Volutttc 21+0. number I, 134=116 FEBS 094?? M'~r¢h 1991 .~ I~1~1 F~deration o f i~urOpea, Biodt¢luieal So++ietic~ (X)I4$+/~/~I/$).$IJ
ADONIS ix014 J,"/9~t~)10Ol a'?O
The effects of glucocorticoid hormone on the expression of c-jun
H c n g Lee t, Y u c h - T s e r n Shaw t. Shean-Tai C h i o u t , W e n . C h a n g C h a n g t'a a n d M i n g . D c r g LM ~ 'a ' ln.tlitute o f Biochemical Science.t, Co11¢g¢ of Science, Natiamd Tatwaa University, TatpeL Taiwalt. Republit" of Ctlina, ~ h~sltmt¢ ofBiological Ctt¢mtstr).. Academia Stalely. PO Box 23.106, Toipei, Tatwan, Republic of Chino t~nd ~ Department af Biod~¢mi~lry, Co11¢t1¢ o f Medt¢i~le, Notional Chetlg Ktmfl University, 7"aina~t, Taiwem. Republic of Chhtu
Received 2 Janmtry 1991
The effects of #ucocortit:oid hormone on the expression of c.jun in the fibrobla~ts were ~tudicd. The expression ore.jim was repressed by dexametha. sone in the NIH3T3 cell~ hut not in the transformed [tl04. I or EJ.Ra~ calls, Tlte repression was not relieved by the addition ofeycloh~ximide.
Glucocorticoid: Oncogcnc: Jun
1. INTRODUCTIOI'I
The oncogene jun was first identified as the transfor- ming component o f avian sarcoma virus 17 which causes fibrosarcomas in chickens [1]. Nueleotide se- quence analysis revealed that the C-terminus of v-jun strafes more than 40~0 homology with the C-terminus o f yeast transcriptional activator GCN4, which is the DNA binding domain of GCN4 [2,3]. This finding leads to the suggestion that the v-jun oncogene product acts as a transcriptional factor to influence the cellular gene expression pattern. Later studies have shown that the human AP-1 (activator protein 1) gene is the cellular homologue of the transforming v-jun gene [4,5]. The AP-I complexes consist of several distinct proteins in- eluding those encoded by the proto-onco.~,-nes c-jun and c-fos, and other members in these two gene families [6-10]. At least two other members, jun-B and jun-D, have been identified in the jun family. Jun-B was shown to inhibit the
trans.activating
activities of c-jun when both were eo-transfected into F9 cells [11-14].Collagenase gene is one of the many genes known to be activated by Jun [15]. The AP-I site in the promoter of the collagenase gene mediates biological responses induced by phorbol esters, growth factors and steroid hormones [16-21]. Direct interaction between the glucocorticoid hormone receptor and the AP-1 complex on the AP-I site has recently been demonstrated, and was shown to be responsible for the inhi0ition of col- lagenase expression by glucocorticoid hormone in CV-1 cells and other cells [18-21]. As the expression of c-jun is regulated by itself, the amount of c-jun mRNA
Correspondence and present address: M.-D, Lai, Department of Biochemistry, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China
should be affected by glucocorticoid hormone accor- ding to the model proposed by several groups [18-211. In this report, we investigated the effects of glucocor- ticoid hormone on the expression o f c.jun in the fibroblasts The results indicated that the expression of c-jun is indeed inhibited by dexamethasone in NIH3T3
fibrobMsts
2. MATERIALS AND METHODS 2,1, Celt culture
NIH3T3, BI04.1 and EJ.Ras cells were cultured in DMENI sup. plemented with 100/0 fetal calf serurn in a humidified atmosphere con. raining 5Olo CO~, 95O1o air on 10-cm plastic dishes.
2.2, RNA isolation and analysis
Total RNA was isolated from cell~ according to the single step method published by Chomczynski and Sacchi [22], and subjected to agarose gel electrophoresis and Northern blot analysis as described [23]. The plasmid pSV-jun containing eDNA of c-jim was used to prepare labeled probes for hybridization, pCJ125 probe containing a ribosomal RNA gene was used as all internal control,
3. RESULTS AND DISCUSSION
3, 1. The effects o f dexamethasone on the expression o f
C-jt.ln10- 6M dexamethasone was added at time zero to the
confluent NIH3T3 cells or B104-1 cells Cells were harvested at different time intervals and 20 #g total RNA was applied each lane. After 12 h, the expression of c-jun decreased significantly in NIH3T3 cells (lane 1 and lane 3, Fig. 1A), and remained low for at least another 24 11 (lane 4, Fig. IA). While the expression of c-jun stayed constant or increased a little in the neu- transformed fibroblasts cells, F~104-1 cells, as shown in Fig. 1B (the relative level has been determined by den- sitometry scanning). Similar results were observed in
Villum¢ 211ii, number t I+EI.:L LET<I'ElitS M a r d t 1991
J u n = ~',~gl
1 2 3 4
,...- m l l m O
t 2 3 4 rig, =, = ne etsects oI uexametltasone on me expressmn o~ c.jun m NIH3T3 cells (AI and BI04.1 ~:dls (B), Cel;s were harvesied at 0 h (lane I), 4 h (lane 21, I1 h (lane ,t}, and 36 h (lane 4) after treatment with dexamettiasone. Total RNA isolation anti Northern blot an=tly~i~
were carried out ax de~cribetl in ~cction 2,
E J - R a s cells w h i c h a r e N I H 3 T 3 cells t r a n s f o r m e d w i t h o n c o g e n e r a s ( d a t a n o t s h o w n ) ,
3.2.
Cycloheximide cannot relieve the repression
cat:sed by dexamethasone
T h e e f f e c t s o f g l u c o c o r t i c o i d h o r m o n e o n t h e e x p r e s . s i o n o f s e v e r a l o t h e r g e n e s r e q u i r e o n g o i n g p r o t e i n s y n . t h e s i s . W e w i s h e d t o s t u d y t h e r o l e o f d e n e r o p r o t e i n s y n t h e s i s d u r i n g t h e r e p r e s s i o n o f c - j u n b y alex. a m e t h a s o n e , C y c l o h e x i m i d e h a s b e e n s h o w n t o i n c r e a s e t h e s t e a d y - s t a t e level o f t h e c - j u n m R N A c o n c e n t r a t i o n by i n h i b i t i n g t h e s y n t h e s i s o f a h y p o t h e t i c l a b i l e p r o t e i n w h i c h c a n d e s t a b i l i z e t h e c - j u n m R N A . A s i m i l a r e f f e c t w a s o b s e r v e d in o u r e x p e r i m e n t s ( l a n e 1 a n d 4, F i g . 2), C o m p a r i n g t h e m R N A l e v e l s o f c - j u n in t h e p r e s e n c e o f d e x a m e t h a s o n e a n d t h a t in the a b s e n c e o f d e x - a m e t h a s o n e w h e n c y c l o h e x i m i d e w a s a d d e d ( l a n e 3 a n d 4, F i g . 2), t h e r e p r e s s i o n o f t h e e x p r e s s i o n o f c - j u n w a s c l e a r l y n o t a f f e c t e d b y t h e a d d i t i o n o f c y c l o h e x i m i d e ,J u n +
=~merRNA---
~ r / ~ ~
1
2
3
4
Fig. 2, The effect of cyclohcximide on the repression of c-jun by dex- amethasone. NIH3T3 cells were treated with dexamethasone or cyclohe×imide or both for 12 h, and total RNA was isolated, elec- trophoresesed, blotted and hybridized as described in section 2, In the cells treated with both dexamethasone and cyclohe×imide, cyclohex= imide was added 1 h prior to the addition of dexamethasone. RNA from cells untreated (lane 1), cells treated with de×amethasone (lane 2), cells treated with dexamethasone and cycloheximide (lane 3), and
cells treated with cycloheximide (lane 4),
I n o t t r r e p o r t , w e d e m o n s t r a t e d ih~ti th© o x p r ~ s s i o n o f c . j u n is d o w n . r e t ~ u l a t e d b y t h e I j l u c o c o r i i c o i d h o r m o n e directly in normal flb~'oblast~ but n o l i n l r a n s f o r m e d f i b r o b l a s l ~ (i.c, B104.1 cells), T h e r e p r e s s i o n o b s e r v e d i~ N I H 3 T 3 cells is p r o b a b l y m e d i a t e d t h r o u i ! h t h e in. i e r a c d o n b e t w e e n the t d u c o c o r i i c o t d h o r m o n e r e c e p t o r a n d t h e A P - I c o m p l e x o n t h e A P - ! s i t e i n : t h e p r o m o t e r o f c - j u n [241, T h e d i f f e r e n c e o f r e s p o n s e t o d~x- a m e t h a s o n e b e t w e e n t r a n s f o r m e d c e l l s and n o r m a l f i b r o b l a s t cells p o s s i b l y r e f l e c t s t h e r a t i o o f f u n c t i o n a l J u n a n d F o s in e a c h cell line a c c o r d i n g t o t h e m o d e l p r o p o s e d by Y a m a m o t o [181. H o w e v e r , other e x p l a n a - t i o n s , s u c h a s d e c r e a s i n g n u m b e r o f 8 1 u c o c o r t i c o i d h o r - m o n e r e c e p t o r in B I 0 4 - I c e l l s , c a n n o t b e e x c l u d e d , O u r r e s u l t s a n d a recent r e p o r t b y H e r r l i c h et al. [191 i n d i c a t e d t h a t t h e e x p r e s s i o n o f c - j u n c a n b e r e p r e s s e d , i n d u c e d , o r u n a f f e c t e d b y t h e t r e a t m e n t o f g l u c o c o r . t i c o i d h o r m o n e tn d i f f e r e n t cell t y p e s . T h e r e f o r e , t h e in- t e r a c t i o n b e t w e e n t h e A P - I c o m p l e x a n d t h e g l u c o c o r - t i c o i d h o r m o n e r e c e p t o r o n t h e A P - I s i t e n o t o n l y a f - f e c t s t h e e x p r e s s i o n o f o t h e r g e n e s b u t a l s o a f f e c t s t h e e x p r e s s i o n o f c - j u n itself.
Acknowledgements:
This work wlts supported by Grant NSC79,0203.B002.03 from the National Science Council in Taiwan, Republic or China, i am grateful to Dr, Inder Verma for the ~it't of the pSV.jun plasmid and to Dr. Nlien.Chie Hunlt for provldin8 all cell lines used in this study,R E F E R E N C E S
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