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Cell Growth & Differentiation Vol. 11, 475-483, September 2000
© 2000 American Association for Cancer Research


Articles

p202 Levels Are Negatively Regulated by Serum Growth Factors1

Yanbiao Geng, Sanjay D’Souza, Hong Xin, Scott Walter and Divaker Choubey2

Department of Radiation Oncology, Stritch School of Medicine, Loyola University Medical Center, Maywood, Illinois 60153

Abstract

p202 is an IFN-inducible phosphoprotein (Mr 52,000) whose expression in transfected cells retards proliferation. Interestingly, the reduced levels of p202 in fibroblasts (in consequence of the expression of antisense to 202 RNA), under reduced serum conditions, increase the susceptibility of cells to apoptosis. To identify the functional role of p202 in cell growth regulation, we tested whether serum growth factor levels in the culture medium affect p202 levels. Here we report that, under reduced serum conditions, the p202 levels were increased in fibroblasts, and the increase was seen at both the mRNA and protein levels. Moreover, an increase in p202 levels was correlated with cell growth arrest in the G1 phase of the cell cycle. Interestingly, the presence of platelet-derived growth factor AB, basic fibroblast growth factor, or transforming growth factor ß1 in the culture medium abrogated the increase in p202 levels seen under reduced serum conditions. We found that the increase in p202 levels was accompanied by an increase in JunD/activation protein 1 (AP-1) levels, and transfection of a JunD-encoding plasmid along with a reporter plasmid in which transcription of the reporter gene (luciferase) was driven by the 5'-regulatory region of the 202 gene resulted in an increase in the activity of luciferase. Additionally, stable overexpression of JunD in cells, under reduced serum conditions, also resulted in an increase in p202 levels. Interestingly, one of the AP-1-like DNA-binding sequences present in the 5'-regulatory region of the 202 gene could selectively bind to the JunD/AP-1 transcription factor. Taken together, our observations reported herein suggest that in fibroblasts, under reduced serum conditions, the increased levels of JunD/AP-1 contribute to the transcriptional up-regulation of p202 levels, which may be important for the regulation of apoptosis.

Introduction

p202 is an IFN-inducible phosphoprotein (Mr 52,000) whose ectopic expression in fibroblasts retards proliferation (1, 2, 3, 4, 5, 6, 7) . p202 has the ability to bind retinoblastoma protein (pRb) and other members of the "pocket" protein family (4 , 8) . Furthermore, p202 modulates the transcriptional activity of several factors including p53 (9) , AP-13 (c-Fos and c-Jun; Ref. 6 ), and E2F (E2F-1/DP-1 and E2F-4/DP-1) (5 , 8) . Whereas the ability of p202 to inhibit the transcriptional activity of these factors provides a potential mechanism to regulate cell proliferation and apoptosis, it remains unclear how p202 negatively regulates cell proliferation. Additionally, it remains to be seen whether growth-regulatory molecules other than IFNs regulate the expression of p202.

The Jun family transcription-regulatory proteins are components of the AP-1 transcription factor, which includes three members: (a) c-Jun; (b) JunB; and (c) JunD (10 , 11) . These proteins share a high degree of homology at the primary sequence level, and the genes encoding them undergo similar but not identical regulation. Both c-jun and junB are considered "classical" immediate early response genes based on their rapid, large, and transient increase in transcription after stimulation of cells with mitogenic agents. By contrast, the junD gene is relatively refractory to such growth-stimulatory signals (12) . Furthermore, JunD gene is constitutively expressed in quiescent fibroblasts (13 , 14) .

The JunD protein has been implicated as a negative regulator of cell proliferation, and serum growth factors are shown to modulate JunD activity in a variety of apparently independent ways to overcome its negative regulatory effect in controlling cell proliferation (12 , 14 , 15) . These include the induction of a transient increase in JunD phosphorylation, repression of JunD/AP-1 DNA binding activity, and down-regulation of JunD expression (11 , 12 , 16) .

When NIH 3T3 fibroblasts become quiescent, the levels of c-Jun decrease, whereas the levels of JunD increase (12) . Furthermore, overexpression of JunD results in slower growth and an increase in the percentage of cells in G0-G1 (12 , 17) . Together, these observations suggest that an important function of JunD is to negatively regulate the proliferation of fibroblasts. Although JunD has been reported to activate transcription of several genes (18) , it is not known how JunD retards cell proliferation.

Our previous studies have revealed that a decrease in p202 levels in murine fibroblasts, under reduced serum conditions, increases susceptibility to apoptosis (19) . Therefore, to elucidate the functional role of p202 in the regulation of cell proliferation, we tested whether the serum growth factor levels could regulate p202 levels.

Here we report that, under reduced serum conditions, p202 levels are increased in murine fibroblasts. The increase was found at both the mRNA and protein levels and was correlated with growth arrest of cells in the G1 phase of the cell cycle. Interestingly, we found that an increase in p202 levels was accompanied by increase in JunD levels, and one of the AP-1-like DNA binding sequences present in the 5'-flanking region of the 202 gene selectively bound to the JunD/AP-1 transcription factor in gel mobility shift assays. Additionally, the exit of serum-starved fibroblasts from the G0-G1 phase of the cell cycle after serum stimulation correlated with the decrease in p202 levels. Moreover, the addition of PDGF-AB, bFGF, or TGF-ß1 to the culture medium, under reduced serum conditions, resulted in a decrease in p202 levels. In transient transfection assays, transfection of JunD-encoding plasmid along with a reporter plasmid in which the expression of luciferase reporter was driven by the 5'-regulatory region of the 202 gene increased the activity of luciferase about 4-fold. Taken together, our observations reported herein suggest that, under reduced serum conditions, increased levels of JunD/AP-1 contribute to the transcriptional up-regulation of p202 levels.

Results

A Decrease in Serum Growth Factor Levels Results in an Increase in p202 Levels.
The reduced basal levels of p202 in murine AKR-2B fibroblasts (in consequence of the expression of antisense to the 202 RNA expression), under reduced serum conditions, were found to increase susceptibility to apoptosis (19) . Therefore, we tested whether serum growth factor levels in the culture medium affect the basal levels of p202. For this purpose, we incubated subconfluent cultures of AKR-2B cells in growth medium supplemented with reduced serum (1% or less). As shown in Fig. 1ACitation , incubation of cells under reduced serum conditions for the indicated time resulted in an increase in p202 levels (compare Lane 2 with Lanes 3 and 4). Furthermore, fractionation of cells into the nuclear and cytoplasmic fractions revealed that the increased levels of p202 were primarily nuclear (data not shown). Because incubation of cells in culture medium supplemented with 1% serum resulted in an increase in p202 levels, we chose this serum concentration for additional studies. Incubation of cells under reduced serum conditions for increasing lengths of time revealed that an increase in p202 levels was evident after 2 days of incubation (Fig. 1BCitation , compare Lane 3 with Lane 4), and further incubation resulted in a 6-fold increase in p202 levels (Fig. 1BCitation , compare Lane 3 with Lanes 5 and 6). Similarly, incubation of NIH 3T3 cells under reduced serum (1% or less) conditions also resulted in an increase in p202 levels (data not shown).



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Fig. 1. p202 levels increase under reduced serum condition. A, subconfluent cultures of murine AKR-2B cells were incubated in growth medium supplemented with 10% (Lane 2), 1% (Lane 3), or 0.01% (Lane 4) FBS for 4 days. As a control, AKR-2B cells were treated with IFN for 48 h (Lane 1). Cells extracts were prepared as described in "Materials and Methods," and equal amounts of proteins were analyzed by immunoblotting using anti-p202 antibody. An arrow indicates the p202 protein band. B, subconfluent cultures of murine AKR-2B cells were incubated in growth medium supplemented with 1% FBS for 0 day (Lane 2), 1 day (Lane 3), 2 days (Lane 4), 3 days (Lane 5), or 4 days (Lane 6). As a control, AKR-2B cells were treated with IFN for 48 h (Lane 1). Cells extracts were prepared as described in A, and equal amounts of proteins were analyzed by immunoblotting using the anti-p202 antibody. An arrow indicates the p202 protein band. C, the steady-state levels of 202 mRNA increase after incubation of AKR-2B cells under reduced serum conditions. Top panel, subconfluent cultures of cells were incubated in growth medium supplemented with either 10% serum (Lanes 1, 3, and 5) or 1% serum (Lanes 2, 4, and 6) for 0 day (Lanes 1 and 2), 2 days (Lanes 3 and 4), or 4 days (Lanes 5 and 6). Total cytoplasmic RNA was analyzed by Northern blot analysis using the mouse 202-specific cDNA probe as described in "Materials and Methods." An arrow indicates the 202 mRNA band. Bottom panel, cytoplasmic RNA applied on the agarose gel was visualized with ethidium bromide staining to control for equal amounts of RNA loading.

 
We next tested whether the increase in p202 levels was due to an increase in the 202 RNA levels. As shown in Fig. 1CCitation , incubation of AKR-2B cells under reduced serum conditions resulted within 2 days in an increase in the steady-state levels of the 202 RNA (compare Lane 3 with Lane 4), and the levels stayed higher even after 4 days (compare Lane 5 with Lane 6). Taken together, these observations indicated that, under reduced serum conditions, the steady-state levels of the 202 RNA and protein were increased in these cells.

Because incubation of cells in 10% serum (but not 1% serum) for 3–4 days in the above-mentioned experiments (Fig. 1)Citation resulted in cultures containing cells at high density, we also tested whether the cell density in cultures had any effect on the basal levels of p202. As shown in Fig. 2Citation , incubation of AKR-2B cells in medium supplemented with 10% serum for the indicated time, which resulted in cells at higher density, did not result in a decrease in p202 levels. Instead, p202 levels increased about 50% (Fig. 2Citation , compare Lane 2 with Lane 5). Thus, this experiment indicated that the cell density in cultures did not contribute significantly to the increase in p202 levels under reduced serum conditions.



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Fig. 2. Increase in p202 levels under reduced serum conditions is independent of the cell density in cultures. Subconfluent cultures of AKR-2B cells were incubated in growth medium supplemented with either 10% (Lanes 2–5) or 1% FBS (Lanes 6–9) for 2 days (Lanes 2 and 6), 3 days (Lanes 3 and 7), 4 days (Lanes 4 and 8), or 5 days (Lanes 5 and 9). As a control, AKR-2B cells were treated with IFN for 48 h (Lane 1). Cells extracts were prepared as described in "Materials and Methods," and equal amounts of proteins were analyzed by immunoblotting using the anti-p202 antibody. An arrow indicates the p202 protein band.

 
To the best of our knowledge, murine AKR-2B fibroblasts and NIH 3T3 cells are not known to produce IFN under reduced serum conditions; however, we tested whether, under reduced serum conditions, AKR-2B cells produced IFN, which can contribute to the increased levels of p202. For this purpose, we collected the conditioned medium after incubation of cells in 1% serum for 3 days, supplemented the medium with 9% serum, and incubated fresh cultures of AKR-2B cells for 3 days. The analysis of p202 levels in extracts prepared from these cells revealed that p202 levels did not increase under these culture conditions (data not shown). This observation makes it unlikely that the increase in p202 levels under reduced serum conditions in AKR-2B and NIH 3T3 cells was due to production of IFNs (or other cytokines).

Addition of Growth Factors Abrogates the Increase in p202 Levels under Reduced Serum Conditions.
Because a decrease in serum growth factor levels resulted in an increase in the basal levels of p202 (Fig. 1)Citation , we sought to determine whether the addition of growth factors such as PDGF, bFGF, or TGF-ß1, which are known to act as mitogen for fibroblasts (10 , 11) , to the culture medium under reduced serum conditions abrogates the increase in p202 levels. As shown in Fig. 3Citation , the addition of increasing amounts of PDGF-AB (Fig. 3A)Citation , bFGF (Fig. 3B)Citation , or TGF-ß1 (Fig. 3C)Citation to the culture medium inhibited the increase in p202 levels. These experiments suggested that the presence of PDGF-AB, bFGF, or TGF-ß1 in the culture medium of these fibroblasts negatively regulates p202 levels.



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Fig. 3. Addition of serum growth factors to the culture medium, under reduced serum conditions, abrogates the increase in p202 levels. A, subconfluent cultures of murine AKR-2B cells were incubated in growth medium supplemented with either 10% (Lane 2) or 1% (Lanes 3–5) FBS along with 10 (Lane 4) or 100 ng/ml human recombinant PDGF-AB (Lane 5) for 3 days. As a control, AKR-2B cells were treated with IFN for 48 h (Lane 1). Cells extracts were prepared as described in Fig. 1Citation A, and equal amounts of proteins were analyzed by immunoblotting using the anti-p202 antibody (top panel). The same membrane was subsequently probed with antibodies to ß-actin (bottom panel) to control loading of equal amounts of proteins in the lanes. An arrow indicates the p202 protein band. B, subconfluent cultures of murine AKR-2B cells were incubated in growth medium supplemented with either 10% (Lane 1) or 1% (Lanes 2–4) FBS along with 10 (Lane 3) or 20 ng/ml recombinant bFGF (Lane 4) for 3 days. Cells extracts were prepared as described in Fig. 1Citation A, and equal amounts of proteins were analyzed by immunoblotting using the anti-p202 antibody (top panel). The same membrane was subsequently probed with antibodies to ß-actin (bottom panel) to control loading of equal amounts of proteins in the lanes. An arrow indicates the p202 protein band. C, subconfluent cultures of murine AKR-2B cells were incubated in growth medium supplemented with either 10% (Lane 2) or 1% (Lanes 3–5) FBS along with 4 (Lane 4) or 8 ng/ml recombinant TGF-ß 1 (Lane 5) for 3 days. As a control, AKR-2B cells were treated with IFN for 48 h (Lane 1). Cells extracts were prepared as described in Fig. 1Citation A, and equal amounts of proteins were analyzed by immunoblotting using the anti-p202 antibody. An arrow indicates the p202 protein band.

 
Because the addition of growth factors in cultures of AKR-2B cells resulted in a decrease in p202 levels, we sought to determine whether p202 levels decrease when the cells exit the G0-G1 phase of the cell cycle in response to serum stimulation. For this purpose, we incubated AKR-2B cells for 3 days in medium supplemented with 1% serum (under these conditions, more than 85% cells accumulated in G0-G1) before stimulating the cells with 10% serum. As shown in Fig. 4Citation , serum stimulation of cells resulted in an about 40% decrease in p202 levels within 8 h (compare Lane 3 with Lane 5), and the levels of p202 decreased to almost the basal levels found in these cycling cells within 24 h (compare Lane 2 with Lane 7). Because these cells start to enter into S phase only after about 16 h of serum stimulation, this observation raised the possibility that a decrease in the basal levels of p202 after serum stimulation may be important for passage of the cells through the restriction point and G1-S-phase progression.



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Fig. 4. Serum stimulation of serum-starved AKR-2B cells results in a decrease in p202 levels. Subconfluent cultures of murine AKR-2B cells were incubated in growth medium supplemented with either 10% (Lane 2) or 1% (Lanes 3–7) FBS for 3 days. Cultures in Lanes 4–7 were washed with warm PBS, and cells were serum stimulated with growth medium supplemented with 10% serum for 4 (Lane 4), 8 (Lane 5), 14 (Lane 6), or 24 h (Lane 7). As a control, cells were treated with IFN for 48 h (Lane 1). The total cell extracts were prepared as described in "Materials and Methods." Extracts containing equal amounts of proteins were analyzed by immunoblotting using the anti-p202 antibody (top panel). The same membrane was subsequently probed with antibodies to ß-actin (bottom panel) to control loading of equal amounts of proteins in the lanes. An arrow indicates the p202 band.

 
The Increase in p202 Levels Correlates with Cell Growth Arrest.
Our previous studies have revealed that expression of p202 in AKR-2B cells retards cell proliferation (20) . Therefore, we determined whether the increase in p202 levels under reduced serum conditions correlated with the inhibition of cell proliferation. As shown in Fig. 5ACitation , incubation of cells in 1% serum significantly reduced number of cells (as compared with incubation of cells in 10% serum) within 2 days, and the decrease in number of cells was more pronounced after 4 days of incubation. To determine whether the decrease in cell number was due to the accumulation of cells in the G0-G1 phase of the cell cycle, we subjected the cells to flow cytometry. As shown in Fig. 5BCitation , incubation of AKR-2B cells under reduced serum conditions for 2 days, which we found resulted in an increase in p202 RNA (see Fig. 1CCitation ) and protein levels (see Fig. 1A and BCitation ), correlated with the accumulation of cells in the G1 phase of the cell cycle. Interestingly, incubation of cells in 10% serum for 4 days, which allowed cells to reach high density (Fig. 2)Citation , also resulted in an accumulation of cells in the G1 phase of the cell cycle (Fig. 5B)Citation . However, these cells did not have a significant (only 50%) increase in the levels of p202 (see Fig. 2Citation ). These results clearly indicated that the cell density-dependent growth arrest of AKR-2B cells seen after the incubation of cells in 10% serum for 4 days (Fig. 5B)Citation differs from the growth arrest due to the reduced serum in the culture medium, and only the latter resulted in a 6-fold increase in p202 levels.



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Fig. 5. An increase in p202 levels under reduced serum conditions correlates with cell growth inhibition. A, subconfluent cultures of AKR-2B cells (in duplicates) were incubated in growth medium supplemented with either 10% ({blacksquare}) or 1% serum ({square}) for 0 day, 2 days, or 4 days. Cells were counted three times in a hemocytometer for each time point, and the average cell number is indicated. B, subconfluent cultures of AKR-2B cells (in duplicates) were incubated in growth medium supplemented with either 10% (left panels) or 1% FBS (right panels) for 2 days (top panels) or 4 days (bottom panels). Harvested cells (5 x 105) were processed for flow cytometry as described in "Materials and Methods." The samples were subjected to flow cytometry (FACStar PLUS; Becton Dickinson) and analyzed using ModFit LT version 2.0 software (Variety Software House). The G1:S-phase ratio is indicated.

 
Furthermore, as reported previously (19) , under reduced serum conditions, cultures of AKR-2B cells did not have a significant number of apoptotic cells (as determined by flow cytometry). However, we observed that under these conditions, cultures of NIH 3T3 cells had a significant (10–20%) number of apoptotic cells (data not shown).

The Increase in p202 Levels Is Accompanied by an Increase in JunD Levels.
It has been shown that under reduced serum conditions, increased levels of JunD in fibroblasts negatively regulate cell proliferation (12 , 14) . Therefore, to begin to elucidate the molecular mechanism(s) by which the 202 RNA and protein levels are increased under reduced serum conditions, we considered the possibility that an increase in JunD levels under reduced serum conditions might contribute to the increase in p202 levels. To test this possibility, we determined whether an increase in p202 levels, under reduced serum conditions, accompanies an increase in JunD levels. As shown in Fig. 6Citation , incubation of AKR-2B fibroblasts for the indicated time under reduced serum conditions resulted in an about 2-fold increase in the nuclear levels of JunD (compare Lane 1 with Lane 2 or Lane 3 with Lane 4). Further incubation of cells after 3 days resulted in a marked increase in JunD levels (compare Lane 7 with Lane 8). It is of note that at this time, the nuclear levels of JunD decreased in cells incubated in 10% serum (compare Lane 5 with Lane 7). Although it remains to be seen why nuclear levels of JunD decrease at this time, it is possible that increased cell density of AKR-2B cells also regulates the nuclear levels of JunD. This observation raised the possibility that the increased nuclear levels of JunD may directly or indirectly regulate the expression of the 202 gene.



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Fig. 6. Under reduced serum conditions, JunD levels increase in AKR-2B cells. Subconfluent cultures of murine AKR-2B cells were incubated in growth medium supplemented with 10% (Lanes 1, 3, 5, and 7) or 1% FBS (Lanes 2, 4, 6, and 8) for 1 day (Lanes 1 and 2), 2 days (Lanes 3 and 4), 3 days (Lanes 5 and 6), and 4 days (Lanes 7 and 8). Nuclear extracts were prepared, and extracts containing equal amounts of proteins were analyzed by immunoblotting using the anti-JunD antibody. The same membrane was subsequently probed with antibodies to ß-actin (bottom panel) to control loading of equal amounts of proteins in the lanes. An arrow indicates the JunD protein band.

 
Expression of JunD in Transient Transfection Assays Increases the Activity of the Luciferase Reporter Gene Driven by the 5'-regulatory Region of the 202 Gene.
To further examine the possibility that the increased levels of JunD, under reduced serum conditions, contribute to the transcriptional activation of the 202 gene, we transiently transfected subconfluent cultures of AKR-2B cells (grown in 10% serum) with increasing amounts of plasmid encoding murine JunD along with a reporter plasmid (p202-luc; see Fig. 7ACitation ) in which the transcription of the reporter gene (luciferase) was driven by the 5'-regulatory region of the 202 gene. As shown in Fig. 7BCitation , transfection with the JunD-encoding plasmid, in three independent experiments, increased luciferase activity 3–4-fold (it is of note that the IFN treatment of these cells after transfection with p202-luc reporter plasmid resulted in only a 2-fold increase in the activity of luciferase reporter). Similarly, transfection of JunD-encoding plasmid along with p202-luc reporter plasmid in NIH 3T3 cells also resulted in a 3-fold increase in luciferase activity (data not shown). Additionally, transfection of AKR-2B cells with a plasmid encoding antisense to JunD did not result in a decrease in 202-luc reporter activity. In fact, it resulted in a moderate (about 2-fold) increase in the reporter activity (data not shown). Although it remains to be seen how a decrease in JunD levels under these experimental conditions affects p202 levels, these experiments revealed that JunD regulates the transcription of the 202 gene.



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Fig. 7. Transfection of JunD-encoding plasmid in transient transfection assays increases the luciferase activity driven by the 5'-regulatory region of the 202 gene. A, schematic of the location of AP-1-like sites within the HindIII and PstI fragment (~0.8 kb) from the 5'-regulatory region of the 202 gene, which is linked to the luciferase reporter gene. The location of the "GA-box," which contains the IFN-responsive elements, is also shown. The upstream and downstream ends of the transcription initiation region in the 202 gene are indicated by t-> and ->t. B, subconfluent cultures of murine AKR-2B cells were transfected with the reporter plasmids pGL3–202luc (5 µg) and pRL-TK (0.5 µg) along with the JunD expression plasmid (0–4 µg) or vector alone (in the same amounts) using the calcium phosphate transfection system as described in "Materials and Methods." Cells were harvested 42–48 h after transfections, and the firefly luciferase and Renilla luciferase activities were determined using the Dual-Luciferase Reporter Assay kit (Promega). The firefly luciferase activity was normalized to the Renilla luciferase activity to control for variations in transfection efficiencies. The luciferase activity in control vector-transfected cells is shown as 1. SD and the results from a representative experiment are shown.

 
Overexpression of JunD in a Stable Cell Line Significantly Increases the Endogenous Levels of p202.
Because expression of JunD in transient transfection assays increased the activity of a reporter gene whose expression was driven by the 202 gene promoter, we tested whether the stable overexpression of JunD increases p202 levels. For this purpose, we established a stable AKR-2B cell line allowing the constitutive overexpression of JunD. Interestingly, overexpression of JunD in transfected cells, but not in vector-transfected cells, resulted in increased nuclear accumulation of JunD (data not shown), and incubation of the cells overexpressing JunD, under reduced serum conditions, resulted in increased levels of p202 (Fig. 8Citation , compare Lane 3 with Lane 6). It is interesting to note that overexpression of JunD in cells grown in the presence of 10% serum did not result in increased levels of p202 (compare Lane 2 with Lane 5). Our observation is thus consistent with earlier studies that found that the presence of growth factors in the culture medium functionally inactivates JunD (12 , 14 , 15) . Additionally, serum stimulation of cells transfected with vector or JunD-encoding plasmid for 24 h resulted in a decrease in p202 levels (compare Lane 3 with Lane 4 and Lane 6 with Lane 7, respectively). Together, these observations are consistent with the possibility that the increased levels of JunD contribute to an increase in p202 levels only under reduced serum conditions.



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Fig. 8. Constitutive overexpression of murine JunD in a stable cell line results in an increase in p202 levels. Cells overexpressing JunD (Lanes 5–7) or control cells (transfected with vector; Lanes 2–4) were incubated in growth medium supplemented with 10% (Lanes 2 and 5) or 1% (Lanes 3, 4, 6, and 7) for 3 days. Cells incubated in 1% serum (Lanes 4 and 7) were also stimulated with 20% serum for 24 h. As a control, AKR-2B cells were treated with IFN for 48 h (Lane 1). Cells extracts were prepared as described in "Materials and Methods," and equal amounts of proteins were analyzed by immunoblotting using the anti-p202 antibody. An arrow indicates the p202 protein band.

 
One of the AP-1-like DNA-binding Sequences Present in the 5'-flanking Region of the 202 Gene Can Bind to the JunD/AP-1 Transcription Factor.
Because the AP-1 family of transcription factors has been shown to participate in the regulation of cell growth and apoptosis in response to signaling from serum growth factor receptors (10 , 11) , we searched the regulatory region of the 202 gene for the presence of an AP-1 DNA-binding element (TGACTCA; Ref. 21 ) to further elucidate the molecular mechanism(s) by which the decrease in serum growth factor levels regulates the 202 RNA and protein levels. This search revealed that the 5'-regulatory region of the 202 gene contains more than one AP-1-like DNA-binding element within 600 bp of the transcriptional start sites [see Fig. 7ACitation (1 , 21) ]. Therefore, we tested whether one of the AP-1-like DNA-binding elements present upstream of the transcription start sites in the 202 gene could bind to transcription factor AP-1 in gel mobility shift assays. As shown in the right panel of Fig. 9ACitation , oligonucleotide containing the AP-1-like sequence (TGACTAA) from the 202 gene regulatory region selectively bound to the transcription factor AP-1 in gel mobility shift assays (Lane 5) and was competed well with a 20-fold mole excess of the cold oligonucleotide containing the AP-1 DNA-binding consensus sequence [TGACTCA; purchased from Santa Cruz Biotechnology (right panel, compare Lane 5 with Lane 8)]. This observation indicated that this AP-1-like sequence from the 202 gene regulatory region has the ability to bind AP-1 in gel mobility shift assays (21) . However, it remains to be seen whether other AP-1-like elements present in the regulatory region of the 202 gene also have the affinity to bind transcription factor AP-1 in gel mobility shift assays. Additionally, it remains to be seen whether more than one AP-1 element present in the 5'-regulatory region of the 202 gene is required for transcriptional regulation of the 202 gene.



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Fig. 9. An AP-1-like element present in the 5'-flanking region of the 202 gene can bind to JunD/AP-1 transcription factor. A, left panel, subconfluent cultures of murine AKR-2B cells were incubated in growth medium supplemented with 10% FBS, and total cell extracts were prepared as described in "Materials and Methods." Samples containing equal amounts of proteins were analyzed by gel mobility shift assays using labeled oligonucleotide containing the AP-1 DNA-binding consensus sequence (probe; Santa Cruz Biotechnology). Extracts with labeled probe (Lane 2) or with labeled probe along with a 50-fold mole excess of cold oligonucleotide (Lane 3). As a control, probe without an extract was also run in Lane 1. Right panel, extracts were subjected to gel mobility shift assays using labeled probe containing the AP-1-like DNA-binding sequence present in the 202 gene promoter (Lanes 4–8). Probe without any extracts (Lane 4), with extracts along with cold oligonucleotide (containing the AP-1 consensus sequence) 2.5-fold mole excess (Lane 5), with 5-fold mole excess (Lane 6), with 10-fold mole excess (Lane 7), and with 20-fold mole excess (Lane 8). An arrow indicates the AP-1-specific band. B, under reduced serum conditions, increased levels of JunD are detected in the AP-1 DNA-binding complex. Subconfluent cultures of AKR-2B cells were incubated in growth medium supplemented with either 10% (Lanes 1–4) or 1% serum (Lanes 5 and 6) for 0 day (Lanes 1 and 2) or 2 days (Lanes 3–6). The nuclear extracts were prepared as described in "Materials and Methods," and samples containing equal amounts of proteins were analyzed by gel mobility shift assays using labeled oligonucleotide probe containing the AP-1 consensus sequence (same as that described in A above). The samples were incubated with anti-JunD antibody before subjecting to gel mobility shift assays (Lanes 2, 4, and 6) as described in "Materials and Methods." A thin arrow indicates the AP-1-specific band. A thick arrow indicates the JunD/AP-1 protein supershifted by the anti-JunD antibodies.

 
Because the increase in p202 levels, under reduced serum conditions, was accompanied by an increase in JunD levels (Fig. 6)Citation , we tested whether the JunD/AP-1 DNA-binding complex could be detected in extracts prepared from AKR-2B cells after incubation under reduced serum conditions. As shown in Fig. 9BCitation , incubation of the cells under reduced serum conditions for 2 days increased (about 3-fold) the proportion of JunD in the DNA-binding complexes of AP-1 (compare Lane 4 with Lane 6). This observation indicated that in these cells, under reduced serum conditions, increased levels of JunD are found in AP-1 DNA binding complexes. Whereas this observation is consistent with the previous report that in mouse fibroblasts, under reduced serum conditions, the transcription factor AP-1 contains JunD (and not c-Jun or JunB; Ref. 14 ), further work is needed to identify and characterize the components of the AP-1 DNA-binding activity in AKR-2B cells.

Discussion

In response to IFN treatment of murine fibroblasts, including AKR-2B cells, the p202 levels increase up to 16-fold (2) . However, in transfected cells, a 2-fold increase in p202 levels (above the basal levels) results in retardation of cell proliferation (2 , 4 , 5) , indicating that the increased levels of p202 negatively regulate cell proliferation. Our observation that p202 levels were markedly increased in fibroblasts in response to a decrease in serum growth factor levels but not in response to an increase in cell density supports the notion that p202 may have a functional role in growth arrest induced by decrease in serum growth factor levels. Moreover, a transient decrease in p202 levels below a threshold level and/or its functional inactivation after serum stimulation of serum-starved fibroblasts may be required for passage of cells through the restriction point and entry into the S phase. Consistent with this view is the observation that proliferation of quiescent NIH 3T3 cells on stimulation by serum addition is strongly inhibited by constitutive overexpression of p202 (22) . Additionally, when growth-arrested cells are serum stimulated to proliferate, overexpression of p202 inhibits G0-G1 progression into the S phase, and the cells accumulate with a DNA content that is equivalent to cells arrested in the G0-G1 phase of the cell cycle (22) .

Our observations reported herein that the presence of growth factors (for example, PDGF-AB, bFGF, and TGF-ß1) in culture medium supplemented with 1% serum results in a decrease in p202 levels (see Fig. 3Citation ) indicate that signaling from the growth factor receptors negatively regulates p202 levels. Consistent with this notion is the observation that transformation of NIH 3T3 cells with SV-40 large T-antigen or activated H-ras inhibited the increase in p202 levels under reduced serum conditions (data not shown).

Furthermore, our observations provide support to the hypothesis that, under reduced serum conditions, increased levels of p202 contribute to the inhibition of cell proliferation by inhibiting the transcriptional activity of E2F and AP-1, the two transcription factors implicated as positive regulators of cell proliferation and G1-S-phase progression (23 , 24) . Consistent with this prediction, we found that increased levels of p202 under reduced serum conditions correlated with inhibition of the sequence-specific DNA-binding activity of E2F and AP-1 (data not shown).

The IFN treatment of cultured murine fibroblasts (including NIH 3T3, AKR-2B, BALB/c 3T3, and L929) and C2C12 myoblasts results in an increase in p202 levels (2 , 25) . However, IFN treatment of fibroblasts derived from the C57 black strain of mice does not result in the induction of p202 (2) . It has been proposed that mouse strain-specific factors contribute to the regulation of the 202 gene in response to the IFN treatment (2 , 3) .

Incubation of murine C2C12 myoblasts under reduced serum conditions was shown to result in an increase in p202 levels (25) . Similarly, we found that, under reduced serum conditions, p202 levels are also increased in AKR-2B and NIH 3T3 fibroblasts. However, p202 levels did not increase in BALB/c 3T3 and L929 cells (data not shown). These observations raise the possibility that factors such as the strain-specific variations also contribute to the regulation of the 202 gene under reduced serum conditions.

Our observations reported herein that: (a) under reduced serum conditions, an increase in p202 levels was accompanied by an increase in JunD levels; (b) ectopic expression of JunD resulted in an increase in p202 levels; (c) in transient transfection assays, transfection of the JunD-encoding plasmid increased the activity of luciferase driven by the 202 gene promoter; and (d) under reduced serum conditions, there was a significant increase in the DNA-binding activity of the JunD/AP-1 complex; support the hypothesis that JunD retards the proliferation of fibroblasts, in part, by up-regulating the p202 levels.

Ectopic expression of p202 in murine L929 cells inhibits AP-1 (c-Fos and c-Jun)-stimulated transcription (6) . Furthermore, p202 binds to c-Jun and c-Fos and inhibits the DNA-binding activity of AP-1 (6) . At present, it is not known whether p202 interacts with JunD. However, based on the high degree of sequence homologies among the Jun family of proteins, it is conceivable that p202 also binds to JunD. Further work is in progress to test this possibility.

The data presented here reveal that, under reduced serum conditions, p202 levels increase in fibroblasts independent of IFN production. Additionally, in transfected cells, expression of JunD positively regulates the expression of the 202 gene. Whereas our observations described herein provide support to the notion that, under reduced serum conditions, increased levels of JunD/AP-1 transcriptionally regulate p202 levels, it remains to be seen whether the binding of the JunD/AP-1 transcription factor to the regulatory region of the 202 gene results in cooperation with other transcription factors to regulate the expression of the 202 gene.

The ability of p202 to negatively regulate the transcriptional activity of E2F and p53 makes it conceivable that increased levels of p202 in fibroblasts contribute to the regulation of growth arrest and apoptosis. Because decreased levels of p202 in AKR-2B fibroblasts, under reduced serum conditions, were found to increase the susceptibility to apoptosis (19) , the data presented here also raise the possibility that increased levels of p202 in cells contribute to the regulation of apoptosis. However, the molecular mechanism(s) by which p202 regulates apoptosis remains to be identified.

The work presented here will facilitate efforts toward understanding the functional role and regulation of p202 in normal and cancer cells in response to the activation of a variety of signaling pathways, including mitogenic signaling.

Materials and Methods

Cell Lines.
AKR-2B (originally a gift from Dr. H. L. Moses; Vanderbilt University, Nashville, TN), NIH 3T3 (from American Type Culture Collection), and L929 (a generous gift from Dr. B. B. Aggarwal; The University of Texas M. D. Anderson Cancer Center, Houston, TX) were grown in DMEM with high glucose supplemented with 10% FBS in a 37°C incubator with 5% CO2. Recombinant IFN (Universal Type-1; 1000 units/ml; Research Diagnostic, Inc., Flanders, NJ) was added as indicated to subconfluent cultures as described previously (26) . To reduce serum concentrations in the medium, cells were first washed with warm (37°C) PBS and then incubated with the medium supplemented with 1% or 0.01% FBS.

Plasmid Constructs and Reporter Assays.
The 202 reporter plasmid (pGL3–202luc) containing the 5'-regulatory region (0.8 kb; see Fig. 7ACitation ) from the 202 gene was constructed by ligating the HindIII-PstI fragment (nucleotides 408-1222 in Fig. 3Citation in Ref. 1 ) from plasmid pBA (27) into the pGL3 basic (without any enhancer and promoter sequences) vector (from Promega, Madison, WI). pRL-TK reporter vector allowing the expression of Renilla luciferase was purchased from Promega. Mouse JunD cDNA in plasmid pcDNA3.1 was generously provided by Dr. Sunita Agarwal (NIH/National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD).

For reporter assays, subconfluent cells in 6-well plates were transfected with the reporter plasmids pGL3–202luc (5 µg) and pRL-TK (0.5 µg) along with the expression plasmid (0–4 µg) using the calcium phosphate transfection system (Life Technologies, Inc., Rockville, MD), as suggested by the supplier. Cells were harvested 42–48 h after transfections, and the firefly luciferase and Renilla luciferase activities were determined using the Dual-Luciferase Reporter Assay kit (from Promega) and a TD-20/20 luminometer (from Turner Designs). The firefly luciferase activity was normalized to the Renilla luciferase activity to control for variation in transfection efficiencies. The luciferase activity in control vector-transfected cells is shown as 1.

Cell Cycle Analyses.
To determine cell cycle distribution, cells (5 x 105) were washed with PBS, fixed with 70% ethanol, and stored at -20°C. For staining, the fixed cells were washed with PBS and incubated for 5 min at room temperature in buffer containing 0.2% Triton X-100 and 1 mM EDTA in PBS. The cells were stained for 2 h at room temperature with buffer containing 50 µg/ml propidium iodide and 50 µg/ml RNase. The samples were subjected to flow cytometry (FACStar PLUS; Becton Dickinson) and analyzed using ModFit LT version 2.0 software (Variety Software House, Topsham, ME).

Northern Blot Analysis.
AKR-2B cells were grown in the presence of the indicated concentrations of serum in the growth medium for the indicated number of days, and cytoplasmic RNA was prepared as described previously (20) . RNA samples were subjected to Northern blot analysis as described previously (20) . A labeled the 202 gene-specific cDNA probe was hybridized to RNA at the desired temperature for 1–1.5 h using rapid hybridization buffer (Clontech) as suggested by the supplier.

Western Blot Analysis.
To detect p202 levels, cells were collected from plates in PBS, resuspended in modified radioimmunoprecipitation assay lysis buffer (2) supplemented with protease inhibitors (50 µg/ml leupeptin, 50 µg/ml pepstatin A, and 1 mM phenylmethylsulfonyl fluoride), and incubated at 4°C for 30 min. Lysates were sonicated briefly before centrifugation at 14,000 x g in a microfuge for 10 min. Supernatants were collected, and equal amounts of proteins (60 µg) were processed for immunoblotting as described previously (2) . The polyclonal anti-p202 antiserum directed against full-length p202 has been described previously (2) . Polyclonal antibodies directed against the NH2 terminus (M-20) and COOH terminus (S-19) peptides of p202 were purchased from Santa Cruz Biotechnology.

To detect JunD levels, the nuclear extracts were prepared as described previously (28) , and the extracts containing equal amounts of proteins were analyzed by immunoblotting using a polyclonal anti-JunD antibody (C-19; Santa Cruz Biotechnology).

Electrophoretic Mobility Shift Assays.
Cells were either treated with {alpha}-IFN or incubated under reduced serum (1% or lower) conditions for the indicated time. The total cell extracts were prepared for mobility shift assays as described previously (5) . For nuclear extracts, cells were processed essentially as described previously (28) , and a protease inhibitor mixture from Sigma was added. Oligonucleotide containing the consensus sequence to bind AP-1 transcription factor was purchased from Santa Cruz Biotechnology. The oligonucleotide containing the AP-1 binding consensus sequence 5'-GTGAGCCTGACTAAGCTGTGA-3' and the complementary sequence 5'-TCACAGCTTAGTCAGGCTCAC-3' in the regulatory region of the 202 gene (from nucleotide 889–908 in Fig. 3Citation in Ref. 1 ) was synthesized. The oligonucleotides were labeled with polynucleotide kinase as described previously (29) .

Acknowledgments

We thank Drs. Manuel O. Diaz, Richard M. Schultz, and Andrew Vaughan for helpful discussions and Sunita Agarwal for providing JunD expression plasmid. We also thank Bonnie Kalemba for secretarial 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 Supported by NIH Grant CA69031. Back

2 To whom requests for reprints should be addressed. Present address: Department of Radiation Oncology, Loyola University Medical Center-Hines, Fifth Avenue and Roosevelt Road, Building 1, Room F-317, Hines, IL 60141. Phone: (708) 202-8387. Back

3 The abbreviations used are: AP-1, activation protein 1; PDGF, platelet-derived growth factor; bFGF, basic fibroblast growth factor; TGF, transforming growth factor; FBS, fetal bovine serum. Back

Received for publication 2/28/00. Revision received 6/15/00. Accepted for publication 7/25/00.

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