Molecular and Cellular Biochemistry68, 79 85 (1985) © 1985 Martinus Nijhoff Publishers, Boston. Printed in The Netherlands

Glucocorticoids induce a 29 000 M r protein in DDT1 MF-2 smooth muscle cells but not in the DDT1 MF-2 GR glucocorticoid resistant variant James S. Norris 1,2, Lawrence E. Cornett 2, Peter O. Kohler 1, Stewart L. MacLeod 1, Allan J. Syms 3 and Roy G. Smith 3,4

Departments of Medicine 1, Physiology-Biophysics 2, University of Arkansas for Medical Sciences, Little Rock, A R 72205; and Departments of Urology 3 and CelI Biology 4 Baylor College of Medicine, Houston, TX 77030, USA

Abstract We have demonstrated that glucocorticoids induce in D D T 1 MF-2 cells by a glucocorticoid mediated mechanism the synthesis of a methionine-cysteine rich protein of 29 000 M r (p29). Induction of p29 is not observed in D D T 1 M F-2 G R glucocorticoid resistant variants which have only 7% of glucocorticoid receptor site per cell compared to wild type cells. Increased synthesis of p29 is specific to glucocorticoids since neither androgens, estrogens, progesterone nor the glucocorticoid antagonist dexamethasone mesylate are effective inducers. Stimulation of p29 synthesis in wild type cells is observed at 10-10 M triamcinolone acetonide, reaching a m a x i m u m at a concentration of 1 × l0 8 M. The induction of p29 is not a function of glucocorticoid arrest of D D T 1 MF-2 cells since D D T 1 MF-2 cells promoted to re-enter the cell cycle by 50 n g / m l platelet derived growth factor ( P D G F ) continue synthesis of p29. Finally, increased levels of p29 translation products are observed in cell free translation assays carried out utilizing poly A + R N A transcripts isolated from glucocorticoid treated cells. These data suggest that the glucocorticoid stimulation of p29 synthesis is a transcriptional a n d / o r RNA processing event controlled by glucocorticoid receptor complexes.

Introduction The ductus deferens derived smooth muscle cell line, D D T I MF-2, has been documented to encode the proto-oncogene c-sis (1, 2). We demonstrated that the expression of c-sis is closely correlated with progression of D D T l MF-2 cells through the cell cycle and that glucocorticoids which block D D T 1 MF-2 cells in Go/Gl (3 5) also inhibit expression of c-sis poly A + R N A transcripts (l, 2). We believe that c-sis m R N A encodes for a protein, hamster platelet de,rived growth factor, which acts as a mitogen for D D T 1MF-2 cells, and that inhibition of its synthesis by glucocorticoids is, at least in part, responsible for cell cycle blockade of the cells in Go/G 1. Regulation of c-sis gene expression is suggested to be under the control of the glucocorticoid receptor since we are able to demonstrate in the D D T l MF-2 variant cell line that glucocorticoids

do not block c-sis expression (6). DDT1 MF-2 G R cells are glucocorticoid receptor poor, having only 7% of the total glucocorticoid receptor binding sites present in wild type DDTI MF-2 cells (7) and are not growth inhibited by glucocorticoids (6). The above results have led us to the present study in which we demonstrate that a 29,000 M r protein (p29) is glucocorticoid inducible in D D T 1 MF-2 cells but not in D D T 1 MF-2 G R cells, a glucocorticold receptor-poor variant. This paper demonstrates a protein domain in D D T I MF-2 cells upregulated by glucocorticoids at a suggested transcriptional a n d / o r processing level. These data, coupled with the observation that there is no detectable stimulation of p29 synthesis by glucocorticoids in the D D T l MF-2 G R variant supports the hypothesis that p29 regulation is a glucocorticoid receptor mediated event.

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Experimental procedures Materials Tissue culture reagents were obtained from Grand Island Biologicals, Inc., Grand Island, NY. [3sS]-methionine (1 100 Ci/mmole) and [35S]-cysteine (952 Ci/mmole) were purchased from New England Nuclear, Boston, MA. Platelet derived growth factor was obtained from Collaborative Research, Inc., Boston, MA. All other reagents were of the highest purity obtainable.

Cell culture Cells were cultured as previously described (8).

Analysis of p29 induction Cells were grown to 60% confluency in Dulbecco's modified Eagles medium with high glucose, 2% fetal bovine serum and 0.5% penicillin-streptomycin. [35S]-methionine or [35S]-cysteine, 62 #Ci/ml and ethanol vehicle, steroids and 50 ng/ml platelet derived growth factor (where indicated) were added and the culture dishes incubated for 24 hr at 37 °C. Following incorporation, the cells were mechanically harvested, washed 1X in ice cold PBS and lysed in 20 mM Tris, pH 7.4, 1.5 mM E D T A by 2

cycles of freezing and thawing. Extracts were clarified by centrifugation at 100 000 X g, diluted 1 to 1 with 2X SDS sample buffer and electrophoresed on 5-15% acrylamide gels under reducing conditions (10). Samples were loaded on the basis of trichloroacetic acid precipitable counts (1 X 106 cpm/lane, or as indicated).

Cell-free translation 0.75 #g of poly A + RNA isolated as previously described (2) was translated in a micrococcal nuclease-treated rabbit reticulocyte lysate exactly as described by the supplier (Promega Biotec). The reaction was stopped at 30 min and 10 #1 of translation products were analyzed in a 10.5% polyacrylamide gel under reducing conditions (10).

Protein determination Protein was assayed by the method of Bradford (11) with bovine serum albumin as the standard.

Results

Glucocorticoid induction of p29 is demonstrated in Fig. 1. In the wild type D D T 1 MF-2 cells al-

Fig. 1. [35S]-methionine and [35S]-cysteine labeling of a 29 000 D protein. DDTj MF-2 (MF-2) or DDT! MF-2 GR (GR) cells were labeled with 135S]-methionine (Panel A) or [35S]-cysteine (Panel B) for 24 hr with the following additions: Control (lane A); 1 X 10 7 M testosterone (lane B); 1 X 10-7 M triamcinolone acetonide (lane C), and 1 X 10-7 M testosterone with 1 X 10-7 M triamcinolone acetonide (lane D). Cells were harvested as described in 'Methods'.

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though p29 synthesis is observed at a constitutive level, no induction of p29 synthesis in [35S]-methionine (panel A) or [35S]-cysteine (panel B) treated cultures was observed in the presence of ,<0.1% ethanol (control, lane A) or testosterone (lane B). However, inthe presence ofglucocorticoid (lane C), or glucocorticoid plus testosterone (lane D), increased levels of p29 synthesis was observed. Conversely, no induction of p29 synthesis above constitutive levels was seen in D D T 1 MF-2 GR cells, the glucocorticoid resistant variant of the wild type D D T 1 MF-2 cell line. Since D D T 1 MF-2 cells were derived from an androgen-estrogen induced leiomyosarcoma (12) we examined induction of p29 in the presence of various steroids, all at a final concentration of 1 X 10 -7 M. The specificity of p29 induction by glucocorticoids is demonstrated in Fig. 2. Only glueocorticoids (Fig. 2, lanes B, C, E) stimulated p29

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Fig. 3. Dose related induction of p29 synthesis by triamcinolone acetonide and cortisol. D D T I MF-2 cells were labeled with [35S]-methionine for 24 hr in the presence of the indicated concentration of either triamcinolone acetonide (O O) or cortisol (e e). Cytosol was prepared and analyzed by electrophoresis under reducing conditions (10). Autoradiograms were developed and scanned, and the area under each peak was integrated. The data are plotted as the percent of maximal stimulation (maximal was the average of 3 consecutive intervals of the same magnitude, 6, 8, 24 hrs). Control values were subtracted before the calculation was made. (Controls were cells grown 24 hr in the presence of 1 × 10 7 M triamcinolone acetonide and 62 ~Ci/ml [35S]-methionine.)

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Fig. 2. Five to 15% laemmli gel pattern of 35S-methionine labeled DDT~ MF-2 cells in the presence of the following steroids at 1 X 10-7 M: Control (lane A), triamcinolone acetonide (lane B), dexamethasone (lane C), dexamethasone mesylate (lane D), cortisol (lane E), progesterone (lane F), 17-/3-estradiol (lane G), testosterone (lane H), diethylstilbestrol (lane I), and the synthetic androgen R;[881 (lane J). Other conditions as in 'Methods'.

Fig. 4. Time dependence of glucocorticoid stimulation of p29 synthesis. Cells were grown to 60% confluency as described in Fig. 1 and 'Methods'. Triameinolone acetonide at 1 × 10 8 M was added at time 0. Incorporation of [35S]-methlonine was monitored at the indicated 2-hr intervals by scanning the autoradiograph and integrating under the p29 peak. Data are plotted as in Fig. 3 except that control values were not subtracted prior to the calculation.

82 synthesis. N e i t h e r t e s t o s t e r o n e (lane H, J) (a p r o v e n m i t o g e n for D D T 1 M F - 2 cells) (5), estrogens (lanes G, I) n o r p r o g e s t e r o n e (lane F) increased p29 p r o d u c t i o n significantly. The g l u c o c o r t i c o i d a n t a g o nist d e x a m e t h a s o n e mesylate also was w i t h o u t effect (lane D). We were u n a b l e to s t i m u l a t e p29 synthesis in D D T I M F - 2 G R cells u n d e r a n y of the a b o v e t r e a t m e n t p r o t o c o l s ( d a t a not shown). I n d u c t i o n of p29 b y g l u c o c o r t i c o i d s was dose related (Fig. 3). The c a l c u l a t e d EDs0 for s t i m u l a tion of p29 i n d u c t i o n was 1.8 n M for t r i a m c i n o l o n e a c e t o n i d e a n d 13 n M for cortisol. These values a r e

close to p r e v i o u s l y published affinities of t r i a m c i n o l o n e a c e t o n i d e ( K d = 1 . 0 n M ) a n d cortisol (K d = 4.5 n M ) for the g l u c o c o r t i c o i d r e c e p t o r in r e c e p t o r assays (7, 8). W e also m o n i t o r e d i n d u c t i o n of p29 as a function o f time in response to 1 × 10 8 M doses o f t r i a m c i n olone acetonide. These d a t a are p l o t t e d (Fig. 4) as a percent of m a x i m a l i n d u c t i o n which was determ i n e d to have been reached by the 6-8 hr labeling interval, p29 synthesis was first observed by 2 hr a n d reached a m a x i m u m by 4 - 6 hr. • The a b o v e d a t a clearly indicate that glucocorti-

Fig. 5. PDGF does not induce p29 synthesis nor block glucocorticoid induction of p29. DDT l MF-2 cells were grown to 60% confluency. Cells were then treated with ethanol vehicle, glucocorticoids and/or platelet derived growth factor (1 U/ml, Collaborative Research) and allowed to incorporate [35S]-methionine for 24 hr. Cytosols were prepared as described in Fig. 1 and analyzed on 5 15% reducing gels (10). Lane A, control; lane B, PDGF; lane C, 1 × 10-8 M triamcinolone acetonide; lane D, 1 × 10 8 M triamcinolone acetonide and I U/ml PDGE

83 coids induce p29 synthesis. However, we were concerned a b o u t the possibility that induction of p29 was a cell cycle dependent event where p29 concentration was highest in glucocorticoid induced G O/ G 1 blockade (g]ucocorticoids block D D T 1 MF-2 cells in G o / G 1 stage of the cell cycle) (1). This was examined by treatment of D D T 1 M F - 2 cells with 1 × 10-8 M triamcinolone acetonide for 24 hr, a time previously determined to block cell cycling (I). At this time glucocorticoid blocked cells were divided into two groups and 50 n g / m l P D G F was added to one group. Two additional groups, untreated with glucocorticoid, were alsosplit at this time and 50 n g / m l P D G F was added to one group. 35S-methionine (62 t~Ci/ml) was added to all four groups and allowed to incorporate for 24 hr. Cells were harvested and cytosols prepared as described. Under these conditions it is clear that P D G F alone is unable to induce p29 synthesis (Fig. 5, lane B) and, as previously observed in Fig. 1, p29 synthesis was not a p p a r e n t in control cells (lane A). However, in T A treated cells (lane C) and T A plus P D G F treated cells (lane D), p29 was present. Since P D G F under these conditions promotes re-entry of D D T l M F - 2 cells into the cell cycle (1), it is unlikely that p29 is a cell cycle dependent protein induced in G 1. It is apparent in Fig. 1 that the major induced protein in glucocorticoid treated cells labeled by both [35S]-cysteine and [35S]-methionine is p29 and in Fig. 4 it is clear that p29 induction begins within 2 hr of glucocorticoid administration which suggests that the m R N A for p29 m a y be in relative abundance. Therefore, poly A + R N A was isolated f r o m control and steroid treated cells, and analyzed in a cell-free translation assay (Fig. 6). [35S]-methionine labeled translation products f r o m poly A + R N A isolated f r o m cells treated with ethanol (lanes A, E), 1 × 10_8 M testosterone (lane B), 1 × 10-8 M triamcinolone acetonide (lanes C, F), and 1 × 10 8 M triamcinolone acetonide and 1 × 10.8 M testosterone (lane D) were analyzed by gel electrophoresis under reducing conditions (10). As can be seen, p29 induction occurred only in glucocorticoid treated wild type cells (lanes C, D). A very faint band in the D D T 1 MF-2 G R cells at the same molecular weight as p29 is Observed but clearly is not glucocorticoid regulated. By scanning densitometry, 9.9% of the total [35S]-methionine incorporated in lane C is in p29.

Fig. 6. Glucocorticoid stimulation of poly A+ RNA encoding p29 analyzed by cell-free translation. 0.75 ~g of poly A+ RNA isolated as previously described (2) was translated in a micrococcal nuclease-treated rabbit reticulocyte lysate exactly as described by the supplier (Promega Biotec). The reaction was stopped at 30 min. 35S-methionine labeled translation products (10 gl) were analyzed by gel electrophoresis under reducing conditions (10). Poly A+ RNA was isolated (described in ref. 2) from cells treated with ethanol (lanes A, E), 1 × 10 8 M testosterone (lane B), 1 × 10 8 M triamcinolone acetonide (lanes C, F), and 1 × 10 8 M testosterone (lane D).

Discussion

Previously, at least two protein domains were shown to be regulated by glucocorticoids in a hepat o m a cell line (13, 14). We have f o u n d that glucocorticoids also regulate two protein domains in the ductus deferens s m o o t h muscle cell line D D T l M F 2. Specifically, we have shown that expression of a gene (c-sis) encoding the autocrine growth factor, platelet derived growth factor ( P D G F ) is inhibited by glucocorticoids (2). Platelet derived growth factor is a k n o w n mitogen for D D T 1 M F - 2 cells (1). In a separate study we have also examined glucocorticoid regulation of a n d r o g e n receptor synthesis (4). These data demonstrated that androgen receptor synthesis was also inhibited by glucoeorticoids. Since androgens are mitogenic for D D T 1 cells (5),

84 this suggests a second level of growth control may be defined by these results. In this paper we describe a second protein domain where protein synthesis is stimulated by glucocorticoids. Specifically, the synthesis of a protein termed p29 is reproducibly stimulated in glucocorticoid treated cells. This protein has a molecular weight, by several different gel systems, of 29 000 M r and appears to be rich in methionine and cysteine residues. Furthermore, we have demonstrated that glucocorticoids specifically induce this protein (Fig. 2) in D D T 1 MF-2 cells while in the variant D D T 1 MF-2 GR cell line no glucocorticoid regulated induction of p29 is observed although low levels of p29 are constitutively expressed in D D T 1 MF-2 GR cells. Our observations on p29 induction in the wild type and variant cell lines are consistent with earlier observations (6) that there are only 0.9 × 10-10 moles of receptor/100 #g of D D T l MF-2 GR D N A compared to 13.6 × 10-j° moles of receptor/100 ~g of D D T 1 MF-2 DNA. No difference in the dissociation constant of receptor for triamcinolone aceto. nide was observed between the two lines (6). Because p29 is not glucocorticoid-inducible in GR cells phenotypically resistant to the G0/G t block induced by glucocorticoids and further since glucocorticoids blocked D D T 1 MF-2 cells in the Go/G I stage of the cell cycle we were concerned that p29 was accumulating as a function of cell cycle stage. In order to evaluate this question we grew DDTj MF-2 cells in triamcinolone acetonide for 24 hr to block them in Go/G1. Subsequently, p29 induction was measured in cells treated with 50 ng/ ml platelet derived growth factor, a dose which causes re-entry of D D T 1 MF-2 cells into the cell cycle (1). Under these conditions, no apparent decrease in the labeling of p29 is seen when analyzed 18 hr later. Furthermore, p29 is not induced by P D G F alone. Thus, p29 induction is a specific event mediated by glucocorticoids and its induction appears to be unrelated to the observed Go/G 1 block although further studies are underway to confirm this conclusion. Finally, we chose to examine the cellular site of glucocorticoid regulation of p29 induction by preparing poly A + R N A transcripts and measuring the apparent p29 encoding m R N A levels using a nuclease treated reticulocyte translation system. These results clearly indicated that a m R N A species tenta-

tively identified as encoding p29 was elevated in glucocorticoid treated cells. Consequently, transcriptional a n d / o r processing events seem to be under receptor-steroid control in D D T t cells. However, the data at present does not allow us to discriminate between stimulation of either transcription or R N A processing as the primary glucocorticold regulated event. This assumes some importance in view of early results of Firestone et al. (17) and the recent results on al-acid glycoprotein (AGP) regulation by glucocorticoids in rat hepatoma cells (18) where a processing event was identified as the step controlling the cytoplasmic appearance of functional AGP mRNA. Since our studies are currently directed at isolating the p29 genes, we hope to eventually answer similar questions. In summary, we have described a protein whose synthesis is stimulated by glucocorticoids. Our evidence strongly supports the role of glucocorticoid receptor in mediating this event. These data coupled with our previous observation that glucocorticoids mediate inhibition of the expression of two genes involved in growth regulation of D D T l MF-2 cancer cells (2, 16) suggest that glucocorticoid receptors have complex interactions with target cells affecting both stimulation and inhibition of different populations of genes within a similar time frame.

Acknowledgments We acknowledge the secretarial assistance of Mrs. Margaret Morrison. This research was supported by the National Institutes of Health GM 30669 and the National Science Foundation ISP 8011447. References I. Syms AJ, Norris JS, Smith RG: Autocrine regulation of growth. I: Glucocorticoidinhibition is overcomeby exogenous platelet derived growth factor. Biochem Biophys Res Commun 122:68-74, 1984. 2. Norris JS, Cornett LE, Hardin JW, Kohler PO, MacLeod SC, Srivastava A, SymsJS, Smith RG: Autocrineregulation of growth. II: Glueocorticoidsinhibit transcription of c-sis proto-oncogenespecificRNA transcripts. BiochemBiophys Res Commun 122:124 128, 1984. 3. Smith RG, Syms A J, Norris JS: Differential effects of androgens and glucocorticoidson regulation of cell growthand androgen receptor concentration. J Steroid Biochem 20: 277 281, 1984.

85 4. Syms A J, Norris JS, Smith RG: Androgen stimulated elevation in androgen receptor levels is inhibited by the synthetic glucocorticoid triamcinolone acetonide. Biochem Biophys Res Commun 116:1020 1025, 1983. 5. Syms A J, Norris JS, Smith RG: Proliferation of a highly androgen-sensitivecloned cell line (DDT] MF-2) is regulated by glucocorticoids and modulated by growth on collagen. In Vitro 19:929-936, 1983. 6. Syms AJ, Norris JS, Smith RG: Oncogene expression and growth in the transplantable glueocorticoid-sensitive(DDT I MF-2) and resistant (DDT1 MF-2 GR) ductus deferens tumor cell lines in vitro and in vivo (submitted 1984). 7. Norris JS, Kohler PO: Partial purification of the glucocorticoid receptor from DDTr cells and characteristics of its interaction with DNA. J Biol Chem 258:2350-2356, 1983. 8. Norris JS, Kohler PO: The coexistance of androgen and glucocorticoid receptors in the DDT I cloned cell line. Endocrinology 100:613 618, 1977. 9. Norris JS, Bowden C, Kohler PO: Description of a hamster ventral prostate cell line containing androgen receptors. In Vitro 13:I08-114, 1977. 10. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970. 11. Bradford MM: A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248 254, 1973.

12. Norris JS: Tumors of the accessory sex glands of male Syrian hamsters and hormone action. In: Spring-Mills E, Hafez ESE (eds), Male Accessory Sex Glands, Elsevier/North Holland Biomedical Press, Amsterdam, pp. 609 616, 1980. 13. Ivarie RD, O'Farrell PH: The glucocorticoid domain: Steroid mediated changes in the rate of synthesis of hepatoma proteins. Cell 13:4I 55, 1978. 14. Phelps DS, Litwack G: An electrophoretic characterization of the glucocorticoid response of the Fu5-5 rat hepatoma cell line. Eur J Biochem 126:407-415, 1982. 15. Sporn MB, Todaro GJ: Autocrine secretion and malignant transformation of cells. N Engl J Med 303:878-880, 1980. 16. Syms AJ, Norris JS, Panko WB, Smith RG: Mechanism of androgen receptor augmentation: Analysis of receptor synthesis and degradation by the density-shift technique. J Biol Chem (in press). 17. Firestone GL, Payvar F, Yamamoto KR: Glucocorticoid regulation of protein processing and compartmentalization. Nature 300:221-225, 1982. 18. Vannice JL, Taylor JM, Ringold GM: Glucocorticoid-mediated induction of a j-acid glycoprotein: Evidence for hormone-regulated RNA processing. Proc Natl Acad Sci USA 81:4241-4245, 1984.

Received in revised form 23 May I985.