Tài liệu Báo cáo khoa học: A novel splice variant of occludin deleted in exon 9 and its role in cell

A novel splice variant of occludin deleted in exon 9 and its

role in cell apoptosis and invasion

Jin-Mo Gu1

, Seung Oe Lim1

, Young Min Park2 and Guhung Jung1

1 Department of Biological Sciences and Seoul National University, Korea

2 Hepatology Center and Laboratory of Hepatocarcinogenesis, Bundang Jesaeng General Hospital, Kyungkido, Korea

Occludin is a tight junction (TJ) protein and the first

identified TJ-associated molecule [1]. Occludin is the

product of a single gene located on human chromo￾some band 5q13.1 and produces several different

mRNAs as a result of alternative splicing [2].

Recently, several variants of occludin, such as

occludin 1B and a fourth transmembrane domain

(TM4)-deleted variant, have been discovered [3,4].

Occludin 1B contains a 193 bp insertion correspond￾ing to an alternatively spliced exon in the gene

encoding a unique N-terminus. Conversely, the TM4-

deleted variant has a missing fourth TM correspond￾ing to exon 4. In addition to these variants, there is

evidence to show that two distinct promoters, P1 and

P2, confer separate transcriptional start sites [5]. Pro￾moter P2 is located downstream of promoter P1, and

both promoter regions are regulated by tumor nec￾rosis factor-a [5].

Occludin ( 65 kDa) is composed of two extracellu￾lar loops that form four membrane-spanning domains;

occludin specifically forms TJ complexes with phos￾phoproteins such as zonula occludens protein 1, zonula

occludens protein 2, and P-130. Other possible signal￾ing molecules include Ga subunits, tyrosine kinase,

small GTPases (Rab ⁄Rho), and junctional adhesion

molecules [6]. Interactions of these proteins with the

actin cytoskeleton are major determinants of the TJ

complex and play significant roles not only in cell–cell

contact but also in signal transmission [7].

Accumulating evidence points to TJ disruption in

malignant phenotypes, including local tumor growth,

invasion, and metastasis [8]. In humans, TJ disruption

Keywords

apoptosis; calcium; invasion; occludin; splice

variant

Correspondence

G. Jung, Department of Biological Sciences

and Seoul National University, 56-1 Shillim￾dong, Kwanak-gu, Seoul 151-747, Korea

Fax: +82 2 872 1993

Tel: +82 2 880 7773

E-mail: [email protected]

(Received 18 February 2008, revised 11

April 2008, accepted 15 April 2008)

doi:10.1111/j.1742-4658.2008.06467.x

The tight junction protein occludin participates in cell adhesion and migra￾tion and has been shown to possess antitumorigenic properties; however,

the exact mechanism underlying these effects is poorly understood. In liver

cell lines, we identified an occludin splice variant deleted in exon 9

(OccDE9). Furthermore, comparison analysis of wild-type occludin (OccWT)

and OccDE9 revealed that exon 9 played important roles in the induction of

mitochondria-mediated apoptosis and the inhibition of invasion, along with

the downregulation of matrix metalloproteinase expression. In addition, by

using the calcium indicator X-rhod-1, and the inositol trisphosphate recep￾tor inhibitor 2-aminoethoxydiphenyl borate, we found that OccWT but not

OccDE9 increased calcium release from the endoplasmic reticulum. In con￾clusion, our results showed that occludin mediates apoptosis and invasion

by elevating the cytoplasmic calcium concentration and that exon 9 of

occludin is an important region that mediates these effects.

Abbreviations

2-APB, 2-aminoethoxydiphenyl borate; 5¢-aza-dC, 5¢-aza-2¢-deoxycytidine; BrdUTP, 5-bromo-2¢-deoxyuridine-5¢-triphosphate; ER, endoplasmic

reticulum; ERK1 ⁄ 2, extracellular signal-regulated kinase 1 ⁄ 2; HA, hemagglutinin; IP3, inositol trisphosphate; IP3R, inositol trisphosphate

receptor; JNK, Jun N-terminal kinase; MMP, matrix metalloproteinase; MSP, methylation-specific PCR; shRNA, short hairpin RNA; TJ, tight

junction; TM, transmembrane domain; TSA, trichostatin A; TUNEL, terminal deoxyribonucleotide transferase-mediated nick-end labeling.

FEBS Journal 275 (2008) 3145–3156 ª 2008 The Authors Journal compilation ª 2008 FEBS 3145