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Allergy driven Alternative Splicing of Interleukin 13 Receptor

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Allergy driven Alternative Splicing of Interleukin 13 Receptor Powered By Docstoc
					Allergy-driven Alternative Splicing of Interleukin-13 Receptor 2 Yields Distinct Membrane and Soluble Forms
Yasuhiro Tabata, Weiguo Chen, Manoj R. Warrier, Aaron M. Gibson, Michael O. Daines, and Gurjit K. Khurana Hershey
Division of Allergy & Immunology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio

IL-13: a Central Mediator of Allergic Inflammation
Collagen, Fibrosis eot axin-3 Fibroblast

Kerat inocyt e Chemokine expression Mucus Hypersecret ion Goblet cell met aplasia

Respirat ory Epit helium VCAM expression Chemokine expression

Vascular Endot helium

IL-1 3
Smoot h Muscle

Smoot h Muscle Proliferat ion Cholinergic-induced cont ract ions

Recruit ment Act ivat ion Eosinophil IgE Product ion

Ag Ag

B Cell

Modulat ion of FcRI IgE priming

CD23 expression Class II MHC expression Inhibit ion of proinflammat ory gene expression Macrophage/ monocyt e

Mast Cell

IL-13 binds to two distinct IL-13 receptors
IL-13

IL-13

?
IL-13 IL-13R1 TYK2

Soluble form

IL-4R

IL-13R2

?

JAK1

Membrane form
IL-13 receptor / Type II IL-4 receptor
STAT6

P P STAT6 STAT6

P STAT6

P
STAT6

Cytoplasm

P
STAT6

P STAT6

Transcription

Nucleus

Background
• IL-13R2 knockout mice have high serum IgE and enhanced IL-13 responsiveness.
(Wood et al. JEM 197:703,2003)

• Soluble IL-13R2 has been measured at high levels in mouse serum (10 ~ 20 ng/ml at baseline).
(Morimoto et al. JI 176:491,2006)

• Soluble IL-13R2 regulates serum and tissue levels of IL-13, and inhibits IL-13-induced liver fibrosis.
(Chiaramonte et al. JEM 197:687,2003)

• Soluble IL-13R2 is induced by parasitic infection in both mouse and human serum.
(Mentink-Kane et al. PNAS 101:586,2004)

Hypothesis

Alternative splicing may yield a secreted form of IL-13R2

3’-RACE revealed two distinct transcripts for mouse IL-13R2
Mouse IL-13R2 gene
ATG TAA

1 Exon:

2

3

4

5

6

7

8

9

10

11

99bp
2803bp

108bp
2841bp

109bp
1623bp

152bp
931bp

154bp

121bp
3295bp

185bp

146bp
1304bp

145bp
3655bp

116bp
752bp

200bp

Intron:

568bp

1030bp

Untranslated region Signal peptide Extracellular domain Transmembrane domain Intracellular domain

F1

F2

Tail adaptor

Mouse spleens express two transcripts of IL-13R2 (nested RT-PCR result)
M 1 2
a (387 bp) b (271 bp)

The shorter transcript lacks exon 10, which encodes transmembrane motif
Full-length transcript
ATG TAA

TM

3

4

5

6

7

8

9

10

11

Ex10 transcript
ATG TGA

(from position 952 to 1152) exon9 GAA TGG AGT GAA E W S E TAC ACA GGG CCA Y T G P ATA GTA CCA GTT exon10 I V P V CTT TTG TTA CTT L L L L AAG GAA GAA CCT K E E P exon11 CAT GTG GAT CTG H V D L TAT GAA GAT ACC Y E D T

GAG E GAC D TGT C CTT L GAA E AAC N CTC L

GAA E TCA S CTT L TGC C CCC P AAA K TGT C

TGT C AAG K TTC F CTT L ACA T GAA E TAA *

TGG W ATT I TTT F ATT I TTG L GTG V

GAA E ATT I ATA I GTG V AGC S TGT C

GGT G TTC F TTC F GAG E CTC L GCT A

3

4

5

6

7

8

9

11

(from position 952 to 1036) exon9 GAA TGG AGT GAA GAG GAA TGT TGG GAA GAG E W S E E E C W E E exon11 CCT CCA TGT GGA TCT GAA CAA AGA AGT GTG P P C G S E Q R S V
TGC TTA TGA AGA TAC CCT CTG TTA A C L *

Full-length transcript encodes membrane IL-13R2
Surface IL-13R2 Untransfected Full-length Ex10

Surface IL-13 binding Untransfected Full-length Ex10

Ex10 transcript encodes soluble IL-13R2

Ex10 transfectants secreted high levels of IL-13R2 in supernatants.

Ex10 transcript encodes biologically active soluble IL-13R2

UT: Supernant from untransfected cells DE: Supernant from Ex10 transfectants IL-4 : murine IL-4 5ng/ml IL-13 : murine IL-13 5ng/ml

Distribution of two IL-13R2 transcripts in mouse organs at baseline

a: full-length transcript b: Ex10 transcript

(496 bp) (380 bp)

House dust mite (HDM) challenge results in allergic reaction in a mouse asthma model
Day: 0 7 14 21 25 26

Intraperitoneal sensitization HDM 10g

Intraperitoneal sensitization HDM 10g

Intratracheal challenge HDM 100g

Intratracheal challenge HDM 100g

BAL

AHR Measurement

Airway hyperresponsiveness (AHR)

Eosinophil counts in BALF

Allergen challenge increased soluble IL-13R2 in BALF and serum in the mouse asthma model

Soluble IL-13R2 in BALF

Soluble IL-13R2 in serum

Expression of full-length and Ex10 transcripts in the mouse asthma model.
Full-length Ex10

Day 1

Day 5

Ex10 decreased in spleen but not in bone marrow in the mouse asthma model 1 day after the challenge
Full-length Ex10

Spleen

Bone marrow

Summary
• Mouse IL-13R2 gene has two transcripts: longer full-length and shorter Ex10 transcript lacking the transmembrane motif. • The full-length transcript encodes functional membrane IL-13R2, which can bind to IL-13. • The Ex10 transcript encodes biologically active soluble IL-13R2, which can effectively block IL-13-dependent STAT6 DNA binding activity. • Expression of the two IL-13R2 transcripts was differentially regulated in vivo in the mouse asthma model. • The alternatively spliced variants of IL-13R2 may have distinct biologic functions in vivo.

Thank You

Confirmation of two sequences from HDM-treated mouse lungs

exon9

exon10

exon9

exon11

IL-13R2 derived from Ex10 transcript has 2.3-fold higher IL-13 binding ability compared with that from full-length transcript.


				
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