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• W2115178051 abstract "Transforming growth factor type β (TGFβ) is a pleiotropic factor that regulates different cellular activities including cell growth, differentiation, and extracellular matrix deposition. All the known effects of TGFβ appear to be mediated by its interaction with cell surface receptors that possess a serine/threonine kinase activity. However, the intracellular signals that follow receptor activation and lead to the different cellular responses to TGFβ are still largely unknown. On the basis of the different sensitivity to the protein kinase inhibitor 2-aminopurine and the phosphatase inhibitor okadaic acid, we identified two distinct pathways through which TGFβ activates a genomic response. Consistently, 2-aminopurine prevented and okadaic acid potentiated the induction of JE by TGFβ. The induction of PAI-1 and junB was instead potentiated by 2-aminopurine, after a transient inhibition and was unaffected by okadaic acid. The superinducing effect of 2-aminopurine required the presence of a functional RB protein since it was abolished in SV40 large T antigen-transfected cells, absent in the BT549 and Saos-2 RB-defective cell lines, and restored in BT549 and Saos-2 cells after reintroduction of pRB. The effects of 2-aminopurine on the TGFβ inducible junB expression occur in all the cell lines examined suggesting that junB, and possibly other genes, can be regulated by TGFβ through a distinct pRB-dependent pathway. Transforming growth factor type β (TGFβ) is a pleiotropic factor that regulates different cellular activities including cell growth, differentiation, and extracellular matrix deposition. All the known effects of TGFβ appear to be mediated by its interaction with cell surface receptors that possess a serine/threonine kinase activity. However, the intracellular signals that follow receptor activation and lead to the different cellular responses to TGFβ are still largely unknown. On the basis of the different sensitivity to the protein kinase inhibitor 2-aminopurine and the phosphatase inhibitor okadaic acid, we identified two distinct pathways through which TGFβ activates a genomic response. Consistently, 2-aminopurine prevented and okadaic acid potentiated the induction of JE by TGFβ. The induction of PAI-1 and junB was instead potentiated by 2-aminopurine, after a transient inhibition and was unaffected by okadaic acid. The superinducing effect of 2-aminopurine required the presence of a functional RB protein since it was abolished in SV40 large T antigen-transfected cells, absent in the BT549 and Saos-2 RB-defective cell lines, and restored in BT549 and Saos-2 cells after reintroduction of pRB. The effects of 2-aminopurine on the TGFβ inducible junB expression occur in all the cell lines examined suggesting that junB, and possibly other genes, can be regulated by TGFβ through a distinct pRB-dependent pathway. The three mammalian isoforms of the transforming growth factor type β (TGFβ1, TGFβ2, and TGFβ3), 1The abbreviations used are: TGFβtransforming growth factor type βpRBretinoblastoma susceptibility gene product2-AP2-aminopurineTPAphorbol esterFACSfluorescence-activated cell sorter. belong to a superfamily of related polypeptides involved in the control of a large number of biological activities, including cell growth, differentiation, and development (1Roberts A.B. Sporn M.B. Sporn M.B. Roberts A.B. Handbook of Experimental Pharmacology, Peptide Growth Factors and Their Receptors. Springer, Heidelberg1990: 419-431Google Scholar). Originally identified as a factor able to induce growth of normal rat kidney fibroblast in soft agar, TGFβ was subsequently shown to be a potent growth inhibitor for most epithelial cells (1Roberts A.B. Sporn M.B. Sporn M.B. Roberts A.B. Handbook of Experimental Pharmacology, Peptide Growth Factors and Their Receptors. Springer, Heidelberg1990: 419-431Google Scholar). transforming growth factor type β retinoblastoma susceptibility gene product 2-aminopurine phorbol ester fluorescence-activated cell sorter. Most of the reported actions of TGFβ were shown to be dependent on its binding to at least two specific membrane-bound proteins, each belonging to a recently discovered family of serine/threonine kinase receptors (2Kingsley D.M. Genes Dev. 1994; 8: 133-146Google Scholar, 3Segarini P.R. Biochim. Biophys. Acta. 1994; 1155: 269-275Google Scholar), that are active as hetero-oligomeric complexes (4Moustakas A. Lin H.Y. Henis Y.A. Plamondon J. O'Connor-McCourt M.D. Lodish H.F. J. Biol. Chem. 1993; 268: 22215-22218Google Scholar, 5Yamashita H. ten Dijke P. Franzen P. Miyazono K. Heldin C-H. J. Biol. Chem. 1994; 269: 20172-20178Google Scholar). Although a few candidate transducing molecules have been identified (6Chen R-H. Miettinen P.J. Maruoka E.M. Choy L. Derynck R. Nature. 1995; 377: 548-552Google Scholar, 7Wang T. Donahoe P.K. Zervos A.S. Science. 1994; 265: 674-676Google Scholar), the biochemical pathways acting downstream of these receptors are still largely uncharacterized. With few exceptions (8Ong G. Sikora K. Gullick W. Oncogene. 1991; 6: 761-763Google Scholar), in epithelial cells TGFβ-mediated growth inhibition correlates with the G1 inhibition of the phosphorylation of the retinoblastoma gene product, pRB (1Roberts A.B. Sporn M.B. Sporn M.B. Roberts A.B. Handbook of Experimental Pharmacology, Peptide Growth Factors and Their Receptors. Springer, Heidelberg1990: 419-431Google Scholar, 9Laiho M. DeCaprio J.A. Ludlow J.W. Livingston D.M. Massague J. Cell. 1990; 62: 175-185Google Scholar). Several events contribute to preventing pRB phosphorylation in TGFβ-treated cells: suppression of CDK4 synthesis (10Ewen M.E. Sluss H.K. Whitehouse L.L. Livingston D.M. Cell. 1993; 74: 1009-1020Google Scholar), down-regulation of cyclins and cdks expression (11Geng Y. Weinberg R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10315-10319Google Scholar), inhibition of cycE-cdk2 complexes by p27Kip1 binding (12Koff A. Ohtsuki M. Polyak K. Roberts J.M. Massague J. Science. 1993; 260: 536-539Google Scholar); induction of p21CIP1, and p15Ink4B with consequent inhibition of cdk4 and cdk6 kinases (13Datto M.B. Li Y. Panus J.F. Howe D.J. Xiong Y. Wang X-F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5545-5549Google Scholar, 14Reynisdottir I. Polyak K. Iavarone A. Massague J. Genes Dev. 1995; 9: 1831-1845Google Scholar). Most of the other cellular responses to TGFβ, including control of extracellular matrix protein deposition, wound healing, and immune suppression are proposed to be mediated by controlling the expression of specific genes (1Roberts A.B. Sporn M.B. Sporn M.B. Roberts A.B. Handbook of Experimental Pharmacology, Peptide Growth Factors and Their Receptors. Springer, Heidelberg1990: 419-431Google Scholar). In many cases this is regulated at the transcriptional level through the binding of specific transcription factors to stimulatory sequences, as in the case of PAI-1 (15Chang E. Goldberg H. J. Biol. Chem. 1995; 270: 4473-4477Google Scholar), JE (16Takeshita A. Chen Y. Watanabe A. Kitano S. Hanazawa S. J. Immunol. 1995; 155: 419-426Google Scholar), p21CIP1 (17Datto M.B. Yu Y. Wang X-F. J. Biol. Chem. 1995; 270: 28623-28628Google Scholar), p15Ink4B (18Li J-M. Nichols M.A. Chandrasekharan S. Xiong Y. Wang X-F. J. Biol. Chem. 1995; 270: 26750-26753Google Scholar), and α2(I) collagen (15Chang E. Goldberg H. J. Biol. Chem. 1995; 270: 4473-4477Google Scholar, 19Rossi P. Karsenty G. Roberts A.B. Roche N.S. Sporn M.B. de Crombrugghe B. Cell. 1988; 52: 405-414Google Scholar) gene regulation. The expression of other genes appears to be regulated through TGFβ inhibitory sequences (20Kerr L.D. Miller D.B. Matrisian L.M. Cell. 1990; 61: 267-278Google Scholar–22Pientepol J.A. Munger K. Howley P.M. Stein R.W. Moses H.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10227-10231Google Scholar). In the case of c-myc the same promoter region, the TGFβ control element, is required for down-regulation of c-myc by both TGFβ and the retinoblastoma protein (21Pientepol J.A. Stein R.W. Moran E. Yaciuk P. Schlegel R. Lyons R.M. Pittelkow M.R. Munger K. Howley P.M. Moses H.L. Cell. 1990; 61: 777-785Google Scholar, 22Pientepol J.A. Munger K. Howley P.M. Stein R.W. Moses H.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10227-10231Google Scholar). Furthermore, pRB is required for TGFβ-dependent c-myc down-regulation and growth inhibition, in skin keratinocytes (21Pientepol J.A. Stein R.W. Moran E. Yaciuk P. Schlegel R. Lyons R.M. Pittelkow M.R. Munger K. Howley P.M. Moses H.L. Cell. 1990; 61: 777-785Google Scholar) and for down-regulation of N-myc in embryonic lung organ cultures (23Serra R. Moses H.L. Development. 1995; 121: 3057-3066Google Scholar). In contrast, in Mv1Lu cells, pRB appears not to be required for PAI-1, junB, and fibronectin induction by TGFβ (24Laiho M. Ronnstrand L. Heino J. DeCaprio J.A. Ludlow J.W. Livingston D.M. Massague J. Mol. Cell. Biol. 1991; 11: 972-978Google Scholar, 25Zentella A. Weis F.M.B. Ralph D.A. Laiho M. Massague J. Mol. Cell Biol. 1991; 11: 4952-4958Google Scholar). 2-Aminopurine (2-AP) is a serine/threonine protein kinase inhibitor initially described for its ability to inhibit the double-stranded RNA-dependent protein kinase (26Zinn K. Keller A. Whittmore L-A. Maniatis T. Science. 1988; 240: 210-213Google Scholar). It was also shown to inhibit expression of interferon-induced genes, to block serum and platelet-derived growth factor induction of c-fos and c-myc (27Tiwari R.K. Kusari J. Kumar R. Sen G.C. Mol. Cell. Biol. 1988; 8: 4289-4294Google Scholar, 28Mahadevan L.C. Wills A.J. Hirst E.A. Rathjen P.D. Heat J.K. Oncogene. 1990; 5: 327-335Google Scholar) and to modulate the expression of a group of genes specifically expressed in growth-arrested cells (29Ciccarelli C. Philipson L. Sorrentino V. Mol. Cell. Biol. 1990; 10: 1525-1529Google Scholar). Okadaic acid is a fatty acid that has tumor promoter activity on mouse skin (30Cohen P. Holmes C.F.B. Tsukitani Y. Trends Biochem. Sci. 1990; 15: 98-102Google Scholar); however, it does not activate or bind protein kinase C. It is, on the contrary, a potent inhibitor of protein phosphatases 1 and 2A (30Cohen P. Holmes C.F.B. Tsukitani Y. Trends Biochem. Sci. 1990; 15: 98-102Google Scholar). Although their broad and still not completely characterized activity may limit the interpretation of the results obtained by their means, 2-AP and okadaic acid have successfully been employed for testing the involvement of protein kinases and phosphatases in several signal transduction pathways (26Zinn K. Keller A. Whittmore L-A. Maniatis T. Science. 1988; 240: 210-213Google Scholar–30Cohen P. Holmes C.F.B. Tsukitani Y. Trends Biochem. Sci. 1990; 15: 98-102Google Scholar). To better understand the role of post-translational modifications, such as phosphorylation and dephosphorylation of pre-existing proteins, on the signal transduction pathway(s) activated by TGFβ we have analyzed the effect of 2-AP and okadaic acid on the activation of gene expression and the inhibition of cell proliferation induced by TGFβ. In particular we report here on the regulation of three genes involved in different biological effects initiated by TGFβ: (i) JE, a monocyte chemoattractant (16Takeshita A. Chen Y. Watanabe A. Kitano S. Hanazawa S. J. Immunol. 1995; 155: 419-426Google Scholar); (ii) PAI-1, whose induction by TGFβ is an important step in the control of extracellular matrix deposition (1Roberts A.B. Sporn M.B. Sporn M.B. Roberts A.B. Handbook of Experimental Pharmacology, Peptide Growth Factors and Their Receptors. Springer, Heidelberg1990: 419-431Google Scholar, 15Chang E. Goldberg H. J. Biol. Chem. 1995; 270: 4473-4477Google Scholar); and (iii) junB, an early responsive gene involved in the control of growth and differentiation which is regulated by TGFβ in most cell types (1Roberts A.B. Sporn M.B. Sporn M.B. Roberts A.B. Handbook of Experimental Pharmacology, Peptide Growth Factors and Their Receptors. Springer, Heidelberg1990: 419-431Google Scholar). The effects of 2-AP on TGFβ-induced pRB dephosphorylation and growth inhibition were also investigated. According to the sensitivity to 2-AP and okadaic acid we identified two different pathways through which TGFβ stimulates gene expression. 2-AP, which is able to prevent TGFβ-inducible expression of several genes, potentiates the induction of PAI-1 and junB by TGFβ, after a transient inhibition. In addition we show that potentiation of TGFβ-inducible junB expression by 2-AP requires a functional RB protein. Therefore 2-AP unravels a role for pRB in a distinct pathway leading to the up-regulation of junB and possibly other genes by TGFβ. All the different cell types were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, 10 units/ml penicillin, and 10 units/ml streptomycin, with the only exception of Saos-2 requiring RPMI 1640 medium. pPVU-01.5.3, Saos-2 #84, Saos-2 #1, and BTB5V4-RB were cultured in the presence of Geneticin or Geneticin and hygromicin as specified elsewhere (9Laiho M. DeCaprio J.A. Ludlow J.W. Livingston D.M. Massague J. Cell. 1990; 62: 175-185Google Scholar, 31Gjetting T. Lukas J. Bartek J. Strauss M. Biol. Chem. Hoppe Seyler. 1995; 376: 441-446Google Scholar, 32Fung Y-K.T. T'Ang A. Linn Murphree A. Zhang F-H. Qui W-R. Wang S-W. Shi X-H. Lee L. Driscoll B. Wu K-J. Oncogene. 1993; 8: 2659-2672Google Scholar). For stimulation studies, unless differently specified, cells were grown to subconfluency and then transferred in serum-free medium containing 10 μg/ml bovine serum albumin and incubated with TGFβ1 (Sigma; 1-5 ng/ml) and/or 2-AP (10 mM), cycloheximide (10 μg/ml), phorbol ester (TPA, 100 ng/ml), and okadaic acid (10 ng/ml). Cells were washed twice with ice-cold phosphate-buffered saline, lysed in guanidinium thiocyanate buffer, and total RNA was isolated by CsCl gradient centrifugation. 20 μg of total RNAs were denatured with formamide and formaldehyde, fractionated by denaturing agarose gel electrophoresis, and transferred to nylon Gene Screen Plus hybridization membranes (DuPont) by overnight blotting. Filters were hybridized overnight with 2 × 106 cpm of 32P-labeled DNA probes/ml. DNA probes were labeled by random priming to an efficiency of 0.5-1 × 109 cpm/μg. Filters were washed to a final concentration of 0.1 × SSC, 0.1% SDS and autoradiographed at −70°C with intensifying screens. Cells were washed twice in ice-cold phosphate-buffered saline, scraped off plates into hypotonic lysis buffer (20 mM Tris-HCl, pH 7.4, 25 mM NaCl, 1 mM sodium orthovanadate, 10 mM sodium orthophosphate, 0.25 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 1% aprotinin) and then flash frozen in liquid nitrogen. After three cycles of freeze-thaw, the lysates were passed several times through a 25-gauge needle. Lysates were cleared by centrifugation at 15,000 × g for 30 min and protein concentrations were determined using Bio-Rad protein assay reagent. Equal amounts of protein (usually 30-60 μg) were separated by SDS-polyacrylamide gel electrophoresis (8.5%), electrophoretically transferred onto nitrocellulose (Schleicher & Schuell), and probed with mouse monoclonal anti-RB (IF8, Santa Cruz Biotechnology, CA). Immunoreactive bands were visualized by enhanced chemoluminescence (ECL, Amersham Corp.). Cell cycle analysis was performed as described previously (33Screpanti I. Scarpa S. Meco D. Bellavia D. Stuppia L. Frati L. Modesti A. Gulino A. J. Cell Biol. 1995; 130: 183-192Google Scholar). Briefly, 1 × 106 cells for each sample were fixed in 70% cold ethanol for 30 min at 4°C and, after washes in cold phosphate-buffered saline, treated with RNase (0.5 mg/ml) and stained with 40 μg/ml propidium iodide. Cells were then kept in the dark at 4°C for 30 min and immediately analyzed by flow cytometry in a linear scale using a FACscan cytometer (Becton Dickinson, Mountain View, CA). Cell debris and doublets were excluded from the analysis by appropriate gating using physical parameters. Fluorescence data were analyzed by the Consort 30 software. The induction of PAI-1 and junB mRNAs is a relatively early response of different cell types to TGFβ treatment, while induction of JE mRNA is considered a late effect. Despite the differences in their kinetic, the accumulation of JE, PAI-1, and junB mRNAs upon treatment with TGFβ is known to be regulated at the transcriptional level and does not require ongoing protein synthesis (1Roberts A.B. Sporn M.B. Sporn M.B. Roberts A.B. Handbook of Experimental Pharmacology, Peptide Growth Factors and Their Receptors. Springer, Heidelberg1990: 419-431Google Scholar, 16Takeshita A. Chen Y. Watanabe A. Kitano S. Hanazawa S. J. Immunol. 1995; 155: 419-426Google Scholar). To verify the possible role of serine/threonine protein kinases on TGFβ-specific pathway(s), mink lung epithelial Mv1Lu cells were treated with 2-AP both in the absence and presence of TGFβ for 1-6 h. Treatment of cycling Mv1Lu cells with TGFβ resulted in a biphasic up-regulation of the JE mRNA with an initial peak after 1 h, a decline to the control level around 3 h, followed by a second gradual increase beginning at 6 h (Fig. 1A) and reaching the plateau level by 12-24 h (not shown). Addition of 2-AP diminished both the basal and the TGFβ-stimulated levels of JE mRNA irrespective of the length of the treatment (Fig. 1A). As expected PAI-1 and junB expression was up-regulated within 1 h of treatment with TGFβ and gradually decreased between 3 and 6 h (Fig. 1A). Addition of 2-AP showed an inhibitory effect on the TGFβ-mediated induction of PAI-1 and junB mRNAs after 1 h of treatment. However, when applied for either 3 or 6 h, 2-AP appeared to specifically superinduce PAI-1 and junB expression activated by TGFβ, without a significant effect on their basal level (Fig. 1A). Neither the inhibitory nor the stimulatory effect of 2-AP were inhibited by contemporary addition of cycloheximide (not shown). To better characterize the described effect of 2-AP on PAI-1 and junB expression induced by TGFβ, Mv1Lu cells were exposed to 2-AP for time intervals of different length, before they were stimulated with TGFβ for 1 h. Addition of 2-AP within 5 min prior to the stimulation with TGFβ consistently reduced the up-regulation of JE, PAI-1, and junB mRNAs (Fig. 1B). Inhibition of JE induction could also be observed in cells that were exposed to 2-AP for 2 or 5 h (Fig. 1B). On the contrary, up-regulation of PAI-1 and junB mRNA by TGFβ was higher in cells subjected to such a long-term pre-exposure to 2-AP compared to control cells (Fig. 1B). To investigate on the specificity of action of 2-AP, we also studied its effect on the genomic response initiated by phorbol ester (TPA), a known activator of protein kinase C. Treatment of Mv1Lu cells with TPA for 6 h also increased the level of JE, PAI-1, and junB mRNAs (Fig. 1C). Contemporary addition of 2-AP slightly reduced JE up-regulation in cells treated with TPA (Fig. 1C). On the other hand, 2-AP did not modify the response of PAI-1 and only modestly affected the response of junB to TPA, suggesting that the late superinducing effect on PAI-1 and junB expression is specific to the regulation of these genes by TGFβ. Given the opposite sensitivity to 2-AP of PAI-1 and junB compared to JE expression induced by TGFβ, we tested the effect of okadaic acid, an inhibitor of phosphatase 1 and 2A. Treatment of cells with okadaic acid did not affect expression of the basal levels of PAI-1 mRNA in Mv1Lu cells, while it slightly increased the level of junB and JE after 6 h (Fig. 2). However, while okadaic acid did not interfere with the induction of PAI-1 and junB expression by either TGFβ (Fig. 2) or TGFβ plus 2-AP (not shown), it caused a small but reproducible increase of the level of JE mRNA induced by TGFβ (Fig. 2). In cells stimulated with TGFβ and okadaic acid, 2-AP induced a reduction of the JE mRNA (not shown), although less severe than that induced in cells treated with TGFβ alone, meaning that also the superinduction due to okadaic acid of the TGFβ-stimulated expression of JE is negatively affected by 2-AP. The retinoblastoma susceptibility gene product, pRB, is considered one of the most important targets for TGFβ action in several cell lines, including Mv1Lu. In this cell type the growth inhibitory effect of TGFβ was shown to be correlated to the inhibition of pRB phosphorylation (9Laiho M. DeCaprio J.A. Ludlow J.W. Livingston D.M. Massague J. Cell. 1990; 62: 175-185Google Scholar). To ascertain if treatment with kinase inhibitor 2-AP could also affect the TGFβ-activated pathways leading to pRB dephosphorylation and/or to the cell cycle arrest, actively growing Mv1Lu cells were treated with TGFβ, 2-AP, or a combination of the two substances for 1-24 h. The relative protein extracts were analyzed by Western blot for the detection of pRB isoforms. In the same experiment duplicates of the different cell culture samples were stained with propidium iodide and subjected to cell cycle FACS analysis. One representative experiment is shown in Fig. 3. As previously reported (9Laiho M. DeCaprio J.A. Ludlow J.W. Livingston D.M. Massague J. Cell. 1990; 62: 175-185Google Scholar), in actively proliferating Mv1Lu cells the majority of pRB appeared in its hyperphosphorylated form (Fig. 3). Hypophosphorylated pRB started to accumulate within 3-6 h of treatment with TGFβ and after 24 h virtually all pRB was in its hypophosphorylated form in TGFβ-treated cells. Although Mv1Lu cells are very sensitive to contact inhibition, a condition in which accumulation of hypophosphorylated pRB is also observed, this did not limit our analysis since pRB was still fully phosphorylated in control cells which were maintained in culture for 24 h without any treatment (Fig. 3, last lane). The addition of 2-AP to TGFβ did not prevent the accumulation of the hypophosphorylated isoform of pRB induced by TGFβ, independent of the length of the exposure to both agents (Fig. 3). 2-AP itself was able to induce accumulation of the hypophosphorylated pRB isoform (Fig. 3), with a maximum effect after 24 h. However, after such a long treatment a considerable amount of pRB was still hyperphosphorylated in 2-AP-treated cells, but not in cells treated with both TGFβ and 2-AP. The cell cycle analysis of the duplicate samples revealed that treatment with TGFβ induced accumulation of the cells in the G1 and a strong reduction of cells in the S and G2/M phases of the cell cycle. This effect was maximum after 24 h and clearly distinct from the spontaneous increase in the number of G1 cells due to the contact inhibition observed in control cells maintained in culture for 24 (Fig. 3, last lane) (9Laiho M. DeCaprio J.A. Ludlow J.W. Livingston D.M. Massague J. Cell. 1990; 62: 175-185Google Scholar). In contrast, 2-AP induced an increase in the number of cells with a G2/M DNA content detected as early as 1-3 h after the beginning of the treatment and maximum after 6 h (Fig. 3). This effect was also accompanied by the reduction in the number G1 cells, whereas S phase cell number was only modestly affected by 2-AP, under these conditions. In the presence of both TGFβ and 2-AP the majority of the cells progressively accumulated in a G2/M DNA content state, similar to 2-AP-treated cells. However, after 24 h we observed a strong reduction in the number of cells in the S phase, which suggests that even under these conditions Mv1Lu cells are sensitive to TGFβ growth inhibitory signals. To study whether pRB could be involved in the specific response generated by 2-AP on TGFβ-induced gene expression, we investigated the effects of 2-AP on the induction of JE, PAI-1, and junB mRNAs by TGFβ in the pPVU-01.5.3, a Mv1Lu clone transfected with the SV40 large T antigen (9Laiho M. DeCaprio J.A. Ludlow J.W. Livingston D.M. Massague J. Cell. 1990; 62: 175-185Google Scholar). In these cells, large T antigen binding to pRB has been related to the loss of response to the growth inhibitory effect of TGFβ (9Laiho M. DeCaprio J.A. Ludlow J.W. Livingston D.M. Massague J. Cell. 1990; 62: 175-185Google Scholar), although they retain the capability to up-regulate junB and extracellular matrix proteins upon TGFβ treatment (24Laiho M. Ronnstrand L. Heino J. DeCaprio J.A. Ludlow J.W. Livingston D.M. Massague J. Mol. Cell. Biol. 1991; 11: 972-978Google Scholar). In keeping with these observations we found that TGFβ induced PAI-1, junB, and JE expression in pPVU-01.5.3 clone (Fig. 4) with a similar kinetic when compared to the parental Mv1Lu cell line (Fig. 1A). Contemporary addition of 2-AP was able to inhibit the induction of JE throughout the 1-6 h treatment and the induction of PAI-1 and junB mRNAs after 1 h of treatment (Fig. 4), as previously observed for Mv1Lu cells. However, we failed to detect the expected superinducing effect of 2-AP, which instead inhibited the TGFβ-inducible expression of both PAI-1 and junB in the pPVU-01.5.3 clone even after 3 and 6 h of treatment (Fig. 4). These results indicate that, while the early inhibitory effect of 2-AP on TGFβ-induced expression of JE, PAI-1, and junB is unaffected by the presence of the large T antigen, the late superinducing effect of 2-AP is negatively regulated by the large T antigen possibly through its binding to pRB. Since large T antigen binds and possibly inactivates several cellular proteins in addition to pRB, we sought to investigate the effect of 2-AP on TGFβ-induced gene expression on different cell lines expressing a functional pRB (A549, PMC42, and HaCat) compared to cell lines harboring inactive RB alleles (BT549 and Saos-2) (Ref. 34Lukas J. Muller H. Bartkova J. Spitkovsky D. Kjeruff A.A. Jansen-Durr P. Strauss M. Bartek J. J. Cell Biol. 1994; 125: 625-638Google Scholar and references therein). The above mentioned cell lines were treated with TGFβ, 2-AP, or a combination of the two for 1-6 h and junB expression was investigated by Northern blot and quantified by densitometric scanning of the x-ray films. To ensure a better comparison of the rough data, the absolute numeric values were converted to junB percent fold induction, with 100% arbitrarily assigned to the highest value of junB induction after TGFβ treatment. On the contrary we could not evaluate the effect of 2-AP on TGFβ-inducible expression of the PAI-1 mRNA since several cell lines did not up-regulate PAI-1 expression in response to TGFβ. In the RB-positive A549, PMC42 (Fig. 5A), and HaCat cells (not shown) 2-AP prevented the induction of junB mRNA occurring after 1 h of treatment with TGFβ, but it superinduced the same mRNA after 3 and 6 h. On the contrary 2-AP inhibited the TGFβ-regulated junB expression even after 3 and 6 h in RB-defective Saos-2 and BT549 cell lines (Fig. 5B).Fig. 5The superinducing effect of 2-AP on the TGFβ-inducible junB expression correlates with the expression of a functional pRB. PMC42, A549, BT549, and Saos-2 cell lines, selected for their property to own either a functional or a deleted pRB, were treated with 5 ng/ml TGFβ1 (solid symbols) or TGFβ1 and 2-AP (open symbols) for the indicated times. Total RNA was isolated and analyzed for junB expression by Northern blot. Data resulting from densitometric scanning are expressed as percent of the junB fold induction. 100% was arbitrarily assigned to the highest value of junB induction after TGFβ treatment. A, RB positive cells, A549 (diamonds) and PMC42 (triangles) are compared to the parental Mv1Lu cells (squares, graphical representation of Northern blot shown in Fig. 1A). B, RB negative cells BT549 (triangles) and Saos-2 (diamonds) are compared with the pPVU-O1.5.3 clone (squares, graphical representation of Northern blot shown in Fig. 4).View Large Image Figure ViewerDownload (PPT) To confirm the involvement of pRB in the superinduction of junB mRNA upon treatment with both TGFβ and 2-AP, we also analyzed the response of three different RB-reconstituted clones (Fig. 6). The BTB5V4-RB is a BT549 stable transfectant in which the full-length human RB cDNA is located under a tetracycline-controlled promoter (31Gjetting T. Lukas J. Bartek J. Strauss M. Biol. Chem. Hoppe Seyler. 1995; 376: 441-446Google Scholar). As previously observed (31Gjetting T. Lukas J. Bartek J. Strauss M. Biol. Chem. Hoppe Seyler. 1995; 376: 441-446Google Scholar), tetracycline-treated cells expressed a discrete amount of pRB, which could be increased after 48 h of culture in the absence of tetracycline (Fig. 6A). Saos-2 cells express a truncated and nonfunctional p95RB (Fig. 6B). Saos-2 number 1 and Saos-2 number 84 are two RB-reconstituted clones (32Fung Y-K.T. T'Ang A. Linn Murphree A. Zhang F-H. Qui W-R. Wang S-W. Shi X-H. Lee L. Driscoll B. Wu K-J. Oncogene. 1993; 8: 2659-2672Google Scholar) expressing low levels of the exogenous p105RB (Fig. 6B). All these cell lines responded to TGFβ with an early up-regulation of junB mRNA (Fig. 6C). Addition of 2-AP to the treatment prevented junB induction by TGFβ after 1 h, whereas it clearly showed a superinducing effect after 3 h of treatment in RB-reconstituted BTB5V4-RB cells which was further enhanced under conditions in which pRB expression was further induced by the removal of tetracycline from the culture medium (Fig. 6C; compare with parental BT549 in Fig. 5B). A similar response to 2-AP was found in the pRB-reconstituted Saos-2 #1 and Saos-2 #84 clones (Fig. 6C; compare with parental Saos-2 in Fig. 5B), confirming that the introduction of a functional RB gene is sufficient to reconstitute the TGFβ-dependent superinduction of junB mRNA in response to 2-AP. Taken together these observations clearly indicate that the late superinducing effect of 2-AP on TGFβ inducible junB expression is dependent on the presence of functional pRB while its early inhibi" @default.
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