nmda ap-1 - marianna uigakukai.marianna-u.ac.jp/idaishi/www/335/10-33-5kouhei kuribayashi... ·...
TRANSCRIPT
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��� c-Jun��������� ��������������������13�14�� � ���c-Jun ����� ���������!�"�#$��%&'���$()%&'���*����+,-./0��� AP-1 ���1������� �&'�15�� 2�� AP-1 DNA�3���������$�45����� ��JNK 67 p38 ������16�� 8�9��NMDA �:;�� <=� JNK 67 p38 �����������>� ?� JNK 67 p38$�=5�������@�A"����$��%�9��%B%? C�DE�F� AP-1� NMDA � <=�G �H�I�5�B�� �&'"'� ?��� NMDA � <=JK,�:'&��������5� AP-1 �#$� B�5��L�� MN9�� AP-1 DNA �3��$O�%? PQRFST&O�%��
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���� ���UV� “Association for Research in Vision
and Ophtalmology, Statement for Use of Animals
in Ophtalmic and Vision Research” �WX%&YZ�� [��UV��\]^��_`a���b�$�&'�� ]^��� 8 c� Wistar d��ef�$�'�� ����\�g]^��hij UVk!"� lm 23�1 �C� n# 55�5�� o� 6pB oq 6p[� �r�s$t� h$u%&vChi��&'������ ��NMDA �Sigma, St. Louis, MO, U.S.A� wE'
(��8�9��YZ�)*�+, x�)�YZ�17�� y'-��z/�{,|-�, �./�01�Osaka, Japan� �35 mg�kg, intraperitoneal �i. p.�� 2}C� �~��.� ��301� Osaka, Japan� 5 ml
4��&�5%� � 67��8 1 mm ������ 9� wE'"� 40 mM NMDA �in 0.01M phosphate bu#erd salin �PBS�� $ 5 ml �:; 200nmol� ��(�%� control ���<9� wE'"�=; PBS 5 ml $(�%������ Hematoxylin-eosin ��ef� �n�10� �z/�{,|-�, �35 mg�kg,i. p. �2}C� wE'"� NMDA$(�%� 24p�q� :�7 7/>���$?�%� �,���@
��& 3p��@%� At� A�$�&�e��/BCq� 4 mm ���$DZ�� E9$�F%�� ? q hematoxylin-eosin G�$Y'� ����� 1.01.5 mm �r$H7�I�C���$��%������ TUNEL �ef� �n�25� �z/�{,|-�, �35 mg�kg,
i. p.�2}C� wE'"� NMDA$(�%� 0� 3�6� 12� 24p�q���$?�%� 10��,� /�& 24p��@%� At� A�$�&�e��/BCq� 4 mm ���$DZ�� E9$�F%�� TUNEL G�� DeadEndTM Fluorometric
TUNEL System �Promega, Madison, WI, U.S.A�$¡�%&¢~�£�,J��YZ�� E9$PBS�K¤q 20 mg�ml proteinase K� 5L¥M%� terminal dUTP transferase enzyme �& 37�C� 60L�n%�q� 2saline sodium citrate �SSC�� 15 L¥M%N¦$§O%�� FITC P¨TUNELQ�©R�ª«4�I� �LSM510META;Carl Zeiss, Jena, Germany� $�'� S¬�%&488 nm T�$� ��,-��{/�����,-�$¡�%&����� 1.01.5 mm �r�� $®U%������ ���TUNEL G�q� E9$ PBS �K¤%� block-ing solution �3� Bovine serum albumin �BSA� inPBS� � 30L¥M%� ¯°±' AP-1 ±' �SantaCruz Biotechnology, Santa Cruz, CA, U.S.A,
1 : 100� � 24p�¥M%�� V°±'�~�²./P¨±³´µ IgG �Cappel, Research Products,NC, U.S.A, 1 : 100� ±'�lm�& 1p�¥M%�TUNEL G��+,���$�Z�� S¬�%& 543 nm W�$�'� ~/+����,-�$¡�%������ Electrophoretic Gel Mobility Shift Assay�EMSA�ef� �n�10� �z/�{,|-�, �35 mg�kg,
i. p.� 2}C� wE'"� NMDA $(�%� 24p�q�� $?�%� ¶XY$ef�� B ¶©R·Z�¸f� �NE-PER Nuclear and Cytoplas-mic Extraction Reagents, Pierce Chemical, IL,
U.S.A� $�'&Z�%�� XY[#� Bio-RadProtein Assay kit �BioRad, Hercules, CA, U.S.A�$�'&U@%�� AP-1 �3N¦�� 10 mg ¶
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��� binding bu#er �20 mM HEPES-NaOH pH
7.90� 1 mM dithiotreitol, 0.3 mM EDTA, 0.2 mMEGTA, 80 mM NaCl, 10� glycerol, 0.2 mM
PMSF� ����� 2 mg poly �dI-dC�� �10,000 cpm 32 P � ���� AP-1 double-
stranded oligonucleotides �AP-1 oligonucleotides��Promega, Madison, WI, U.S.A�� 32P� ������� AP-1 oligonucleotides � 100������������������ ��� bindingbu#er� 32P ���� AP-1 oligonucleotides� !"� 32P� ������� AP-1 oligonucleotides�#$� 30 %&'()�*� DNA ��+�,�6� polyacrylamide gel �0.5 mM EDTA, 25 mMTris-borate bu#er� � apply �� 100V� 4�C� 2-./012��� 3��45()� X 678�9�:;<=()�� 78�9�>?*� Densito-graph �ATTO Corporation, Tokyo, Japan� ��@������� ���ABCBDEFG�HIJK�L��� MND
Analysis of Variance �ANOVA� �OP�*� %Q���RS�D Fisher-PLSD T�� %Q�����S�DMannWhitney-U test���� UVW 5�XY�Z[����
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��� Hematoxylin and eosin �Fig. 1 �L\"]� NMDA ^_,`ab 24
-.*�D control c� NMDA c�!��defghijkijl�`dmkno�pqDrstu�vP�� �v�� Fig. 2�L\"]�� wxy�z{�|}� NMDA ^_,`ab* 7~� NMDA c�D control c���defghijkijl������`dmkno��q�rs� �Fig. 1, 2����� TUNELFig. 3�L\"]�� NMDA ^_,`ab 24
-.*�!��� controlc�D TUNEL ��ijDrstu�vP�� ��� NMDA ^_,`ab 0-.*!"� 3-.*�!�� TUNEL ��ijDrstu�vP�� �v�� NMDA abc�Dab 6 -.*"�fghijk�`��k�TUNEL ��ij�rstu� ������ 24-.*�����P� �Fig. 3B�C����� EMSAFig. 4�L\"]� AP-1 ��� DNA ����
D� 10���D 100��� 32P� ������� AP-1 oligonucleotide������(u����"��r��� � AP-1��� DNA ����DNMDA^_,`ab 24-.*� control������������� AP-1 �� �Fig. 5�L\"]�� NMDA ^_,`ab 24
-.*�!�� AP-1 ��ijDfghijk�`��k�rstu� �Fig. 5B�� ���� NMDA^_,`ab 24 -.*� AP-1 ��ijD con-
Fig. 1. Representative light microscopic photographs of rat retina at 24 hours after
intravitreal injection of phosphate bu#ered saline �PBS� as a control �A� or 200 nmolN-methyl-D-aspartate �NMDA� �B�. Scale bar�50 mm. RGCL: retinal ganglion cell
layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform
layer, ONL: outer nuclear layer, RPE: retinal pigment epithelium.
NMDA �"R� ¡B¢£ 437
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Fig. 2. Representative light microscopic photographs of rat retina at 7 days after intravitreal
injection of phosphate bu#ered saline �PBS� as a control �A� or 200 nmol N-methyl-D-aspartate �NMDA� �B�. Note loss of inner retinal thickness with 200 nmol NMDA injectionand decreased number of cell in retinal ganglion cell layer �RGCL�. Scale bar�50 mm. IPL:
inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer
nuclear layer, RPE: retinal pigment epithelium.
Fig. 3. In situ TUNEL assay. TUNEL-positive cells in the inner retina 24 hours after phosphate
bu#ered saline �PBS� as a control �A� or 200 nmol N-methyl-D-aspartate �NMDA� �B�injection. TUNEL-positive cells were localized in the retinal ganglion cell layer �RGCL� andthe inner nuclear layer �INL� of the NMDA-treated retina �B�. �C� The number ofTUNEL-positive cells in the inner retina at di#erent times after NMDA injection. Data are
expressed as means�SEM. �n�5�. IPL: inner plexiform layer, OPL: outer plexiform layer,ONL: outer nuclear layer.
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trol� �A����� NMDA� �B� ������Fig. 5A�B� � ��� TUNEL �B,� ����� �A, � � � 2�� �C, � � ��� AP-1 ��������� TUNEL � ������� �Fig.6��
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��� !�"�� #��NMDA$%&'()�*�+,�����"�� -./0�12�34�$5678�9:��� 4�-./0�1
2�3���"� AP-1� DNA ;<=�>=�?@A��$�BC���� ��4� AP-1 �DEF���"�-.G��H�(H�IJK34�>�BC�L��� @B�� AP-1 �TUNEL ������MN>:OBA��PQ�RS�T���� 200 nmol� NMDA $%&'(�)�* 7U��� VWL��-.G��X�YZ�(�[H� \?>:OBA��NMDA ]�'�^_L`a���( Ca2� bc�de�fg-.���hiFjkl$14K�mnBA�3� NMDA �f3��-./0�hiFjkl$op��Lqr>��L�s$tu�3�mnBA�3� vw�xy��NMDA�%&'(z�)��-.G���"�� p53{N��hiFjkl.�$=�?K3���318��p53>hiFjkl$|GK3}�~�-.�������BA�3� ��� ���"��(/�"�3-.G�����hiFjkl���>��@A�3�O19�� 4AB�.����>����-.�������������3�mnBA3���� !�"�� hiFjkl�����3TUNEL �������� NMDA )� 6��*�,��� 24 ��*�v��L��� �C�� ��8�� NMDA )� 24��*��-.G��X�(�[H���?�:OBALC������NMDA )�* 7U��"�-.G��X�YZ�(�[H� \?>:OBA�� Pd�4�CB�����-.G��X�YZ�(�[H� \?>VJ?K3���� TUNEL ����>��K3�������>�3���� hiFjkl>����3���>mnBA��^��RS��� -.¡�"�¢£� ¤¥¦§��LlF¨l�fg AP-1 =��de>RS@A�320�21�� ��� ���©`a�f3ª���>AP-1��«$¬K34�>RS@A�3>22��-.G����� AP-1 �����K3RS�ZL� PQ#��� NMDA �f3 AP-1 �®¯&��°��3 c-Jun �=��de$9:�� �un-published data� � ��#�� NMDA ��-./0$14�����"�T�� AP-1� DNA ;<=�>de��34�$�BC���� 4AB�4��±²���"�³´µ¶·¸>NMDA ]�'$¬�� c-Jun ¹º AP-1 $=�?@»�hiFjkl$�«K3�¼RS��½�
Fig. 4. Electrophoretic mobility shift assay of activator
protein-1 �AP-1� DNA binding activity in the ratretina at 24 hours after intravitreal injection of
phosphate bu#ered saline �PBS� as a control�cont.� or 200 nmol N-methyl-D-aspartate
�NMDA�. Ten excess, 10-fold excess of the AP-1unlabeled oligonucleotide; 100 excess, 100 -fold
excess of the AP-1 unlabeled oligonucleotide.
NMDA �f3hiFjkl 439
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���23�� ���� AP-1 �� ������������������ c-Jun����� !����"#���� AP-1 c-Jun$�% c-fos&���'��()*+,-���� !&!� AP-1��.���������� c-fos �/0�12 3���45�&���24�� 36� NMDA ���'�� AP-1 �����789:;��<=��>��?@A?� AP-1 9BC'D�EFGHI�!� NMDA � MAP kinase JKLMN� O�c-Jun N terminal kinase �JNK�PQ9��'D����"#�����25�� JNK c-Jun � N-terminal transactivation RS9MTU�!� AP-19��'D�� VWX� NMDA�Y-Z[\
6 6]^�_P+`abc%[deb� JNKc% p-JNK BC!fg� 24]^h�ij0���9kl!=9�� 3m NMDA \ 6� _P+`abc%[de`ab� AP-1 n�`a�ij!=�����b NMDA � TUNEL n�`a�o�pq'��b��� ��&#�r_P+`a�stuMT`a�sv@Nw?�x�;����"#���� h=]^y�� AP-1 DNA z{� NMDA \ 24]^6�BC!=�� ��]|��}~[b� TUNEL n�`a������� ��6�����0����������10�� ���.� JNK ���� AP-1 ���� c%sv@Nw?������ TUNEL n�`a��� ]^�
Fig. 5. Immunohistochemistry of activator protein-1 �AP-1� in rat retinas at 24 hours after intravitreal injection ofphosphate bu#ered saline �PBS� as a control �A� or 200 nmol N-methyl-D-aspartate �NMDA� �B�. Asubstantial increase in immunoreactivity after NMDA injection was noted in the retinal ganglion cell layer
�RGCL� and inner nuclear layer �INL�. Scale bar�50 mm. IPL: inner plexiform layer, ONL: outer nuclear
layer.
Fig. 6. Inner retina double-labeled with TUNEL staining and activator protein-1 �AP-1� immunostaining afterintravitreal injection of 200 nmol N-methyl-D-aspartate �NMDA�. �A� AP-1 �red�. �B� TUNEL-positive cells�green�. �C� Merged images. Scale bar�50 mm. RGCL: retinal ganglion cell layer, IPL: inner plexiform layer,
INL: inner nuclear layer.
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������� JNK � AP-1 �� �������������� ���� ��� c-Jun����� JNK ������ !�"#$����26��%��&'�()*+,-./� 0/� 12345��6789:� AP-1 ��;�<=>��� ?.�@ABCDEF3� granular dentate gyrus ��GH��I*J� AP-1 ���*BCDEF3�K�LMNO-.�PO����*"#=.���27�� �Q�� RST�� TUNEL* AP-1�UVWX��� AP-1Y�Z[�\*]^� TUNELY�Z[*_��/� I�I*J AP-1 �����/` Z[�� ab�BCDEF3;c�/Z[d�e�AI*�fg=.��AP-1 �BCDEF3�hi�j68kl*��
FasL* TNF-a��<�+,-.���� FasL*TNF-a �*$� 5’� �mnoEpEqr� AP-1 Lstu;vwI*�x-.���24�� FasL �^� Fas yz9{�?�|}~�Ls�� caspase8 ;���i�I*�JBCDEF3;��i�� �Q� AP-1 DNA Ls����<� Bcl-2family �BCDEF3�������i� Bcl-Xl;���������� Bim ���;��i�I*�"#=.���28�� 0/� dominant-negativeJun mutant ;Z[���=>�*�D�9{yB�-� cytochrome c �����=.�I*$"#=.���29�� I.-�I*�-BCDEF3�Fas yz9{���� TNF-a ;c�/ caspase 8 !*�D�9{yB;c�/�Q� !�+,-.�� RS� NMDA �J AP-1 �����/` Z[* TUNEL Y�Z[�\�_��/I*�- NMDA �J���=./ AP-1 � FasL �TNF-a � 5’� mnoEpEqr�Ls�� ��;��/L�� I.-�Q� !�BCDEF3;���I�/������� I��Z�R�� ���/��L�*��� NMDA �J��������$AP-1 !�BCDEF3� b¡¢���AP-1 ����BCDEF3���e�A*+,-./�
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Abstract
NMDA Stimulates AP-1 Pathway in the Rat Retina
Kohei Kuribayashi1, Yasushi Kitaoka1, Toshio Kumai2, Yasuhiro Hayashi1,
Yasue Yomura1, Hiroyuki Takeda1, and Satoki Ueno1
Activator protein-1 �AP-1� is known to be involved in apoptotic pathways for the various stimuli. Weexamined expression of AP-1 in N-methyl-D-aspartate �NMDA�-induced neurotoxicity in the rat retina.Apoptotic cell death in the inner retina estimated by terminal deoxynucleotidyl transferase-mediated
dUTP-biotin nick-end labeling �TUNEL� staining started to appear at 6 hours and peaked at 24 hours afterintravitreal injection of NMDA �200 nmnol�. Electrophoretic mobility shift assay �EMSA� showed anincrease of AP-1 DNA binding activity in NMDA-treated retina. AP-1 immunoreactivity was observed in
the inner retina and co-localized with TUNEL positive cells after NMDA injection. These results suggest
that activation of AP-1 pathway may be pro-apoptotic in NMDA-induced neuronal cell death in the rat
retina.
1 Department of Ophthalmology St. Marianna University School of Medicine
2 Department of Pharmacology St. Marianna University School of Medicine
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