NF-kappa B-dependent Induction of Cyclin D1 by Retinoblastoma Protein (pRB) Family Proteins and Tumor-derived pRB Mutants*

Tetsuro TakebayashiDagger , Hideaki HigashiDagger , Hideki SudoDagger , Heita OzawaDagger , Etsu Suzuki§, Osamu Shirado, Hiroyuki Katoh||, and Masanori HatakeyamaDagger **

From the Dagger  Division of Molecular Oncology, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, the § Division of Nephrology and Endocrinology, Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Tokyo 113-8655, the  Department of Orthopedic Surgery, and the || Department of Surgical Oncology, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan

Received for publication, October 23, 2002, and in revised form, January 27, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The retinoblastoma protein (pRB) and its homologues, p107 and p130, prevent cell cycle progression from G0/G1 to S phase by forming complexes with E2F transcription factors. Upon phosphorylation by G1 cyclin-cyclin-dependent kinase (Cdk) complexes such as cyclin D1-Cdk4/6 and cyclin E-Cdk2, they lose the ability to bind E2F, and cells are thereby allowed to progress into S phase. Functional loss of one or more of the pRB family members, as a result of genetic mutation or deregulated phosphorylation, is considered to be an essential prerequisite for cellular transformation. In this study, we found that pRB family proteins have the ability to stimulate cyclin D1 transcription by activation of the NF-kappa B transcription factor. The cyclin D1-inducing activity of pRB is abolished by adenovirus E1A oncoprotein but not by the deletion of the A-box, the B-box, or the C-terminal region of the pocket, indicating that multiple pocket sequences are independently involved in cyclin D1 activation. Intriguingly, tumor-derived pRB pocket mutants retain the cyclin D1-inducing activity. Our results reveal a novel role of pRB family proteins as potential activators of NF-kappa B and inducers of G1 cyclin. Certain pRB pocket mutants may give rise to a cellular situation in which deregulated E2F and cyclin D1 cooperatively promote abnormal cell proliferation.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The retinoblastoma protein (pRB)1 and its homologues, p107 and p130, are important regulators of the mammalian cell cycle (1-11). pRB (and probably p107 as well) plays an essential role in the growth decision-making at the late G1 restriction point, whereas p130 is thought to be involved in G0-to-G1 phase transition (10, 11). Results of recent studies have further suggested that pRB and p107 play a regulatory role in the S phase in response to DNA damage (12, 13). The cell cycle-controlling activities of the pRB family proteins are dependent on the functions of the shared structure termed the "pocket" that is composed of the A-box, the spacer region, the B-box, and the C-terminal region (10, 14-17). The pocket is capable of binding a number of cellular proteins (18). Among those, E2F family transcriptional factors are thought to be physiologically relevant targets of pRB family proteins (10, 19-21). Upon complex formation, pRB family proteins inhibit transcriptional activation of E2F-dependent genes, whose products are essentially required for cell cycle progression. A pRB family protein-E2F complex also acts as a repressor against promoters containing E2F-binding sites, thereby actively repressing transcription of E2F-responsive genes in the G0/G1 phase (20-22). Stimulation of resting (or G0) cells with mitogenic signals gives rise to the induction of G1 cyclins (cyclin D1, D2, D3, and E), which in turn activate cyclin-dependent kinases (Cdk) such as Cdk2, 4, and 6 (1, 2, 23). The resulting G1 cyclin-Cdk complexes phosphorylate pRB family proteins and abolish their ability to form physical complexes with E2F, thereby leading to cell cycle progression and subsequent cell division.

The function of pRB is lost through mutations of the RB gene in retinoblastomas, small cell lung carcinomas, osteosarcomas, and bladder carcinomas (1, 2, 24). Although mutations of the RB gene occur in less than 20% of whole cancer cell types, loss of p16INK4A, a specific inhibitor of cyclin D1-Cdk4/6, cyclin D1 overexpression, or production of Cdk4 mutants that cannot bind p16INK4A is observed in most, if not all, cancer cells (25-30). All of these changes lead to inappropriate phosphorylation and, hence, functional inactivation of pRB family proteins. Thus, the frequent alterations in upstream regulators that constitute the "p16INK4A-pRB family" pathway appear to be an essential step in cancer development.

Cyclin D1 is inducibly expressed upon mitogenic stimulation and forms a physical complex with Cdk4 or Cdk6 (2, 31, 32). Since the cyclin D1-Cdk4/6 complex is a major kinase for pRB family proteins, cyclin D1 is recognized as an upstream regulator of pRB family proteins. Furthermore, cyclin D1 binds pRB through the pocket domain (33, 34), indicating that cyclin D1-Cdk4/6 may be a downstream target of pRB as well. A complex interaction between cyclin D1 and pRB has also been suggested by the finding that pRB negatively regulates p16INK4A (35). These findings indicate the presence of a regulatory network among pRB family proteins, p16INK4A and cyclin D1. Such a regulatory loop may play a crucial role in the physiological regulation of pRB family proteins.

In addition to the above described interaction between pRB and cyclin D1, Müller et al. reported that pRB has the ability to induce cyclin D1 (36). We have extended their work by demonstrating that all of the pRB family proteins are capable of inducing cyclin D1. We also found that pRB family proteins transcriptionally activate the cyclin D1 gene by stimulating the NF-kappa B transcription factor. The cyclin D1-inducing activity is dependent on the pocket structure but is independent of pocket function for inhibition of cell growth. Intriguingly, tumor-derived pRB pocket mutants, which cannot bind E2F and therefore cannot inhibit progression of the cell cycle, are still capable of inducing cyclin D1. Stimulation of cyclin D1 by pRB family proteins may play an important role in the physiological regulation of cell cycle progression as well as in the development of cancer.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cells-- The human osteosarcoma line SAOS-2 was provided by Dr. Phil Hinds (Harvard Medical School). The human cervical carcinoma line C33A and the human osteosarcoma line U2-OS were obtained from the American Type Culture Collection (Manassas, VA). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS).

cDNAs and Plasmids-- Expression vectors for the human pRB, p107, and p130 were constructed as described previously (37). A cDNA encoding the phosphorylation-resistant pRB mutant, pRBDelta S/T-P, was described previously (38). cDNAs encoding tumor-derived pocket mutants, pRBDelta 22 and pRB706CF, were described previously (39). The pRBDelta N-HA and pRBN392 mutants were generated by subcloning pRB fragments generated by PCR using human RB cDNA as a substrate. The PCR primers employed were as follows: for pRBDelta N-HA, sense primer 5'-GGGCGGCCGCGCCGCCATGGCAAGTGAT-3' and antisense primer 5'-CTGGGTCTGGAAGGCTGAGGTTGC-3'; for pRBN392, sense primer 5'-GGTTCACCTCGAACACCCAGGCGAGG-3' and antisense primer 5'-GGGGTACCTCATGCAGAATTTAAAATCATCATTAATTGTTGG-3'. A series of deletion mutants for the pRB pocket were generated by oligonucleotide-mediated mutagenesis with the use of the Chameleon site-directed mutagenesis system (Stratagene). The RB mutant cDNAs were subcloned into pSP65-SRalpha vector (40). The human cyclin D1 promoter-luciferase construct, pGL2-944 cycD1-luc, was obtained from Rolf Müller (Philipps-Universitat Marburg, Germany). Construction of the cyclin D1 promoter mutants (pGL2-707Delta NF-kappa B2, -229Delta STAT2, -95Delta SP1, -23Delta NF-kappa B3, -229Delta STAT2/CREB mut, and -229Delta STAT2/NF-kappa B3 mut) has been described previously (41). p65 NF-kappa B cDNA and p55-Igkappa -luc reporter plasmid were gifts from Dr. Takashi Fujita (Tokyo Metropolitan Institute for Medical Science, Tokyo, Japan) (42). pME-Ikappa Balpha and pME-Ikappa Balpha S32AS36A were gifts from Dr. Jun-Ichiro Inoue (University of Tokyo, Tokyo, Japan). pSV-E1A-12S and pSV-E1A-928 were described previously (37).

Transfection and Immunoblotting-- Expression plasmids (18 µg) were transfected together with the 2 µg of the puromycin resistance gene (pBabe-puro) into 1.5 × 106 SAOS-2 cells or 4 × 106 C33A cells in a 100-mm plate by the calcium phosphate precipitation method as described previously (43). In Ikappa B experiment, 5 µg of pSP65-SRalpha -pRB and 15 µg of pME-Ikappa Balpha or pME-Ikappa Balpha S32AS36A were transfected together with the 2 µg of pBabe-puro into 1.5 × 106 SAOS-2 cells. In the E1A experiment, 10 µg of pSP65-SRalpha -pRB and 10 µg of pSV-E1A-12S or pSV-E1A-928 mutant were transfected together with the 2 µg of pBabe-puro into 1.5 × 106 SAOS-2 cells. After 72 h of culture in DMEM with 10% FCS in the presence of 0.5 and 2 µg/ml puromycin for SAOS-2 cells and C33A cells, respectively, the transfected cells were harvested and lysed in E1A cell lysis buffer as described (43). Cell lysates were subjected to SDS-PAGE, transferred to polyvinylidene difluoride filters (Millipore Corp.), and immunoblotted with appropriate antibodies. Proteins were visualized using the enhanced chemiluminescence detection system (ECL; PerkinElmer Life Sciences). The antibodies used were anti-cyclin D1 (H-295, sc-753; Santa Cruz Biotechnology), anti-pRB (G3-245; Pharmingen), anti-HA monoclonal antibody (12CA5), and anti-Cdk2 (M2, sc-163; Santa Cruz Biotechnology). Intensities of chemiluminescence on the immunoblotted filters were quantitated by using a luminescent image analyzer (LAS-1000; Fuji).

Transfection of Small Interfering RNAs (siRNAs)-- Synthetic siRNAs were purchased from Greiner Bio-One. The pRB-specific siRNA molecules used in this study have the following sequences: 5'-cuguggggaaucuguaucu TT and 3' TT-gacaccccuuagacauaga. The U2-OS cells were grown in DMEM supplemented with 10% FCS. Synthetic siRNA transfection was carried out in a 60-mm dish (1.5 × 105 cells/4 ml of DMEM with 10% FCS) by using Oligofectamine Reagent (Invitrogen) according to the manufacturer's instructions. At 72 h after the transfection, cells were harvested and cell lysates were prepared. Aliquots of the cell extracts containing equal amounts of proteins were analyzed by immunoblotting.

Luciferase Assay-- SAOS-2 cells or C33A cells were transiently co-transfected with a luciferase reporter plasmid and an expression vector by the calcium phosphate precipitation method. Transfected cells were cultured in DMEM plus 10% FCS for 36 h, and luciferase activities were measured using the luciferase reporter assay system (Promega) as described previously (44).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

pRB, p107, and p130 Stimulate Expression of Cyclin D1-- Ectopic expression of pRB in RB-deficient cells has been reported to induce cyclin D1 (36). We carried out an experiment to determine whether this cyclin D1-inducing activity is a biological property shared by other pRB family members. An expression vector for pRB, p107, or p130 was transiently transfected together with the puromycin resistance gene (pBabe-puro) into the RB-defective human osteosarcoma line SAOS-2. Transfected cells were selected by puromycin, and the cell lysates were prepared 72 h after the drug selection. Anti-cyclin D1 immunoblotting of the cell lysates exhibited that, in addition to pRB, ectopic expression of p107 or p130 is capable of inducing cyclin D1 (Fig. 1A). To exclude the possibility that the cyclin D1 induction is simply due to G1 cell cycle arrest provoked by pRB family proteins, we ectopically expressed a p27Kip1 Cdk inhibitor, which exerts a potent action to arrest the cell cycle at the G1 phase, and confirmed that p27Kip1 did not induce cyclin D1 in SAOS-2 cells (Fig. 1B). An increase in cyclin D1 protein levels induced by pRB family proteins was also observed in the human cervical carcinoma cell line C33A (Fig. 1A).


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Fig. 1.   Induction of cyclin D1 by ectopic expression of pRB, p107, and p130. A, whole cell extracts were prepared from SAOS-2 cells or C33A cells transfected with each of the pRB family expression vector and were immunoblotted with the described antibodies. The plasmid-directed pRB family proteins were HA epitope-tagged at the C terminus and thus were detectable by anti-HA. B, whole cell extracts were prepared from SAOS-2 cells transfected with HA-tagged p27Kip1 (p27-HA) or a control empty vector. Cell lysates prepared were immunoblotted with the described antibodies.

Effect of pRB-specific siRNA on Cyclin D1 Expression-- We next used U2-OS human osteosarcoma cells in an experiment to determine whether the level of cyclin D1 is reduced by a decrease in endogenous pRB level. In U2-OS cells, virtually all of the pRB species are present in the hyperphosphorylated form (Fig. 2A) due to loss of the p16INK4A Cdk inhibitor as previously reported (37, 45). Treatment of U2-OS cells with pRB-specific siRNA (pRB-siRNA) resulted in specific inhibition of pRB expression (Fig. 2B). Quantitation of the pRB bands using the Cdk2 bands as controls revealed that pRB expression was reduced by 50% in cells treated with pRB-siRNA. This pRB inhibition was concomitantly associated with reduced levels of cyclin D1 (~50% reduction) (Fig. 2B). The result indicates that endogenous pRB plays an important role in the maintenance of cellular cyclin D1 levels. Since pRB-siRNA treatment did not alter the phosphorylation status of pRB, the result also suggests that hyperphosphorylated pRB, which is incapable of binding to E2F and several other pocket-binding proteins, retains the ability to induce cyclin D1.


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Fig. 2.   Effect of pRB knockdown by siRNA on the levels of cyclin D1. A, phosphorylation status of pRB in U2-OS cells. Whole cell extracts were prepared from U2-OS cells transfected with control vector (control) or expression vector for the phosphorylation-resistant pRB, pRBDelta S/T-P, and were immunoblotted with anti-pRB antibody. The positions of the hypophosphorylated pRB (pRB) and the hyperphosphorylated pRB (ppRB) as determined by the band corresponding to pRBDelta S/T-P are indicated. B, whole cell extracts were prepared from U2-OS cells treated with (+) or without (-) pRB-specific siRNA for 72 h and were immunoblotted with the indicated antibodies.

Activation of the Cyclin D1 Promoter by pRB, p107, and p130-- pRB stimulates cyclin D1 through transcriptional activation of the cyclin D1 gene (36). To determine whether the same is also true in the cases of p107 and p130, we examined their effects on the human cyclin D1 promoter. Transient co-expression of a reporter plasmid that has a luciferase gene fused to the human the cyclin D1 promoter together with each of pRB family expression vectors in C33A cells revealed that, like pRB, both p107 and p130 are capable of transactivating the cyclin D1 promoter (Fig. 3A). Since these proteins are characterized by a shared structure termed the "pocket" that is involved in their interaction with target proteins, the results indicate that pRB family proteins induce cyclin D1 through a shared pocket function, most probably through pocket-protein interaction.


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Fig. 3.   pRB family proteins transcriptionally activate cyclin D1 through a proximal NF-kappa B site. A, C33A cells were transiently cotransfected with a reporter plasmid containing the cyclin D1 promoter-luciferase gene (-944 cycD1-luc) and pRB-HA, p107-HA, p130-HA, or a control empty vector. The promoter activation was shown as a ratio of the luciferase activities in the presence and absence of individual pRB family protein. The graphs represent the means and 2× S.D. values from three individual experiments. B, schematic representations of cis-elements in the human cyclin D1 promoter as well as the mutant cyclin D1 promoter-luciferase constructs used in the experiment (upper panel). SAOS-2 or C33A cells were transiently cotransfected with a reporter plasmid containing the mutant cyclin D1 promoter-luciferase gene and pRB-HA or a control empty vector. The promoter activation was shown as a ratio of the luciferase activities between the presence and absence of pRB-HA. The graphs represent the means and 2× S.D. values from three individual experiments (lower panel). C, C33A cells were transiently cotransfected with a reporter construct with three tandemly repeated kappa B motifs upstream of a minimal interferon-beta promoter (p55-Igkappa -luc) and pRB-HA, p107-HA, p130-HA, or a control empty vector. The promoter activation was shown as a ratio of the luciferase activities in the presence and absence of individual pRB family members. The graphs represent the means and 2× S.D. values from three individual experiments.

Involvement of NF-kappa B in Cyclin D1 Induction by pRB-- The cyclin D1 promoter is known to be regulated by multiple cis-acting elements, each of which plays a distinct role in promoter activation. They include SP1 sites, a cAMP-responsive element (CRE), and distal and proximal NF-kappa B sites. To delineate cis elements that are required for transcriptional activation of the cyclin D1 promoter by pRB, we transfected a series of luciferase reporter constructs (41) that contained various lengths of the cyclin D1 promoter together with the RB expression vector in SAOS-2 cells. As shown in Fig. 3B, the 5'-boundary of the functional sequence required for cyclin D1 induction was located between -95 and -23 from the transcription initiation site of the cyclin D1 gene. The identified sequence contained CRE and the proximal NF-kappa B site, both of which are reported to be important for cyclin D1 induction in other cell types. Accordingly, we next examined a pGL2/-229 derivative in which either the CRE or NF-kappa B site was specifically destroyed by introducing a point mutation. The promoter activity was not affected when CRE was mutated but was totally abolished by mutation of the proximal NF-kappa B site (Fig. 3B, lower panel, left). Our results thus indicate that the proximal NF-kappa B binding site on the cyclin D1 promoter is required for the induction of cyclin D1 by pRB. The same conclusion was also obtained with the use of C33A cells (Fig. 3B, lower panel, right).

The above observation suggested that the NF-kappa B transcription factor is involved in the induction of cyclin D1 by pRB family proteins. To investigate this, we co-transfected p55-Igkappa -luc, which has a luciferase reporter gene fused to three tandem repeats of the NF-kappa B binding sites from the immunoglobulin kappa  light chain enhancer, with the expression vector for pRB, p107, or p130 into C33A cells. Upon ectopic expression, all of the pRB family proteins were capable of stimulating the NF-kappa B-dependent promoter, indicating that the pocket proteins indeed stimulated transcriptional activity of NF-kappa B (Fig. 3C). As reported (46-48), ectopic expression of the p65 (RelA) subunit of NF-kappa B transactivated the cyclin D1 promoter and increased cyclin D1 protein levels in C33A cells.2

Since activity of NF-kappa B is inhibited by interaction with Ikappa B, we next addressed the effect of Ikappa Balpha on the induction of cyclin D1 by pRB. Co-expression of the NF-kappa B inhibitor, Ikappa Balpha , together with pRB inhibited pRB-dependent cyclin D1 induction (Fig. 4). An Ikappa B up-mutant, Ikappa Balpha S32AS36A, which has serine-to-alanine substitutions at the amino acid residues 32 and 36 and thus is resistant to phosphorylation-dependent degradation (49), exhibited greater activity to inhibit cyclin D1 induction by pRB than wild-type Ikappa B. Based on these observations, we concluded that pRB, and probably the pRB-related p107 and p130 as well, stimulated NF-kappa B and that the stimulated NF-kappa B in turn induced transcriptional activation of the cyclin D1 gene.


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Fig. 4.   Inhibition of pRB-dependent cyclin D1 induction by Ikappa Balpha . SAOS-2 cells were transfected with expression plasmids encoding pRB and Ikappa Balpha or its up-mutant, Ikappa Balpha S32AS36A, in which serine 32 and serine 36 were replaced by alanine residues, together with the puromycin resistance gene. After selection with puromycin for 72 h, cells were harvested, and the cell lysates prepared were immunoblotted with anti-cyclin D1 and anti-Cdk2, respectively.

Structural Requirement for pRB in the Induction of Cyclin D1-- To determine whether the cyclin D1-inducing activity of pRB is due to the pocket domain that is shared among pRB family proteins, we examined the effect of adenovirus E1A 12S product, which specifically binds to the pockets of all of the pRB family proteins (50-52). As shown in Fig. 5A, ectopic co-expression of E1A completely inhibited induction of cyclin D1 by pRB. On the other hand, a mutant E1A (E1A 928) defective for pRB binding (53) did not exhibit any effect on the cyclin D1-inducing activity of pRB. The results indicate that the pocket structure is required for the cyclin D1-inducing activity of pRB (and probably that of p107 and p130 as well) and suggest the involvement of a cellular protein(s) that physically interacts with the pocket for cyclin D1 induction. This notion was further supported by the observation that NF-IL-6 (CCAAT/enhancer-binding protein-beta ), which also binds to the pRB pocket (54-56), again competitively inhibited the induction of cyclin D1 by pRB.3


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Fig. 5.   Involvement of the pRB pocket domain for cyclin D1 induction. A, SAOS-2 cells were cotransfected with pRB and adenovirus E1A (either 12 S product or 928 mutant) expression plasmids. Cell lysates were immunoblotted with anti-cyclin D1 and anti-Cdk2, respectively. B, schematic representations of pRB Delta N-HA and pRBN392 (upper panel). Black and gray rectangles represent the A-box and the B-box of pRB, respectively. HA indicates the HA-epitope tag. Whole cell lysates were prepared from SAOS-2 cells transfected with pRB-HA, pRBDelta N-HA, pRBN392, or a control empty vector and were subjected to immunoblottings with the described antibodies (middle and lower panels). Note that pRBN392 does not have the HA epitope and therefore is detected by anti-pRB but not by anti-HA. C, C33A cells were transiently cotransfected with -944 cycD1-luc and pRB-HA, pRBDelta N-HA, pRBN392, or a control empty vector. The cyclin D1 promoter activation was shown as a ratio of the luciferase activities in the presence and absence of wild-type or each pRB mutant. The graphs represent the means and 2× S.D. values from three individual experiments.

To elucidate the structural basis of pRB involved in cyclin D1 induction, we generated two pRB mutants: pRBN392, which consists of N-terminal amino acid residues 1-392 of pRB, and pRBDelta N-HA, which is composed of the pocket spanning amino acid residues 392-928 of pRB (Fig. 5B, upper panel). The pRBDelta N-HA mutant retained the ability to induce the formation of flat cells (37, 57), a well known pocket activity, when expressed in SAOS-2 cells, and, as expected from the results of the E1A experiment, it was also capable of stimulating cyclin D1. In contrast, pRBN392 did not have the cyclin D1-inducing activity (Fig. 5, B (middle, lower panel) and C). To further clarify the pRB regions that are responsible for cyclin D1 induction, we generated a series of pocket deletion mutants (Fig. 6A). These pRB mutants were expressed in SAOS-2 cells, and their activity to induce cyclin D1 was examined by the use of anti-cyclin D1 immunoblotting. Surprisingly, each of the pRB pocket mutants, which independently lacks the A-box, the spacer region, the B-box, or the C-terminal region, was still capable of inducing cyclin D1 (Fig. 6B). These pRB mutants exhibited comparable levels of cyclin D1 induction, although the expression levels of the Delta C mutant were significantly higher than those of other mutants. Hence, Delta C may be less effective in inducing cyclin D1 than other pocket mutants. To determine the role of the C-terminal region of pRB in cyclin D1 induction, we also generated pRBDelta AB (Fig. 7A), in which the A-box and the B-box were simultaneously deleted. As shown in Fig. 7, B and C, the mutant retained the ability to induce cyclin D1. These results indicate that the "A/B-boxes" and the "C-terminal region" are independently capable of inducing cyclin D1.


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Fig. 6.   Determination of the pRB pocket subregions required for cyclin D1 induction. A, schematic representations of pRB and its pocket mutants. Black and gray rectangles represent the A-box and the B-box of pRB, respectively. B, total cell lysates were prepared from SAOS-2 cells transfected with each of the pRB deletion mutants or a control empty vector and were immunoblotted with the described antibodies.


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Fig. 7.   The role of the pRB C-terminal region in cyclin D1 induction. A, schematic representations of pRB and pRBDelta AB. Black and gray rectangles represent the A-box and the B-box of pRB, respectively. B, whole cell extracts were prepared from SAOS-2 cells transfected with pRB, pRBDelta AB, or a control empty vector and were immunoblotted with the described antibodies. C, C33A cells were transiently co-transfected with -944 cycD1-luc and pRB, pRBDelta AB, or a control empty vector. The promoter activation was shown as a ratio of the luciferase activities in the presence and absence of wild-type pRB or pRBDelta AB. The graphs represent the means and 2× S.D. values from three individual experiments.

Tumor-derived pRB Pocket Mutants Retain the Ability to Induce Cyclin D1-- The pRB pocket mutants described above are incapable of binding E2F and are "functionally inactive" in inhibiting cell growth. It is therefore thought that the cyclin D1-inducing activity of pRB does not require structural integrity of the pocket domain that is specifically involved in cell growth inhibition. This notion prompted us to investigate the activity of tumor-derived pRB pocket mutants, pRBDelta 22 and pRB706CF, in inducing cyclin D1. pRBDelta 22 is an exon 22-skipping pRB mutant, and pRB706CF has a cysteine-to-phenylalanine point mutation at residue 706. Upon expression in SAOS-2, both the pRBDelta 22 and pRB706CF mutants were capable of inducing cyclin D1 (Fig. 8A). Consistent with this observation, both of the mutants activated NF-kappa B and stimulated the cyclin D1 promoter (Fig. 8, B and C). Accordingly, we concluded that the tumor-derived pRB mutants, while incapable of inhibiting cell proliferation, still possess the ability to activate cyclin D1 transcription.


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Fig. 8.   Induction of cyclin D1 by tumor-derived pRB pocket mutants. A, whole cell extracts were prepared from SAOS-2 cells transfected with pRB, pRBDelta 22, pRB706CF, or a control empty vector and were immunoblotted with the described antibodies. B, C33A cells were transiently co-transfected with -944 cycD1-luc and pRB, pRBDelta 22, pRB706CF, or a control empty vector. The promoter activation was shown as a ratio of the luciferase activities between the presence and absence of wild-type or the tumor-derived pRB pocket mutant. The graphs represent the means and 2× S.D. value from three individual experiments. C, C33A cells were transiently co-transfected with p55-Igkappa -luc and pRB, pRBDelta 22, pRB706CF, or a control empty vector. The promoter activation was shown as a ratio of the luciferase activities in the presence and absence of wild type or the tumor-derived pRB pocket mutant. The graphs represent the means and 2× S.D. values from three individual experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this work, we demonstrated that the retinoblastoma family of pocket proteins, consisting of pRB, p107, and p130, are capable of inducing cyclin D1. Our finding extends the results of previous work by Müller et al. (36) showing that pRB induces cyclin D1 and reveals the existence of a "pRB-NF-kappa B-cyclin D1" pathway. Since cyclin D1 forms complexes with Cdk4 or Cdk6 and inactivates the growth-suppressive activity of pRB family proteins by phosphorylation, our results indicate the existence of a complicated functional interplay between pRB family proteins and cyclin D1-Cdk4/6. From the results of overexpression and knockdown experiments, it appears that pRB plays the greatest role in regulation of cyclin D1 among the pRB family proteins. Our present work therefore provides a molecular basis for the reduction in the level of cyclin D1 expression in RB-deficient cells (35, 58-62) as well as in cells expressing simian virus 40 large T antigen, adenovirus E1A, or human papilloma virus E7 oncoprotein (36).

Induction of cyclin D1 by pRB family proteins is due to transcriptional activation of the cyclin D1 gene. Whereas the cyclin D1 promoter is regulated by multiple transcription factors, pRB family-dependent induction is specifically mediated by the NF-kappa B transcription factor through the proximal NF-kappa B binding site on the cyclin D1 promoter. Consistent with our observation, recent studies have shown the importance of NF-kappa B in the activation of cyclin D1 (46-48, 63-65). Proteins of the pRB family can cooperate with transcription factors, such as SP-1 (66-68), CCAAT/enhancer-binding protein family members (54-56), and MyoD (69), to transcriptionally activate genes. Although NF-kappa B has also been reported to interact with pRB (70), we were not able to detect complex formation between the p65 subunit of NF-kappa B and pRB4 (data not shown). Accordingly, the molecular mechanisms through which pRB family members stimulate NF-kappa B are still not known. However, the finding that the adenovirus E1A 12 S RNA product, which specifically interacts with the pocket domains of pRB family proteins (50-52), inhibited cyclin D1 induction by pRB indicates that the activity requires a cellular molecule(s) that physically interacts with the pocket domain.

The cell cycle effect of ectopic pRB family proteins depends on cell context. In cells such as SAOS-2 and C33A that possess high levels of p16INK4A due to a lack of endogenous pRB (35), ectopic expression of pRB, p107, or p130 induces cyclin D1 but arrests cells in G1 (37). This G1 block is mediated by the pRB family proteins, which are kept in their hypophosphorylated forms as a result of cyclin D1-Cdk4/6 inhibition by elevated p16INK4A. In such cells, ectopically expressed pRB family proteins may be in molar excess to E2F and therefore not only neutralize E2F but also interact with other pocket-binding proteins, including those involved in NF-kappa B activation. As a result, cells arrest in G1 but induce cyclin D1. On the other hand, in cells such as U2-OS that do not express p16INK4A (45), ectopically expressed pRB family proteins are instantly hyperphosphorylated and inactivated by deregulated cyclin D1-Cdk4/6 and thus cannot halt the cell cycle (71).

The potential role of the hyperphosphorylated pRB in cyclin D1 induction is indicated by the observation that knockdown of pRB by RNA interference in U2-OS cells, in which pRB is totally hyperphosphorylated, significantly reduced cyclin D1 levels. Furthermore, pRB pocket mutants such as pRBDelta 22 and pRB706CF, both of which are derived from cancer patients, retain the ability to induce cyclin D1. Like hyperphosphorylated pRB, these tumor-derived mutants are unable to bind to E2F and thus are unable to inhibit cell growth. Hence, the cyclin D1-inducing activity of a pRB family member is separable from the pocket function that has been well characterized as growth-suppressive activity. Accordingly, hyperphosphorylated pRB and certain pRB pocket mutants may share a "phosphorylation-insensitive" pocket structure that is involved in cyclin D1 induction. The results of a series of pRB pocket mutant analyses suggested that the presence of either A/B-boxes or the C-terminal region is sufficient for the induction of cyclin D1. Müller et al. (36) reported that a pRB mutant that lacks the B-box and the C-terminal region failed to induce cyclin D1. Hence, the presence of the A-box alone may not be sufficient for cyclin D1 stimulation. These observations collectively suggest that a pocket-binding protein(s) involved in cyclin D1 induction, if it exists, has at least two independent pocket-binding sites, one for the A/B-boxes and the other for the C-terminal region. Binding to one of these pRB sites may be sufficient for significant activation of NF-kappa B.

Given that both the growth-suppressive activity and the cyclin D1-inducing activity of pRB require the pocket domain, through which pRB binds multiple cellular targets, the two seemingly counterintuitive pRB activities may be mutually exclusive. Furthermore, the cyclin D1-inducing activity of pRB family proteins may be insensitive to phosphorylation. Accordingly, we suggest that pRB family proteins are converted from inhibitors to stimulators of the cell cycle and vice versa, depending on their phosphorylation status. In hypophosphorylated forms, they block cell cycle progression at G0/G1 by interacting with E2F as well as other cell cycle-regulating proteins via the pocket domain. Once cells have been committed to cell cycle progression, they undergo hyperphosphorylation, release E2Fs, and then interact with a distinct cellular protein(s) via the pocket domain. By doing so, pRB family proteins may activate NF-kappa B and thereby induce cyclin D1, enforcing irreversible cell cycle progression to the S phase in cells that have passed the G1 restriction point. In this regard, recent studies have indicated that non-pRB substrates of cyclin D1-Cdk4/6 play crucial roles in G1/S transition (72, 73). Elevated cyclin D1-Cdk4/6 may also promote cell cycle progression by squelching Cdk inhibitors such as p27Kip1 (74, 75).

Finally, our results suggest a heretofore unexplored function of pRB in tumor development. Certain tumor-derived pRB pocket mutants, such as pRB706CF and pRBDelta 22 mutants, are unable to bind to E2F and are thus unable to induce cell growth inhibition, yet they are biologically active in inducing cyclin D1. Accordingly, in cells harboring such a pRB mutant, cyclin D1 levels may be higher than those in RB-null cells. Whereas inactivation of pRB as a cell growth inhibitor may be sufficient to provoke cellular transformation, certain levels of cyclin D1 induced by pRB pocket mutants should accelerate abnormal cell proliferation by phosphorylation of p107, p130, and other non-pRB family substrates. Hence, such RB pocket mutants may be more potent than RB-null mutants in promoting transformation. Furthermore, increases in the levels of pRB have been reported in colorectal carcinomas (76). In such cells, constitutive hyperphosphorylation of elevated pRB, occasionally due to loss of p16INK4A, may also play a role in cellular transformation by aberrantly increasing cyclin D1 levels.

    ACKNOWLEDGEMENTS

We thank Dr. Takashi Fujita for p65 NF-kappa B cDNA and Igkappa -reporter plasmids, Drs. Rolf Müller and Brian Elenbaas for cyclin D1 promoter, Dr. Jun-Ichiro Inoue for Ikappa Balpha cDNAs, and Yuhki Ishikawa for technical assistance.

    FOOTNOTES

* This work was supported by grants-in-aid for science research from the Ministry of Education, Science, Sports, and Culture of Japan, by a research grant from the Human Frontier Science Program Organization, and by a grant of the Virtual Research Institute of Aging of Nippon Boehringer Ingelheim.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: Division of Molecular Oncology, Institute for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815, Japan. Tel./Fax: 81-11-706-7544; E-mail: mhata@imm.hokudai.ac.jp.

Published, JBC Papers in Press, February 19, 2003, DOI 10.1074/jbc.M210849200

2 T. Takebayashi, H. Sudo, and M. Hatakeyama, unpublished observation.

3 Y. Ishikawa, H. Sudo, T. Takebayashi, and M. Hatakeyama, unpublished observation.

4 Y. Ishikawa, H. Sudo, T. Takebayashi, and M. Hatakeyama, unpublished observation.

    ABBREVIATIONS

The abbreviations used are: pRB, retinoblastoma protein; Cdk, cyclin-dependent kinase; HA, hemagglutinin; siRNA, small interfering RNA; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; CRE, cAMP-response element.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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