Post-transcriptional Regulation Factor-#I1 Gene* of the Human ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 267, No. 19, Issue of July 5, pp. 13702-13707, 1992 Printed in U.S.A.

Post-transcriptional Regulationof the Human Transforming Growth Factor-#I1 Gene* (Received for publication, November 1, 1991)

Seone-Jin Kimk. KeunchilPark. David KoellergV, Kyung YoungKim)),Lalage M. Wakefield, Michiel B. Sporn, and Anita B. Roberts’ From the Laboratory of Chemoprevention, National Cancer Institute and the §Cell Biologyand Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892

Since many lines of evidence suggest that expression of the transforming growth factor-81 (TGF-81) gene may be regulated post-transcriptionally, we examined the effect of the 6’-untranslated region (UTR) of this gene on TGF-j31 expression. For this purpose, fragments of the 840-nucleotide highly GC-rich TGF-81 5’-UTR were inserted into the 5’-UTR of the structural gene for human growth hormone driven by the simian virus 40 early promoter. A portion of the 5‘UTR of TGF-81 mRNA spanning the sequences from +11 to +147 was shown to inhibit growth hormone expression by as much as 22-fold.This effect was cellspecific; growth hormone production was inhibited in PC-3 human prostate adenocarcinoma and A-549 human lung adenocarcinoma cells, while no effect was seen in rat pheochromocytomaPC12 cells,which show efficient translation of endogenous TGF-81 mRNA. Computer analysis showed that this region of the 5‘UTR contained a stable secondary stem-loop structure spanning sequences +49 to +76. This stem-loop region alone is sufficient to inhibit expression of the growth hormone gene, suggesting that it plays an important role in post-transcriptional regulation of TGF-81 gene expression.

Several studiessuggest that post-transcriptionalregulation of TGF-P1 is another important control point in regulating the activity of this growth factor. Thus, Assoian et al. (1987) reported that TGF-P1 mRNA expressed is at similar levels in unstimulated monocytes and in monocytes activated to become macrophages; however, TGF-P1 protein is secretedonly by activated macrophages, suggesting that expression is controlled at the level of translation. In addition, activation of both B and T lymphocytes results in a significant increase in TGF-P1 mRNA expression as early as 2 h following stimulation, whereas the rate of secretion of TGF-Pl gradually increased over a period of 4 days, again suggesting post-transcriptional regulation (Kehrl et al., 1986). Recently, it has been reported that PC-3 human prostate adenocarcinoma cells secrete predominantly TGF-P2 butrelatively little TGF-PI protein, whereas the TGF-P1 mRNA level is higher than that of TGF-P2 (Ikeda et al., 1987). On the other hand, PC12 rat pheochromocytoma cells constitutively secrete thehigh levels of TGF-P1 even though the TGFP l mRNA level is relatively low compared with thatin other cell lines such as PC-3cells.’ TGF-Pl secretion is also stimulated incellular the response to steroids and related molecules. TGF-P1 transcription was increased after treatment of keratinocytes with retinoic acid, but there was no accompanying increase in secretion of the TGF-P1 peptide (Glick et al., 1989). In contrast, secretion of Transforming growth factor-pl (TGF-Bl)l is a multifunc- the TGF-Bl peptide was increased in response to treatment tional regulator of cellular activity (Roberts and Sporn, 1990; of fibroblasts with the progestin gestodene with little or no et al., 1990). These Massague, 1987) belonging to a larger family of polypeptides increaseinTGF-P1mRNA(Colletta which regulate cell growth, cell differentiation, andcell func- findings again suggest that TGF-P1 expression may be regution. Expression of TGF-P1 is tightlyregulated during devel- lated post-transcriptionally. The TGF-P1 mRNA contains an unusually long 5”untransopment (Heineet al., 1987) and following viral transformation of cells (Anzano et al., 1985; Jakowlew et al., 1988; Kim et al., lated sequence that is highly rich in GC content (Derynck et 1990a; Birchenall-Roberts et al., 1990; Geiser et al., 1991), al., 1985; Kim et al., 1989). Thus, as a first step to understand treatment with phorbol ester (Akhurst et al., 1988), or treat- the translational control of TGF-P1 gene expression, we exregion of TGF-P1 mRNA ment of cells with TGF-P1 itself (Van Obberghen-Schilling et amined whether the 5”untranslated al., 1988; Kim et al., 1990b). Following the recent cloning of plays a role in regulating expression of this importantpeptide. et al., 1989), several of the target the TGF-Pl promoter (Kim MATERIALS AND METHODS sequencesregulating transcription of the gene havebeen identified and characterized (Kim et al., 1990b). Cell Culture-PC-3 human prostate adenocarcinoma cells (Kaighn

* This research was supported in part by a grant from Schering AG. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 8 To whom correspondence should be addressed. ll Present address: Dept. of Medicine and Medical Genetics, University of Washington, Seattle, WA 98195. 11 Supported by grants from Johnson & Johnson. The abbreviations used are: TGF-j3, transforming growth factor8; UTR, untranslated region (equivalent to UTS, untranslated sequence); DMEM, Dulbecco’s modified Eagle’s medium.

et al., 1979) were grown in Dulbecco’s modified Eagle’s medium (DMEM) and Ham’sF-12 medium (1:l) supplemented with 10% fetal bovine serum. Human lung adenocarcinoma (A-549) cells were maintained in DMEMwith high glucose supplemented with 5%fetal bovine serum. Rat pheochromocytoma (PC12) cells were grown in DMEM containing 7% horse serum and 7% fetal bovine serum on rat tail collagen and polylysine-coated tissue culture plates. Construction of Plasmids-The plasmid pSVGH used here hasbeen described elsewhere (Casey et al., 1988). The plasmids TR1-1, TRl2, TR1-3, and TR1-4 were created by insertion of fragments of the

13702

S.-J. Kim, manuscript in preparation.

Post-transcriptional Regulation of the TGF-Dl Gene

13703

5'-untranslated sequence of the human TGF-81 gene generated by polymerase chain reaction amplification, using oligonucleotides designed to generate ends with the Hind111 and XbaI restriction sites, into the pSVGH vector cut with the same enzymes. The plasmid TR1-1L was constructed by inserting the double-stranded oligonucleotides corresponding to the region of +49 to +77 of the human 4 P1 4 P2 TGF-Dl B'-UTR. The plasmids phTG5-1 and phTG5-2 were constructed by digesting phTGl2 (Kim et al., 1989) with HincIIISstII or +432 phTG5-1 HincII/AuaI, respectively, filling in by Klenow fragment, and blunt +453 I +453 I 1 +95 phTG5-2 ligating with T4 DNA ligase. 1 - 1 +11 phTG5 Transfections-Cell lines were transfected by the calcium phos- +453 phate coprecipitation method using 10 pg of the appropriate plasmids purified by banding inCsCl. Transfections into A-549 and PC-3cells were performed by the calcium phosphate precipitation method, but PC12 cells were transfected by electroporation (Bio-Rad). Transfection frequencies were monitored by cotransfection with 1 pgof Ac-Cm 0 pSVGH, a growth hormone expression vector, or 3 pgof pCHllO (Pharmacia LKB Biotechnology Inc.), a B-galactosidase expression vector. Cells were harvested 48 h after addition of DNA, and extracts were assayed for chloramphenicol acetyltransferase activityaccording to themethod of Gorman et al. (1982). Growth hormone production was measured by radioimmunoassay using a growth hormone assay kit (Nichols Institute Diagnostics, San Juan Capistrano, CA).All transfections were repeated a t least three times. Analysis of TGF-Dl Expression-Total cellular RNA was isolated by the method of Chirgwin et al. (1979). For Northern blot analysis, equal amounts of RNA (10 pg) were electrophoresed on 1% agarose gels containing 0.66 M formaldehyde and transferred to Nytran. Blots were hybridized with '"P-labeled probes to a nick-translated cDNA for TGF-01 by the method of Church and Gilbert (1984). Production of TGF-@Z-TGF-@l in the collected conditioned medium was quantitated by a sandwich enzyme-linked immunosorbent 0 assay using antisera specific for TCF-01 (Danielpour et al., 1989). W 0 SI Nuclease Protection Analysis-For the probe, two oligonucleotides were synthesized (3'-oligonucleotide, 5'-CACATTGTGCCCAAAGGCATTTTAGGG-3' (Casey et al., 1988); 5'-oligonucleotide, 5'CCGCATATGGTGCACTCTCAGTAC-3' (Gorman et al., 1982)). Using these oligonucleotides, the fragment for S1 (the probe) was amplified according to the standard protocol for the GeneAmp Kit FIG. 1. Influence of the 5'-UTR on the activity of human (Perkin-ElmerCetus Instruments). This fragment was then endlabeled with "P and cut with NdeI. The resulting 32P-labeled 800- TGF-Bl promoter-chloramphenicolacetyltransferasechibase pair fragment was gel-purified, and 5 X lo6cpm of the fragment meric constructs. A-549 cells were transientlytransfected with was coprecipitated with 100 pg of total RNA from PC-3 cells trans- DNA from the TGF-Dl promoter-chloramphenicol acetyltransferase fected with pSVGH or TR1-1. The precipitate was dissolved in 40 pl constructs. The DNA fragment from the promoter region of the human TGF-Dlgene was used to constructplasmids phTG5, phTG5of hybridization buffer (80% formamide, 20 mM Tris, pH 7.4, 0.4 M NaCl, 1 mM EDTA),heated to 75 "C for 15 min, and incubated 1, and phTG5-2, represented on the upper panel by a solid bar. The overnight at 48 "C. The unhybridized fragment was digested by S1 blacksolid bar shows the 5"untranslated sequence (UTS) of the nuclease, and the protected fragments were recovered by ethanol TGF-Dl mRNA. Arrows (PZand P2) denote the positions of the two precipitation and then denatured and analyzed on a 4% sequencing major transcriptional start sites. Transfection frequencies were monitored by cotransfection with 1 pg of pSVGH, a growth hormone gel. expression vector. Growth hormone production was measured by radioimmunoassay using a growth hormone assay kit (Nichols InstiRESULTS tute Diagnostics).

-

-

Effectsof the 5'-Untranslated Sequence of the TGF-01 Gene on ChloramphenicolAcetyltransferase Activity Driven by the TGF-PI Promoter-To examine the influence of its 5'-UTR on chloramphenicol acetyltransferase expression driven by the human TGF-B1 promoter, we constructed several plasmids in which regions of the human TGF-01 5'-UTR and promoter were inserted into the pGEM4-SVOCAT vector; these constructs were then transfected into PC-3 cells. Previously, we reported that the plasmid phTG5 (-453/+11) showed the highest basal promoter activity. In contrast, the plasmids phTG5-1 and phTG5-2, which contain the additional 5'-UTR sequencesbetween +11and +432, and between +11 and +95, respectively, showed low basal chloramphenicol acetyltransferase activities compared with phTG5 (Fig. 1). Deletion of the sequences from position +95 to +11 (original phTG5) in the 5'-UTR resulted in a 10-fold increase in expression of chloramphenicol acetyltransferase activity. These results suggested that the 5'-UTR of the human TGF-P1 genenegatively influences the expression of the chloramphenicol acetyltransferase gene drivenbythe TGF-B1 promoter.

7-

LA

Effects of the B'-UTR of the TGF-81 Gene on Growth Hormone Production Driven by the SV40 Promoter-We have also taken anotherapproach to thequestion of putative translational inhibition of TGF-Bl expression. To demonstrate the sufficiency of the region between +11and +95 to confer posttranscriptional inhibition, we inserted fragments containing various portions of the human TGF-Pl 5'-UTR into the 5'UTR of the structural gene for human growth hormone (Fig. 2). Computer-aided analysis of the region of the human TGF81 mRNA corresponding to bases +1to +840 of the TGF-P1 cDNA identified two putative sequences capable of forming stem-loop structures, one from +49 to +77 (see Fig. 8) and another from +750 to +786. Based on this analysis, we constructed a set of specific deletional mutants of the 5'-UTR of the TGF-B1 gene. For plasmids TR1-1 and TR1-2, the 5'oligonucleotide used in amplifications was synthesized corresponding to the region +11 to +31 of the TGF-P1 cDNA in the region immediately downstream from the transcription start site. The 3'-oligonucleotides used corresponded to the

13704

Post-transcriptional RegulationTGF-@I of the

Gene B. TGF-01

A.

r u. TGF-01 (ng/lOe Cells)

1

+6057+701 0

+E10 TRI-3 +810 TRI-4

0

A-549

6.13 2 0.1 (1)

PC3 2.90

f10.47) 0.1

PC12

6.93 2 0.1 (1.13)

region +126 to +146 for TR1-1 and theregion +801 to +820 for TR1-2. For plasmid TR1-4, the 5’-oligonucleotide corresponded to the region +711 to +733, and the 3’-oligonucleotide corresponded to the region +801 to +820. The plasmid TR1-3 was constructed by digesting the TR1-2 with NaeI and PflMI, and blunt ligating after filling by the Klenow fragment. First, we examined the accumulated levels of TGF-B1 mRNA and secretion of TGF-Bl in A-549, PC-3, and PC12 cells. Amongthese threecell lines, the level of TGF-B1 mRNA was the highest in A-549 cells and the lowest in PC12 cells. However, the level of peptide secreted into conditioned medium from PC12cells was higher than thatof either A-549 or PC-3 cells. Because these results suggest that theexpression of TGF-B1is post-transcriptionally inhibited in PC-3and A549 cells (Fig. 3), we transfected them with the TGF-B1 5’UTR-SVGH chimeric deletion mutants described above and assayed for growth hormone production (Fig. 4). The plasmid TRl-1, which contained the sequences from +11 to +147 of the B’-UTR of TGF-P1 mRNA, produced a 22-fold lower level of growth hormone than that of pSVGH, which lacked the 5’-UTR. The plasmid TR1-2, which contained almost the entire 5’-UTR, and theplasmid TR1-3, which had an internal deletion of +91 to +605 compared with TR1-2, produced an inhibition of translation similar to thatof TR1-1. In contrast, the plasmid TR1-4, which contained only the 3’-end of the 5’-UTR of the TGF-Bl mRNA from +701 to +810, had no effect on translation even though the computer analysis showed stable stem-loop formation in this region. These findings again demonstrated that the5’-UTR of human TGF-B1 mRNA contained the sequences responsible for the inhibition of translation. A time course experiment showed that the secretion of growth hormone into the conditioned medium of cells transfected with pSVGH increases steadily from time 0 to 45 h in the period studied. In contrast, incells transfected with TR11, secreted levels of growth hormone remained unchanged

6k 50

g

40

k at

30

4

t5 ?r a m 5

n

20

10

0

A-549

hGH

FIG. 2. Structure of TGF-@15’-UTRpSVGH chimeric plasmids. The DNA fragments generated by polymerase chain reaction using phTGl2 (Kim et al., 1989) as a templatewerecloned into HindIII/XbaI sites of pSVGH (SVGH), a SV40 promoter-derived growth hormoneexpressionvector. P2 denotes one of the major transcriptional start sites.

5

PC3

PC12

FIG. 3. Accumulated levels of TGF-Bl mRNA and its peptide in A-549, PC-3, and PC12 cells. A, secretion of TGF-@Iin A-549,PC-3,andPC12 cells. Subconfluentcellsweregrown in complete medium for 24 h, washed with serum-free medium twice, andcultured in serum-freemediumcontaining100 pg/ml bovine serum albumin. After 24 h, conditioned medium was collected, and the level of TGF-@lwas measured using a sandwich enzyme-linked immunosorbent assay (Danielpour et al., 1989). The data expressed with A-549 cells are shown in parenrelative to the activity obtained theses. B, steady state levels of TGF-@lmRNA in A-549, PC-3, and PC12cells. Total RNAs wereisolatedfrom the A-549,PC-3,and PC12 cells; separated by electrophoresis(10 pg); and hybridized to a radiolabeled TGF-@lprobe. C, quantitativeanalysis of levels of mRNA for TGF-@lby densitometric scanningof the autoradiogram in B.

FIG. 4. Effects of the5’-UTR of the human TGF-81 mRNA on the expression of the human growth hormone (hGH) gene. TheTGF-@I 5’-UTRpSVGH (SVGH) chimeric constructs were transfected into PC-3 cells, and the transient expression of growth hormone in the medium was assessed as described in the manufacturer’s instructions (Nichols Institute Diagnostics). Datashown are representative of at least three independent transfectionswith these constructs. Transfection frequencies were monitored by cotransfection with 3 gg of a pCHllO (Pharmacia LKB Biotechnology Inc.), a @-galactosidase expression vector. throughout the entiretime course and were 30-fold lower than that of pSVGH-transfected cells 44 h after the transfection (Fig. 5A). To demonstrate that the mRNA transcribed from the plasmid TR1-1 contained the expected 5’-UTR insertion, total RNA was isolated from cells transfected with pSVGH or TR1-1 and examined in an S1 nuclease assay (Fig. 5B). One major S1 nuclease-resistant fragment was evident after hybridization of each of the RNAs with the 32P-labeledprobe described under “Materials and Methods” (Fig. 5B). The size

Post-transcriptional Regulation of the TGF-PI Gene

13705

of the S1-protected fragment from the RNA extracted from the cells that transfected with TR1-1 was approximately 142 nucleotides larger than thatof pSVGH-transfected cells, suggesting that growth hormone mRNAfrom the cells that transfected with TR1-1 contains the appropriate sequence of the TGF-P1 5’-UTR. Importantly, the intensity of the protected band of TR1-transfected cells was &fold greater than that of pSVGH, suggestingthat sequences +11to +147 of the TGF-B1 5’-UTR contribute to thestability of mRNA as well as to theinhibition of translation. When the level of growth hormone secreted in TR1-1 transfected cells was normalized to the level of its mRNA, the secretion of growth hormone from TR1-1 wasshown to be approximately 90-100-fold lower 0 1 0 2 0 3 0 4 0 5 0 than thatof pSVGH, whichlacked the 5’-UTR. To examine whether the 5’-UTR of TGF-Bl mRNA confers the translational inhibition in a cell-specific manner, we transfected the pSVGH, TR1-1, or TR1-4 plasmids into AB 549, PC-3, or PC12 cells and assayed for the production of 1 2 3 growth hormone. As shown in Fig. 6, PC-3 and A-549 cells transfected with the plasmid TR1-1, which contains the 130Probe (-800 n . t P base pair GC-rich sequence located immediately downstream 622of the TGF-Dl transcription start site, produced approximately 20- and 4.5-fold lower levels, respectively, of growth 527 hormone. However, transfection of PC12 cells with this same construct led to no significant difference in the growth hormone level compared with the plasmid pSVGH lacking the 5’-UTR, whereas the plasmid TR1-4 enhanced the produc427 tion of growth hormone slightly compared with pSVGH. Moreover, the relative degree of translational inhibition in these cell types correlated with the ratio of secreted TGF-Dl 309to TGF-Dl mRNA levels in these same cells (See Fig.3). These results are consistent with the conclusion that the 5’UTR contains a cis-acting translational modulator. It is pos242 +TRl-1 ProtectedFragment (242 n.t.1 217sible that this element interacts with a trans-acting RNA200binding protein that is lacking or inactive in PC12 cells. 160147 The Sequenceand Proposed Secondary Structure of the 123Post-transcriptionally Inhibitory Element of the Human TGF110cSVGH ProtectedFragment D l mRNA-Computer-aided analysis of the sequence of the (100 n.t.) 90human TGF-Bl mRNA corresponding to bases 1-839 revealed 76 that this sequence has the potential to form a number of stem-loop structures. Of particular note are the sequences from +49 to +77 shown in Fig.7. This 5’-UTR sequence element, which is contained entirely within the post-transcriptionally inhibitory TR1-1 construct, is capable of folding to form the stable stem-loop structure shown in Fig. 7. ComTGF-01 parison of the sequence of this stem-loop of the human TGF5 U T R GH t 7, Probe (-EO0 nucleotides) Pl5’-UTR with those of the porcine and murine TGF-Pl5’t i: TR1-1 mRNA UTR sequences demonstrates that this sequence element is TR1-1 Protected Fragment (242 n.t.) well conserved in these species. The sequences from +49 to SVGH mRNA +77 were sufficient to confer the inhibitory effect on growth + SVGH Protected Fragment ( 1 0 0 n.t.) FIG. 5. Influence of the 5’-UTR of TGF-01 mRNA on the hormone production post-transcriptionally (Fig. 8), suggestexpression of SV40 growth hormone gene. A , time course of the ing that itmay play an important role in the post-transcripeffect of the 5’-UTR on the expression of the SV40 growth hormone tional regulation of the TGF-P1 gene. I

gene. The plasmids, pSVGH (SVGH) and TR1-1, were transfected into PC-3 cells, and the transient expression of human growth hormone (hGH) in the medium was assessed. Data shown representthe average ofthreeindependent transfections with these constructs. Growthhormoneproductionwasnormalizedfor transfection efficiency by cotransfection of3 pg of a @-galactosidaseexpression vector (pCH110). RIA, radioimmunoassay. B, S1 nuclease mapping of the SVGH or TR1-1 start sites. 48 h after transfection of 10pg of SVGH or TR1-1, total RNA was isolated from the transfectants. The transcriptional start sites of SVGH and TR1-1 were determined by S1 nuclease mapping using an end-labeled polymerase chain reaction-3 amplified fragment as described under“Materialsand Methods.”The SVGH transfectant RNAprotectedpredominantly a fragmentof

DISCUSSION

Long and/or GC-rich 5’-UTRs have been observedfor several genes that are involved in regulation of cellular proliferation (Ratner et al., 1987; Rao et al., 1988; Kozak, 1989; Klausner and Harford, 1989). TGF-01, TGF-P2, and TGF-P3 have profound effects on cellular proliferation and share the property of having unusually long 5’-UTRs (Kim et al., 1989; about 100 base pairs whereas the TR1-1 transfectant protected a fragment of about 242 basepairs. n.t., nucleotides.

13706

Post-transcriptional Regulationof the TGF-01 Gene A-549

PC3

24 22 20

-

24 22

PC12

iI

18 -

-

E 1 Is)

c

1

ATG

P2

v/////,//7//,/,5 F". " _" "- "_"urs/////////A +11

16 -

"_

t +49

3

+147

7

I +77

TRI-1 TRI 1L

1412

-

SVGH TRI-1 T R I 4

SVGH TRI-1 TR14

SVGH TRI-1 TRI4

FIG. 6. Influence of the 5'-UTR of the TGF-j31 mRNA on the expression of growth hormone gene in A-549,PC-3, and PC12 cells. The plasmids, pSVGH (SVGH), TR1-1, and TR1-4, were transfected into A-549, PC-3, and PC12 cells, and the transient expression of human growth hormone (hGH) in the medium was assessed as described above. PC12 cells were transfected by electroporation (Bio-Rad). Results represent the average of three independFIG. 8. The region of +49 to +87 is sufficient to inhibit the ent experiments. Transfection efficiency was monitored by cotransgrowth hormone production. The oligonucleotides corresponding fection of 3 pg of a (3-galactosidaseexpression vector (pCH110). to the region of +49 to +77 were synthesized, annealed, and inserted into the HindIIIIXbaI site of pSVGH (SVGH). The resulting conU C struct, TR1-lL, was transfected into PC-3 cells, andthe growth G C hormone in the medium was assayed after 48 h. Data shown are C-G C-G representative of at least three independent transfections with these C-G C-G constructs. hgh, human growth hormone; RIA, radioimmunoassay. G-C A

C A

G -C

growth hormone production in PC-3 andA-549 cells, respectively, whereas this same sequence failed to inhibit growth hormone production in PC12 cells. These results correlated G-C+n well with observed ratios of TGF-61 mRNA levels compared FIG. 7. The sequence and proposed secondary structure of cells. the post-transcriptionally inhibitory element of the human with levels of the secreted peptide in these Recently, we have generated the stable transfectants of TGF-Bl mRNA. The nucleotide sequences of the stem-loop element of the human (Derynck et al., 1985), murine (Geiser e t al., 1991), and TR1-1 or pSVGH plasmid in C2CIz myoblasts. By treating porcine(Kondaiah et al., 1988) mRNAs are aligned for maximal the stable transfectants with actinomycin D, we demonstrated homology. Matched nucleotides are underlined. Nucleotide not pres- that the TR1-1 region of the TGF-Dl 5'-UTR increases the ent in murine TGF-Dl is shown as a black dot. The proposed secondary message stability much as we have shown in the transient structure of this stem-loop element is shown at the right. transfections (Fig. 5B). The presence of the 5'-UTR of the TGF-Pl mRNA in the TR1-1 stable transfectants reduced Lafyatis et al., 1990; Noma et al., 1991), all of which appear the growth hormone production by 50-f0ld.~ These results t o play a role in post-transcriptional regulation3 (Arricket al., suggest that in both of these cases the reduction of growth 1991). TGF-P1 mRNA has an 840-nucleotide-long 5'-UTR hormone production in the TR1-1 plasmid is not caused by and is highly GC-rich. The role of such sequences in the the increase of the message degradation. modulation of TGF-P1 gene expression has yet to be inComputer analysis identified two highly stable stem-loop vestigated. structuresinthe5'-UTR of thehumanTGF-P1 mRNA The studiesdescribed here were based on the accumulating (sequencesbetween+49 and +77 and between +750 and evidence that expression of the TGF-Dlgene maybe regulated +786). Intramolecular duplex structures that are positioned post-transcriptionally (Kehrl et al., 1986; Assoian et al., 1987; close to the 5'-end of an mRNA have been shown to inhibit Glick et al., 1989; Colletta et al., 1990) andthe need to the initiation of translation of several eukaryotic genes (Kounderstand the mechanisms that result either in increased zak, 1989; Klausner and Harford, 1989), presumably by preTGF-(31 secretion with no corresponding change in mRNA venting binding of a 40 S ribosomal subunit (Kozak, 1986). levels or in increased TGF-Pl mRNA levels with no correHowever, these same studies also showed that a stem-loop sponding increase in TGF-Pl secretion. We have identified a structure did not inhibit growth the hormone productionwhen new regulatory control affecting TGF-P1 expression and have it was located further downstream from the mRNA start site. demonstrated that the 5'-UTR of the human TGF-P1 mRNAOur findings thattheGC-rich region immediatelydownexerts a strong inhibitory effect on growth hormone produc- stream from the transcriptional start site of the TGF-P1gene tion. This inhibition is specific for certain cell types. Thus, inhibited the growth hormone production, but that another insertion of the 5'-UTR sequence that islocated immediately GC-rich region furtherdownstream failed toinhibitthe downstream of the TGF-P1 transcription start site into the growth hormoneproduction,supportthese observations. sequences between the SV40 promoter and the growth hor- However, since the second stem-loop had no effect on the mone coding region resulted in a 22- and 4-fold inhibition in growth hormone production when moved close to the start G -C

G-C G-C G-C

+N

,' K. Park, manuscript

in preparation.

D. Romeo, manuscript in preparation.

Post-transcriptional Regulation site, theinhibition of growth hormone production by the first stem-loop may be very specific. Moreover, it is also likely that the proposed secondary structures present in the 5'-UTR of the TGF-PlmRNA may serve as binding sitesfor cytoplasmic factors that arecell type-specific. Recently, other examples of regulation by secondary structures in eukaryotic mRNAs have been reported. Translation of ferritin mRNA seems to be modulated by a stem-loop structure called an iron-responsive element (Hentze et al., 1987). The presence of an iron-responsive element in the 5'UTR of ferritin mRNA decreases its in vitro translation by serving as a translational inhibitor (Caughman et al., 1988; Dickey et al., 1988; Walden et al., 1989). The iron-responsive element-binding protein has been purified and characterized though the molecular details of its binding remain to be determined (Rouault et al., 1989). Preliminary data suggest that cytoplasmic proteins of PC3 cells bind to sequences between +11 and +147 of the 5'UTR of TGF-Pl mRNA. While PC12 cytoplasmic extracts also contain proteinsthat interactwith these sequences, they are different from those of PC-3 cells? It is likely that binding of these cellular factors regulates translation of the TGF-P1 mRNA negatively and/or positively. Further studies are needed to understand the molecular details of the mechanisms by which such binding proteins might altertheTGF-Pl expression post-transcriptionally. Acknowledgments-We

thank L. Mullen, R. Holland, and J. Cubert

for oligonucleotides. W e a r e g r a t e f u l to Drs. J. Harford, T. Rouault, D. Romeo, and R. Klausner for helpful discussions. REFERENCES Akhurst, R. J., Fee, F., and Balmain, A. (1988) Nature 3 3 1 , 363-365 Anzano, M. A,, Roberts, A. B., De Larco, J. E., Wakefield, L. M., Assoian, R. K., Roche, N. S., Smlth, J. M., Lazarus, J. E., and Sporn, M. B. (1985) Mol. Cell. Bid. 5 , 242-247 Arrick, B. A., Lee, A. L., Grendell, R. L., and Derynck, R. (1991) Mol. Cell. Bid. 11,4306-4313 Assoian, R. K., Fleurdelys, B.E., Stevenson, H. C., Miller, P. J., Madtes D. K., Raines, E. W., Ross, R., and Sporn, M. B. (1987) Proc. Natl. Acad. kci. U. S. A. 84,6020-6024 Birchenall-Roberts, M. C., Ruscetti, R. W., Kasper, J., Lee, H.-D., Friedman, R:, Geiser, A., Sporn, M. B., Roberts, A. B., and Kim, S.-J. (1990) Mol. Cell. Brol. 10,4978-4983 Casey, J. L., Hentze, M. W., Koeller, D. M., Caughman, S. W., Rouault, T. A,, Klausner, R. D., and Harford, J. B. (1988) Scrence 240,924-928 Caughman, S. W., Hentze, M. W., Rouault, T. A., Harford, J. B., and Klausner, R. D. (1988) J. Biol. Chem. 2 6 3 , 19048-19052

TGF-PI Gene of the

13707

Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 18,5294-5299 Church, G., and Gilbert, W. (1984) Proc. Natl. Acad. Sci. U. S. A. 8 1 , 19911995 Colletta, A.A., Wakefield, L. M., Howell, F. V., Van Roozendaal, K. E. P., Danielpour, D., Ebbs, S. R., Sporn, M. B., and Baum,M. (1990) Br. J. Cancer 62,405-409 Danielpour, D., Kim, K. Y., Dart, L. L., Watanabe, S., Roberts, A. B., and Sporn, M. B. (1989) Growth Factors 2 , 61-71 Derynck, R., Jarret, J. A., Chen, E. Y., Eaton, D. H., Bell, J. R., Assoian, R. K.! Roberts, A. B., Sporn, M. B., and Goeddel, D. V. (1985) Nature 3 1 6 , IUI-IUS

Dickey, L. F., Wang, Y.-H., Shull, G. E., Wortman, I. A., 111, and Theil, E. C. (1988) J. Biol. Chem. 2 6 3 , 3071-3074 Geiser, A. G., Kim, S.-J., Roberts, A. B., and Sporn, M. B. (1991) Mol. Cell.

"."

R i d 1 1 . 84-92

"

Glick, A. B., Flanders, K. C., Danielpour, D., Yuspa, S. H., and Sporn, M. B. (1989) Cell Regul. 1,87-97 Gorman, C. M., Moffat, L. F., and Howard, B. H. (1982) Mol. Cell. Biol. 2 , 1044-1051 Heine, U. I., Munoz, E. F., Flanders, K. C., Ellingsworth, L. R., Lam, H.-P. P., Thompson, N. L., Roberts, A. B., and Sporn, M.B. (1987) J. Cell Biol. 1 0 5 , 2861-2876 Hentze, M. W., Rouault, T. A,, Caughman, S. W. Dancis, A,, Harford, J. B., and Klausner, R. D. (1987) Proc. Natl. Acad. Sc; U. S. A . 84,6730-6734 Ikeda, T., Lioubin, M. N., and Marquardt, H. (1987) Biochemistry 2 6 , 24062410 Jakowlew, S. B., Kondaiah, P., Flanders, K. C., Thompson, N. L., Sporn, M. B., and Roberts, A. B. (1988) Oncogene Res. 2,135-148 Kaighn, M. E., Narayan, K. S., Ohnuki, Y., Lechner, J. F., and Jones, L. W. (1979) Inuest. Urol. 1 7 , 16-23 Kehrl, J. H., Wakefield, L. M., Roberts, A. B., Jakowlew, S., Alvarez-Mon M., Derynck, R., Sporn, M. B., and Fauci, A. S. (1986) J. Exp. Med. 1 6 3 , lb371 nnn

Kk;S.-J., Glick, A,, Sporn, M. B., and Roberts, A. B. (1989) J. Biol. Chem. 264,402-408 Kim, S.-J., Kehrl, J. H., Burton, J., Tendler, C. L., Jeang, K.-T., Danielpour, D., Thevenin, C., Kim, K. Y., Sporn, M. B., and Roberts, A. B. (1990a) J. Exp. Med. 1 7 2 , 121-129 Kim, S.-J.,Angel, P., Lafyatis, R., Hattori, K., Kim, K.Y., Sporn, M. B., Karin, M., and Roberts, A. B. (1990b) Mol. Cell. B i d . 10,1492-1497 Klausner, R. D., and Harford,J. B. (1989) Science 246,870-872 Kondaiah, P., Van Obberghen-Schilling, E.,Ludwig, R. L., Dhar, R., Sporn, M. B., and Roberts, A. B. (1988) J. B i d . Chem. 263,18313-18317 Kozak, M. (1986) Proc. Natl. Acad. Sci. U. S. A. 83,2580-2584 Kozak, M. (1989) Mol. Cell. Biol. 9,5134-5142 Lafyatis, R., Lechleider, R., Kim, S.-J.,Jakowlew, S., Roberts, A. B., and Sporn, M. B. (1990) J. Biol. Chem. 265,19128-19136 Massague, J. (1987) Cell 49,437-438 Noma, T., Glick, A. B., Geiser,A. G., O'Reilly, M. A,, Miller, J., Roberts, A. B., and Sporn, M. B. (1991) Growth Factors 4,247-256 Rao, C. D., Pech, M., Robhins, K. C., and Aaronson, S. A. (1988) Mol. Cell. Biol. 8.284-292 _ ,"_ ~ ~ ~ Ratner, L., Thielan, D., and Collins, T. (1987) Nucleic Acids Res. 1 5 , 60176036 Roberts, A. B., and Sporn, M. B. (1990) in Handbook of Experimental Pharmacology, Peptide Growth Factors and Their Receptors (Sporn, M.B. and Roberts, A. B., eds) Vol. 95, Part 1, p.p. 419-472, Springer-Verlag,Heidelberg, corman"

Rouault, $. A,, Hentze, M. W., Haile, D. J., Harford, J. B., and Klausner, R. D. (1989) Proc. Natl: Acad. Sci. U. S. A . 8 6 , 5768-5772 Van Obberghen-Schllllng E., Roche, N. S. Flanders K. C., Sporn, M. B., and Roberts, A. B. (1988) J,' Biol. Chem. 2 6 3 , 7741-77k Walden, W. E., Patino, M. M., and Gaffield, L. (1989) J. Bid. Chem. 2 6 4 , 13765-13769