Induction of CRMP-2 by GDNF and analysis of the CRMP-2 promoter region Yoshinori Kodama, Yoshiki Murakumo, Masatoshi Ichihara, Kumi Kawai,Yohei Shimono, and

1
Induction of
CRMP-2 by GDNF and analysis of
the CRMP-2 promoter
region Yoshinori Kodama, Yoshiki
Murakumo, Masatoshi Ichihara,
Kumi Kawai,Yohei Shimono, and Masahide
Takahashia
Received 21
April 2004

Available
online 7 June 2004

• ????
• ??????
• ??????? ??

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Introduction

• CRMP-2 (Collapsin response mediator protein-2)
• ??????????,??????
• CRMP-2???????????(axon)???????
• Dominant negative mutation of CRMP-2

3
Introduction

• RET gene was identified from activated oncogene.
• RET encode a receptor tyrosine kinase.
• Mutation of RET cause human diseases.
• Ex Papillary thyroid carcinoma-???????
• Hirschsprungs disease-???????
• RET is activated by GFLs (GDNF family ligands)
• ex GDNF(glial cell line-derived neurotrophic
  factor),
• GFLs requires GFRas (GDNF family receptor
  aligands).
• As ligand binding components.

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(No Transcript)
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activation
Ras signaling pathway
ERK pathway
AKT pathway
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Introduction

• From other studies, revealed that
• 1) GDNF/RET signaling pathway play a important
  role in development
• of the kidney and enteric nervous system.
• 2) CRMP-2 expression increased after nerve
  injury and accelerated
• axon regeneration in rat motor neurons
• Study direction
• 1) GDNF can enhance CRMP-2 expression in
  neuronal cells ?
• 2) Mechanism of GDNF induced CRMP-2 ?
• 3) Analysis CRMP-2 promoter gene.

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Materials and methods

• Cell culture
• -TGW human neuroblastoma cells
• Western blotting
• Inhibitors and antibodies
• -PD98059, Ly294002, Wortmannin, PP2, PP3
• Construction of luciferase reporter plasmid
• Transfection and luciferase assay
• Immunostaining
• - Anti-CRMP-antibody, normal rabbit
  immunoglobulin.

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Result
?GDNF????48??, CRMP-2?????????? ??,?????????
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??PD98059,?????,???GDNF???CRMP-2???,?????????,????
????????
PD98059-MEK inhibitor Wortmannin-PI3K
inhibitor LY294002-PI3K inhibitor PP2-Src family
kinase inhibitor
?? ERK pathway??GDNF??CRMP-2???,??????
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Analysis of CRMP-2promoter region
CRMP-2 promoter region ??CRMP-2????????
1kb fragement?
Transcription factor binding site Sp1, E2F,
Oct-1, GATA-1/2 ????region????
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PD98059-ERK inhibitor
???? ERK pathway??GDNF??CRMP-2???,????????
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? transcription start point???-214-48 bp
?region, ??CRMP-2 transcription?????
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(No Transcript)
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???????, ??transcription factors??? CRMP-2
transcription ??? ???
??GDNF??????mutated binding site?,????????,???35?
???GDNF?????mutated binding site?,??????2230?
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???,??GDNF??,???????????? ???????,????CRMP-2??????
??
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???,?GDNF???,?????????????,GDNF??????????????????,
CRMP-2??????????
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??(E)?,PP2??????GDNF???????????
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(No Transcript)
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????????? Src family kinase??,? ???ERK?????
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Conclusion

• CRMP-2???????????????
• CRMP-2??????GDNF/RET signaling
• pathway????ERK???????
• ??GDNF??????,CRMP-2??????
• ???axon????
• CRMP-2 transcriptional start point??21448
  bp???,??CRMP-2 transcription ??????
• E2F, Sp1, Oct-1, GATA-1/2, p300?transcription
  factors?????CRMP-2 transcription????
• ?????????Src family kinase??,????ERK?????
• ???????CRMP-2???,?????????????