Apr 12, 1990 - system is completely blocked by prior treatment of the cells with TPA. These data identify a new .... human cell lines by Northern (RNA) analysis. Cultures of ... 10,000 x g, 4°C) separated the nuclear pellet from the extract; the upper ..... Angel, P., M. Imagawa, R. Chiu, B. Stein, R. J. Imbra, H. J.. Rahmsdorf, C.
MOLECULAR AND CELLULAR BIOLOGY, Nov. 1990, p. 5983-5990 0270-7306/90/115983-08$02.00/0 Copyright C) 1990, American Society for Microbiology
Vol. 10, No. 11
A Phorbol Ester-Regulated Ribonuclease System Controlling Transforming Growth Factor 13 Gene Expression in Hematopoietic Cells RUTH E. WAGER AND RICHARD K. ASSOIAN* Department of Biochemistry and Molecular Biophysics and Center for Reproductive Sciences, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York, New York 10032 Received 12 April 1990/Accepted 22 August 1990
12-Tetradecanoylphorbol-13-acetate (TPA)-induced differentiation of U937 promonocytes leads to a 30-fold increase in transforming growth factor ,Bl (TGF-,1l) gene expression, and this effect results from a stabilized mRNA. Similar up-regulation was detected in TPA-treated K562 erythroblasts but was absent from cell lines that do not differentiate in response to TPA. Related studies in vitro showed that postnuclear extracts of U937 promonocytes contain a ribonuclease system that degrades TGF-11 mRNA selectively and that this system is completely blocked by prior treatment of the cells with TPA. These data identify a new mechanism for regulating TGF-pl mRNA levels and allow us to establish the overall basis for control of TGF-,B1 gene expression by activation of protein kinase C. Our results also provide a new basis for understanding the long-term up-regulation of TGF-,Bl gene expression that can accompany hematopoietic cell differentiation.
Transforming growth factor 13 (TGF-,11) has widespread and complex effects on cells, including both stimulation and inhibition of growth and differentiation (37, 42, 61, 63). The specific effect of TGF-1l depends on many variables, including cell type and the presence of other growth factors (7, 16, 49, 58). Potent effects are particularly evident in blood cells; TGF-131 regulates monocyte chemotaxis and inhibits T-cell, B-cell, and NK cell function (26, 27, 50, 65). Several differentiated hematopoietic cell types (6, 19, 26, 27) express relatively high levels of TGF-,1l mRNA, suggesting the potential for autocrine and paracrine effects of the growth factor on blood cell physiology and pathology. The pervasive effects of TGF-131 emphasize the need to understand mechanisms that regulate expression of this protein. Several studies have shown that biosynthesis of TGF-p1 can be regulated by controls on transcription (2830, 33, 56). Two active promoters have been characterized in the human TGF-pl gene (20) by transfection studies with chimeric chloramphenicol acetyltransferase constructs and by structural analysis of TGF-131 mRNAs. Enhancer domains for several established transcription factors have also been identified in these promoter regions. Phorbol esterresponsive elements (TREs) have been identified in both the upstream and downstream domains of the TGF-13 gene. The existence of these TREs explains, at least in part, the observation that activators of protein kinase C such as 12-tetradecanoylphorbol-13-acetate (TPA) and platelet-derived growth factor stimulate expression of TGF-,11 mRNA in target cells (1, 56, 64). TGF-131 stimulates transcription of its own gene, and the cis-acting sequences mediating this effect appear to coincide with upstream TREs (28). The importance of transcriptional regulation notwithstanding, many studies have described clear examples in which posttranscriptional controls play major roles in determining overall gene expression (13, 14). For example, his*
tone mRNAs are selectively stabilized in S phase of the cell cycle (18, 60), transcripts encoding colony-stimulating factors are stabilized during activation of monocytes and macrophages (22, 55), c-myc mRNA stability is regulated by growth factors and beta interferon (12, 17, 31), iron levels control turnover of ferritin and transferrin receptor mRNAs (32, 38, 40), and 1-tubulin mRNA degradation is determined by the concentration of free 13-tubulin subunits (43, 69). A recent study indicates that TGF-1l may stabilize its own transcript in epithelial and fibroblastic cells (11). The potential consequence of altered posttranscriptional control is demonstrated by experiments showing that the aberrant forms of fos and myc transcripts associated with transformation are more stable than their normal counterparts (21, 39). In the studies reported here, we have used TPA-treated U937 promonocytes (48) as a tool to identify mechanisms that regulate TGF-p1 gene expression during the latter stages of hematopoietic cell differentiation. A combined analysis with intact and extracted cells has allowed us to identify a ribonuclease system that regulates TGF-p1 mRNA turnover during TPA-induced U937 cell differentiation. Our results provide a new basis for understanding the persistent up-regulation of TGF-p1 gene expression that can accompany differentiation of hematopoietic cells. MATERIALS AND METHODS Cell culture. U937 (clone 7, a generous gift from Joseph DeLarco) and K562 cell lines were cultured in RPMI 1640 medium containing 5% heat-inactivated newborn calf serum and gentamicin and maintained at a cell density of 0.1 x 106 to 0.5 x 106 cells per ml. Clone 7 cells represent a weakly adherent subpopulation of U937 promonocytes. W138, A549, and A431 cell lines were cultured in Dulbecco modified Eagle medium supplemented with 10% heat-inactivated fetal calf serum (FCS). In some experiments, cells were treated with TPA (160 nM, LC Services) or purified human TGF-,1l (250 pM) (8).
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FIG. 1. Biphasic stimulation of TGF-p1 mRNA levels in hematopoietic cells by treatment with TPA. Selected human cell lines of distinct lineages were serum starved (see Materials and Methods) and exposed to TPA (160 nM) for selected times as indicated in the figure. Cells were collected, RNA was extracted, and 10-,ug aliquots of the purified RNAs were fractionated on formaldehyde-agarose gels before Northern analysis, using TGF-p1 and GAPDH cDNA probes. The figure shows TPA-induced alterations in the steadystate levels of TGF-p1 and GAPDH mRNA in U937 promonocytes (A) and W138 fibroblasts (B). The bar graphs show results of time-course studies that compare (by densitometric scanning of X-ray films) the time-dependent effects of TPA on TGF-pl mRNA levels detected in U937 promonocytes (C), K562 erythroblasts (D), A431 epithelial cells (E), and W138 fibroblasts (F). Changes in TGF-pl mRNA levels were corrected for small differences in sample loading by normalizing hybridization signals relative to GAPDH mRNA. The horizontal arrows in each panel show the basal level of TGF-pl mRNA expression. For all experiments, TPA was dissolved in dimethyl sulfoxide and diluted into medium such that the final dimethyl sulfoxide concentration was 0.01%. Control studies showed that 0.01% dimethyl sulfoxide had no effect on TGF-pl mRNA levels.
Analysis of RNA levels and stability in whole cells. Timedependent alterations in the level of TGF-pl mRNA in response to TPA and TGF-,1 were assessed in several human cell lines by Northern (RNA) analysis. Cultures of U937 promonocytes and K562 erythroblasts (107 cells in 20 ml) were prepared as described above (except that the medium contained 0.5% FCS) and incubated in 0.5% serum for 24 h. Monolayer cells (WI38, A549, and A431) were grown to confluence in 150-mm-diameter tissue culture dishes (one dish per time point); cells were washed once with phosphate-buffered saline and incubated (24 h) in 20 ml of Dulbecco modified Eagle medium-0.5% FCS. TPA or TGF-pl was added to the serum-starved cells; RNA was isolated at selected times (refer to Fig. 1 and 2) and fractionated on formaldehyde-agarose gels as described below. For experiments examining RNA turnover, cells were treated with dactinomycin (5 ,ug/ml); see the legend to Fig. 3. Analysis of RNA stability in vitro. Purified, total RNA (10-,ug aliquots) was incubated with selected concentrations of the U937 cell postnuclear extract for 0 to 30 min at 4°C (see the legends to Fig. 4 to 6) in 50 RI (final volume) of extract buffer (0.01 M Tris hydrochloride [pH 7.9], 0.15 M NaCl, 0.5% Nonidet P-40). The reactions were terminated by phenol-chloroform extraction. Samples were supple-
FIG. 2. Different effects of TPA and TGF-1l on TGF-1l mRNA levels in U937 cells. Serum-starved U937 cells were prepared and treated with TGF-11 (250 pM) or TPA (160 nM) for the times indicated in the figure (see Materials and Methods for details). Cells were collected for isolation of RNA and Northern analysis of TGF-11 and GAPDH mRNA levels as described in Materials and Methods and the legend to Fig. 1. (A and B) Steady-state levels TGF-,B1 and GAPDH mRNAs after short and long-term exposure to TGF-pl, respectively. (C) Levels of TGF-pl and GAPDH mRNAs after 48 h of treatment of U937 cells with TPA. The concentration of TGF-p1 used in this experiment is similar to that used in other studies demonstrating autoinduction of TGF-pl mRNA (30, 64).
mented with tRNA (10 ,ug) as carrier, precipitated with ethanol, and fractionated on formaldehyde-agarose gels. The stability of individual RNAs was assessed by Northern analysis with defined cDNA probes. To prepare the postnuclear extract, U937 cells (108) were collected by centrifugation, washed once with phosphate-buffered saline, and suspended in 1 ml of ice-cold extract buffer by vortexing briefly. After a 5-min incubation on ice, the sample was carefully layered over an equal volume of extract buffer containing 24% sucrose and 1% Nonidet P-40. Centrifugation (20 min, 10,000 x g, 4°C) separated the nuclear pellet from the extract; the upper phase (postnuclear extract) was collected and stored on ice before use. When specifically noted, reaction mixtures were supplemented with 50 mM EDTA. Isolation of RNA and Northern analysis. Total RNA was isolated from cells by extraction in guanidinium isothiocyanate-containing solution and centrifugation through a cesium chloride cushion (9). The recovered RNA was extracted with phenol-chloroform, precipitated with ethanol, and dissolved in water (1 to 2 mg/ml). RNA was fractionated on 1% agarose gels containing formaldehyde (36), transferred electrophoretically to Nytran filters (Schleicher and Schuell), and fixed by exposure to UV light. The positions of 28S and 18S rRNAs were identified by ethidium bromide staining. Filters were prehybridized (1 h at 42°C) in a solution consisting of Sx SSPE, 50% formamide, 5x Denhardt reagent, 0.1% sodium dodecyl sulfate, and 0.1 mg of low-molecular-weight DNA per ml (36). Hybridization proceeded (16 h at 42°C) in the same solution containing nick-translated cDNA probes (2 x 106 cpm/ml) for TGFp1 (56) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (23). Filters were washed at 0.l5x SSPE-0.1% SDS at 67°C and exposed to X-ray film. In some experiments, hybridization signal intensities were quantitated by densitometry. The broad hybridization signals detected for TGF-pl mRNA reflect the presence of two transcripts (see Fig. 7) that are poorly resolved on Northern blots.
RESULTS Cell-specific up-regulation of TGF-I1 gene expression accompanies TPA-induced differentiation of U937 promono-
CONTROL OF TGF-,1l mRNA STABILITY
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degradation of was incubated with the U937 cell postnuclear extract, and the reactions were terminated at 10, 20, and 30 min (see Materials and Methods). RNA, which had not been exposed to extract, was processed in parallel and used as the positive control (shown as 0-min incubation). Samples were fractionated on formaldehyde-agarose gels, and the relative degradation of TGF-pl mRNA (A), GAPDH mRNA (B), and 28S and 18S rRNAs (C) were assessed by hybridization of the filters with specific cDNA probes and ethidium bromide staining, respectivety. Panels D to F, respectively, compare the levels of TGF-p1 mRNA, GAPDH mRNA, and rRNAs after a 30-min incubation with U937 postnuclear extract in the absence (-) and presence (+) of 50 mM EDTA. Note that EDTA had no effect on transcript levels in the absence of extract (shown as 0-min incubation).
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Period of Incubation (hr) FIG. 3. TGF-p1 mRNA is stabilized in TPA-differentiated U937 cells. U937 cells (107 in 20 ml of RPMI 1640-5% NCS) were incubated in the absence (open symbols) or presence (closed symbols) of TPA (160 nM) for 24 h before the addition of dactinomycin. Incubation with dactinomycin (5 ,ug/ml) proceeded for 0, 6, 12, 24, or 48 h after which RNA was isolated, fractionated (20% of each sample), and subjected to Northern analysis. Control studies showed that cell viability exceeded 90% throughout the experiment, as determined by trypan blue exclusion and that 5 Lg of dactinomycin per ml inhibited [3H]uridine incorporation into RNA by >95%. The figure shows the effect of TPA on in vivo turnover of TGF-p1 mRNA (A) and 28S rRNA (B). The half-life of 18S RNA (not shown) resembled that of 28S RNA in the absence and presence of TPA. The graph was generated by densitometric scanning of X-ray films and photographic negatives; signal intensities were plotted relative to the positive control (RNA levels in cells incubated for 0 min with
dactinomycin). cytes. We have used TPA-treated U937 promonocytes (48) as a model to examine mechanisms that may regulate
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gene expression during hematopoietic cell differentiation. Initial experiments monitored changes in steadystate TGF-q1 transcript levels after U937 cells were exposed to TPA. As assessed on Northern blots (Fig. 1A), this approach revealed two distinct time periods (0 to 6 and 24 to 48 h) in which TPA up-regulated the expression of TGF-pl mRNA. The early effect was transient, whereas the late effect persisted for at least 24 h and greatly exceeded the early effect in magnitude. GAPDH mRNA levels (Fig. 1A) were unaltered by TPA and reflected sample loading efficiency as assessed by ethidium bromide-stained rRNAs (data not shown). Others have also shown that GAPDH mRNA levels are unaffected by TPA (3). We have repeated this experiment with U937 cells cultured in several concentrations of calf serum (0.5 to 10% [vol/vol]), and similar transient and long-term TPA-mediated stimulation of TGF-pl mRNA levels was observed. This result was not unexpected, since monocytes are not a major target for platelet-derived growth factor (the major activator of protein kinase C in serum) (53).
The rapid and transient stimulation of TGF-pl mRNA levels we observed in TPA-treated U937 cells was also detected in serum-starved W138 fibroblasts, but interestingly, these cells did not respond to TPA with long-term up-regulation of the TGF-p1 transcript (Fig. 1B). Longer incubation times (up to 96 h) or alterations in the percentage of cell confluence (30 versus 100%) did not yield the biphasic TPA effect observed in U937 cells (data not shown). These results raised the possibility that the long-term stimulatory effect of TPA on steady-state TGF-p1 mRNA levels might be cell specific. Several human cell lines of distinct lineage were examined for short- and long-term stimulation of TGF-1l mRNA levels in response to TPA. Figure 1, panels C through F, shows densitometric scans of autoradiograms that allowed us to assess magnitudes of the changes we detected. The biphasic TPA response we observed in U937 promonocytes (panel C) was also detected in K562 erythroblasts (panel D), whereas A431 epithelial cells (panel E) resembled W138 fibroblasts (panel F) in showing only the early, transient response. We have also tested the parental strain of U937 cells from which our clone was derived (see Materials and Methods) and A549 epithelial cells. The time-dependent TPA responses on TGF-p1 transcript levels in these two cell lines (data not shown) were in complete agreement with the other hematopoietic and epithelial cells tested (Fig. 1). Although this study was not exhaustive, it is clear that the early, transient TPA effect is detectable in a variety of cell types, whereas the long-term effect is restricted and accompanies TPAinduced cell differentiation. In the hematopoietic cells we
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FIG. 5. Dose-dependent identification of a ribonuclease activity selective for TGF-pl mRNA. Total RNA was incubated for 30 min with selected concentrations of the U937 cell postnuclear extract (0.03, 0.1, 0.3, 0.9, 2.7, 8% [vol/vol] per reaction; lanes 1 to 6, respectively, in panels A to C). Northern blots were prepared and analyzed for levels of TGF-f1 mRNA (A), GAPDH mRNA (B), and rRNAs (C). The graph (D) plots signal intensity relative to the positive control (see the legend to Fig. 4) for TGF-pl mRNA (0), GAPDH mRNA (A), and 28S rRNA (A) as determined by densitometric scanning of X-ray films and photographic negatives. Control studies showed that the level of RNA endogenous to the highest concentration of postnuclear extract was negligible and did not contribute to hybridization signal intensities (data not shown).
tested, the long-term TPA effect resulted in a 10- to 30-fold increase in steady-state TGF-pl transcript levels, and the magnitude of this effect exceeded that of the early response fivefold. Others have shown that TGF-pl can regulate the expression of its own transcript and that this regulation can occur transcriptionally and posttranscriptionally in fibroblasts and epithelial cells (11, 30, 64). To determine whether TPA stimulates TGF-31 mRNA levels in U937 cells by inducing secretion of the growth factor (with consequent autocrine stimulation of TGF-pl gene expression), U937 cells were cultured with TGF-pl directly. The effect of the growth factor on TGF-pl mRNA levels was assessed on Northern blots. Slight (two- to threefold) increases in the level of TGF-pl transcript were detected without discernible pattern after both short- (Fig. 2A) and long-term (Fig. 2B) exposure of U937 cells to the growth factor. These TGF-pl effects do not account for the large change in TGF-pl mRNA levels we observed after 24 to 48 h of exposure of U937 cells to TPA (Fig. 1 and 2C). Several cell types respond to TPA by increasing steadystate levels of TGF-pl mRNA (Fig. 1) (1, 6, 56), but this effect has been explained solely in terms of the TREs in the TGF-pl gene (28-30, 56). Since TRE-stimulated gene transcription is a rapid event (4, 5, 34), the early TPA effect we detected in all cell types (Fig. 1) was completely consistent with TRE-based transcriptional stimulation; we have not examined this effect further. In contrast, the delayed and long-term stimulation of TGF-pl gene expression associated with TPA-induced differentiation of U937 and K562 cells was more difficult to explain kinetically on the basis of transcriptional controls alone, and we considered it the likely consequence of a novel mechanism controlling TGF-pl gene expression posttranscriptionally. TPA inhibits TGF-j1 mRNA turnover in U937 cells. To examine the potential for posttranscriptional regulation of TGF-,1 mRNA directly, U937 cells were cultured in the absence or presence of TPA (24 h) before transcription arrest
by addition of dactinomycin. Decay rates of existing RNAs were assessed by Northern analysis. The decay rate of TGF-p1 mRNA decreased approximately fivefold in TPAdifferentiated U937 cells (Fig. 3A). The decay of rRNA was unaffected by TPA (Fig. 3B). Note that TGF-p1 mRNA was not subject to rapid turnover in the absence of TPA (half-life, approx. 6 h); its stabilization resulted in a very long-lived transcript with a half-life similar to that of rRNA (compare Fig. 3A and B). Since the 24-h TPA pretreatment period used in this experiment corresponds to the long-term stimulatory TPA effect in Fig. 1, stabilization of TGF-1l mRNA provides the likely explanation for the delayed and persistent up-regulation of TGF-pl gene expression associated with TPA-differentiated U937 cells. Control studies showed that the large majority of TGF-pl mRNA was associated with polysomes in the absence and presence of TPA (data not shown). Thus, steric protection of the transcript, by an increased association with ribosomes, does not account for TPA action in this system. Selective degradation of TGF-Il1 mRNA in vitro. To examine the ribonuclease system controlling TGF-pl mRNA turnover and assess its potential regulation during TPAinduced U937 cell differentiation, we developed a simple in vitro system in which total, deproteinated RNA was added to a U937 cell postnuclear extract (see Materials and Methods). Degradation of individual transcripts was monitored after fractionation of the reaction products and Northern blot hybridization with specific cDNA probes. Initial experiments with this system compared the time-dependent degradation of TGF-pl mRNA, GAPDH mRNA, and rRNAs (Fig. 4A to C). Although the concentration of postnuclear extract used in this experiment (5% [vol/vol]) resulted in the degradation of all three transcripts, we noticed that TGF-pl mRNA was degraded more extensively by the end of the 30-min incubation (approx. 80, 40, and 25% degradation of the TGF-pl, GAPDH, and rRNA transcripts, respectively, as determined by densitometric scanning). We examined the divalent cation requirement for degra-
CONTROL OF TGF-,B1 mRNA STABILITY
VOL. 10, 1990
by the results shown in Fig. 5. In five separate experiments, 28S rRNA and GAPDH mRNA were degraded similarly, whereas TGF-p1 mRNA was degraded at least 10-fold more
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dation of TGF-pl mRNA, GAPDH mRNA, and rRNAs using a high concentration (10% [vol/vol]) of the U937 cell postnuclear extract that yielded total transcript degradation (Fig. 4D to F). The results show that degradation of all the transcripts was blocked completely by addition of EDTA to the incubation mixture. The requirement for divalent cations in our in vitro system distinguishes the responsible U937 cell enzyme(s) from noncyclizing RNases and the acid lysosomal RNase characterized to date (10, 54). The potential for differential degradation of TGF-pl mRNA, GAPDH mRNA, and 28S rRNA (28S and 18S rRNAs were always degraded similarly) was examined in detail by incubating total U937 cell RNA with selected concentrations of the postnuclear extract (Fig. 5). Results from this experiment allowed us to identify two concentration ranges that were associated with distinct patterns of RNA degradation. High concentrations of the extract (>10% [vol/vol]) degraded all three RNAs to completion (data not shown). However, degradation profiles of the three transcripts were distinguishable with lower concentrations (0.2 to 8% [vollvol]) of the extract (Fig. 5A to C). As determined by densitometric scanning of the autoradiograms (Fig. SD), the concentration of extract that resulted in half-maximal degradation of TGF-pl mRNA (0) was 10-fold less than that required to degrade either 28S rRNA (U) or GAPDH mRNA (A). We find that the relative level of in vitro ribonuclease activity varies somewhat in different preparations of the U937 cell postnuclear extract, but the overall pattern of selective and nonselective degradation is well-represented
efficiently. Note that our initial studies showing smaller differences in the degradation of these transcripts (Fig. 4) used a single concentration of the U937 cell extract, which was sufficient to degrade transcripts through both the selective and nonselective mechanisms. TPA-induced differentiation of U937 cells blocks selective degradation of the TGF-I1 transcript in vitro. We considered the possibility that stabilization of TGF-pl mRNA in TPAdifferentiated U937 cells could result from inhibition of the ribonuclease activity identified in Fig. 5. To test the validity of this potential mechanism, U937 promonocytes were cultured in the absence or presence of TPA for 24 h (consistent with the kinetics from Fig. 1 and 3) before preparation of the postnuclear extracts. The preferential degradation of TGF-pl mRNA observed in the extract prepared from untreated cells (Fig. 6A) was lost when the extract was prepared from TPA-differentiated cells (Fig. 6B). Note that the TPA-induced change in degradation efficiency is restricted to the TGF-pl transcript; 28S rRNA and GAPDH mRNA were degraded similarly by extracts prepared from control and TPA-differentiated U937 cells (compare Fig. 6A and B). The results of Fig. 6 demonstrate that TPA-mediated differentiation of U937 cells inhibits in vitro ribonuclease activity towards TGF-1l mRNA selectively. Moreover, these in vitro results describe a mechanism for regulating TGF-pl mRNA turnover, and the mechanism correctly predicts that the corresponding effect of TPA in intact U937 cells would be detected as up-regulation of a stabilized TGF-,B1 transcript. DISCUSSION Several studies indicate that long-term up-regulation of TGF-pl gene expression is associated with differentiation of certain hematopoietic cells and hematopoietic cell lines (6, 19, 27). For example, TGF-pl transcript levels remain elevated for days in stimulated T cells, and atypically high levels of the mRNA are expressed in mature blood monocytes. Long-term increases in TGF-1l gene expression have also been reported during megakaryoblastic differentiation of K562 erythroblasts (2, 3). Transcriptional control of the TGF-i1 gene may well play an important role in this phenotype. However, the results described in this report establish an alternative basis for up-regulating TGF-fi1 gene expression during hematopoietic cell differentiation that is posttranscriptional and associated with inhibition of a transcript-selective ribonuclease system. The validity of the mechanism, identified in studies with cell extracts, is supported by its ability to predict and explain the long-term stimulatory effect of TPA on TGF-pl transcript levels in intact cells. Taken together with previous studies of TRE-stimulated TGF-pl gene transcription (28-30, 56), the results in this report now allow us to assemble the overall framework by which TPA-induced activation of protein kinase C regulates TGF-1l gene expression. The diagram in Fig. 7 outlines the metabolism of TGF-,1l mRNA and identifies (with solid triangles) the sites regulated by TPA. A comparison of the transcriptional and posttranscriptional effects emphasizes differences between the respective mechanisms with regard to kinetics, directionality, and cell specificity (right-hand side of the figure). Despite these apparent dissimilarities, a consequence of this multistep regulation is the coordinated
5988
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FIG. 7. Coordinate regulation of TGF-31 expression by activated protein kinase C. The figure shows a diagrammatic representation of TPA action on TGF-pl gene expression. Solid triangles in the top part of the figure identify the approximate positions of TREs in the human TGF-pl gene. The single solid triangle in the bottom part of the figure identifies the posttranscriptional site of TPA action. The intron-exon structure, the two transcription start sites, and the primary polyadenylation signal in the human TGF-pl gene are indicated (19, 20, 28, 29, 56). The hatched area in TGF-pl mRNA depicts the difference between transcripts initiated from start sites 1 and 2.
short- and long-term up-regulation of TGF-,11 transcript levels. The cellular requirement for such coordination is not yet established, but we speculate that long-term up-regulation of the TGF-,B1 mRNA in hematopoietic cells (assuming subsequent secretion and activation of the growth factor) could play important roles in generating or maintaining differentiated phenotypes as well as mediating TGF-pl effects on inflammation, wound repair, chemotaxis, and the immune response (61). We cannot exclude the possibility that the posttranscriptional stabilization of TGF-pl mRNA accompanying TPAdependent U937 cell differentiation results from cell specificity in the pattern of protein phosphorylations, rather than the differentiating effect of activated protein kinase C in hematopoietic cell lines. Appropriate examination of this complex question will require the use of primary cells and a variety of physiologic activators. Studies in other mammalian systems have shown that posttranscriptionally regulated gene expression usually requires a complex interaction of cis- and trans-acting RNA elements with one or more ribonucleases. cis elements include multiple AUUUA-like motifs in the 3' untranslated domains of short-lived mRNAs and specific stem-loop structures (24, 25, 40, 44, 47, 55, 57, 59). trans-Acting factors that bind to AUUUA multimers (35) and the transferrin receptor stem-loop (32, 41) have recently been reported. Examination of the human TGF-pl cDNA structure (19, 56) shows that the predominant transcript (terminating at position 2136) contains one copy of AUUUA in the 3' untranslated region rather than a large AU-rich domain as is found in rapidly degraded transcripts (57). Moreover, TGF-pl mRNA has a long half-life (Fig. 3) relative to prototype AUUUA-regulated mRNAs. Thus it is unlikely that the single AUUUA sequence functions as the cis element mediating TPA-regulated turnover of TGF-pl mRNA. We are currently trying to identify domains of the
TGF-pl mRNA which confer TPA-regulated turnover to the transcript. Although we cannot exclude the potential presence of acid lysosomal RNAse in our in vitro system, we can exclude a functional role for this enzyme by the observation that degradation of all transcripts tested with the U937 cell extract requires divalent cations (10, 54). In fact, the requirement for divalent cations is a hallmark of ribonucleases that regulate mammalian mRNA turnover (45, 46, 52, 62). In our in vitro system, similar incubation periods degrade GAPDH mRNA and rRNA (Fig. 5 and 6) despite the established stability of rRNA in cells. This paradox is reconciled by other studies showing that the in vivo half-lives of transcripts are not necessarily reproduced in vitro, even though the same mechanisms are used to degrade transcripts in intact and extracted cells (15, 45, 51, 62). The results presented here establish a mechanism for posttranscriptional control of TGF-pl gene expression that is consistent with the long-term increases in TGF-i1 mRNA levels observed during TPA-induced differentiation of hematopoietic cells (2, 3, 6). Since TGF-,11 mRNA is one of several mammalian transcripts that are stabilized in TPAtreated blood cells (66-68), it is possible that recognition by the phorbol ester-mediated ribonuclease system described here will define a new criterion for the classification of transcripts. Expression of the corresponding proteins, as a group, might then be predictably up-regulated by activated protein kinase C in appropriate target cells. ACKNOWLEDGMENTS We thank Joseph DeLarco (Monsanto Company) for the generous gift of U937 promonocytes (clone 7). These studies were supported by Public Health Service grant HL38884 (to R.K.A.) from the National Institutes of Health. R.E.W. was supported by National Institutes of Health predoctoral
CONTROL OF TGF-,B1 mRNA STABILITY
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