regulation of ferredoxin gene in steroidogenic and nonsteroidogenic cells

Pergamon J. Steroid Biochem. Molec. Biol. Vol. 53, No. 1-6, pp. 47-51, 1995

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Regulat ion of Ferredoxin Gene in Steroidogenic and Nonsteroidogenic Cells

J ing-Ruey Yeh, 1,2 C o r y H u a n g , 1,2 D u - A n Wu, 3 I n g - C h e r n g G u o , t W i l l i a m E. R a i n e y 4 a n d B o n - c h u C h u n g 1,2.

t Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan, 2Institute of Biochemistry, Yang-Ming University, Shi-Pai, Taipei, Taiwan, 3Division of Metabolism and Endocrinology, Tri-Service General Hospital, Taipei, Taiwan, China and 4Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology,

University of Texas Southwestern Medical Center, Dallas, TX 75235, U.S.A.

F e r r e d o x i n is a n e l e c t r o n t r a n s p o r t i n t e r m e d i a t e f o r al l t h e m i t o c h o n d r i a l c y t o c h r o m e s P450. I t is e s p e c i a l l y a b u n d a n t in s t e r o i d o g e n i c o r g a n s w h e r e i t f u n c t i o n s in s t e r o i d b i o s y n t h e s i s . T h e r e g u - l a t i o n o f f e r r e d o x i n gene e x p r e s s i o n was s t u d i e d in b o t h s t e r o i d o g e n i c a n d n o n s t e r o i d o g e n i c cell l ines . In s t e r o i d o g e n i c cell l ine Y1, t h e e x p r e s s i o n o f f e r r e d o x i n was s t i m u l a t e d b y c A M P a n d r e p r e s s e d s l i gh t ly b y a n g i o t e n s i n I I a n d p h o r b o l e s t e r P M A . T h e s e d r u g s e x h i b i t e d t he s a m e ef fec t on t h e b a s a l p r o m o t e r o f t he f e r r e d o x i n gene , w h i c h i n c l u d e s one T A T A b o x a n d a n SP1 si te . In h u m a n a d r e n o c o r t i c a l cell l ine H295, t h e s t i m u l a t i o n o f t he f e r r e d o x i n gene b y c A M P w a s b l o c k e d b y c y c l o h e x i m i d e , as o b s e r v e d in b o v i n e a d r e n o c o r t i c a l cell c u l t u r e . In n o n s t e r o i d o g e n i c cell l ines s u c h as H e L a a n d C O S - l , t he s t i m u l a t i o n o f f e r r e d o x i n gene e x p r e s s i o n b y c A M P was n o t o b s e r v e d , a l t h o u g h b a s a l e x p r e s s i o n was s t r o n g . T r a n s f e c t i o n s t u d i e s s h o w e d t h a t t he f e r r e d o x i n p r o m o t e r c o u l d n o t be s t i m u l a t e d b y c A M P in n o n s t e r o i d o g e n i c cells. T h e r e f o r e t he s t e r o i d o g e n i c c e l l - spec i f i c r e g u l a t i o n a n d the g e n e r a l e x p r e s s i o n p a t t e r n a p p e a r s to be a p r o p e r t y u n i q u e to t he f e r r e d o x i n gene .

J. Steroid Biochem. Molec. Biol., Vol. 53, No. 1-6, pp. 47-51, 1995

I N T R O D U C T I O N

Ferredoxin is a m e m b e r of the electron t ransport chain in the mitochondria. Electrons are t ransported f rom N A D P H , via ferredoxin reductase and ferredoxin, to terminal cytochrome P450, which use electrons and oxygen in a monooxygenase reaction to add a hydroxyl group to their substrates. Therefore , ferredoxin is important for the metabol ism of substrates for mi- tochrondrial cytochrome P450.

Ferredoxin is found most abundant ly in the adrenal cortex, where steroids are synthesized, therefore it is also called adrenodoxin. I t is also found in the liver and kidney, for the metabol ism of vi tamin D and bile acid. Hence the name of hepatoredoxin and renodoxin. But sequencing of the c D N A showed that there is only a single protein species for all these proteins found in all these tissues, therefore ferredoxin may be a more appropriate te rm for this protein [1]. In addition to

Proceedings of the I X International Congress on Hormonal Steroids, Dallas, Texas, U.S.A., 24-29 September 1994.

*Correspondence to B-c. Chung at the Inst i tute of Molecular Biology.

47

these tissues, ferredoxin m R N A was also found in every tissue at low levels [2].

T h e ferredoxin gene contains four exons and three introns [1, 3]. I t is located on human chromosome 11 with two pseudogenes at chromosomes 20 and 21 [4]. Mult iple polyadenylation sites are used resulting in m R N A species of different sizes. Since its major function is the participation of steroid synthesis in the adrenal cortex, its regulation pat tern follows that of steroidogenic cytochrome P450. In bovine adrenal cortical cells [5], mouse adrenal tumor cell line Y1 [6], and human choriocarcinoma cell line JEG-3 [2], expression of ferredoxin m R N A is stimulated by cAMP in a delayed manner. I t takes at least 6 h before this increase of ferredoxin m R N A is apparent.

T h e stimulation of steroidogenic cytochrome P450 is inhibited by cycloheximide [5], indicating that the synthesis of a labile intermediate protein may be required. Ablation by cycloheximide of cAMP st imu- lation of ferredoxin expression was observed only in the bovine adrenal cortical cell culture [5], but not in granulosa cells [7], Y1 [6], or JEG-3 cells [2]. Th is discrepancy may reflect an inherent difference of pat- terns of regulation in cells originated f rom different

48 Jing-Ruey Yeh et al.

tissues or species. But more likely, it indicates that regulation of ferredoxin expression may be altered after cells were cultured.

Besides the work ment ioned above, regulation of ferredoxin gene in nonsteroidogenic tissues, or by other reagents, has never been studied. This report presents our work on the study of ferredoxin gene regulation in both steroidogenic and nonsteroidogenic cells. We showed that in Y1 cells, cAMP was the major s t imulator, while calcium slightly represses expression. We also demonstra ted that the major cis-acting regulatory element of the ferredoxin gene is at the basal promoter , in the proximity of the T A T A box. In addition, we found that in nonsteroidogenic cells, ferredoxin m R N A levels were not regulated by cAMP, reflecting the nature of the cell type-specific promoter .

EXPERIMENTAL

Cell culture

Y1, C O S - l , 1-10, H295, or H e L a cells were grown to near confluence before 1 0 0 # M phorbol ester PMA, 10 m M angiotensin I I , 10 # M A23187, 50 ktg/ml cycloheximide, 1 m M 8-Br-cAMP, or 0 . 1 / t M dexamethasone was added to the culture and incubated for the amount of t ime indicated in each figure legend. Plas- mids were transfected into cells using the calcium phosphate precipitation method as described earlier [8]. p W T - O V E C and OVEC have been described [8, 9].

Analysis of R N A

Nor thern-b lo t analysis was per formed as described earlier [6]. T h e probes of human ferredoxin c D N A and G A P (glyceraldehyde-3-phosphate dehydrogenase) c D N A have been published [2]. After transfection, R N A was analyzed by pr imer extension as described earlier [6].

RESULTS

Regulation of ferredoxin in Y1 cells

Mouse adrenal Y1 cells have been used extensively for the studies of regulation of steroidogenic gene expression, including the analysis of ferredoxin promoter. We therefore chose to analyze regulation of ferredoxin gene expression by various stimulators in Y1 using Nor thern-b lo t analysis. As shown in Fig. 1, cAMP was the major st imulator of ferredoxin synthesis. Angiotensin I I and phorbol ester P M A slightly repress ferredoxin m R N A levels. Angiotensin I I is known to alter calcium levels in the adrenal g lomeru- losa cells [10]. Phorbol esters modify protein kinase C activity which also alters intracellular calcium concentration [11]. Therefore , it seems that reagents which change intracellular calcium concentration may have a slight negative effect on ferredoxin levels.

T h e ferredoxin promoter has been located to contain a T A T A and 2 Sp i elements which function in cAMP-

stimulated transcription [8], therefore whether the same element functions in regulation by other reagents was tested. The control region from - 7 6 to - 4 2 was cloned in front of an ovec vector driven by the fl-globin minimal T A T A promoter [9]. This promoter was greatly induced when 8 -Br -cAMP was present (Fig. 2). Other reagents, such as synthetic glucocorticoid dexamethasone, A23187, and phorbol ester PMA, decreased basal transcription by about 20°Jo. These reagents had the same effect whether alone or in combination, show- ing that they acted through the same pathway. There was an additive effect when A23187, phorbol ester, or dexamethasone was added together with 8-Br-cAMP, indicating 8 -Br -cAMP used a different pathway for stimulation, and both pathways were independent of each other. The recapitulation of endogenous regulation by transfection experiment also demonstrated that the basal promoter contained all the control elements necessary for regulation.

Regulation of ferredoxin expression in 1-129.5 cells

A new cell line, N C I - H 2 9 5 , has recently been characterized [12, 13]. This cell line retains multiple steroidogenic function, including P450scc, P450c21, P450c17, P450c11, P450as, P450aro etc., in a regulated fashion. In addition, this human cell line will also be perfect for the study of human ferredoxin gene

co 12. ,,- ,,(

cJ - - ~ < .,( < n o

F d x

1 2 3 4 5

G A P

Fig. 1. Regulation of ferredoxin by calcium-inducing effec- tors in Y1 cells. Y1 cells were treated with 10/~M calcium ionophore A23187, 10 mM angiotensin II (AII), 100/~M phorbol ester PMA, or l m M 8-Br-cAMP for 2h before RNA extraction. RNA was electrophoresed and hybridized with human ferredoxin cDNA probes (Fdx). The same blot was stripped and rehybridized with an internal control probe

GAP.

Transcription of the Ferredoxin Gene 49

n

,,( n Q

O L) < tO D

- o v e c

~ ~ ~ ~ ~ - o v e c - R e f

1 2 3 4 5 6 Fig. 2. Sl ight r e p r e s s i o n of A23187, phorbo l ester , and dexa- m e t h a s o n e on the F d x - W T express ion . Y1 cells were t r a n s - fected wi th F d x - W T and O V E C - R e f p l a s m i d s . A day la ter , the cells were spl i t in to 6, and were s u p p l e m e n t e d wi th n o t h i n g (--) , 20 p M forskol in (C), 100 n M d e x a m e t h a s o n e (D), 0 .5/ iM A239187 (A), 30 n M phorbo l es te r P M A (P), or the i r c o m b i n a t i o n s . RNA was h a r v e s t e d 24 h la te r and was assayed

for r e p o r t e r gene express ion by p r i m e r extension.

expression. Ferredoxin expression in H295 cells was inhibited by cycloheximide and st imulated by forskolin (Fig. 3). T h e combinat ion of cycloheximide and forskolin also exhibited a negative effect, indicating that the st imulatory effect of forskolin was blocked by cycloheximide, the protein synthesis inhibitor. Ex- pression of glyceraldehyde-3-phosphate dehydrogenase (Gap), which was used as an internal control, was not affected by the treatment. T h e effect of cycloheximide to block cAMP-s t imula ted ferredoxin gene expression was unique to H295 cells. In almost all other

G a p

t-.

o

x ~ ' - "1" o o O u_ m

- 1 6 0

1 4 7

F d x 1 2 4

1 2 3 4 Fig. 3. Endogenous express ion o f f erredox in gene in H295 cells. H295 cells were t r e a t e d wi th n o t h i n g ( - - ) , cyclohex- im ide (CHX), Forskol in , or bo th d r u g s for 24 h before RNA was h a r v e s t e d an d ana lyzed by p r i m e r ex tens ion wi th bo th

f e r r ed o x in (Fdx) and Gap p r i m e r s at the s a m e t ime .

steroidogenic cell lines, such as granulosa cells [7], Y1 [6], or JEG-3 cells [2], the expression of ferredoxin was synergistically stimulated by cycloheximide and cAMP. Only in bovine adrenocortical cell culture [5], was the expression of ferredoxin inhibited by cycloheximide, as for all other steroidogenic P450s. Therefore , the fact that human H295 cells can suppress ferredoxin expression in the presence of cycloheximide shows that it retains a proper ty more similar to the in vivo situation.

T h e promoter function of the human ferredoxin gene was tested in H295 cells. Using the same constructs as in Fig. 2, we observed an increase in test m R N A synthesis (ovec), in which either cycloheximide or forskolin was added to the cell culture. These two drugs also superinduced transcription when added together (Fig. 4). T h e empty ovec vector had a very low activity, which was detectable only when both forskolin and cycloheximide were added (Fig. 4, lane 4). Cyclo- heximide, but not forskolin, also has an effect on the internal control plasmid, ovec-Ref, which contains the SV40 promoter and enhancer. These data showed that the ferredoxin promoter , as determined by transfection into H295 cells, was superinduced by cycloheximide and differed from the endogenous expression, which showed cycloheximide sensitivity. Therefore , the transfection data did not parallel the endogenous expression. This reflects our limitation in using transfection experiments to study gene regulation.

Regulation of ferredoxin in nonsteroidogenic cells

T h e expression of ferredoxin was studied by Nor th - ern-blot analysis in different cell lines. Y1 and 1-10 are two steroidogenic cell lines. Y1 has a low level of ferredoxin expression which was stimulated by 8-Br- cAMP (Fig. 5). 1-10, the mouse testis Leyd ig - tumor cell line, had very little basal expression, but could be stimulated by the addition of 8 -Br -cAMP (Fig. 5, Lanes 3 and 4). H e L a and C O S - l , two nonsteroidogenic cell lines, expressed high levels of ferredoxin m R N A , yet they were not stimulated after 8 -Br -cAMP treatment. I t shows that the expression of ferredoxin in these nonsteroidogenic cells is constitu- tive, and not regulated by cAMP.

T h e function of the human ferredoxin promoter in nonsteroidogenic cell line was tested. Figure 6 shows the message levels transcribed f rom clones containing different lengths of the human ferredoxin 5'-flanking region fused to the chloramphenicol acetyltransferase (CAT) reporter gene and R N A transcribed from a constransfected C A T plasmid driven by Rous Sarcoma Virus (RSV) promoter served as an internal control. T h e C A T R N A transcribed from two promoters were marked respectively. In Y1 cells, p romoter activity was readily detectable in the clone containing 94 bp of the 5 '-sequence, and was stimulated by cAMP (Fig. 6). In Rat-1 fibroblast cells, the promoter function was even stronger than in Y1 when the clone was 94 bp long, yet

50

X "I"

O

Jing-Ruey Yeh et al.

t - ¢-

o o

o ~ z o u_ co - 0 u_

OVEC WT-OVEC M

OVEC -

OVEC--. - -Ref

1 2 3 4 5 6 7 8

Fig. 4. Super induc t ion of Fdx-WT t r ansc r ip t ion in H295 cells by cAMP and cycloheximide. Fdx-WT p lasmid in OVEC vector (WT-OVEC) was co t rans fec ted with in te rna l control p l a smid OVEC-Ref into H295 cells. On t h e second day, cells were split into 4 plates, and were t r ea ted with nothing (--) , 10/~g/ml cycloheximide (CHX), 40/iM forskolin, or both d r u g s for 24 h. RNA was analyzed by p r i m e r extension of a globin p r i me r .

Size m a r k e r was shown at the r i g h t o f t h e gel.

it was not stimulated by cAMP, nor the longer clones. Therefore the ferredoxin promoter was active in both steroidogenic and nonsteroidogenic cell lines, but the cAMP stimulation was only steroidogenic cell specific.

Y1 1-10 HeLa COS-1

- - + - - + - - + - - +

1 2 3 4 5 6 7 8 Fig. 5. Fe r redox in express ion in nons te ro idogenic cells is n o t regula ted by cAMP. RNA f r o m Steroidogenic Y1, 1-10, n o n s - t e r o i d o g e n i c HeLa, and COS-1 cells t r ea ted with ( + ) o r u n t r e a t e d ( - - ) wi th 1 m M 8-Br -cAMP was e lec t rophoresed , blot ted, and hybr id ized with r a d i o a c t i v e h u m a n fe r redoxin

eDNA probe.

DISCUSSION

In this report, we have studied the regulation of the human ferredoxin gene in both steroidogenic and nonsteroidogenic cells. Although ferredoxin gene is expressed in both cell types, it is stimulated by cAMP only in steroidogenic cells. In addition, it was inhibited slightly by angiotensin II and phorbol ester PMA in Y1 cells. This active transcription in all cells, yet tissue- specific regulation by cAMP only in steroidogenic cells, is unique for ferredoxin. As ferredoxin is found in many tissues, yet only required to be regulated in steroidogenic tissues, it is reasonable that nature has devised a scheme to regulate ferredoxin only when necessary.

The human ferredoxin promoter started to function when it-was about 94 bp long and contained one Spl site. When there are two Spl sites as in the 209 bp fragment, then there is full promoter function. This promoter is functional in both steroidogenic and nonsteroidogenic cells, consistent with the finding that Spl is present in all cell types. Yet, the promoter responds to cAMP to further activate transcription only in steroidogenic cells. Therefore the factors that partici- pate in cAMP-dependent transcription are present only in steroidogenic cells.

Th e mode of ferredoxin expression was characterized in H295 cells. The stimulation of ferredoxin in H295

(A) Y1 cells

60 94 209 452 - - 4 - - - + - - + - - 4 -

Transc r ip t ion of the Fer redoxin Gene

(B) Rat-1 cells

772 1058 1951 60 94 209 - - + - - 4 - - - + - - + - - 4 - - - 4 -

1058 1951 - 4 - - 4 -

51

l Fdx

RSV

1 2 3 4 5 6 7 8 9 1011 121314 1 2 3 4 5 6 7 8 9 10 Fig. 6. The h u m a n fe r redoxin p r o m o t e r r e sponds to cAMP in Y1 but not Rat-1 and COS-I cells. Y1 (A) and Rat-1 (B) cells were co t rans fec ted with in te rna l cont ro l p l a smid RSVCAT and p lasmids car ry ing CAT gene dr iven by different lengths of the 5 '-f lanking region of the h u m a n fe r redoxin gene as wr i t t en on top of each lane. On the second day, each plate of cells was split into two, 20 t~m forskolin was added to one plate ( + ) but not the o ther ( - - ) . The a m o u n t of CAT RNA was analyzed by p r i m e r extension using a CAT p r i m e r 24 h later.

T ransc r ip t s dr iven by the RSV and fe r redox in (Fdx) p r o m o t e r s were m a r k e d respectively.

cells was suppressed by cycloheximide, a proper ty of the cells similar to in vivo situation. Therefore , this cell line appears to be a good cell line to use because of its proper ty and its human origin. We, however, found a discrepancy in results obtained f rom transfection ex- per iments and endogenous expression. This is one example of problems encountered by transfection studies. I t is possible that plasmid transfected transiently into cells was not incorporated into the chromosome yet, and was not in the same chromatin structure as most genes are in vivo. This could explain the lack of cycloheximide inhibition of transfected genes. Eventu- ally approaches using transgenic mice will have to be taken to definitively answer the questions in vivo.

Acknowledgement- -This work was supported by Academia Sinica and National Science Council, Republic of China, Grant NSC83- 2311-B-001-078.

R E F E R E N C E S

1. Chang C-Y., Wu D-A., Lai C-C., Miller W. L. and Chung B-c.: Cloning and structure of human adrenodoxin gene. D N A 7 (1988) 609-615.

2. Picado-Leonard J., Voutilainen R., Kao L-c., Chung B-c., Strauss J. F. III and Miller W. L.: Human adrenodoxin: cloning of three cDNAs and cycloheximide enhancement in JEG-3 cells. J. Biol. Chem. 263 (1988) 3240-3244.

3. Sagara Y., Sawae H., Kimura A., Sagara-Nakano Y., Morohashi K-i., Miyoshi K-i. and Horiuchi T.: Structural organization of the bovine adrenodoxin gene. J. Biochem. 107 (1990) 77-83.

4. Chang C-Y., Wu D-A., Mohandas T. K. and Chung B-c.: Structure, sequence, chromosomal location and evolution of the human ferredoxin gene family. D N A Cell Biol. 9 (1990) 205-212.

5. John M. E., John M. C., Boggaram V., Simpson E. R. and Waterman M. R.: Transcriptional regulation of steroid hy- droxylase genes by corticotropin. Proc. Natn. Acad. Sci. U.S .A . 8 3 (1986) 4715-4719.

6. Guo I-C., Huang C. and Chung B-c.: Differential regulation of the C Y P I l A I (P450scc) and ferredoxin genes in adrenal and placental cells. D N A Cell Biol. 12 (1993) 849-860.

7. Golos T. G., Miller W. L. and Strauss J. F. III: Human chorionic gonadotropin and 8-bromo cyclic AMP promote an acute increase in cytochrome P450scc and adrenodoxin mRNAs in cultured human granulosa cells by a cycloheximide-insensitive mechanism. J. Clin. Invest. 80 (1987) 897-899.

8. Chang C-Y., Huang C., Guo I-C., Tsai H-M., Wu D-A. and Chung B-c.: Transcription of the human ferredoxin gene through a single promoter which contains the 3',5'-cyclic adenosine monophosphate-responsive sequence and Sp 1-binding site. Molec. Endocr. 6 (19,92) 1362-1370.

9. Westin G., Gerster T., Mfiller M. M., Schaffner G. and Schaffner W.: OVEC, a versatile system to study transcription in mammalian cells and cell-free extracts. Nucl. Acids Res. 15 (1987) 6787-6798.

10. Barrett P. Q., Bollag W. B., Isales C. M., McCarthy R. T. and Rasmussen H.: Role of calcium in angiotensin II-mediated aldosterone section. Endocrine Rev. 10 (1989) 496-518.

11. Nishizuka Y.: Studies and perspectives of protein kinase C. Science 233 (1986) 305-312.

12. Staels B., Hum D. W. and Miller W. L.: Regulation of steroido- genesis in NCI-H295 cells: a cellular model of the human fetal adrenal. Molec. Endocr. 7 (1993) 423-433.

13. Rainey W. E., Bird I. M., Sawetawan C., Hanley N. A., McCarthy J. L., McGee E. A.~ Wester R. and Mason J. I.: Regulation of human adrenal carcinoma cells (NCI-H295) pro- duction of C19 steroids. J. Clin. Endocr. Metab. 77 (1993) 731-737.

regulation of ferredoxin gene in steroidogenic and nonsteroidogenic cells

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