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Cell Growth & Differentiation Vol. 12, 9-18, January 2001
© 2001 American Association for Cancer Research


Articles

Mechanism of Induction of Transforming Growth Factor-ß Type II Receptor Gene Expression by v-Src in Murine Myeloid Cells

Seok Hee Park, Maria C. Birchenall-Roberts, Youngsuk Yi, Byoung Ick Lee, Dug Keun Lee, Daniel C. Bertolette, Tao Fu, Frank Ruscetti and Seong-Jin Kim1

Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892-5055 [S. H. P., Y. Y., B. I. L., D. K. L., S-J. K.], and Intramural Research Support Program, Science Applications International Corporation-Frederick [M. C. B-R., T. F.], Laboratory of Leukocyte Biology, Frederick Cancer Research and Development Center [D. C. B., F. R.], Frederick, Maryland 21702

Abstract

Transforming growth factor (TGF)-ß1 plays an important role during hematopoiesis. Previously, we had shown that the growth of a v-Src-transformed myeloid cell line was markedly more inhibited by TGF-ß treatment when compared with the wild-type myeloid cell line. To investigate the increased growth sensitivity of the v-Src-transformed myeloid cell line, 32D-src, to TGF-ß, we examined expression of the TGF-ß type II receptor (TGF-ß RII) gene in myeloid cell lines. Northern blot analysis showed that expression of ~8- and 6-kb species of TGF-ß RII transcripts was markedly increased in the 32D-src cell line. The expression of the TGF-ß RII promoter linked to a reporter gene was increased 23-fold by v-Src. DNA transfection and electrophoretic mobility shift assay revealed that v-Src induces TGF-ß RII promoter activity through an AP1/ATF2-like sequence (-219 to -172), ETS binding sites (+1 to +36), and the inverted CCAAT box (-81 to -77). Novel DNA-protein complexes with ETS binding sites are significantly increased in v-src-transformed cell lines compared with the control cell line. These results suggest that v-Src induces activity of the TGF-ß RII promoter through multiple elements by inducing expression of nuclear proteins interacting with these elements.

Introduction

Expression and function of the TGF-ß RII2 is necessary for the growth-inhibitory effects of TGF-ß on proliferating epithelial cells (1, 2, 3, 4) . Inactivation of the TGF-ß signaling pathway contributes to carcinogenesis and can occur through one of several mechanisms involving mutation or transcriptional repression of either the receptors or intermediates in the signal transduction pathway (5, 6, 7, 8) . For example, a specific TGF-ß RII mutation is associated with DNA microsatellite instability in hereditary nonpolyposis colon cancer (9 , 10) and in human gastric cancer (11) . These mutations result in a frameshift mutation leading to premature chain termination and the expression of a receptor lacking the kinase domain. Other mutations and deletions in TGF-ß RII have been described that inactivate receptor binding or functions (5 , 12) . In contrast, there are few examples of defects in the TGF-ß RI gene. A structural alteration in the TGF-ß RI gene that correlates loss of TGF-ß RI expression with insensitivity to TGF-ß1 was reported in a human prostate cancer cell line LNCaP (13) . A defect in the intracellular signaling machinery downstream of the TGF-ß RII and RI, such as the SMAD proteins, can also render a cell insensitive to TGF-ß (14, 15, 16, 17, 18, 19, 20) .

Transcriptional repression of TGF-ß receptor genes is another, perhaps more common, mechanism leading to TGF-ß resistance. Many human cancer cell lines harbor normal TGF-ß RII gene and downstream signaling proteins but express significantly reduced or undetectable levels of TGF-ß RII mRNA (5 , 21) . Transcriptional regulation of TGF-ß RII expression plays an important role in modulating TGF-ß responsiveness, because transformation of cells by the product of the adenovirus E1A gene (22 , 23) or overexpression of cyclin D1 (24) in epithelial cells has been associated with down-regulation of TGF-ß RII expression and TGF-ß resistance. Recently, we have demonstrated that Ewing sarcoma EWS-FLI1 fusion gene suppresses transcription of the TGF-ß RII gene (25) .

Our laboratory has previously cloned and sequenced the 5' flanking region of TGF-ß RII and has characterized two positive and two negative regulatory elements as well as the core promoter region (26) . We have isolated the epithelial-specific ets family of transcription factor, ERT/ESX/ESE-1/ELF3/jen (27, 28, 29, 30, 31) , which binds to the second positive regulatory element of the TGF-ß RII promoter (26) . ERT induces TGF-ß RII promoter activity, suggesting that ets family members may be the major transcriptional factors involved in regulation of TGF-ß RII gene expression (27) .

We have demonstrated previously that murine myeloid cells stably transfected with the src oncogene demonstrate enhanced sensitivity to TGF-ß (32) . This increased sensitivity may attributable to up-regulation of TGF-ß RII expression. In this study, we examined whether the v-src oncogene product induces transcription of the TGF-ß RII gene. Northern blot analysis and cross-linking assays showed that TGF-ß RII mRNAs and protein are markedly increased in v-src-transformed myeloid cell lines. DNA transfection and EMSA demonstrated that v-Src induces TGF-ß promoter activity through the PRE1 and PRE2 elements and an inverted CCAAT box.

Results

v-Src Increases Transcription of TGF-ß RII in Murine Myeloid Cell Line.
The retrovirally infected IL-3-independent cell line, 32D-src, expresses higher levels of TGF-ß receptors than the parental 32D-123 murine myeloid cell line and responds to TGF-ß1 with increased sensitivity (Fig. 1ACitation ; Ref. 32 ). As shown in Fig. 1, BCitation and C, expression of the v-src gene was only detected in 32D-src cell line. Therefore, we first examined whether 32D-src cells express higher levels of TGF-ß RII transcripts than the parental cell. In parental cells, a major ~5.0-kb message and a second mRNA species of ~8.0 kb are present. In the 32D-src cell line, the level of the ~5.0-kb message was not changed, but the ~8.0-kb transcript was induced 8-fold. Interestingly, a third species of ~6.0 kb is also present in 32D-src cell line but not in parental cells (Fig. 1B)Citation . We did not find the reason why these transcripts with the sizes of 6 and 8 kb are detected in the 32D-src cell line thus far. However, these results suggest that src protein may be involved in the transcriptional regulation of the TGF-ß RII gene.



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Fig. 1. Effects of v-Src on the expression of TGF-ß RII in myeloid cell lines. A, cross-linking of 125I-labeled TGF-ß1 to receptors on 32D-123 and 32D-src murine myeloid cell lines. Aliquots of 5 x 106 cells were suspended in binding buffer with 125I-labeled TGF-ß1 in the presence or absence of an excess of unlabeled TGF-ß1, followed by incubation with 100 µg/ml of disuccinimidyl suberate. Affinity-labeled sample were resuspended in lysis buffer and analyzed on SDS-PAGE. A 40-fold molar excess of unlabeled TGF-ß1 was added into the reaction. Numbers at the left of 32D-123 and at the right of 32D-src indicate the positions of molecular weight size markers. B, Northern analysis of the expression of TGF-ß RII gene. Poly(A)+ RNAs of 32D murine myeloid cell lines were isolated for Northern analysis (Lane 1, 32D-123; Lane 2, 32D-src cell line). A 1.5-kb fragment of RII gene was used to detect the mouse RII transcript. The actin gene was used as an internal control. A 1.5-kb fragment was used as a probe to show the expression of the v-src gene. C, Western blot analysis of v-Src protein (Mr 60,000) in 32D myeloid cell lines (Lane 1, 32D-123 cell line; Lane 2, 32D-src cell line). Protein extracts were separated by SDS-PAGE, and Western blot analysis was performed with mouse monoclonal anti-v-Src antibody.

 
Characterization of v-Src-responsive Elements in TGF-ß RII Promoter.
Previous characterization of the TGF-ß RII promoter region identified two discrete DNA binding sites (PRE1 and PRE2) for an AP1/ATF2-like transcription factor and an ets family transcription factor, respectively (26) . The 5'-flanking region of the human TGF-ß RII gene also contains an inverted CCAAT box. To characterize the elements responsible for induction by v-Src, several deletion mutants linked to a reporter gene were transiently transfected into 32D-123 and HepG2 (human hepatoblastoma) cells so that we could analyze their activities when cotransfected in the presence or absence of the v-Src expression vector (pMvsrc). Serial deletion mutants of TGF-ß RII promoter shown in Fig. 2ACitation were constructed in pGL2 reporter plasmid. Cotransfection with the v-Src expression vector increased expression of pTßRIIP-1670/+36-GL2luc about 23-fold in HepG2 cells. The pTßRIIP-219/+36-GL2luc was induced 13.5-fold by v-Src, and pTßRIIP-76/+36-GL2luc was still induced ~4.5-fold (Fig. 2A)Citation . These results indicate that the region from -219 to +36 contains multiple elements responsive to v-Src. To ensure that these observations were not unique to HepG2 cells, we also examined the expression of the deletion constructs containing v-src-responsive elements in 32D-123 cells. In 32D-123 cells, cotransfection with the v-Src expression vector also increased the activity of reporter gene of the plasmids, pTßRIIP-219/+2-CAT and pTßRIIP-+2/+36-CAT (Fig. 2B)Citation . These results demonstrate that v-Src activates the promoter of the TGF-ß RII receptor gene through cis-acting elements located in the regions from -219 to +36. To further characterize v-src-responsive elements in the regions from -219 to +36, serial deletion mutants were constructed into pGL3-basic vector for reporter gene assay (See "Materials and Methods") and transiently cotransfected with a v-Src expression vector into HepG2 cells. As shown in Fig. 2Citation C, expression of pTßRIIP-219/+36-luc was increased 30-fold by v-Src. Deletion of the PRE1 element containing an AP1/ATF2-like sequence (pTßRIIP-139/+36) caused a 50% reduction in reporter gene activity. When the promoter was further deleted to -100 (pTßRIIP-100/+36), a further 50% reduction in induction level was seen, whereas deletion of the region from -100 to -45, which contains the inverted CCAAT box, dramatically decreased expression of the reporter gene. However, expression of pTßRIIP-45/+36 plasmid was still induced ~7.5-fold by v-Src, and this region contains only the PRE2 element with ETS binding sites. These results indicate that the promoter of the TGF-ß RII gene contains multiple cis-acting elements responsible for the induction by v-Src and that the region containing the inverted CCAAT box (-100 to -45) plays a major role for the induction by v-Src. To confirm that elements other than the inverted CCAAT box in the TGF-ß RII promoter are involved in the v-Src-mediated transactivation, the region from -130 to -8 sequence, which contains CCAAT box, was deleted (plasmid pTßRIIP{Delta}-130/-8-luc). This plasmid contains only the PRE1 and PRE2 elements. As shown in Fig. 2Citation D, expression of pTßRIIP{Delta}-130/-8-luc was induced 11-fold, although the inverted CCAAT box was deleted. Taken together with other results, this suggests that v-Src induces TGF-ß RII promoter activity through multiple promoter elements including the PRE1, an inverted CCAAT box, and the PRE2.



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Fig. 2. Transactivation of the deletion mutants of the TGF-ß RII promoter by v-Src. A, schematic representation of TGF-ß RII promoter deletion is shown. Serial deletion mutants of the 5'-flanking region of the TGF-ß RII gene were ligated to luciferase gene (pGL2-basic vector) and cotransfected with v-Src expression vector (pMvsrc) into HepG2 cell. B, pTßRIIP-219/+2-CAT and pTßRIIP+2/+36-CAT, which contain v-src-responsive elements, were cotransfected with v-Src expression vector into the 32D-123 cell line, and cells were harvested after 48 h. The results show a representative CAT assay. C, a series of deletion mutants from the regions -219 to +36 of the RII gene were subcloned into the promoterless pGL3-basic vector and transiently cotransfected with v-Src expression vector into HepG2 cell. Numbers indicate the position relative to transcription start site. D, to characterize other v-Src-responsive elements except for the inverted CCAAT box, the inverted CCAAT region was deleted from pTßRIIP-219/+36 by PvuII restriction enzyme, generating pTßRIIP{Delta}-130/-8-luc. This construct was cotransfected with v-Src expression vector into HepG2 cell. Results from these experiments are averages of at least triplicate experiments of three independent transfections. Transfection efficiencies were normalized by ß-gal activity as described in "Materials and Methods."

 
v-Src Induces PRE1 Transcriptional Activity through an AP-1/ATF2-like Site.
Our results prompted us to examine the v-Src responsiveness of each element individually. To characterize the v-Src responsiveness in the PRE1, we generated chimeric constructs containing one copy of the PRE1 sequence of the TGF-ß RII promoter between -219 and -180 linked to the adenovirus E4 minimal promoter (-38/+38)-luciferase reporter construct. Mutated forms of the two nuclear protein recognition sequences within the PRE1 were also generated by nucleotide substitution as described in Table 1Citation . The M7 oligonucleotide contains a four base substitution of the AP1/ATF-2-like sequence of PRE1. In cotransfection assays, v-Src induced the transcription of control E4{Delta}38-luc construct 2.7-fold (Fig. 3A)Citation . However, v-Src up-regulated the transcription of wild-type construct (PRE1 WT-E4{Delta}38-luc) 21-fold. Mutation of nucleotides -203 to -200 slightly decreased the v-Src inducibility of the PRE1 transcriptional activity, whereas mutation of nucleotides -195 to -192 (PRE1 M7-E4{Delta}38-luc) abolished the effect of v-Src on PRE1 transcriptional activity (Fig. 3A)Citation . This result suggests that v-Src induced the activity of PRE1 through an AP1/ATF2-like sequence.


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Table 1 Double-stranded oligonucleotides

 


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Fig. 3. Identification of the first regulatory element (PRE1) required for v-Src induction of TGF-ß RII promoter. A, one copy of the PRE1 sequence from -219 to -180 was linked to adenovirus E4 minimal promoter (-38/+36)-luciferase reporter construct. WT, wild-type sequence. M5 and M7 possess the same sequences except for the 4-nucleotide substitution. M7 mutation contains the base substitution of AP1/ATF2-like sequence. These chimeric constructs were cotransfected with v-Src expression vector into HepG2 cell, which were harvested after 48 h and assayed for luciferase activity. Transfection efficiencies were normalized by ß-gal activity as described in "Materials and Methods." Results are averages of at least triplicate experiments of three independent transfections. B, electrophoretic mobility shift assay was performed with labeled PRE1 oligonucleotide and nuclear extracts of 32D myeloid cell lines. Competitions were performed with a 50-fold excess of the indicated unlabeled oligonucleotides. Lanes 1 and 6, free probe. Lanes 3–5 and Lanes 8–10, competition with unlabeled wild-type and mutant sequences in both cell lines. In Lanes 2 and 7, competitors were not added into the binding reaction. Arrows, specific bands increased by v-Src protein.

 
Expression of v-Src Is Associated with Increased Interaction of Complexes with PRE1.
We have described previously two discrete nuclear protein complexes that interact specifically with target sequences within PRE1 (26) . To determine whether v-Src expression affects interaction of transcription factor complexes, EMSA was performed using a labeled PRE1 probe and nuclear extracts from both 32D-123 and 32D-src cells. As shown in Fig. 3Citation B, interaction of complexes a, b, and c is markedly increased in v-Src-expressing 32D-src cells compared with control. Lanes 2 and 7 represent reaction mixtures containing 5'end-labeled oligonucleotide representing PRE1 (-219 to -172) incubated with 10 µg of purified 32D-123 or 32D-src nuclear extract, respectively. Lanes 3 and 8 demonstrate specificity of binding and represent identical reaction mixtures in competition with unlabeled PRE1 oligonucleotide. Previous characterization of the PRE1 identified two discrete DNA binding protein complexes, an AP-1/ATF2-like transcription factor complex and a putative novel transcription factor complex (26) . To determine whether these two protein complexes were similarly present in 32D murine myeloid cells, EMSA was performed using a radiolabeled PRE1 probe incubated with 32D-123 and 32D-src nuclear extracts with the M5 or M7 mutant oligonucleotides for the competition of binding. M7 mutant oligonucleotides were unable to compete complexes a and b (Fig. 3Citation B, Lane 9). Because M7 mutation contains an AP-1/ATF2 binding site, the results of competition assay demonstrate that this sequence and its binding factor(s) are responsible for the induction of PRE1 element by v-Src protein. To identify the protein bound to the AP-1/ATF2-like site, we performed antibody supershift assay with c-Jun antibody and ATF2 antibody commercially available. However, we did not clearly detect the supershift band (data not shown). The reason is likely to be attributable to the weak intensity of complexes a and b, although these complexes were enriched in 32D-src nuclear extract.

The Transcription Factor NF-Y Complex Increases the Activity of TGF-ß RII Promoter in v-src-transformed Cell Line.
To determine whether the inverted CCAAT box is involved in the up-regulation of the TGF-ß RII promoter by v-Src, site-directed mutations were introduced (5'-ATTGG-3' to 5'-AGGTT-3') in the inverted CCAAT box sequence of pTßRII-132/+2, which does not contain either PRE1 or PRE2 elements. We also generated the right orientation of CCAAT box (5'-ATTGG-3' to 5'-CCAAT-3'). v-Src markedly induced the luciferase activity of the original, inverted CCAAT box construct, whereas mutation resulted in a decrease in luciferase activity of the construct (Fig. 4A)Citation . Interestingly, v-Src further activated the luciferase activity of the right orientation of CCAAT box construct compared with the inverted CCAAT box construct.



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Fig. 4. Identification of the inverted CCAAT box required for v-Src induction of the TGF-ß RII promoter. A, site-directed mutations were introduced in the inverted CCAAT sequence of pTßRIIP-132/+2, which does not contain both PRE1 and PRE2 elements. WT, wild-type sequence. R, right orientation of CCAAT box; M, change of the inverted CCAAT sequence to AGGTT. These constructs were cotransfected with v-Src expression vector into HepG2 cell, harvested after 48 h and assayed for luciferase activity. Results are averages of at least triplicate experiments of three independent transfections. ß-gal expression vector (pRSV-ßgal) was also cotransfected to normalize the transfection efficiencies. B, electrophoretic mobility shift assay was performed with labeled oligonucleotide containing the inverted CCAAT box (-100 to -67; Table 1Citation ) and nuclear extracts of 32D myeloid cell lines. Competitions were performed with a 75-fold molar excess of the indicated unlabeled oligonucleotides. Unlabeled oligonucleotide from -100 to -67 was used as specific competitor (S). Oligonucleotides of PRE2 elements (+2 to +36) was used as nonspecific competitor (N). Lane 1, free probe. Lanes 3–7, competition with unlabeled wild-type and nonspecific oligonucleotides in both cell lines. Arrows, specific bands in both cell lines. C, for antibody supershift assay, the oligonucleotides containing the inverted CCAAT box were incubated either with 10 µg of nuclear extracts of 32D myeloid cell lines alone (Lanes 2 and 7), with normal goat IgG (Lanes 3 and 8), with 0.5 µg of anti-NF-YB (Lanes 4 and 9), or with 0.5 µg of anti-NF-YB and 0.5 µg of competitor peptide against NF-YB antibody (Lanes 5 and 10), respectively. Supershifts with anti-NF-YB antibody are shown as indicated.

 
To identify nuclear components that might mediate v-Src effects on TGF-ß RII transcription, EMSA was performed using nuclear extracts from both 32D-123 and 32D-src cells and a double-stranded 32P-labeled oligonucleotide containing the sequence for the inverted CCAAT box (CCAAT-WT). The reaction mixture was then electrophoresed on a polyacrylamide gel and viewed by autoradiography. The results are shown in Fig. 4BCitation . Two nuclear protein complexes (a and b) from 32D-123 cells interacted specifically with a 34-mer oligonucleotide probe of the region around the CCAAT box (Fig. 4B)Citation . Interestingly, these protein complexes are not present in the nuclear extract of 32D-src cells. In 32D-src cells, two major complexes (c and d), distinct from a and b, were revealed. Complexes c and d showed lower mobility and higher intensity than complexes a and b. The results from competition assays show that these complexes are specific to the CCAAT box (Fig. 4B)Citation .

To identify which CCAAT box-binding transcription factor from 32D murine myeloid cells recognizes the CCAAT box of the TGF-ß RII promoter, antibodies against NF-YB was used in the EMSA assay. The NF-YB antibody was found to selectively supershift the a complex from the 32D-123 nuclear extract and the c complex from the 32D-src nuclear extract (Fig. 4C)Citation . Addition of competitor peptide against the NF-YB antibody abolished the supershifted band, demonstrating the specificity of the supershift assay (Fig. 4C)Citation . The antibody against the other CCAAT box binding protein, C/EBP, did not show any shifted band (data not shown). NF-Y is a heterotrimeric complex made up of three subunits (A, B, and C) that are collectively required for DNA binding. Consequently, the a and c complexes likely represent the NF-Y protein bound to the CCAAT boxes of the TGF-ß RII promoter. The b and d complexes were not changed by supershift assay, indicating that these complexes are formed by other nuclear protein(s). The gel shift assay shows that the c complex has lower mobility than the a complex, suggesting that the c complex might contain another unknown component interacting with the NF-Y components, or that the NF-Y components are posttranslationally modified in the 32D-src cell line.

Induction of the PRE2 Element by v-Src Is Dependent on the Nuclear Proteins Interacting with ETS Binding Sites.
Because the pTßRII-45/+36 construct was activated by the v-src gene product, we further characterized the sequences of this construct responsible for v-Src transactivation. Plasmids PRE2WT-E4{Delta}38-luc and PRE2M4-E4{Delta}38-luc were generated by inserting double-stranded oligonucleotides into the E4{Delta}38-luc vector. We have shown previously that mutation of nucleotides +16 to +20 (AAGTG, M4) abolishes the competition for binding to the PRE2, and that an epithelial-specific ets-related transcription factor, ERT, cannot induce the PRE2 promoter activity when these sequences are mutated (26 , 27) . The level of luciferase expression by the PRE2 WT-E4{Delta}38-luc construct was increased 11-fold by the v-src gene, whereas there was no induction of the PRE2 M4-E4{Delta}38-luc construct (Fig. 5A)Citation . These results strongly suggest that the v-src gene product induces an element(s) that binds to the ETS binding site of the TGF-ß RII promoter.



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Fig. 5. Identification of the second regulatory element (PRE2) required for v-Src induction of TGF-ß RII promoter. A, one copy of the PRE2 sequence from +2 to +36 was linked to adenovirus E4 minimal promoter (-38/+36)-luciferase reporter construct. WT, wild-type sequence. M4 possesses the same sequence except for the 5-nucleotide substitution of ETS binding site (+16 to +20). These chimeric constructs were cotransfected with v-Src expression vector into HepG2 cell, which were harvested after 48 h and assayed for luciferase. Results are averages of at least triplicate experiments of three independent transfections. ßgal expression vector (pRSV-ß-gal) was also cotransfected to normalize the transfection efficiencies. B, EMSA was performed with labeled PRE2 oligonucleotide and nuclear extracts of 32D myeloid cell lines. Competitions were performed with a 80-fold molar excess of the indicated unlabeled oligonucleotides. Lanes 1 and 6, free probe. Lanes 3–5 and Lanes 8–10, competition with unlabeled wild-type (S), nonspecific oligonucleotide (N), and M4 mutant sequence (M4) in two cell lines, respectively. Competitors were not added in Lanes 2 and 7. Unlabeled PRE1 element was used as nonspecific competitor. Arrows, specific bands in both cell lines. C, for antibody supershift assay, the oligonucleotide containing PRE2 element was incubated either with nuclear extracts alone of the 32D-src cell line (Lane 2), with normal rabbit IgG (Lane 3), with 0.5 µg anti-PU.1 antibody (Lane 4), or with 0.5 µg of anti-PU.1 antibody and 0.5 µg of competitor peptide against anti-PU.1 antibody, respectively. Supershifts with anti-PU.1 antibody are shown as indicated. PU.1 antibody, which is made against the DNA binding domain of the PU.1 protein, was purchased from Santa Cruz Biotechnology, Inc.

 
To see whether the level of PRE2 binding proteins is regulated by the v-src gene product, we performed the EMSA with 32P-labeled PRE2 using 32D-123 and 32D-src nuclear extracts (Fig. 5B)Citation . In 32D-123 extracts, one strong lower band (complex c) and two moderate bands (complexes a and b) appeared and represented specific binding because these bands disappeared with the addition of unlabeled competitor. However, in 32D-src nuclear extracts, the level of complex d, which it was little detected in the 32D-123 nuclear extract, was markedly increased. There were also several extra complexes (e, g, h, and i) in the 32D-src nuclear extracts compared with the 32D-123 nuclear extracts. The intensity of complex c in the 32D-123 nuclear extracts was significantly decreased in the 32D-src nuclear extracts (complex j). PRE2 M4 oligonucleotides did not compete for binding to the PRE2, suggesting that the v-src gene product induces expression of nuclear proteins interacting with ETS binding sites in the PRE2 of the TGF-ß RII promoter.

PU.1 is a transcription factor that is a member of the Ets family of proteins and is expressed specifically in myeloid and B-lymphoid cells of the hematopoietic system (33) . To examine whether PU.1 is one of nuclear proteins interacting with PRE2 element, we performed antibody supershift assay with PU.1 antibody. Supershift assay shows that complex g, h, i, and j are formed by PU.1 protein, not complex d in the 32D-src cell line (Fig. 5C)Citation . Because PU.1 antibody in this assay is made against the DNA binding domain of PU.1 protein, DNA-protein complexes are not formed in the supershift assay. Addition of competitor peptide against anti-PU.1 antibody revealed DNA-protein complexes again (Fig. 5C)Citation . In the 32D-123 parental cell line, complex c was not formed by addition of PU.1 antibody (data not shown). These results indicate that complex d, showing strong intensity in the 32D-src cell line, might be a novel Ets binding protein induced by v-Src protein, not PU.1 protein.

Discussion

The murine myeloid progenitor cell lines, the parental IL-3-dependent 32D-123 cells, and the v-src-transformed, IL-3-independent 32D-src cells have markedly different responses to growth inhibition by TGF-ß1, with 32D-src cells being 58-fold more sensitive than the parental cell line (32) . Cross-linking studies have shown that 32D-src cells express higher numbers of TGF-ß receptors than 32D-123 cells. To better understand the differences in the responsiveness of these cell lines to TGF-ß1, we examined the level of TGF-ß RII transcripts. The level of the ~8.0-kb species was markedly induced, and a new ~6.0-kb species appeared in 32D-src cells compared with the parental cells, suggesting that the v-src gene product may regulate expression of the TGF-ß RII gene transcriptionally.

We have identified previously two PREs (PRE1 and PRE2) in the promoter of TGF-ß RII. Deletional analysis suggests that the v-src gene product induces TGF-ß RII promoter activity through PRE1 and PRE2, as well as the inverted CCAAT box. PRE1 (-219 to -172) interacts with two distinct nuclear protein complexes from HepG2 cells, at least one of which appears to be an AP-1 or ATF2 protein. Nuclear extracts isolated from 32D-123 or 32D-src cells contain at least five protein complexes interacting with the PRE1. The intensity of the protein complexes in the 32D-src nuclear extracts (a and b) is much higher than in the 32D-123 nuclear extracts, suggesting that 32D-src induces expression of proteins in these complexes. These complexes were unable to compete with M7 mutation containing the mutation of the AP-1/ATF2 sequence (Fig. 3B)Citation . Therefore, our results suggest the possibility that v-Src directly or indirectly stimulates the binding proteins for the AP-1/ATF2-like sequence to induce its transcriptional activity.

It has been shown that v-Src can stimulate gene transcription through the CCAAT box. The mouse osteopontin promoter is also stimulated by v-Src through a CCAAT box-binding factor (34) . Kim and Sodek (34) have also demonstrated that v-Src increased the transcriptional activity of rat bone sialoprotein through a mechanism mediated by the NF-Y transcription factor, which targets an inverted CCAAT box. They were unable to demonstrate any significant differences in the amount of NF-Y that bound to the CCAAT box in nuclear extracts of src -/- and src +/- cells, nor were the levels significantly elevated or suppressed in cells transfected with v-Src and c-Src expression vector. In the present study, we have demonstrated that the binding of NF-Y components is dramatically increased in 32D-src nuclear extracts compared with the 32D-123 nuclear extracts. However, we do not know whether v-Src stimulates transcription of NF-Y components or activates NF-Y components posttranslationally. Because the NF-Y complex in 32D-src cells forms larger complexes than those in 32D-123 cells, it is possible that v-Src induces a protein that forms a complex with NF-Y components.

In a previous study, we demonstrated that PRE2 contained at least one nuclear protein recognition sequence from +11 to +29 (26) . This region contains two direct repeats of the purine-rich sequences (GGAAAC) in a reverse orientation. These purine-rich sequences are the binding sites for the ets family of transcription factors. We have isolated an epithelial-specific, ets family member, ERT/ESX/ESE, which is a potent transcriptional activator of the TGF-ß RII gene (27) . Expression of exogenous ERT increases the level of transcription from the TGF-ß RII promoter, implying an activating role for ERT in TGF-ß RII expression. The expression patterns of different members of the ets gene family vary between tissues (35, 36, 37) . Many members of this family are expressed in hematopoietic cells, suggesting a role for these members of the ets family in hematopoietic cell growth and differentiation (33 , 38) . All of the ets family genes (39, 40, 41, 42, 43) , with the exception of yan (44) and ERF (45) , are known to be potent transcriptional transactivators. v-Src also induces TGF-ß RII promoter activity through the PRE2 element. Mutation of the ETS binding sites abolishes the v-Src activation of PRE2 activity (Fig. 5)Citation , suggesting that v-Src stimulates the PRE2 enhancer activity of the TGF-ß RII gene through the ETS binding sites. Our preliminary results show that other ets family members, including Ets-1 and Ets-2, also induce TGF-ß RII promoter activity, suggesting that ets family members may be one of the major transcription factors involved in regulation of TGF-ß RII gene expression. Because 32D-src cells express high levels of nuclear proteins interacting with the PRE2 and these complexes are specific to the ETS binding site, it is possible that v-Src stimulates expression of distinct ets family members expressed in mouse myeloid cells. Antibody supershift assay with PU.1 antibody indicates strongly that complex d, showing strong intensity in EMSA with PRE2 element, may be a novel Ets binding protein specifically induced by v-Src protein in myeloid cell line because complex d was not shifted by anti-PU.1 antibody (Fig. 5C)Citation .

In summary, we have shown that v-Src induces activity of the TGF-ß RII gene through the PRE1, PRE2, and the CCAAT elements, supporting our hypothesis that the enhanced activity of TGF-ß of these cells may result primarily from direct effects of v-Src on transcriptional regulation of the TGF-ß RII gene. It is noteworthy, however, that these sites by themselves are not sufficient to activate the promoter fully, because mutational analysis reveals that the first PRE (PRE1) of the TGF-ß RII promoter cooperates with the second PRE (PRE2) to sustain basal levels of promoter activity (26) . Our findings suggest that several enhancer elements and transcription factors may contribute to the increased expression of TGF-ß RII upon v-Src transformation and the possibility that v-Src induces novel ETS binding proteins in myeloid cell lines (Fig. 6)Citation .



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Fig. 6. Schematic representation of the multiple v-Src regulatory elements within the TGF-ß RII promoter. Relative positions of the three v-Src regulatory elements have been mapped to the regions shown. PRE1 and PRE2 indicate the first and second PREs, respectively. Arrow, +1 transcriptional start site.

 
Materials and Methods

Cell Culture, Transfections, and Reporter Assays.
The 32D-123 parental cell line and 32D-src cell line were cultured as described (32 , 46 , 47) . HepG2 cells were grown in minimal medium supplemented with 10% fetal bovine serum. For transient transfections, 2 x 105 cells were plated in each well of a six-well dish and transfected using Lipofectin reagent (Life Technologies, Inc.) according to manufacturer’s protocol. Cells were harvested 40–48 h after transfection. Luciferase assays were performed with commercially available reagents. All test plasmids were analyzed in triplicate cultures in at least three separate experiments. ß-gal expression vector (pRSV-ßgal) was cotransfected to normalize the transfection efficiencies. Transfection efficiencies were measured by ßgal activity in cell extracts as described previously (48) .

Cross-Linking Assay of 125I-TGF-ß1 to Receptors and Western Blot Analysis.
Affinity binding and cross-linking of 125I-labeled TGF-ß1 to cells were performed as described previously (32) . 125I-labeled TGF-ß1 (specific activity, 82–150 µCi/µg) was purchased from Biomedical Technology. To show the expression of v-Src protein, protein extracts were separated by 4–20% SDS-PAGE, and Western blot analysis was performed with mouse monoclonal anti-v-Src antibody, which is purchased from Oncogene Science. v-Src protein was visualized by chemiluminescence detection according to manufacturer’s protocol (Pierce).

Northern Blot Analysis.
Poly(A)+ RNAs of myeloid cell lines (32D-123 and 32D-src) were isolated by Oligotex mRNA kit purchased from Qiagen. For Northern blot analysis, RNA was electrophoresed on 1.0% agarose-formaldehyde gel and transferred to nylon membrane. To detect the mouse type II receptor transcript, a 1.5-kb fragment of human TGF-ß RII gene were labeled by PRIME-IT random primer labeling kit purchased from Stratagene.

Plasmids and Site-directed Mutagenesis.
Deletion mutants of TßRII promoter shown in Fig. 2ACitation were cloned into the luciferase plasmid (pGL2) using BglII and SacI restriction sites, whereas the mutants in Fig. 2CCitation were cloned into the promoterless luciferase vector (pGL3-basic) using HindIII and SacI sites. The sequences of the PCR-generated portions of all constructs were verified by DNA sequencing. To generate pTßRII{Delta}-130/-8-luc, the region containing the inverted CCAAT box (-81 to -77) was deleted from pTßRII-219/+36-luc by restriction enzyme PvuII. Double-stranded oligonucleotides of PRE1(-219/-172) and PRE2 (+2/+36) were cloned into NheI and XhoI sites of adenovirus E4 minimal promoter (-38/+38)-luciferase reporter construct. Oligonucleotides of PRE1, invert CCAAT box, and PRE2 used in this study were described in Table 1Citation . Site-directed mutagenesis of the inverted CCAAT box was performed by QuickChange site-direct mutagenesis kit purchased from Stratagene. Oligomers for site-directed mutagenesis of the inverted CCAAT box were described in Table 1Citation . The mutations of the inverted CCAAT box were also verified by DNA sequencing.

Nuclear Extracts.
Nuclear extracts of 32D-123 and 32D-src cell lines were prepared as described (26) with minor modification. Cells were harvested by centrifugation, washed in cold PBS, and incubated in two packed cell volumes of buffer A [10 mM HEPES (pH 8.0), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiotreitol, 1 mM phenylmethanesulfonyl fluoride, 1x protease inhibitor mix (Sigma), and 0.5% NP-40] for 5 min at 4°C. The crude nuclei released by lysis were collected by microcentrifugation, rinsed twice in buffer A, resuspended in two-thirds packed cell volume of buffer C [20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 1 mM phenylmethanesulfonyl fluoride, and 1x protease inhibitor mix]. Nuclei were incubated on a rocking platform at 4°C for 30 min and clarified by microcentrifugation for 5 min. The resulting supernatants were diluted 1:1 with buffer D [20 mM HEPES (pH 7.9), 100 mM KCl, 0.2 mM EDTA, 20% glycerol, 1 mM DTT, 1 mM phenylmethanesulfonyl fluoride, and 1x protease inhibitor mix].

EMSA and Supershift Antibody.
Three oligonucleotides, PRE1 (-219/-172), CCAAT box region (-100/-67: CCAAT-WT), and PRE2 (+2/+36), were labeled with [{gamma}-32P]ATP and polynucleotide kinase. The fragments were then gel purified using 10% polyacrylamide gel. Binding reactions contained 10 µg of nuclear extract protein, buffer [10 mM Tris (pH 7.5), 50 mM NaCl, 1 mM DTT, 1 mM EDTA, and 5% glycerol], 1.5 or 2 µg of poly(deoxyinosinic-deoxycytidylic acid), and 30,000 cpm of 32P-labeled DNA in a volume of 20 µl. Reactions were incubated at room temperature for 20 min. For competition assay, unlabeled oligonucleotides were added to binding reactions as specific competitor. Binding reactions were electrophoresed on a 6% nondenaturing polyacrylamide gel at 180 V for 2 h in 0.5x TBE buffer and subjected to autoradiography. For antibody supershift assay, the reactions were performed by preincubating nuclear extracts with 0.5 µg of antibody at 4°C for 1 h. After addition of the labeled oligonucleotide and 20 min of incubation at room temperature, the products were resolved by 6% nondenaturating PAGE. The anti-NF-YB and anti-PU.1 antibodies were purchased from Santa Cruz Biotechnology, Inc. To show the specificity of antibody supershift assay, competitor peptide for anti-PU.1 antibody was purchased from Santa Cruz Biotechnology and competitor peptide for anti-NF-YB antibody was synthesized form American Peptide Company based on the sequence of COOH-terminal region of NF-YB protein.

Acknowledgments

We thank Dr. Anita B. Roberts for helpful discussion and critical review of manuscript and Cecile Lee and Danielle Poulin for excellent editorial assistance.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, Building 41, Room B1106, Bethesda, MD 20892. Phone: (301) 496-8350; Fax: (301) 496-8395; E-mail: kims{at}dce41.nci.nih.gov Back

2 The abbreviations used are: TGF-ß RII, transforming growth factor-ß type II receptor; EMSA, electrophoretic mobility shift assay; IL, interleukin; PRE, positive regulatory element; ERT, ets-related transcription; ß-gal, ß-galactosidase. Back

Received for publication 8/ 1/00. Revision received 11/15/00. Accepted for publication 11/16/00.

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