift assay for the detection of nuclear NF-kB
4. Results
4.1. TGase 2 and TNF-a mediated NF-k B activation and its reducing by cystamine, TGase 2 inhibitor
4.2. Role of TGase 2 on I-k Ba degradation in TNF-a induced NF-k B signaling pathway
4.3. The elongate effect of TGase 2 on maintenance of TNF-a mediated NF-k B activation
4.4. Inhibitory effect of TNF-a induced NF-k B activation by I-k Ba mutation at TGase 2 targeting sites
4.5. Reduction of TNF-a induced NF-k B translocation to nucleus with I-k Ba mutants at TGase 2 targeting sites
4.6. Profound NF-k B activation in mouse liver by TNF-a injection together with TGase 2 over-expression
5. Discussion
5.1. TGase 2 expression is increased in inflammatory diseases and cancers
5.2. The prolongation effect of TGase 2 on TNF-a -induced NF-k B activation
5.3. Reduction of TNF-a induced NF-k B activation by TGase 2 inhibition
5.4. Exacerbation of TNF-a -mediated inflammation in the mouse liver by TGase 2 over-expression
5.5. Model for TGase 2 in NF-k B activation
6. Acknowledgments
7. References

1. ABSTRACT

Increased levels of transglutaminase 2 (TGase 2) expression have been reported in many inflammatory diseases, as well as in drug resistant cancer cells. Previous reports have shown that TGase 2 is capable of inducing nuclear factor-kappaB (NF-kappaB) activation via depletion of inhibitor of kappaB (I-kappaB)alpha through polymerization in the absence of I-kappaBalpha kinase activation. This raises the question of whether increased expression of TGase 2 can extend NF-kappaB activation mediated by a canonical activation pathway. In the TGase 2-inducible EcR23/TG cell line, TGase 2 over-expression resulted in sustained activation of NF-kappa B in the presence of TNF-alpha, for up to 24 hrs, while in the absence of TGase 2 induction, NF-kappaB activity was restored to basal levels within 6 hrs of TNF-alpha treatment. In mice injected with an adenovirus vector expressing TGase 2, NF-kappaB was constitutively activated for up to 5 days, whereas Adeno/GFP-injected mice exhibited attenuated activation of NF-kappaB in response to TNF-alpha stress. Thus, the presence of increased levels of TGase 2 may exacerbate NF-kappa B activation in inflammatory states.