Growth Factors, 1992, Vol. 6, pp. 193-201 Reprints available directly from the publisher Photocopyingpermitted by license only

0 1992 Harwood Academic Publishers GmbH Printed in the United Kingdom

Regulation of Expression of Transforming Growth Factor+ 2 by Transforming Growth Factor+ Isoforms is Dependent upon Cell Type MICHAEL A. OREELY, DAVID DANIELPOUR, ANITA B. ROBERTS and MICHAEL B. SPORN Laboratory of Chemopreventwn, National Cancer Institute, National Institutes of Health, Bethesda, M D 20814

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(Received August 12 1991, Accepted September 3 1991)

The effect of three different isoforms of transforming growth fador-/3 (TGF-/3) on the expression of TGF-/32 mRNA was studied in several continuous tumor cell lines. As previously reported for the mouse fibroblakt cell line AKR-2B, the expression of TGF-/3 2 mRNA transcripts of 5.4, 4.7, 3.7 and 3.0 kb was decreased after a 24 hr treatment with 5 ng/ml of TGF-/3 1, TGF-P or TGF-/33. In A549, HBL-100 and BSC-1 epithelial cell lines, five distinct TGF-/32 mRNA transcripts of 5.8,5.1,4.0,3.8 and 2.8 kb were detected by Northern blot analysis. Treatment of these cells with TGF-/3 1, TGF-j?2 or TGF-/3 3 for 24 hr resulted in a 2-3 fold increase in the 5.8, 4.0 and 3.8 kb transcripts, with little detectable change in abundance of the 5.1 and 2.8 kb transcripts. The effect of the TGF-/3 proteins was dose (5 ng/ml) and time (3-6 hr) dependent. A similar 2-3 fold increase in the level of secreted TGF-/32 was observed following treatment of A549 cells with TGF-/3 1. Basal level and induced expression of TGF-/3 2 mRNA in response to TGF-/3 isoforms was decreased in the presence of actinomycin D. In all cell lines studied, the expression of the 2.5kb TGF-/31 mRNA was relatively unchanged or markedly increased in response to treatment with TGF-j?. These studies support the hypothesis that expression of TGF-/32 is regulated by members of the TGF-/3 family and is dependent upon cell type. KEYWORDS: gene expression, transforming growth factor-/3

INTRODUCTION

Additional TGF-/?-like molecules have been identified based upon unique biologic assays or Transforming growth factor-p (TGF-B) is a multi- cross-hybridization of cDNAs under reduced functional regulator of cell growth and differen- stringency. All of these proteins are 64 to 85% tiation (Roberts and Sporn, 1990; Massagub, homologous among each other. Thus, TGF-/32 1990). TGF-P was purified to homogeneity from was initially identified and purified from differseveral sources as a homodimeric peptide of ent cells and tissues because of its ability to 25,000 daltons. Sequence analysis of cDNA inhibit cell growth (Cheifetz et al., 1987; Ikeda et clones demonstrated that TGF-j3 is synthesized as al., 1987; Hanks et al., 1988), suppress T-cell funca larger preproprotein of approximately 390 tion (Wrann et al., 1987) and induce the formaamino acids, of which the biologically active tion of cartilage (Seyedin et al., 1987). TGF-Ps 3,4 TGF-/?monomer resides in the carboxy-terminal and 5 have been identified by cross-hybridization 112 amino acids (Derynck et al., 1986).The TGF-P of cDNA probes under reduced stringency (ten purified from platelets has been termed TGF-fl . Dijke et al., 1988; Jakowlew et al., 1988; Kondaiah et al., 1990). Although TGF-Bs 1 and 2 were originally identified by their unique biologic activities, they and recombinant TGF-B3 have been Address for correspondence: Michael A. OReilly, Ph.D., shown to display similar biologic activities in Laboratory of Chemoprevention, Building 41, Rm C629, NCI/NIH, Bethesda, h4D 20892. Phone: (301)496-5391; FAX many, but not all assay systems. (301)496-8395. Recent studies have demonstrated complex 193

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and distinct patterns of expression for the TGF-P isoforms in the developing mouse and human. Although each isoform may be expressed differentially among various tissues, in situ hybridization has also demonstrated a complex pattern of expression of TGF-P isoforms within a given tissue (Pelton et al., 1989; Gatherer et al., 1990; Millan et al., 1991; Schmid et al., 1991). Because different TGF-P isoforms have been found to be expressed by cells that lie within close proximity to each other, a variety of studies have been designed to determine whether the expression of a particular TGF-P isoform is regulated by the other isoforms. Previous studies have demonstrated autoinduction of TGF-D mRNA in a variety of cell lines (Van ObberghenSchilling et al., 1988; Bascom et al., 1989); this increase in TGF-fl expression is due to an increase in TGF-P1 gene transcription mediated by the transcription factor AP-1 (Kim et al., 1990). Less is known about autoregulation of TGF-/32 and TGF-@ expression. TGF-/32 mRNA was transiently increased 2-4 fold in response to TGF-/32 in a mouse fibroblast cell line; however, by 24 hr the level of expression was decreased compared to the untreated cells (Bascom et al., 1989). The present work extends these observations by demonstrating complex cell-type dependent regulation of TGF-/32 expression in response to treatment with TGF-P1, TGF-/?2 or TGF-/33.

derived TGF-P1 or TGF-P2 (R&D Systems, Minneapolis, MN), or recombinant chicken TGF-P3 (Roberts et al., 1990). Actinomycin D was purchased from Schweizerhall, Inc., South Plainfield, NJ and solubilized in ethanol. Cells were harvested by scraping in ice-cold phosphate buffered saline and centrifuged at 800xg for 5 min. Cell pellets were either quick-frozen on dry ice and stored at -70 "C until analyzed or cell numbers were quantitated with a hemacytometer. Media was centrifuged at 8OOxg for 5 min and the supernatants stored at -20°C until analyzed. RNA Extraction and Analysis

Total RNA was isolated from frozen cell lysates by sonication in 4 M guanidine thiocyanate, 0.5% N-lauroylsarcosine, 20 mM sodium citrate, 0.1 M P-mercaptoethanol and 0.1% antifoam A. RNA was extracted by centrifugation through a cushion of 5.7 M cesium chloride (Chirgwin et al., 1979). The RNA pellet was dissolved in water, extracted three times with phenol/ chloroform/isoamyl alcohol (25:24:1), once with chloroform/isoamyl alcohol (24:l) and precipitated with ethanol. PolyA' RNA was prepared using oligo dT columns (Pharmacia, Piscataway, NJ). The amount of RNA in an aqueous solution was determined by absorbance at 260 nm. RNA was separated on a 1.2% agarose-formaldehyde gel, transferred to Nytran (Schleicher and Schull, Inc., Keene, NH) and baked at 80°C for MATERIALS AND METHODS 2 hr. Blots were stained in 0.02% methylene blue with 0.3 M sodium acetate to verify the accuracy Cell Culture of transfer. Blots were prehybridized and A549 (human pulmonary adenocarcinoma), HBL- hybridized in 1% bovine serum albumin, 7% 100 (human breast carcinoma), BSC-1 (African sodium dodecylsulfate, 0.5 M sodium phosphate green monkey kidney) and AKR-2B (mouse and 1 mM EDTA at 65 "C (Church and Gilbert, embryo-derived fibroblast) cells were obtained 1984). Hybridized blots were washed in 1%bovfrom American Type Culture Collection. Cells ine serum albumin, 40mM sodium phosphate were incubated at 37°C and 5% COz in and 2 mM EDTA twice at room temperature and Dulbecco's Modified Eagle's media in 10% fetal twice at 65°C for 15min before exposing to bovine serum and 50 Units/ml penicillin and Kodak XAR-2 film. 50 pg/ml streptomycin (GIBCO, Grand Island, NY). Cells were maintained in tissue culture cDNA Probes flasks and routinely passaged every 3 days. Confluent cells were washed twice in phosphate RNA blots were sequentially hybridized with buffered saline and incubated for 12 hr in media 1.2 kb simian TGF-P;I probe (Hanks et al., 1988) without serum, or with 100 pg/ml bovine serum and 1.2 kb rat TGF-P1 cDNA (Qian et al., 1990), albumin for collection of conditioned media. which contained their respective coding regions, Cells were then treated with porcine platelet- and a rat glyceraldehyde 3'-phosphate dehydro-

AUTOREGULATION OF TGF-@ EXPRESSION

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genase probe (Van Obberghen-Schilling et al., 1988). Blots were routinely probed with 3-5x lo6cpm/ml of probe prepared by random primer labeling (Bethesda Research Laboratories, Gaithersburg, MD) to a specific activity of 2x lo9 cpm/,ug. Blots were stripped in 0 . 1 SSPE ~ and 0.2% SDS at 100 "C for 10 min.

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RESULTS Regulation of Expression of TGF-/ll and TGF-fl mRNAs by TGF-/3l, TGF-fl and TGF-fl

Previous studies by Bascom et al. (1989) demonstrated that confluent AKR-2B cells treated with 10 ng/ml TGF-fl or TGF-B;! resulted in increased ELISA levels of TGF-fl mRNA over 24 hr. In the same Conditioned media from control and TGF-PI cells, TGF-/32 mRNA was modestly increased 2-4 treated A549 cells cultured with 100,ug/ml bov- fold by 1-3 hr and was markedly decreased by ine serum albumin (BSA), was collected and 24 hr in response to TGF-/32. Likewise, treatment stored as described above. Media samples were of the cells for 24 hr with TGF-fl resulted in a precipitated with trichloracetic acid and the pel- decrease in TGF-B;! mRNA levels. In order to let washed in ether/ethanol (1:l). Precipitates confirm these results, confluent AKR-2B cells were reconstituted in 4 mM HC1, 150 mM NaCl were treated with 5ng/ml TGF-fl for 24 hr. and 0.05% BSA, and neutralized with an equal AKR-2B cells treated with TGF-fl for 24 hr volume of 200mM Tris-HC1, pH 7.6, 150mM showed a decrease in expression of TGF-B;! NaCl, 0.1% Tween 20 and 1 mg/ml crystalline mRNA transcripts of 5.4, 4.7, 3.7 and 3.0 kb (Fig. BSA, as previously described (Danielpour et al., 1C). In contrast, the level of the 2.5 kb TGF-P 1989). Serially diluted samples were quantitated mRNA transcript was increased greater than 10 with a capture enzyme-linked immunosorbent fold in response to TGF-fl. The level of glycerassay (ELISA) using rabbit anti-TGF-B;! and tur- aldehyde phosphate dehydrogenase (GAPDH) key anti-TGF-fl antibodies and standardized to mRNA expression was not significantly altered purified porcine TGF-B;! (Danielpour et al., 1989; by treatment with TGF-fl. Similar results on the Flanders et al., 1990). This ELISA does not cross- expression of TCF-B, TGF-/32 and GAPDH react with TGF-fl or TGF-@. The amount of mRNAs were observed following a 24 hr treatTGF-B;! secreted into the media was normalized ment with 5 ng/ml TGF-/32 or TGF-@ (data not to cell number. shown).

C.

B.

A.

-5.4

-5.8 -5.1 - 4.0

In

-3.8

-4.7 3.7 -3.0

-

-2.8

81

-2.5

81

-2.5

81

m - 2 . 5

FIGURE 1. Effect of TGF-P isoforms on expression of TGF-fl and TGF-E mRNAs. Confluent A549 (A), BSC-1 (B), or AKR-2B (C) cells were treated with 5 ng/ml of TGF-fl, TGF-@ or TGF-83 for 24 hr and then harvested. Total RNA from BSC-1 cells (15 ,fig) or poly A' RNA from A549 and AKR-2B cells (5 ,fig) were separated on a 1.2%agarose-formaldehyde gel and blotted to Nytran. Northern blots were probed with 32P-labeledTGF-m, TGF-&?or GAPDH cDNAs. The sizes of the specific mRNAs in kilobase pairs are indicated to the right of each panel.

O'REILLY et al

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The effect of TGF-P on the expression of TGF-/3 1 and TGF-@ mRNAs was also investigated in A549 and BSC-1 cells. These cells express 5 distinct TGF-@ mRNA transcripts of 5.8, 5.1, 4.0, 3.8, and 2.8 kb (Fig. 1A and B). The relative intensity of each transcript differs among these cell lines. Treatment of A549 cells with 5 ng/ml TGFpl, TGE@ or TGF-/%I for 24hr resulted in a modest 2-3 fold increase in expression of the 5.8, 4.0 and 3.8 kb mRNA transcripts, with little change in expression of the 5.1 and 2.8 kb transcripts (Fig. 1A). These changes in expression of TGF-@ mRNA in response to treatment with TGF-fl, and with TGF-@ and TGF-/%I(data not shown), were also observed in the BSC-1 cell line (Fig. 1B). While treatment with any of the TGF-/3 isoforms dramatically increased the expression of the 2.5 kb TGF-fl mRNA transcript in the A549 cell line (Fig. lA), the expression of TGF-fl mRNA was not markedly altered in response to TGF-/? in the BSC-1 cell line (Fig. 1B). Similar changes in the expression of TGF-fl and TGE@ mRNAs, as described for the A549 cell line, were also observed with the HBL-100 cell line (data not shown). In all cell lines, treatment with TGF-/? did not significantly alter the level of expression for GADPH mRNA (Fig. 1). Thus, whereas the expression of the TGF-P;! mRNA was decreased in response to TGF-P in the AKR2B cell line, TGF-P;! mRNA was modestly

6.

A.

GAPDH

increased 2-3 fold over the same time period in the A549, BSC-1 and HBL-100 cell lines.

The Changes in TGF-P Expression are Dose and Time Dependent The effect of TGF-P on the expression of TGF-fl and TGF-@ mRNAs was investigated in greater detail in the A549 cell line. TGF-P1, TGF-P;! and TGF-P each increased expression of TGF-pl and TGE@ mRNA in a dose-dependent manner (Fig. 2). Maximal induction of TGF-m and TGF-@ mRNAs was observed with a concentration of 5 ng/ml. All three TGF-P isoforms had similar dose-dependent effects on the expression of TGFf l and TGF-/32 mRNAs. Blots were stripped and probed for GAPDH mRNA to verify equal RNA loading in each lane. Because the dose-response curves of each isoform were similar, the abundance of TGF-fl and TGF-/32 mRNAs was investigated at various times following treatment of A549 cells with 5ng/ml TGF-/31 (Fig. 3). When blots were normalized to the level of GAPDH expression, stimulatory effects on expression of TGF-@ were detected as early as 3-6 hr, with maximal effects detected by 24 hr. A similar timedependent induction of TGF-pl mRNA was also observed (Fig. 3). In order to determine whether TGF-P also stimulated synthesis and secretion of TGF-P;!

C.

-1.4

FIGURE 2. Dose-response of TGF-/I isoforms on expression of TGF-fl and TGF-fl mRNAs. Confluent A549 cells were treatea with TGF-fl (A), TGF-/32 (B)or TGF-@ (C) for 24 hr and the cells harvested. Total RNA was prepared and 15 p g were separated on a 1.2%agarose-formaldehyde gel and blotted to Nytran. Northern blots were probed with =P-labeled TGF-m, TGF-fl or GADPH cDNAs. The sizes of the specific mRNAs in kilobase pairs are indicated to the right of each panel.

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AUTOREGUTION OF TCiF-82 EXPRESSION

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fluence A549 cells were treated with

0

1

3 6 1 2 2 4 HOURS

u)

-!

0CONTROL (D

TGF-P1

5

41-

T

GAPDH cDNAs. The sizes of the specific mRNAs in kilobase pairs are indicated to the right of -each panel.

protein, A549 cells were cultured for 24 and 48 hr in the presence and absence of 5 ng/ml TGF-fl. The amount of TGF-B;! in the conditioned media was quantitated by ELISA and normalized to cell number. TGF-fl treatment increased the amount of TGF-/32 in the media approximately 2 fold over the first: 24hr and approximately 3 fold over 48hr (Fig. 4). Thus, the modest changes in expression of TGF-P;! mRNA in response to TGF/3l are also reflected in an increase in secreted TGF-82.

Effect of Actinomycin D on Induction of

5? Y

s

24 HRS

48 HRS TIME

FIGURE 4. Effect of TGF-fl on expression of TGF-/32. Confluent A549 cells were treated with 5 ng/ml TGF-m, and cells and media were harvested at 24 and 48 hr. The amount of TGF-/92 secreted into the media was quantitated by ELISA and normalized to cell number. The values represent an average of three experimentsfstandard deviation.

TGF-j3 mRNA A549 cells were treated with actinomycin D and TGF-fl in order to determine whether gene transcription was required for the induction of TGF-fl and TGF-D mRNAs. The increase in expression of TGF-P;! mRNA in response to TGFf l ,TGF-P;! and TGF-/33 was inhibited in the presence of actinomycin D (Fig. 5). Moreover, basal level expression of TGF-P;! mRNA was markedly

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decreased in the presence of actinomycin D. Actinomycin D also inhibited basal and induced expression of TGF-/3l mRNA in response to TGF/?I, as previously reported for this cell line (Van Obberghen-Schilling et al., 1988). In contrast, expression of GAPDH was not significantly altered in response to treatment with TGF-p and was only modestly decreased in the presence of actinomycin D.

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DISCUSSION The present work demonstrates that the regulation of the expression of TGF-/Q mRNA in response to treatment with TGF-/3 isoforms is dependent upon the cell type. AKR-2B cells

treated.for 24 hr with TGF-P isoforms resulted in a decrease in the expression of TGF-/Q mRNA. However, TGF-/I2 mRNA was modestly increased 2-3 fold over the same time period in A549, BSC-1 and HBL-100 cells. As shown for the A549 cell line, these changes in the expression of TGF-Bl and TGF-D mRNA were dose and time dependent. The increase in TGF-/%?mRNA correlated with a similar increase in secreted TGF-/Q. Treatment of A549 cells with actinomycin D, which blocks gene transcription, inhibited both basal and TGF-pinduced expression of TGF-/Q mRNA. Although differential effects of TGF-B;! expression in response to TGF-P isoforms was observed in different cell lines, the expression of TGF-fl was relatively unchanged or increased in response to TGF-/3 isoforms. Our findings dem-

FIGURE 5. Effect of actinomycin D on expression of TGF-m and TGF-D &As. Confluent A549 cells were cultured for 24 hr with 5 ng/ml TGF-m, TGF-/32 or TGF-j33 in the presence or absence of 10 pg/ml actinomycin D and then harvested. Total RNA was prepared and 15 pg were separated on a 1.2% agarose-formaldehyde gel and blotted to Nytran. Blots were probed with 32P-labeledTGF-m, TGF-/32 or.GAPDH cDNAs. The sizes of the specific mRNAs in kilobase pairs are indicated to the right of each panel. Con., Control.

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AUTOREGULATION OF TGF-p EXPRESSION

onstrate that autoregulation of the TGF-/3 isoforms is complex and is dependent upon cell type. In a previous study describing autoregulation of TGF-P;! expression, TGF-P;! was shown to increase the expression of TGF-P;! mRNA 2-4 fold in mouse fibroblasts; however, these changes occurred within 1-3hr and by 24hr there was a marked decrease in TGF-P;! mRNA expression (Bascom et al., 1989). The initial increase in expression of TGF-P;! was due to an increase in TGF-/32 gene transcription, as determined by nuclear run-on analyses. The present study confirms these observations by demonstrating a similar decrease in expression of TGF-P;! mRNA following treatment of the cells for 24 hr with TGF-/3 isoforms. However, treatment of additional cell lines with TGF-P isoforms resulted in a modest 2-3 fold increase in TGF-P;! mRNA. These changes occurred later (3-6 hr) and were not followed by a decrease over time, as observed with the AKR-2B cell lines. The modest 2-3 fold increase in TGF-P;! mRNA expression was also detected as a similar increase in the level of TGF-/32 secreted into the media of A549 cells. Furthermore, cells treated with actinomycin D failed to induce TGF-P;! mRNA levels in response to treatment with TGF-/3 isoforms, suggesting that these changes in expression of TGF-P;! required gene transcription. These experiments support the concept that the relatively small change in the expression of TGF-P;! mRNA was due to a cellular change in the expression of TGF-P;!. Future studies will be to determine whether TGF-/3 increased TGF-P;! expression through cis-acting regulatory elements of the TGF-P;! promoter ‘or at the level of TGF-P;! mRNA stability. Molecular mechanisms that regulate expression of the TGF-/3 isoforms are beginning to be elucidated with the recent cloning of the human and murine TGF-fl (Kim et al., 1989; Geiser et al., 19901, human TGF-/32 (Malipiero et al., 1990; Noma et al., 1991) and human TGF-B3 (Lafyatis et al., 1990) gene promoters. Sequence analysis clearly shows the lack of homology among the promoters, supporting the concept that the TGF-P isoforms are differentially regulated. Previous studies have demonstated that autoinduction of TGF-PI is due to an increase in transcriptional activation of the TGF-fl gene promoter mediated by AP-1 (Kim et al., 1990).

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However, TGF-fl increased gene expression of type I collagen through an NF-1 site (Rossi et al., 1988) and post-transcriptionally stabilized fibronectin mRNA through an unknown mechanism (Dean et al., 1988). In contrast, TGF-fl decreased stromelysin gene expression through a fos binding site, which does not interact with jun (Kerr et al., 1990). The TGF-P;! promoter contains a TATA element and an upstream CRE/ATF element that is required for basal level expression (O’Reilly, unpublished observations). Regions upstream and downstream do not contain consensus AP-1, AP-2 or NF-1 sites, suggesting that expression of TGF-P;! in response to TGF-/3isoforms may occur through transcription factors other than those described here. Moreover, the 3I-untranslated region of the TGF-P mRNA contains multiple consensus stability sequences (AUUUA), which may be important in post-transcriptional mRNA stability (Hanks et al., 1988; Madisen et al., 1988; Miller et al., 1989). Thus the mechanism by which TGF-P;! expression is differentially regulated, as described in the present study, remains to be determined, and will most likely involve both transcriptional and post-transcriptional components. Because of the numerous biologic effects the TGF-fi have on the regulation of cell growth and differentiation, it is not surprising to discover that their regulation of expression is complex. Recent in situ analysis of the expression of the TGF-/3 isoforms in the developing mouse and human has identified both distinct and overlapping patterns of expression (Pelton et al., 1989; Gatherer et al., 1990; Millan et al., 1991; Schmid et al., 1991). Moreover, it has been shown that the level of secretion of each TGF-/? isoform may vary between different cells (Danielpour et al., 1989a, 1989b). Based upon these studies, it has been hypothesized that TGF-/3 acts through both autocrine and paracrine modes of regulation (Roberts and Sporn, 1990). TGF-fl RNA is often localized to mesenchymal tissue underlying epithelium that expresses TGF-P;!. The close cellular proximity of TGF-/3 isoforms demands a complex and highly regulated means of controlling TGF-P gene expression. The present study supports this hypothesis by demonstrating differential cell type dependent regulation of TGF-P;! expression. The precise mechanism by which this occurs remains to be determined, as well as the cell type in which this regulation takes place in vivo.

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ACKNOWLEDGMENTS We thank R. W. Holley for supplying the simian TGF-/32 cDNA, and A. Geiser, L. Wakefield and J. Cubert for critical review of this manuscript. We acknowledge the technical assistance of L. Cook in the quantitation of TGF-m protein by ELISA.

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REFERENCES Bascom, C. C., Wolfschohl, J. R., Coffey, R. J., Madisen, L., Webb, N. R., Purchio, A. R., Derynck, R. and Moses, H. L. (1989) Complex regulation of transforming growth factor /,?1,@ and Bj mRNA expression in mouse fibroblasts and keratinocytes by transforming growth factors 191 and @. Mol. Cell. Biol. 9,5508-5515. Bodmer, S., Strommer, K., Frei, K., Siepl, C., De Tribolet, N., Heid, I. and Fontana, A. (1989) Immunosuppression and transforming growth factor-8 in glioblastoma: Preferential production of-transforming .g r o i t h factor-@. 1. Immunol. 143,3222-3229. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. and Rutter, W. J. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemsitry 18, 5294-5299. Church, G. M. and Gilbert, W. (1984) Genomic sequencing. Proc. Natl. Acad. Sci. U S A 81,1991-1995. Danielpour, D., Dart, L. L., Flanders, K. C., Roberts, A. B. and Sporn, M. B. (1989a) Immunodetection and quantitation of the two forms of transforming growth factor-beta (TGF-fl and TGF-@) secreted by cells in culture. 1. Cell. Physiol. 138, 79-86. Danielpour, D., Kim, K. Y., Dart, L. L., Watanabe, S., Roberts, A. B. and Sporn, M. B. (1989b) Sandwich enzyme-linked immunosorbent assays (SELISAs) quantitate and distinguish two forms of transforming growth factor-beta (TGF-fl and TGF-@) in complex biological fluids. Growth Factors 2,61-71. Dean, D. C., Newby, R. F. and Bourgeois, S. (1988) Regulation of fibronectin biosynthesis by dexamethasone, transforming growth factor-/3, and CAMP in human cell lines. ]. Cell Biol. 106,2159-2170. Derynck, R. J., Jararett, J. A., Chen, E. Y., Eaton, D. H., Bell, J. R., Assoian, R. K., Roberts, A. B., Sporn, N. B. and Goeddel, D. V. (1985) Human transforming growth factor-beta cDNA sequence and expression in tumor cell lines. Nature 316,701-705. Flanders, K. C., Cissel, D. S., Mullen, L. T., Danielpour, D., Sporn, M. B. and Roberts, A. B. (1990) Antibodies to transforming growth factor-P peptides: Specific detection of TGF-@ in immunoassays. Growth Factors 3,45-52. Gatherer, D., ten Dijke, P., Baird, D. T. and Akhurst, R. J. (1990) Expression of TGF-P isoforms during first trimester human embryogenesis. Development 110,445-460. Geiser, A. G., Kim, S. J., Roberts, A. B. and Sporn, M. B. (1991) Characterization of the mouse transforming growth factor/,?1 promoter and activation by the Ha-rasckogene. Mol. Cell. Biol. 11,84-92. Hanks, S. K., Armour, R., Baldwin, J. H., Maldonado, F., Spiess, J. and Holley, R. W. (1988) Amino acid sequence of the BSC-1 cell growth inhibitor (mlverein) deduced from the nucleotide gequence of the c ~ N APyoc. . Natl. Acad. Sci. USA 85,79432. Jakowlew, S. B., Dillard, P. J., Sporn, M. B. and Roberts, A. B. (1988) Complimentary deoxyribonucleic acid cloning of a

messenger ribonucleic acid encoding transforming growth factor beta 4 from chicken embryo chondrocytes. Mol. Endocrinol. 2,1186-1195. Kerr, L. D., Miller, D. B. and Matrisian, L. M. (1990) TGF-fl inhibition of transin/stromelysin gene expression is mediated through a Fos binding sequence. Cell 6,267-278. Kim, S . J.,Angel, P., Lagyatis, R., Hattori, K., Kim, K. Y., Sporn, M. B., Karin, M. and Roberts, A. B. (1990) Autoinduction of transforming growth factor f l is mediated by the AP-1 complex. Mol. Cell. Bid. 10,1492-1497. Kim, S . J., Glick, A., Sporn, M. B. and Roberts, A. B. (1989) Characterization of the promoter region of the human transforming growth factor-/3l gene. 1. B i d . Chem. 264, 402-408. Kondaiah, P., Sands, M. J., Smith, J. M., Fields, A., Roberts, A. B. and Sporn, M. B. (1990) Identification of a novel transforming growth factor+ (TGF-P) mRNA from Xenopus laevis. 1. B i d . Chem.265,1089-1093. Lafyatis, R., Lechleider, R., Kim, S. J., Jakowlew, S., Roberts, A. B. and Sporn, M. B. (1990) Structural and functional characterization of the transforming growth factor@ promoter: A CAMP responsive element regulates basal and induced transcription. 1.B i d . Chem. 265,19128-19136. Madisen, L., Webb, N. R., Rose, T. M., Marquardt, H., Ikeda, T., Twardzik, D., Seyedin, S. and Purchio, A. F. (1988) Transforming growth factor-@: cDNA cloning and sequence analysis. D N A 7,143. Malipiero, U.,Moller, M., Werner, U. and Fontana, A. (1990) Sequence analysis of the promoter region of the glioblastoma derived T cell suppressor factor/transforming growth factor (TGF)-@ gene reveals striking differences to the TGF-fl and -83 genes. Biochem. Biophys. Res. Commun. 171, 1145-1151. Massagu6, J. (1990) The transforming growth factor-/,?family. Ann. Rev. Cell B i d . 6,597-641. Millan, F. A., Denhez, F., Kondaiah, P. and Akhurst, R. J. (1991) Embryonic gene expression patterns of TGF-fl, @ and 83 suggest different developmental functions in vivo. Development 111,131-144. Miller, D. A., Lee, A., Pelton, R. W., Chen, E. Y., Moses, H. L. and Derynck, R. (1989) Murine transforming growth factor @ cDNA sequence and expression in adult tissues and embryos. Mol. Endocrinol. 3,1108-1114. Mummery, C. L., Slager, H., Kruijer, W., Feijen, A., Freund, E., Koornneef, I. and Van Den Eijnden-Van Raaij, A. J. M. (1990) Expression of transforming growth factor p2 during the differentiation of murine embryonal carcinoma and embryonic stem cells. Develop. Biol. 137,161-170. Noma, T., Glick, A. B., Geiser, A. G., OReilly, M. A., Miller, J., Roberts, A. B. and Spom, M. B. (1991) Molecular cloning and structure of the human transforming growth factor-@ gene promoter. Growth Factors, 4,247-255. Pelton, R. W.,Nomura, S., Moses, H. L. and Hogan, B. L. M. (1989) Expression of transforming growth factor /32 RNA during murine embryogenesis. Development 106,759-767. Qian, S . W., Kondaiah, P., Roberts, A. B. and Sporn, M. B. (1990) cDNA cloning by PCR of rat transforming growth /,?1. NUC.Acids Res. 18,3059. Roberts, A. B. and Sporn, M. B. (1990) The transforming growth factor-betas. In: Sporn, M. B. and Roberts, A. B. (eds) Handbook of Experimental Pharmacology, vol. 95/1. Peptide Growth Factors and their Receptors. Springer-Verlag, Heidelberg, pp. 419-472. Rossi, P., Karsenty, G., Roberts, A. B., Roche, N. S., Sporn, M. B. and de Crombrugghe, 8 . (1988) A nuclear factor 1 binding site mediates the transcriptional activation of a type 1 collagen promoter by transforming growth factor-/,?. Cell 52,405-414. Schmid, P.,Cox, D., Bilbe, G., Maier, R. and McMaster, G. K. (1991) Differential expression of TGF-PI, p2 and p3 genes

AUTOREGULATION OF TGF-P EXPRESSION

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during mouse embryogenesis. Development 111,117-130. Seyedin, S. M., Segarini, P. R., Rosen, D. M., Thompson, A. Y., Bentz, H. and Graycar, J. (1987)Cartilage-inducing factor-B in a unique protein structurally and functionally related to transforming growth factor-8.1. Bid. Chem.262,1946-1949. ten Dijke, P., Hansen, P., Iwata, K. K., Pieler, C. and Foullces, J. G. (1988)Identification of another member of the transforming growth factor type gene family. Proc. Nutl. Acud. Sci. USA 85,4715-4719.

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Regulation of expression of transforming growth factor-beta 2 by transforming growth factor-beta isoforms is dependent upon cell type.

The effect of three different isoforms of transforming growth factor-beta (TGF-beta) on the expression of TGF-beta 2 mRNA was studied in several conti...
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