2-Aminopurine Unravels a Role for pRB in the Regulation of Gene Expression by Transforming Growth Factor beta *

(Received for publication, July 23, 1996, and in revised form, November 4, 1996)

Giuseppe Giannini Dagger §, Lucia Di Marcotullio Dagger , Francesca Zazzeroni Dagger , Edoardo Alesse Dagger , Massimo Zani , Anne T'Ang par , Vincenzo Sorrentino **§§, Isabella Screpanti , Luigi Frati ¶¶ and Alberto Gulino Dagger

From the Dagger  Department of Experimental Medicine, University of L'Aquila, 67100 L'Aquila, Italy, the  Department of Experimental Medicine and Pathology, University "La Sapienza," 00161 Rome, Italy, the ¶¶ Neurological Mediterranean Institute, Neuromed, Pozzilli, Italy, the par  Department of Pediatrics and Microbiology, School of Medicine, Children's Hospital of Los Angeles, Los Angeles, California 90027, ** Dibit, San Raffaele Scientific Institute, 20132 Milano, Italy, and the §§ Institute of Histology, School of Medicine, University of Siena, 53100 Siena, Italy

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Transforming growth factor type beta  (TGFbeta ) is a pleiotropic factor that regulates different cellular activities including cell growth, differentiation, and extracellular matrix deposition. All the known effects of TGFbeta 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 TGFbeta 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 TGFbeta activates a genomic response. Consistently, 2-aminopurine prevented and okadaic acid potentiated the induction of JE by TGFbeta . 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 TGFbeta inducible junB expression occur in all the cell lines examined suggesting that junB, and possibly other genes, can be regulated by TGFbeta through a distinct pRB-dependent pathway.


INTRODUCTION

The three mammalian isoforms of the transforming growth factor type beta  (TGFbeta 1, TGFbeta 2, and TGFbeta 3),1 belong to a superfamily of related polypeptides involved in the control of a large number of biological activities, including cell growth, differentiation, and development (1). Originally identified as a factor able to induce growth of normal rat kidney fibroblast in soft agar, TGFbeta was subsequently shown to be a potent growth inhibitor for most epithelial cells (1).

Most of the reported actions of TGFbeta 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 (2, 3), that are active as hetero-oligomeric complexes (4, 5). Although a few candidate transducing molecules have been identified (6, 7), the biochemical pathways acting downstream of these receptors are still largely uncharacterized.

With few exceptions (8), in epithelial cells TGFbeta -mediated growth inhibition correlates with the G1 inhibition of the phosphorylation of the retinoblastoma gene product, pRB (1, 9). Several events contribute to preventing pRB phosphorylation in TGFbeta -treated cells: suppression of CDK4 synthesis (10), down-regulation of cyclins and cdks expression (11), inhibition of cycE-cdk2 complexes by p27Kip1 binding (12); induction of p21CIP1, and p15Ink4B with consequent inhibition of cdk4 and cdk6 kinases (13, 14).

Most of the other cellular responses to TGFbeta , including control of extracellular matrix protein deposition, wound healing, and immune suppression are proposed to be mediated by controlling the expression of specific genes (1). 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 (15), JE (16), p21CIP1 (17), p15Ink4B (18), and alpha 2(I) collagen (15, 19) gene regulation. The expression of other genes appears to be regulated through TGFbeta inhibitory sequences (20-22). In the case of c-myc the same promoter region, the TGFbeta control element, is required for down-regulation of c-myc by both TGFbeta and the retinoblastoma protein (21, 22). Furthermore, pRB is required for TGFbeta -dependent c-myc down-regulation and growth inhibition, in skin keratinocytes (21) and for down-regulation of N-myc in embryonic lung organ cultures (23). In contrast, in Mv1Lu cells, pRB appears not to be required for PAI-1, junB, and fibronectin induction by TGFbeta (24, 25).

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 (26). 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 (27, 28) and to modulate the expression of a group of genes specifically expressed in growth-arrested cells (29). Okadaic acid is a fatty acid that has tumor promoter activity on mouse skin (30); however, it does not activate or bind protein kinase C. It is, on the contrary, a potent inhibitor of protein phosphatases 1 and 2A (30). 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 (26-30).

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 TGFbeta 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 TGFbeta . In particular we report here on the regulation of three genes involved in different biological effects initiated by TGFbeta : (i) JE, a monocyte chemoattractant (16); (ii) PAI-1, whose induction by TGFbeta is an important step in the control of extracellular matrix deposition (1, 15); and (iii) junB, an early responsive gene involved in the control of growth and differentiation which is regulated by TGFbeta in most cell types (1). The effects of 2-AP on TGFbeta -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 TGFbeta stimulates gene expression. 2-AP, which is able to prevent TGFbeta -inducible expression of several genes, potentiates the induction of PAI-1 and junB by TGFbeta , after a transient inhibition. In addition we show that potentiation of TGFbeta -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 TGFbeta .


EXPERIMENTAL PROCEDURES

Cell Culture Conditions

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 (9, 31, 32). 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 TGFbeta 1 (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).

RNA Preparation and Northern Blot Analysis

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.

Protein Preparation and Western Blot

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

Cell cycle analysis was performed as described previously (33). 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.


RESULTS

Induction by TGFbeta of JE, PAI-1, and junB mRNAs in Mv1Lu Cells: effect of 2-AP and Okadaic Acid

The induction of PAI-1 and junB mRNAs is a relatively early response of different cell types to TGFbeta 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 TGFbeta is known to be regulated at the transcriptional level and does not require ongoing protein synthesis (1, 16). To verify the possible role of serine/threonine protein kinases on TGFbeta -specific pathway(s), mink lung epithelial Mv1Lu cells were treated with 2-AP both in the absence and presence of TGFbeta for 1-6 h. Treatment of cycling Mv1Lu cells with TGFbeta 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 TGFbeta -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 TGFbeta and gradually decreased between 3 and 6 h (Fig. 1A). Addition of 2-AP showed an inhibitory effect on the TGFbeta -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 TGFbeta , 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).


Fig. 1. Effect of 2-AP on TGFbeta - and TPA-inducible expression of JE, PAI-1, and junB. Growing Mv1Lu cells were treated with TGFbeta 1 (1 ng/ml) or TPA (100 ng/ml) in the presence or absence of 2-AP (10 nM). Total RNA was extracted and analyzed by Northern blot for the expression of JE, PAI-1, and junB. A, TGFbeta 1, 2-AP, or a combination of the two substances were added to the culture at the same time and incubated for 1, 3, or 6 h; B, cells were pretreated with 2-AP for the indicated time before they were stimulated with TGFbeta for 1 h; C, TPA, 2-AP, or a combination of the two substances were added to the culture at the same time and incubated for 6 h. Ethidium bromide staining of ribosomal RNA is included for each panel as a loading control.
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To better characterize the described effect of 2-AP on PAI-1 and junB expression induced by TGFbeta , Mv1Lu cells were exposed to 2-AP for time intervals of different length, before they were stimulated with TGFbeta for 1 h. Addition of 2-AP within 5 min prior to the stimulation with TGFbeta 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 TGFbeta 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 TGFbeta .

Given the opposite sensitivity to 2-AP of PAI-1 and junB compared to JE expression induced by TGFbeta , 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 TGFbeta (Fig. 2) or TGFbeta plus 2-AP (not shown), it caused a small but reproducible increase of the level of JE mRNA induced by TGFbeta (Fig. 2). In cells stimulated with TGFbeta and okadaic acid, 2-AP induced a reduction of the JE mRNA (not shown), although less severe than that induced in cells treated with TGFbeta alone, meaning that also the superinduction due to okadaic acid of the TGFbeta -stimulated expression of JE is negatively affected by 2-AP.


Fig. 2. Effect of okadic acid on TGFbeta -inducible expression of JE, PAI-1, and junB. Total RNA was extracted from Mv1Lu cells treated with TGFbeta 1 (1 ng/ml), okadaic acid (OA) (10 ng/ml), or a combination of the two substances for 1, 3, and 6 h. The expression of JE, PAI-1, and junB was analyzed by Northern blot. Ethidium bromide staining of ribosomal RNA is included as a loading control.
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Effect of 2-AP on the TGFbeta -mediated pRB Dephosphorylation and Cell Cycle Regulation

The retinoblastoma susceptibility gene product, pRB, is considered one of the most important targets for TGFbeta action in several cell lines, including Mv1Lu. In this cell type the growth inhibitory effect of TGFbeta was shown to be correlated to the inhibition of pRB phosphorylation (9). To ascertain if treatment with kinase inhibitor 2-AP could also affect the TGFbeta -activated pathways leading to pRB dephosphorylation and/or to the cell cycle arrest, actively growing Mv1Lu cells were treated with TGFbeta , 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 (9), 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 TGFbeta and after 24 h virtually all pRB was in its hypophosphorylated form in TGFbeta -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 TGFbeta did not prevent the accumulation of the hypophosphorylated isoform of pRB induced by TGFbeta , 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 TGFbeta and 2-AP. The cell cycle analysis of the duplicate samples revealed that treatment with TGFbeta 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) (9). 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 TGFbeta 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 TGFbeta growth inhibitory signals.


Fig. 3. 2-AP does not prevent the inhibition of pRB phosphorylation induced by TGFbeta but affects the cell cycle progression of Mv1Lu cells. Actively proliferating Mv1Lu cells were treated with TGFbeta 1, 2-AP, or a combination of the two substances in medium containing 10% fetal calf serum. After the indicated time, cells were lysed and the protein extract analyzed by Western blot with an anti-RB antibody. The position of hypophosphorylated and hyperphosphorylated forms of pRB are indicated. The cell cycle distribution of duplicate cell samples was studied by propidium iodide staining and FACS analysis. For each sample the percentage of the cells in the G0/G1, S, or G2/M phases of the cell cycle is indicated. These results are representative of at least three separate experiments.
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Superinduction of the TGFbeta -induced junB Expression by 2-AP Requires a Functional pRB

To study whether pRB could be involved in the specific response generated by 2-AP on TGFbeta -induced gene expression, we investigated the effects of 2-AP on the induction of JE, PAI-1, and junB mRNAs by TGFbeta in the pPVU-01.5.3, a Mv1Lu clone transfected with the SV40 large T antigen (9). In these cells, large T antigen binding to pRB has been related to the loss of response to the growth inhibitory effect of TGFbeta (9), although they retain the capability to up-regulate junB and extracellular matrix proteins upon TGFbeta treatment (24). In keeping with these observations we found that TGFbeta 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 TGFbeta -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 TGFbeta -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 TGFbeta -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. 34 and references therein). The above mentioned cell lines were treated with TGFbeta , 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 TGFbeta treatment. On the contrary we could not evaluate the effect of 2-AP on TGFbeta -inducible expression of the PAI-1 mRNA since several cell lines did not up-regulate PAI-1 expression in response to TGFbeta . 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 TGFbeta , but it superinduced the same mRNA after 3 and 6 h. On the contrary 2-AP inhibited the TGFbeta -regulated junB expression even after 3 and 6 h in RB-defective Saos-2 and BT549 cell lines (Fig. 5B).


Fig. 4. The superinducing effect of 2-AP on the genomic response to TGFbeta is abolished in SV40 large T antigen-transfected cells. pPVU-01.5.3 cells, a Mv1Lu clone derived for stable transfection with the SV40 large T antigen, were treated with TGFbeta 1, 2-AP, or a combination of the two substances for the indicated time. Total RNA was extracted and analyzed for JE, PAI-1, and junB expression by Northern blot. Ethidium bromide staining of ribosomal RNA is included as a loading control.
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Fig. 5. The superinducing effect of 2-AP on the TGFbeta -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 TGFbeta 1 (solid symbols) or TGFbeta 1 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 TGFbeta 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).
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To confirm the involvement of pRB in the superinduction of junB mRNA upon treatment with both TGFbeta 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 (31). As previously observed (31), 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 (32) expressing low levels of the exogenous p105RB (Fig. 6B). All these cell lines responded to TGFbeta with an early up-regulation of junB mRNA (Fig. 6C). Addition of 2-AP to the treatment prevented junB induction by TGFbeta 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 TGFbeta -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 TGFbeta inducible junB expression is dependent on the presence of functional pRB while its early inhibitory effect is pRB independent.


Fig. 6. The superinducing effect of 2-AP on the TGFbeta inducible junB expression is restored in RB-reconstituted cell lines. A, protein extracts prepared from parental BT549 and BTB5V4-RB either kept under the repressing effect of tetracycline (V4+) or grown for 48 h in the absence of tetracycline (V4-) were analyzed by Western blot for pRB expression. B, protein extracts prepared from parental Saos-2 cell line (Sa) and from Saos-2 #1 (#1) and Saos-2 #84 (#84) clones were analyzed by Western blot for pRB expression. The partially deleted endogenous p95RB and the exogenous p105RB are indicated. C, RB-reconstituted cell lines were treated with TGFbeta 1 (solid symbols) or TGFbeta 1 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 indicated in the legend to Fig. 5. BTB5V4-RB under the repressing effect of tetracycline (circles); BTB5V4-RB grown for 48 h in the absence of tetracycline (squares); Saos-2 #1 (diamonds) and Saos-2 #84 (triangles).
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The G2/M Arrest Induced by 2-AP Requires a Functional pRB

The temporal association between the RB-dependent superinducing effect of 2-AP on the expression of PAI-1 and junB induced by TGFbeta and its ability to block Mv1Lu cells in the G2/M phase of the cell cycle prompted us to verify whether also the latter effect was dependent on the presence of a functional RB protein. In order to test this possibility, parental Saos-2 cells and the RB-reconstituted Saos-2 #1 and the Saos-2 #84 clones were treated with 10 nM 2-AP for 6 h and subjected to cell cycle FACS analysis. The average values of G2/M and of the sum of G1 and S cells obtained from three different experiments are summarized in Table I. Treatment with 2-AP failed to block pRB-defective Saos-2 cells in the G2/M phase of the cell cycle. Rather it induced a slight decrease in the number of cells with a G2/M DNA content compared to untreated control cells. However, the ability of 2-AP to induce a G2/M cell cycle arrest was restored in the RB-reconstituted Saos-2 #1 (Table I) and Saos-2 #84 (not shown) clones. These results suggest that a functional RB protein is required for the induction of the G2/M arrest as well as for the potentiation of TGFbeta inducible junB expression by 2-AP.

Table I.

The ability of 2-AP to arrest cells in the G2/M phase of the cell cycle depends on the presence of a functional pRB

Cell cycle FACS analysis of propidium iodide-stained Saos-2 parental cells and Saos-2 #1 RB-reconstituted clone, cultured for 6 h in the absence or presence of 2-AP, was performed as described under "Experimental Procedures." The results represent the average percentages (± S.D.) relative to three different experiments. *r = (% G2/M)/(% G1 + S).
Cell lines CTR
2-AP
% G2/M % G1 + S r* % G2/M G1 + S r*

Saos-2 37.7  ± 9.4 62.3  ± 9.4 0.60 25.4  ± 8.4 74.6  ± 8.4 0.34
Saos-2 #1 38.9  ± 3.6 61.1  ± 3.6 0.63 58.1  ± 6.5 41.9  ± 6.5 1.38


DISCUSSION

TGFbeta Activates Two Signaling Pathways with Different Sensitivity to 2-AP and Okadaic Acid

In this report we describe the effect of 2-aminopurine and okadaic acid, an inhibitor of serine-threonine protein kinases and an inhibitor of protein phosphatases, respectively, on TGFbeta transduction pathway(s) leading to the regulation of gene expression. The use of these two drugs allowed us to identify two different pathways involved in TGFbeta signal transduction and gene regulation. Specifically, we found that induction of JE expression by TGFbeta requires the action of a 2-AP-sensitive protein kinase. Similar results were also obtained for TGFbeta -induced expression of c-jun, vimentin, thymosin-beta 4,2 and RYR3 (35). The same kinase may also be required for the basal expression of JE and for its up-regulation by protein kinase C activation. Indeed 2-AP is also able to inhibit basal level as well as TPA-induced JE expression. On the other hand we found that okadaic acid slightly stimulates both basal and TGFbeta -induced JE expression, confirming that JE induction may be mediated by increasing the level of phosphorylation of an unknown protein in the cells. Okadaic acid was also shown to stimulate basal and TGFbeta -induced expression of urokinase receptor and collagenase mRNAs in A549 cells (36, 37) and alpha 2(I) collagen mRNA in CF-37 fibroblasts (38), suggesting that a similar mechanism can be involved in the regulation of several genes by TGFbeta .

Our data indicate that the regulation of other genes by TGFbeta can utilize a different signaling pathway. PAI-1 and junB induction by TGFbeta , in fact, is independent from the action of okadaic acid-sensitive phosphatases. More interestingly, although the early induction of these genes by TGFbeta still rely on the activity of a 2-AP-sensitive kinase, as suggested by the early inhibitory effect of 2-AP, an alternative TGFbeta -dependent pathway is activated after prolonged exposure to this agent and leads to PAI-1 and junB superinduction. Under this condition the pathway leading to the induction of the JE gene is still repressed, suggesting that 2-AP inhibitory potential is still intact. In addition, the delayed action of 2-AP is specific to the TGFbeta -induced pathway since it does not significantly affect PAI-1 and junB basal or TPA-stimulated levels.

Together with the observation that an active kinase domain in both TGFbeta receptor type I and -II are required for signal transduction (39-41), our results also suggest that the inhibitory action of 2-AP on the TGFbeta -induced expression of JE is likely to involve cellular kinases acting downstream from the TGFbeta receptors complex. A complete inhibition of the receptor kinase activity should interfere with any downstream event following TGFbeta treatment. On the contrary TGFbeta is still able to activate an alternative pathway leading to induction of PAI-1 and junB genes even after 2-5 h of exposure to 2-AP. The recent description of a cytoplasmic 78-kDa protein kinase (42) activated within minutes by TGFbeta in PC3 cells and the isolation of TAK1 (43), a member of the MAPKKK family, linked to TGFbeta signal transduction in both Mv1Lu and MC3T3-E1 cells, identifies these proteins as candidate targets for the early inhibitory action of 2-AP on TGFbeta signal transduction pathway(s).

pRB Is Necessary for the Potentiating Effect of 2-AP on the Induction of junB by TGFbeta

TGFbeta generate antiproliferative signals in several cell types, possibly through its ability to prevent pRB phosphorylation in the G1 phase of the cell cycle (9). This event may contribute to the control of S phase-specific genes by TGFbeta . Indeed pRB is required for the TGFbeta -dependent down-regulation of c-myc in skin keratinocytes (21, 22) and of N-myc in lung organ cultures (23). In Mv1Lu cells the pathway activated by TGFbeta and leading to the inhibition of pRB phosphorylation is inhibited by the serine/threonine kinase inhibitors H7, H8, and H9, which can also prevent the induction of PAI-1, junB, and fibronectin expression by TGFbeta (44). On the contrary we found that 2-AP, which by itself caused accumulation of hypophosphorylated pRB isoforms, did not interfere with the induction of pRB dephosphorylation in response to TGFbeta , since TGFbeta -treated cells showed substantially the same content of hypophosphorylated pRB as cells treated with both TGFbeta and 2-AP. The failure of 2-AP to inhibit the TGFbeta growth inhibitory pathway is also indicated by the fact that TGFbeta can consistently reduce the number of Mv1Lu cells entering the S phase of the cell cycle even in the presence of 2-AP. Such observations suggested that pRB could play a role in the superinduction of PAI-1 and junB in cells treated with TGFbeta and 2-AP. Indeed this is demonstrated by three different lines of evidence. 2-AP could no longer superinduce PAI-1 and junB expression regulated by TGFbeta in the pPVU-01.5.3 cell line, in which pRB is inactivated by the presence of the SV40 large T antigen. In addition, although 2-AP potentiated the TGFbeta -inducible junB expression in the RB positive A549, HaCat, and PMC42 cell lines, it failed to do so in the BT549 and Saos-2 tumor-derived cell lines, which were previously shown to lack a functional pRB. In both these cell lines the superinducing effect of 2-AP was restored after transfection of the human RB cDNA. Taken together our data demonstrate that TGFbeta can regulate the expression of junB, and possibly other genes, through a distinct pathway. A similar conclusion is also supported by the evidence that TGFbeta -inducible junB expression, but not c-jun expression, is selectively inhibited by the E1A adenoviral oncogene (45).

Despite its function as a transcriptional repressor, the necessity of a functional pRB for the 2-AP dependent superinduction of junB by TGFbeta suggests that pRB can positively regulate transcription. This conclusion is in agreement with previous reports which demonstrated that pRB stimulates the SP1/SP3-mediated transcription by binding and inactivating a SP1-inhibitory protein (46, 47). More recently pRB was shown to potentiate transcriptional activation induced by glucocorticoid receptor in complex with the hBrm protein (48).

It has been shown that pRB is not necessary for TGFbeta - inducible expression of several genes, including junB (24, 25). This observation is not in conflict with our data which demonstrate that, under particular circumstances, uncovered by 2-AP, pRB can potentiate TGFbeta -inducible junB expression. In contrast to other serine/threonine kinase inhibitors (44), 2-AP did not prevent accumulation of hypophosphorylated pRB induced by TGFbeta , implying that a potentially active pRB is still able to contribute to TGFbeta signal transduction in 2-AP-treated cells and could be responsible for junB superinduction. However, contact inhibited Mv1Lu cells, which are synchronized in the G1 phase of the cell cycle and accumulated hypophosphorylated pRB, do not respond to TGFbeta with a stronger induction of junB mRNA compared to actively proliferating cells (not shown and Ref. 25). Therefore pRB dephosphorylation per se is not sufficient to mimic 2-AP effect on TGFbeta induced expression of junB. Cell cycle analysis of Mv1Lu cells treated with TGFbeta and 2-AP revealed that, coincident with the superinducing effect on the TGFbeta inducible junB expression, 2-AP caused accumulation of the cells in the G2/M phase of the cell cycle. The G2/M arrest induced by 2-AP is not unique to Mv1Lu cells since it was also observed on other cell lines (49). Metheny et al. (50) have shown that release of CV-1P cells from nocodazole block in the presence of 2-AP prevented cells from progressing through methaphase and anaphase concomitant with pRB being in the hypophosphorylated form. Our results also indicate that RB-negative Saos-2 cells, in which 2-AP fails to potentiate the TGFbeta -inducible junB expression, are insensitive to the effect of 2-AP on the cell cycle. However, 2-AP was consistently able to increase the G2/M cell population in Saos-2 #1 and Saos-2 #84 pRB-transfected clones, providing evidence for a role of pRB in 2-AP induced G2/M arrest.

It is accepted that the TGFbeta growth inhibitory activity takes place within mid-late G1 in order to prevent G1-arrested cells from re-entering the cell cycle (9). On the contrary it is still debated how TGFbeta inhibits proliferation of asynchronously growing cells. It was shown that the growth inhibition of Mv1Lu cells by TGFbeta does not occur within the ongoing cycle, but only after actively proliferating cells have undergone division in the presence of TGFbeta (51), indicating that important events are likely to occur in the G2 and/or M phases of the cell cycle. Taken together these observations raise the possibility that as cells enter a restricted point of the cell cycle, in a particular condition in which pRB is mostly in its hypophosphorylated form, as in the case of the 2-AP block, they acquire an increased responsiveness to TGFbeta in terms of induction of specific genes like junB, and perhaps other growth regulatory genes, which in turn may contribute to the regulation of cell cycle progression. Further work is required to ascertain whether the potentiation of junB induction by TGFbeta is selectively occurring in the G2/M cell population which accumulates upon treatment with 2-AP.

In conclusion, we have provided evidence that TGFbeta regulates gene expression through at least two different signaling pathways, one of which is dependent on a functional pRB and possibly restricted to the G2/M phase of the cell cycle.


FOOTNOTES

*   This work was supported in part by the Associazione Italiana per la Ricerca sul cancro (AIRC), the National Research Council (CNR), ACRO project, by MURST 40% and 60%, and by the Istituto Pasteur-Fondazione Cenci-Bolognetti. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed. Present address: Dept. of Experimental Medicine and Pathology, University "La Sapienza," Viale Regina Elena, 324, 00161 Rome, Italy. Tel.: 39-6-44700816; Fax: 39-6-4454820; E-mail: screpant{at}caspur.it.
1    The abbreviations used are: TGFbeta , transforming growth factor type beta ; pRB, retinoblastoma susceptibility gene product; 2-AP, 2-aminopurine; TPA, phorbol ester; FACS, fluorescence-activated cell sorter.
2   G. Giannini, unpublished results.

Acknowledgments

We thank Dr. J. Lukas, Dr. M. Strauss, and Dr. T. Gjetting for providing BTB5V4-RB cells, Dr. Y-K. Fung for Saos-2 number 1 and Saos-2 number 84 clones, and Dr. J. Massague and Dr. M. Lahio for pPVU-O1.5.3 cells. We also thank Dr. B. Cardinali, Dr. C. Thiele, Dr. J. Letterio, Dr. G. Piaggio, Dr. M. Crescenzi, Dr. M. Maroder, and Dr. F. Navid for comments on the manuscript, and Dr. A. Del Nero for artwork.


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