Transcriptional Regulation of Steroid Hydroxylase Genes by Corticotropin MALIYAKAL E

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Proc. Natl. Acad. Sci. USA Vol. 83, pp. 4715-4719, July 1986 Biochemistry Transcriptional regulation of steroid hydroxylase genes by corticotropin MALIYAKAL E. JOHN*t, MANORAMA C. JOHN*, VUAYAKUMAR BOGGARAM*, EVAN R. SIMPSON*t, AND MICHAEL R. WATERMAN*t *Department of Biochemistry and tDepartment of Obstetrics and Gynecology and The Cecil H. and Ida Green Center for Reproductive Biology Sciences, The University of Texas Health Science Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75235 Communicated by Ronald W. Estabrook, March 17, 1986

ABSTRACT Maintenance of optimal steroidogenic capac- bovine adrenocortical cDNA library we have found that the ity in the adrenal cortex is the result of a cAMP-dependent expression of the vast majority of genes in adrenocortical response to the peptide hormone corticotropin (ACTH). The cells in monolayer culture is not affected by ACTH treatment molecular mechanism of this action of ACTH has been exam- (7). The genes whose expression is altered are <2% of the ined by using five recombinant DNA clones specific for enzymes transcribed genes in these cells. To date, only genes encoding of the steroidogenic pathway (P-45O.,,, P-45011p, P-450C21, components of the steroid biosynthetic pathway have been P-45017a, and adrenodoxin). The presence of nuclear precur- found to be induced by ACTH (7-11). sors in steady-state RNA samples derived from cultured bovine This paper describes results ofstudies undertaken to define adrenocortical cells and moderate increases in the number of the role of transcriptional events in the ACTH-mediated RNA chain initiations, as determined by in vitro nuclear run-off regulation of steroid hydroxylase gene expression. These assays, indicate that ACTH controls the expression of the studies indicate that ACTH influences steroid hydroxylase gene(s) for each of these proteins at the transcriptional level. gene expression at the transcriptional level and provide The ACTH-mediated increase in accumulation of transcripts preliminary evidence to suggest that in the case of all four specific for steroid hydroxylases in nuclear RNA can be steroid hydroxylases as well as adrenodoxin, transcriptional specifically blocked by inhibiting protein synthesis in bovine activation is mediated by labile protein factors. adrenocortical cell cultures. The steady-state concentrations of nuclear RNA for control genes show no decrease upon cycloheximide treatment. These studies suggest that a primary MATERIALS AND METHODS action of ACTH in the adrenal cortex is to activate (via cAMP) Preparation and manipulation ofprimary bovine adrenocorti- the synthesis ofrapidly turning over protein factors that in turn cal monolayer cell cultures, RNA and DNA isolation and mediate increased initiation of transcription of steroid hydrox- blotting to nitrocellulose paper, construction of cDNA librar- ylase genes. We propose that these protein factors impart specificity of induction to genes encoding components of this ies, and identification of specific clones have been described pathway in steroidogenic tissues. (7-11). In vitro transcription run-off assays (12, 13) were performed with isolated nuclei (1-2 x 107) in a buffer The peptide hormone corticotropin (ACTH) elicits biochem- containing 150 mM KCl, 5 mM MgCl2, 10% (vol/vol) glyc- ical responses in its target organ, the adrenal cortex, leading erol, 20 mM Hepes (pH 7.8), 5 mM dithiothreitol, 1 mM each to the maintenance of optimal steroidogenic capacity (1). ATP, CTP, and UTP, and 100-200 ,Ci of [32P]GTP (1 Ci = ACTH binding to its cell-surface receptor activates adenylate 37 GBq). Nuclei were incubated at 25°C for 30 min, after cyclase, resulting in increased levels of intracellular cAMP which 32P-labeled RNA was isolated by cesium chloride (2), which are thought to mediate acute (increased centrifugation and hybridized to specific plasmids bound to steroidogenesis) and chronic (increased steroid hydroxylase nitrocellulose paper. Prehybridization (12 hr) and hybridiza- synthesis) actions in the adrenal cortex (3). Thus, the regu- tion (70-90 hr) were carried out in 5x NaCl/Cit (1 x NaCl/Cit lation of steroid hydroxylase gene expression is of general = 0.15 M NaCl/15 mM sodium citrate), 50% formamide, 5X interest with respect to understanding the biochemical mech- Denhardt's solution (14), tRNA (300 ,ug/ml), polyadenylic anisms of action of peptide hormones and cAMP, as well as acid (10 ,ug/ml), 0.1% NaDodSO4, and sonicated denatured of specific interest with respect to the mechanism of main- salmon sperm DNA (50 ,g/ml) at 37°C. Filters were washed tenance of optimal steroidogenic capacity. In the adrenals of twice in 2x NaCl/Cit containing 0.1% NaDodSO4 at room hypophysectomized rats, the activities of the various steroid temperature for 20 min and twice in 0.1 x NaCl/Cit contain- hydroxylases (cholesterol side-chain cleavage cytochrome ing 0.1% NaDodSO4 at 60°C. The filters were then treated P-450, P-450SCC; 17a-hydroxylase cytochrome P-450, with RNase (10 ,g/ml) at 37°C for 10 min in 0.3 M NaCl. A P-45017a; 21-hydroxylase cytochrome P-450, P-450C21; 111- final wash was carried out in 0.3 M NaCl containing 0.1% hydroxylase cytochrome P-450, P-45011,9); and adrenodoxin NaDodSO4, and the filters were autoradiographed. Quanti- (the mitochondrial iron-sulfur protein that reduces P-450,CC fication of the signals was achieved by densitometric scan- and P-4501113) decline and are subsequently restored upon ning. Also, radioactivity on the filters was determined by ACTH administration (4). By using primary cultures of scintillation counting. Nuclear RNA was obtained from bovine adrenocortical cells, it has been established that the isolated nuclei, while cytoplasmic RNA was isolated from ACTH-mediated restoration of these activities is due to postnuclear fractions. The cytoplasmic contamination of the increased synthesis of the steroidogenic enzymes (1, 3, 5, 6), nuclear RNA was estimated (15) by the use of rabbit resulting in large part from increased levels ofmRNA specific reticulocyte lysate and a rabbit globin cDNA clone pRG-1, for these enzymes (7-10). By differential screening of a Abbreviations: ACTH, corticotropin; P-450CC, cholesterol side- The publication costs of this article were defrayed in part by page charge chain cleavage cytochrome P-450; P-45017,, 17a-hydroxylase payment. This article must therefore be hereby marked "advertisement" cytochrome P-450; P-450C21, 21-hydroxylase cytochrome P-450; in accordance with 18 U.S.C. §1734 solely to indicate this fact. P-45011,9, llf-hydroxylase cytochrome P-450; kb, kilobase(s). 4715 Downloaded by guest on October 3, 2021 4716 Biochemistry: John et al. Proc. Natl. Acad. Sci. USA 83 (1986) and it was found to be <0.02%. All other general procedures In vitro elongation in isolated nuclei of RNA chains have been performed according to Maniatis et al. (14). initiated in vivo has also been used in a number of studies to demonstrate transcriptional activation of genes (12, 13, 16, RESULTS 17). We compared the specific RNA chain initiation in cells treated with and without ACTH by nuclear run-offassays and From bovine adrenocortical cDNA libraries we have identi- typical results are shown in Fig. 2. In eight separate assays fied and characterized cDNA clones specific for P-450,cC (7), from different cell cultures, the induction of P-450,cC, P-45011p (8), adrenodoxin (9), P-45017a (10), and P-450C21 (11). P-450C21, P-450113, and adrenodoxin by ACTH ranged be- In each instance, we have used these cDNA clones to tween 2- and 4-fold. The exception is P-45017m, where the determine the size of the corresponding RNA species and to control level is very low, making the fold-induction some- show specific quantitative increases in the total steady-state what larger. Given the limitations of this type of assay for levels of these RNAs in response to ACTH treatment. An genes of low transcriptional activity [steroid hydroxylases increase in the level of primary transcripts in nuclear RNA is each constitute only 0.05-0.1% of total cellular protein, are evidence for transcriptional activation of specific genes. As not rapidly turned over (18), and induction by ACTH in cell seen in Fig. 1, when RNA transfer blots of nuclear RNA cultures is from 4- to 10-fold], this is not an unexpected result isolated from ACTH-treated or control cell cultures were (16, 17). We also determined the relative stability of the hybridized to specific cDNAs for P-450,cc, P-45017a, and various steroid hydroxylase RNAs in the presence and adrenodoxin, increases in high molecular weight RNA in absence of ACTH. Only P-450SCC shows a moderate increase addition to mature RNA were observed in cells treated with in stability as a result ofhormonal treatment, the other RNAs ACTH. The content of three nuclear precursors of adreno- being unaffected (unpublished results). Thus, the discrepan- doxin [ranging in size between 15 and 4 kilobases (kb)] is cy between the -fold increase in RNA steady-state levels increased in nuclear RNA from ACTH-treated cells com- (Fig. 1) and nuclear run-off assays (Fig. 2) is not due to pared to nuclear RNA from control cells (Fig. lA). Two changes in the stability of the RNAs but rather to lack of nuclear precursors ofP-45017a (Fig. 1B) are increased 10-fold in treated cells. In addition, a 3-kb nuclear precursor of A B P-450,cc (Fig. 1C) is increased 3-fold after ACTH treatment. CMC\I~~~~~~~ 6 q' C o ch. Thus, the specific induction of high molecular weight tran- CO a:, O 1% C~ scripts in cells stimulated by ACTH suggests activation of mLcn IL.mn CLz~~~~~~~~~~~~E~% 0. 0. 0.0 steroid hydroxylase genes. QL Q. QQQ

A B C *: , Cont 1 2 1 2 3 12 3 4 Cont 2 -O -o ..* 0 -o 6 hr

6 hr I 9,. .: * 8hr

1 0 hr 9 hr *,-* *f,#W: -4.3 :~tIti FIG. 2. Transcriptional activation of steroid hydroxylase genes. -2.2 In vitro nuclear run-off assays were carried out using nuclei from :* -1.9 control (Cont) or ACTH-treated (1 ,uM for 6, 8, 9, or 10 hr) cells. Isolation ofnuclei and transcription run-offassays have been carried out using different procedures (12, 13) with comparable results. Results shown are from an assay containing -1 x 107 nuclei, as described in Materials and Methods. As shown in B, steroid hydroxylase-specific plasmids (pBSCC-2, pB113-2, pBC21-1, pB17a- 1, and pBADX-4) were bound on nitrocellulose filters. For hybrid- izations, 32P-labeled RNA (3.7 x 106 cpm) was used. In other control experiments when a-amanitin (1 ,ug/ml) was included, the incorpo- ration ofradiolabel into total RNA was inhibited by -40%o ofcontrol, indicating inhibition of polymerase II activity. Inhibition of incor- poration of radioactivity into P-450,SC RNA by a-amanitin was 84% in run-off assays using nuclei from ACTH-treated cells (25 cpm vs. FIG. 1. ACTH-mediated increase in the content of nuclear 161 cpm). When unhybridized RNA was rehybridized to filters transcripts encoding steroid hydroxylases in cultured bovine containing pBSCC-2, no additional RNA binding was observed. adrenocortical cells. Nuclear and cytoplasmic RNA (15 mg) were Nonspecific hybridization was estimated by using pBR322 or simian size-fractionated by formaldehyde/agarose gel electrophoresis and virus 40 (10 ,ug). In A, the nonspecific hybridization to pBR322 is blotted onto nitrocellulose. The blots were then hybridized to shown. Also, results using a control plasmid, pBJF-9, which is nick-translated cDNA probes (5 x 1iO cpm/ml). Arrows indicate nonresponsive to ACTH treatment are shown under the same high molecular weight RNA transcripts and 0 is the origin. A HindIII conditions where pBSCC-2 shows increased transcription initiation digest of DNA was used as a molecular weight marker. (A) at both 6 and 9 hr. Densitometric scanning indicates a 4-fold increase Adrenodoxin. Lanes: 1, nuclear RNA (6 hr of ACTH treatment); 2, in initiation of P-4S0,, mRNA at 9 hr compared to control (Cont). nuclear RNA (control). Mature RNA sizes are 1.75, 1.4, and 0.95 kb When nuclei from fresh adrenal cortex were used for the assay, 3-fold (9). (B) P-45017.. Lanes: 1, cytoplasmic RNA (control); 2, nuclear greater P-450,SC RNA initiation could be measured compared to RNA (6 hr of ACTH treatment); 3, nuclear RNA (control). Mature nuclei from ACTH-treated cultured cells, consistent with the obser- RNA size is 1.9 kb (10). (C) P-450,SC. Lanes: 1, cytoplasmic RNA (6 vation that there is -3- to 5-fold more P-450SC RNA per ,g of total hr of ACTH treatment); 2, cytoplasmic RNA (control); 3, nuclear gland RNA compared to RNA from ACTH-treated cells. This also RNA (6 hr of ACTH treatment); 4, nuclear RNA (control). Mature indicates that the low signal intensities shown are not due to RNA size is 2.0 kb (7). competition of unlabeled RNA. Downloaded by guest on October 3, 2021 Biochemistry: John et al. Proc. Natl. Acad. Sci. USA 83 (1986) 4717 sensitivity in the nuclear run-offassays. we have Thus, relied A C PBSCC-2 on measurements of the steady-state levels of RNA for the Cont characterization ofgene activity in the presence and absence of ACTH and the protein synthesis inhibitor, cycloheximide. B D To further elucidate the molecular events subsequent to the 8@ * elevation of intracellular cyclic AMP levels, which result in 8 0 activation of gene transcription, we have examined whether cycloheximide can block transcriptional activation. Cells in 10 0 culture were incubated with cycloheximide in the presence or absence of ACTH. Total protein synthesis in cycloheximide- 120 treated cells was inhibited by up to 97% as determined by 190 4 radiolabeling with [35S]methionine. RNA blot analysis of the steady-state levels of RNA shows that cycloheximide inhibits NulCI Nuci Cyto Cylto ACTH or dibutyryl cyclic AMP-induced accumulation of P- A C pSARRP-A I I 450scc RNA (Fig. 3 Left). A similar inhibition has been observed Cont go for P-4501113 (8), adrenodoxin (9), and P-45017a (10). Also, as B 0 seen (Fig. 3 Right), cycloheximide inhibits the accumulation of * 0 the detectable primary nuclear transcript ofP450,cc, as well as that of the mature RNA. After 12 hr of treatment, ACTH 8 * . 0 induces the nuclear precursor form 10-fold while increasing the 10 * mature RNA level 5-fold. Cycloheximide treatment (5 ,ug/ml) in *0 conjunction with ACTH reduces these accumulations to 3-fold 12: and 2-fold, respectively. Higher concentrations of cycloheximide lead to greater inhibition (Fig. 3 Left). Because ofthe low * 0 level of transcription of these genes as measured by nuclear Nuol Nucl I yto C~yto run-off, we confirmed this action of cycloheximide for each of pBARP-F1 the enzymes in this study by comparison oflevels ofnuclear and A C cytoplasmic transcripts. Cant O D Dot blot analysis of nuclear RNA suggests that the cyclo- S. * heximide effect is specific for enzymes of the steroidogenic pathway (Figs. 4 and 5). As can be seen, the concentrations 8* * of nuclear RNA species detected by the control plasmid pBARP-A11 remain unchanged upon cycloheximide treat- 0* 0 120 0 1l* * A* 1 2 3 Nuel Nuel Cyto Cyto -o FIG. 4. Specific inhibition of steroid hydroxylase gene transcription by cycloheximide. Cell cultures were incubated for various time ~-28S periods (hr) with ACTH (1 MM) or ACTH/cycloheximide (5 Mg/ml). The control (Cont) cell culture underwent no treatment. Dot blots of equal amounts of nuclear and cytoplasmic RNAs (15 Mg) are presented, although in vivo the majority of the RNA is expected to be in the cytoplasm. Lanes A and C, nuclear (Nucl, lane A) and El~SR cytoplasmic (Cyto, lane C) RNAs from cells treated with ACTH (except control). Lanes B and D, nuclear (lane B) and cytoplasmic (lane D) RNAs from cells treated with ACTH and cycloheximide. :1 8 S; Results utilizing P-450,,c (pBSCC-2) and control (pBARP-A11 and pBARP-F1) hybridization probes are shown. Numbers refer to hours of treatment with modulators. Densitometric scanning indicates a 7-fold increase in P-4505CC nuclear RNA levels and a 3-fold increase in cytoplasmic levels (at 6 hr) in the presence of ACTH. When cycloheximide is also present, the increase is only 2-fold and 1.4-fold, respectively. ment, while those hybridizing to pBARP-F1 are increased by cycloheximide treatment, indicating that specific transcription of these two genes is not inhibited by cycloheximide treatment (Fig. 4). These results were obtained under the A B C D same conditions where cycloheximide strongly inhibits the appearance of nuclear P-450,cc transcripts. Thus, the RNA FIG. 3. Inhibition of cAMP-mediated P-450SCC RNA accumula- polymerase II activity is normal in cycloheximide-treated tion by cycloheximide. (Left) Cell cultures were treated with cyclo- cells (Fig. 4). Dot-blot analysis of nuclear RNA using probes heximide (20 /ig per ml of culture medium) with or without dibutyryl specific for P-45017a, P-450C21, and adrenodoxin cAMP (1 mM) for 24 hr. Total RNA (20 ug) was analyzed for P-450,cC P-45011,, RNA by RNA blot analysis. Lanes: A, control; B, dibutyryl cAMP; shows that cycloheximide inhibits transcription of each of C, dibutyryl cAMP/cycloheximide; D, cycloheximide alone. (Right) these genes (Fig. 5). Densitometric scanning suggests that Nuclear RNA (15 Mg) from cell cultures treated with ACTH (1 M1M) P-450l7a genes are the most responsive to ACTH treatment. for 12 hr. Lanes: 1, cycloheximide (5 Mg)/ACTH; 2, ACTH; 3, At 6 hr, there is a 14-fold increase in the nuclear RNA and a control. Arrow indicates the position of high molecular weight 12-fold increase in the cytoplasmic RNA in this experiment. P-450,,C RNA and 0 is the origin. When cycloheximide is present, the increases are only 1.8- Downloaded by guest on October 3, 2021 4718 Biochemistry: John et A Proc. Natl. Acad. Sci. USA 83 (1986) pBC21-1 pBl 7aX1 Cont A C A C CON B D CON eB D 6 * A 6 * 8 0 *: * 8 0 B 10 0 * 0 * 10 * * * 12 * *..0 * 1 2 a. I* C 19 * '. OH*f *~~~~~~~~~~~~19 J'I f1D; Mum.....CVTO pBADX-4 D .'A.'C. 1C Nucl Cyto con T con tS D 6 I** ,":. 6 .. *..... A FIG. 6. P-450sC RNA is stable in the presence of cycloheximide. 8 . 8 *hBs Cell cultures were treated with ACTH for 6 hr and then cyclohexi- *o. w. 10of 10 *i. * mide (5 t±g/ml) was added and incubations continued for various 1 2 #m periods of time. Nuclear (Nucl) and cytoplasmic (Cyto) RNAs were 12 * * hybridized with 32P-labeled insert ofpBSCC-2. Cont, RNA from cells 1 9alCW ir 0 not treated with ACTH. Lane A, ACTH (1 /iM) treatment for 6 hr. IRA"L . .m Lanes B, C, and D, cells treated with cycloheximide for 4, 6, and 16 hr, respectively, after initial treatment with ACTH for 6 hr. A gradual decrease in the nuclear content of P-450,cc RNA and concomitant FIG. 5. Dot blots of nuclear and cytoplasmic RNA from cell increase in the cytoplasmic content is evident. P-450s1c RNA in the cultures treated in an identical manner to those described for Fig. 4. presence of cycloheximide is stable, since a rapid turnover would The blots were hybridized to the 32P-labeled inserts of pBl7a-1, have significantly decreased the amount of P-450Occ RNA in the pBC21-1, pB11(-2, and pBADX-4. Numbers and other markings are cytoplasmic fractions in lanes C and D. the same as in Fig. 4. a primary response to ACTH-mediated elevation of intracel- and 1.3-fold, respectively. P-450C21, P-45011p, and adreno- lular cAMP levels. Because ofthe action of cycloheximide to doxin show modest increases in nuclear RNA at 6 hr (1.9, 2.3, inhibit steroid hydroxylase RNA accumulation, these factors and 3.4, respectively) that are also inhibited by cyclohexi- appear to be labile protein inducer molecules. Thus, it seems mide. Maximum accumulation of nuclear RNA for P-450C21, likely that the specificity of ACTH induction on genes P-45017a, and adrenodoxin occurs between 12 and 19 hr; for encoding components of the steroid hydroxylase pathway is P-450,cc, between 6 and 12 hr; and for P-45011p, at 12 hr. achieved through labile protein factors. Another member of Among the time points examined, maximum accumulation of the cytochrome-P450 family, the 2,3,7,8-tetrachlorodibenzo- cytoplasmic RNA occurs at 19 hr for P-450c21, P-450scc, p-dioxin (TCDD)-inducible mouse liver protein, P1-450, is P-4501113, and adrenodoxin and at 12 hr for P-45017a. The superinduced upon treatment with cycloheximide in the majority of the nuclear transcripts are mature (Figs. 1 and 3 presence of the positive regulator TCDD (19, 20). A labile Right) and thus ,the lag period in the transport to the trans-acting repressor molecule has been postulated to ac- cytoplasm is an interesting observation. Any influence of count for the superinduction. In the case ofthe P-450,cc gene, ACTH on this process will require detailed kinetic studies. In the profile of ACTH-induced RNA accumulation is not all cases, the specific RNA accumulation in the nucleus was affected by pretreatment ofthe cells with cycloheximide prior inhibited upon treatment with ACTH plus cycloheximide to the addition of ACTH, providing the cycloheximide is (column B in each panel). Since these studies were carried removed at the time of ACTH addition (unpublished obser- out using primary cell cultures, the temporal profiles and vations). However, we do not infer the absence ofa repressor levels of induction and inhibition of both nuclear and cyto- moiety from these results since synthesis of a putative labile plasmic RNA for these enzymes varied somewhat from one protein repressor as well as the labile protein activator will be cell culture to another. Therefore, conclusions concerning inhibited by cycloheximide. the general observations of induction and inhibition are Temporal studies of steady-state levels of nuclear and reproducible, while comparisons of one enzyme to another cytoplasmic RNA from cells treated with ACTH for 6 hr prior cannot be made from these studies. We also examined to addition of cycloheximide, followed by continued incuba- whether the inhibition of ACTH-induced RNA accumulation tion for up to 22 hr, provide no evidence for rapid turnover is a reflection of unusually rapid turnover of steroid hydrox- of steroid hydroxylase RNAs upon cycloheximide treatment ylase RNAs in the presence of cycloheximide. Steady-state (Fig. 6). Similar results were also obtained when cells were levels of nuclear and cytoplasmic P-450CC RNA from cells treated with cycloheximide in the absence of ACTH (Fig. 3 treated with ACTH for 6 hr prior to addition of cyclohexi- Left). Thus, regulation of steroid hydroxylase gene expres- mide, followed by continued incubation for up to 22 hr, are sion by ACTH is suggested to be mediated directly by shown in Fig. 6. These studies show that P-450,c, RNA is positive labile protein factors. Recent studies on rat hepatic stable in the cytoplasm in the presence of cycloheximide for L-type pyruvate kinase gene expression by insulin also show up to 22 hr. that the RNA induction is sensitive to cycloheximide (21). Thus, labile activator protein factors may function in a few DISCUSSION genetic systems. Taken together, these observations lead to a working ACTH influences the transcriptional activation ofthe gene(s) hypothesis for ACTH regulation of steroid hydroxylase gene encoding the four adrenocortical steroid hydroxylases and expression in the adrenal cortex. Binding of ACTH to its adrenodoxin, resulting in the accumulation of RNA specific receptor on the surface of the adrenal cortical cell leads to for these enzymes (Figs. 1-5). It is very likely that this elevation of intracellular cAMP levels. Since internalization process is mediated by protein factors that are synthesized as of the ACTH-receptor complex or its translocation to the Downloaded by guest on October 3, 2021 Biochemistry: John et al. Proc. Natl. Acad. Sci. USA 83 (1986) 4719

nucleus is not known to occur and analogs of cAMP mimic 6. Zuber, M. X., Simpson, E. R., Hall, P. F. & Waterman, the actions of ACTH, we assume that it is cAMP, acting M. R. (1985) J. Biol. Chem. 260, 1842-1848. through some undetermined process, that initiates the tran- 7. John, M. E., John, M. C., Ashley, P., MacDonald, R. J., the a Simpson, E. R. & Waterman, M. R. (1984) Proc. Natl. Acad. scription of gene(s) encoding specific protein activa- Sci. USA 81, 5628-5632. tor(s). cAMP has been implicated in the regulation of expres- 8. John, M. E., John, M. C., Simpson, E. R. & Waterman, sion of several eukaryotic genes (22-27). Phosphorylation of M. R. (1985) J. Biol. Chem. 260, 5760-5767. chromatin-associated proteins in prolactin gene expression is 9. Okamura, T., John, M. E., Zuber, M. X., Simpson, E. R. & a case in point (23). Alternatively, with respect to the steroid Waterman, M. R. (1985) Proc. Natl. Acad. Sci. USA 82, hydroxylases, a cAMP-dependent process may activate 5705-5709. translation of a pre-existing RNA. In any case, after trans- 10. Zuber, M. X., John, M. E., Okamura, T., Simpson, E. R. & lation, the protein factor is translocated to the nucleus where Waterman, M. R. (1986) J. Biol. Chem. 261, 2475-2482. it modulates gene expression. While it is not clear how this 11. John, M. E., Okamura, T., Dee, A., Adler, B., John, M. C., process affects gene transcription, formation of a stable White, P. C., Simpson, E. R. & Waterman, M. R. (1986) Biochemistry, in press. transcription complex comprising RNA polymerase II, the 12. Marzluff, W. F. & Huang, R. C. C. (1984) in Transcription labile protein factor(s), and regulatory regions of the steroid and Translation, a Practical Approach, eds. Hamer, B. D. & hydroxylase genes is an attractive possibility. The degree of Higgins, S. J. (IRL, Arlington, VA), pp. 89-128. response of the steroid hydroxylase genes to ACTH or to 13. Lawson, G. M., Tsai, M. J., Tsai, S. Y., Minghetti, P. P., cycloheximide in primary cell cultures is not uniform (Figs. McClure, M. E. & O'Malley, B. W. (1981) in Laboratory 4 and 5). The working hypothesis can accommodate these Methods Manualfor Hormone Action and Molecular Endocri- differences based on differences in protein factors and/or nology, eds. Schrader, W. T. & O'Malley, B. W. (Baylor their binding to regulatory regions of the genes. However, College of Medicine, Houston, TX), pp. 7-1-7-51. involvement of newly synthesized protein factors as media- 14. Maniatis, T. G., Fritsch, E. F. & Sambrook, J. (1982) Molec- ular Cloning: A Laboratory Manual (Cold Spring Harbor tors of the regulation of eukaryotic gene expression by Laboratory, Cold Spring Harbor, NY). peptide hormones or other modulators is not a general 15. Goldman, M. J., Backs, D. W. & Goodridge, A. G. (1985) J. phenomenon (27-30). Biol. Chem. 260, 4404-4408. Thus, we suggest that the labile protein factor(s) synthe- 16. Israel, D. I. & Whitlock, J. P. (1984) J. Biol. Chem. 259, sized as a primary response to ACTH in the adrenal cortex 5400-5402. exerts a secondary response at the transcriptional level on 17. Mayo, R. E. & Palmiter, R. D. (1981) J. Biol. Chem. 256, steroid hydroxylase genes, thereby resulting in the mainte- 2621-2624. nance of optimal steroidogenic capacity. Whether each 18. Boggaram, V., Zuber, M. X. & Waterman, M. R. (1984) Arch. steroid hydroxylase gene requires a specific protein for Biochem. Biophys. 231, 518-523. 19. Jones, P. B. C., Galeazzi, D. R., Fisher, J. M. & Whitlock, expression or one protein can service all members of this set J. P. (1985) Science 227, 1499-1502. of genes is unknown at present. Finally, this study further 20. Gonzalez, F. J. & Nebert, D. W. (1985) Nucleic Acids Res. emphasizes the role of cAMP in the regulation of synthesis of 113, 7269-7288. transcriptional modulators of eukaryotic gene expression. 21. Noguchi, T., Inoue, H. & Tanaka, T. (1985) J. 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