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Originally published In Press as doi:10.1074/jbc.M400365200 on May 11, 2004

J. Biol. Chem., Vol. 279, Issue 30, 31524-31532, July 23, 2004
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Regulation of Cellular Senescence and p16INK4a Expression by Id1 and E47 Proteins in Human Diploid Fibroblast*

Wenjie Zheng{ddagger}, Heyao Wang§, Lixiang Xue{ddagger}, Zongyu Zhang{ddagger}, and Tanjun Tong{ddagger}

From the {ddagger}Department of Biochemistry and Molecular Biology, Peking University, Health Science Center, 38 Xueyuan Road, Beijing 100083 and the §Beijing Chao Yang Hospital Affiliated to the Capital University of Medical Science, 8 Baijiazhuang Road, Beijing 100020, People's Republic of China

Received for publication, January 13, 2004 , and in revised form, March 24, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Id1, a member of Id family of helix-loop-helix transcriptional regulatory proteins, is implicated in cellular senescence by repressing p16INK4a expression, but the mechanisms and cellular effects in human diploid fibroblasts remain unknown. Here we analyzed the patterns of p16INK4a and Id1 expression during the lifespan of 2BS cells and presented the inverse correlation between these two proteins. Immunoprecipitation assays demonstrated the presence of endogenous interaction of Id1 and E47 proteins that was strong in young 2BS cells and weakened during replicative senescence and, thereby, influenced the transcription activation of p16INK4a by E47. Furthermore, we found that E47 protein could bind to the E-box-containing region in p16INK4a promoter in senescent cells by chromatin immunoprecipitation analyses, suggesting that E47 is indeed ultimately involved in the regulation of p16INK4a transcription in vivo. Silencing Id1 expression in young cells by RNA interference induced an increased p16INK4a level and premature cellular senescence, whereas silencing E47 expression inhibited the expression of p16INK4a and delayed the onset of senescent phenotype. The present study demonstrated not only the capacity of Id1 to regulate p16INK4a gene expression by E47, but also the phenotypic consequence of the regulation on cellular senescence, moreover, raised the possibility of Id1-specific gene silencing for human cancer therapy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The class I basic helix-loop-helix (HLH)1 proteins, also known as E proteins, including E12, E47, HEB, and E2-2, are ubiquitously expressed transcription factors (1–5). They have been shown to play important roles in cell differentiation, lineage commitment, and the expression of many tissue-specific genes. The family of proteins generally contains an HLH domain that primarily mediates homo- or heterodimerization with tissue-restricted HLH proteins, e.g. MyoD (skeletal muscle) or NeuroD (neurons), to regulate cell-specific gene transcription. An adjacent domain that is rich in basic amino acids mediates the binding to DNA when these proteins dimerize (6). Both homodimers and heterodimers have been shown to bind to the consensus DNA sequence CANNTG, referred to as the E-box, and to activate gene transcription. It has been shown that E47 binds to DNA both as a heterodimer and as a homodimer (7, 8), whereas E12 binds to DNA efficiently only as heterodimer, for an inhibitory domain N-terminal to the basic region of E12 prevents E12 homodimers from binding to DNA (7). The E12 and E47 proteins are two products of the E2A gene (7), and they differ only by differential utilization of two alternative exons in E2A.

The Id family is another category of HLH protein that contains a highly conserved HLH domain but lacks the basic region necessary for DNA binding. So, members of this family can heterodimerize with basic HLH proteins and result in a form that is unable to bind to DNA, hence they function as dominant negative regulators to inhibit the transcriptional activation mediated by E proteins (9–11). The term "Id" is conveniently taken to reflect the ability of these proteins to inhibit both DNA binding and differentiation. There are four members of the Id family in mammals, Id1 through Id4 (9, 12–17). Generally, proliferating cells express multiple Id genes, whereas upon differentiation in many cell types, the expression of Id genes is down-regulated (9, 12, 19–21). The expressions of Id1, Id2, and Id3 are induced when the serum or growth factors are added to G0-arrested fibroblasts (13, 16, 22, 23). In addition, abolishing Id proteins synthesis by antisense oligonucleotides blocks the quiescent fibroblasts into the cell cycle (22, 23). These facts suggest that Id proteins play a role in the regulation of the cell cycle.

It has been shown that Id1 and Id2 inhibited DNA binding by E47 very efficiently (9, 12). E2A-encoded proteins (E12 and E47) have the ability to block cells in G1 phase through the transcriptional activation of cyclin-dependent kinase (CDK) inhibitor p21 expression (24, 25). Id1 is able to inhibit E2A-mediated expression of the p21 gene. Overexpression of E2A in 293T cells activates expression of the endogenous p21 gene at both the levels of mRNA and protein (25). Enforced expression of E proteins suppresses the cell colony-forming efficiency of several cell lines. Moreover, they activate transcription of p21CIP1/WAF1, p15INK4b, and p16INK4a promoters, which contain E-boxes (26). Taken together, E proteins may inhibit cell proliferation through the transcriptional activation of CDK inhibitor genes.

Id1 is implicated in the modulation of cell senescence and immortalization (27, 28). Overexpression of Id1 extends the replicative lifespan of the primary human keratinocytes or immortalizes them. A recent report (29) indicates that Id1-null mouse embryo fibroblasts undergo premature senescence and express high levels of p16INK4a. These data imply that Id1 could delay cellular senescence through regulation of p16INK4a expression. p16INK4a is an important tumor suppressor that is inactivated in a large proportion of human tumors (30). p16INK4a elicits its cellular effects by inhibiting CDK4 and CDK6, which regulate cell cycle progression in G1 phase by contributing to the phosphorylation of the retinoblastoma (Rb) protein (31). Despite the significant function of it in cell senescence, immortalization, and tumorigenesis, the transcriptional control of p16INK4a is poorly understood at the present time but is thought to concern diverse nuclear factors. Understanding the mechanisms of p16INK4a regulation may help us to find a way of controlling its expression and inducing senescence in cancer.

Here we take Id1 as a model to explore how the Id proteins are involved in the regulation of p16 expression and cellular senescence. We report here that Id1 inhibits p16INK4a expression by sequestering E47 protein and blocking the transcriptional activation of p16INK4a and, therefore, maintains the proliferation state of young 2BS cells. The reducing Id1 expression may be at least in part one of the causes of the enhancing p16INK4a level in senescent 2BS cells, which in turn contribute to the onset of cellular senescence. Our results provide valuable new insights into the mechanisms of p16INK4a control and cellular senescence.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Synchronization—Human embryonic lung diploid fibroblast 2BS cells (obtained from the National Institute of Biological Products, Beijing, China) were previously isolated from female fetal lung fibroblast tissue and have been fully characterized (32–35). The current expected life span is ~70 population doublings (PDs). 2BS cells are considered to be young at PD30 or below and to be fully senescent at PD55 or above. Cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in 5% CO2.

For synchronization, 2BS cells were rendered quiescent by serum deprivation for 48 h and then stimulated to re-enter the cell cycle by the addition of serum to a final concentration of 10%. G1 phase cells were harvested at 8 h after serum stimulation.

Western Blotting—Cells were lysed in radioimmune precipitation assay buffer (1x PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, 50 kallikrein-inactivating units/ml aprotinin, 1 mM sodium orthovanadate). Protein concentration of each sample was determined by BCA Protein Assay Reagent (Pierce). 60–100 µg of proteins was electrophoresed on 15% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride filters (Millipore). The filter was blocked and then incubated with the primary antibody in 5% nonfat dry milk in TBST (10 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) overnight at 4 °C. After washing, the blots were incubated with secondary antibody conjugated to horseradish peroxidase at 1:40,000 in TBST for 1 h at room temperature. Proteins were visualized with Chemiluminescent Substrate (Pierce) according to the manufacturer's instruction. Antibodies against p16 (SC-9968), Id1 (SC-488), Id2 (SC-489), Id3 (SC-490), Id4 (SC-491), E12 (SC-762), E47 (SC-763), and HEB (SC-357) were purchased from Santa Cruz Biotechnology and used at 1 µg/ml concentration.

Immunoprecipitation—Total proteins of young (PD26), middle-aged (PD42), and senescent (PD60) cells were precleared by incubation with 10 µl of rabbit immunoglobulin (Ig) and 50 µl of protein A-Sepharose beads (Pierce) for 30 min at 4 °C with agitation. For each immunoprecipitation reaction, 500 µl of cleared lysate was incubated with 5 µl of polyclonal anti-Id1 antiserum (SC-488) or 5 µl of Ig and rocked overnight at 4 °C, after which 30 µl of protein A-Sepharose beads was added and the incubation continued for an additional 1 h. The immune complexes were pelleted by centrifugation, washed five times with radioimmune precipitation assay buffer, and finally resuspended and boiled for 5 min in SDS sample buffer. The released proteins were examined by Western blotting with anti-Id1 or anti-E47 antisera.

Chromatin Immunoprecipitation—ChIPs were performed using the Chromatin Immunoprecipitation Assay kit (Upstate, New York, NY) according to manufacturer's instruction. In brief, 1 x 106 cells were cross-linked by adding formaldehyde directly to cell culture media and incubated for 10 min at 37 °C. Cells were washed twice with cold PBS, and then cells were scraped and resuspended in 200 µl of SDS lysis buffer. Chromatin was then sonicated to an average length of 0.5 kb for three 30-s pulses at maximum power. Chromatin extracts were diluted 10-fold in dilution buffer and preincubated for 30 min at 4 °C with 80 µl of Salmon Sperm DNA/protein A-agarose. Twenty microliters of diluted supernatant was kept for isolation of input DNA and to quantitate the DNA in different samples. After pelleting agarose by brief centrifugation, 2 µg of anti-E47 antiserum (test group) or 2 µgof {beta}-actin antibody (irrelevant antibody control) was added to the supernatant fraction and incubated overnight at 4 °C with rotation. In addition, we performed a no-antibody immunoprecipitation by incubating the supernatant fraction with Salmon Sperm DNA/protein A-agarose for 1 h at 4 °C. Then 60 µl of Salmon Sperm DNA/protein A-agarose was added, and the mixture was incubated for 1 h at 4 °C to collect the antibody/antigen-DNA complex. The chromatin bound to the protein A-agarose beads was eluted in 500 µl of freshly prepared elution buffer (1% SDS, 0.1 M NaHCO3). After reversing cross-linking, the samples were deproteinized and phenol-chloroform-extracted, and then DNA was ethanol-precipitated using glycogen as a carrier. Pellets were resuspended in 50 µl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) for PCR analysis.

Each PCR reaction mixture contained 5 µl of immunoprecipitated chromatin in a final reaction volume of 20 µl. PCR mixtures were amplified for 35 cycles of 94 °C for 30 s, 54 °C for 30 s, and 72 °C for 30 s. To amplify E-box-containing regions of p16INK4a promoter, the sequences of the primers used were as follows: E1s, 5'-AGGAATCCTTTGAACTAGGG-3'; E1a, 5'-GGGAAAGTATGGCTTCTTCT-3'; E2s, 5'-GAGTGAACGCACTCAAACAC-3'; and E2a, 5'-ATGTGGCACCCCTGAAGT-3'. All primers were synthesized at AuGCT Biotechnology Co., Ltd. (Beijing, China).

Small Interfering RNAs Preparation and Transfections—siRNAs corresponding to Id1 and E47 mRNAs were designed according to the pSilencer neo instruction manual (Ambion). In brief, the 21-nucleotide potential sequences in the target mRNAs that begin with an AA dinucleotide were found and compared with the human genome data base using BLAST (www.ncbi.nlm.nih.gov/BLAST). Any target sequences with more than 16 or 17 contiguous base pairs of homology to other coding sequences were eliminated from consideration. The hairpin siRNA template oligonucleotides were designed by using a web-based insert design tool (www.ambion.com/techlib/misc/psilencer_converter.html) and chemically synthesized, with 5'-phosphate, 3'-hydroxyl, and two base overhangs on each strand. The following gene-specific sequences were used successfully: Id1, 5'-AACTCGGAATCCGAAGTTGGA-3'; E47, 5'-AAAGACCTGAGGGACCGGGAG-3'. Then the siRNAs were inserted into the BamHI and HindIII sites of pSilencer 2.1-U6 neo vector (Ambion) and referred to as pSilencer-Id1 and pSilencer-E47. They were transfected by LipofectAMINE 2000 reagent (Invitrogen) into 4 x 105 early passage 2BS cells (PD26) in 80–90% confluence. pSilencer NC vector (negative control of the pSilencer 2.1-U6 neo vector that expresses a hairpin siRNA with limited homology to any known sequences in the human, mouse, and rat genomes) was also transfected as a control. Stable transformants were obtained by sustained selection of G418 (Invitrogen).

Cell Cycle Analysis—siRNA-transfected cells and untransfected young, middle-aged, and senescent 2BS cells were detached with 0.25% trypsin, and fixed with 75% ethanol overnight. After treated with 100 mg/ml RNase A (Sigma) at 37 °C for 30 min, cells were resuspended in 0.5 ml of PBS and stained with propidium iodide in the dark for 30 min, and the DNA contents were measured by fluorescence-activated cell sorting on a FACScan flow cytometry system (BD Biosciences). Cell cycle distributions were analyzed using CellFiT software (36).

Senescence-associated {beta}-Galactosidase Staining—siRNA-transfected cells and untransfected young, middle-aged, and senescent 2BS cells were washed twice in PBS, fixed to plates using 3% formaldehyde for 3–5 min, and washed with PBS again. Then cells were incubated overnight at 37 °C without CO2 in a freshly prepared staining buffer (1 mg/ml 5-bromo-4-chloro-3-indolyl-{beta}-D-galactopyranoside (X-gal), 40 mM citric acid/sodium phosphate, pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl2) (37).

Growth Curve—siRNA-transfected cells and untransfected young, middle-aged, and senescent 2BS cells were detached and seeded into 96-well plates 2000 cells per well. At the indicated times, cells were stained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (10 µg/ml in PBS; Sigma) for 3 h, then dissolved with 50% N,N-dimethylformamide and 10% SDS for 3h at 37 °C. The optical density at 570 nm was determined. Each point was determined in triplicate, and each curve was performed twice.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Id1 Expression Inversely Correlated with p16INK4a Expression during the Lifespan of 2BS Cells—Due to the potential role of Id1 and E47 in the modulation of p16INK4a expression, their expression patterns during the progression of successive passages are of interest. Western blot analysis was performed on the 2BS cells cultured to PD26 (young), PD42 (middle-aged), and PD60 (senescent), respectively. To exclude the discrepancy of expression levels due to different stages of the cell cycle, cells were synchronized to G1 phase by serum starvation and harvested at 8 h after serum addition. Then endogenous expression of Id1, E47, and p16INK4a were measured. The results of Id1 in various ages of 2BS cells revealed a high Id1 protein level in young cells compared with the relatively low Id1 level in cells undergoing spontaneous replicative senescence (Fig. 1). As expected, middle-aged and young 2BS cells possessed low p16INK4a levels (Fig. 1). However, there was an increase in the p16INK4a level in senescent cells. There was either no change, or minimal change, in the levels of either E47 or {beta}-actin (Fig. 1). These data indicated that young 2BS cells expressed high levels of Id1, however, Id1 expression declined accompanied by an improving level of p16INK4a and the onset of cellular senescence. In these cells, Id1 expression reduced during the lifespan of 2BS cells and inversely correlated with p16INK4a, which gave the opportunity for Id1 to adjust p16INK4a expression by changing itself. The stable level of E47 suggests it would not be a variable in the regulation of p16INK4a expression, but the data did not rule out the possibility that Id1 could repress p16INK4a expression by sequestering E47 and antagonize its function of transcriptional activation.



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  		FIG. 1.
Id1 expression was inversely correlated with p16INK4a expression during the lifespan of 2BS Cells. A, Western blot analysis of Id1 and E47, and p16 expression in young (Y: PD26), middle-aged (M: PD42), and senescent (S: PD60) 2BS cells. Total proteins were extracted respectively, and Western blotting was performed using specific antibodies against Id1, E47, and p16 as indicated. The {beta}-actin lane serves as loading control. B, ratios of Id1, E47, and p16 message to the {beta}-actin message. The intensity of bands in A was determined by densitometric scanning.

 
Id1 and E47 Interact with Each Other in Young 2BS Cells Stronger Than in Senescent Cells—To discover the probable mechanism by which Id1 can affect p16INK4a expression in 2BS cells, the endogenous protein-protein interaction of Id1 and E47 was determined. Total proteins from young (PD26), middle-aged (PD42), and senescent (PD60) 2BS cells were prepared as aforementioned. The proteins were immunoprecipitated with a polyclonal anti-Id1 antiserum or with normal rabbit immunoglobulin (Ig) as a negative control. Crude cell extract from 2BS cells (PD 26) was run in a parallel lane. Immunodetection was done with polyclonal anti-E47 or anti-Id1 antiserum, as indicated (Fig. 2). The data showed that Id1 functionally interacted with E47, and the strength of binding was diminished with cellular senescence. Evidently, the binding of Id1 to E47 in young 2BS cells was the strongest, second only was that in middle-aged cells, and the combination potential was the weakest in senescent cells, corresponding to the low expression of Id1. As mentioned above, the E47 levels kept constant in young, middle-aged, and old cells, so the more E47 bound to Id1, the less E47 were free to activate transcription of target genes, for example, p16INK4a. Interestingly, the level of p16INK4a was induced in senescent 2BS cells where the binding strength of E47 to Id1 was weak and the E47 proteins had much more possibilities to function as transcriptional regulators. These data demonstrated the existence of a physical interaction between Id1 and E47 in vivo and established the relationship between amounts of free E47 and p16INK4a expression levels. It was conceivable that p16INK4a might be induced at least partly by the E47 protein which couldn't bind to Id1 in senescent cells.



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  		FIG. 2.
Co-immunoprecipitation of endogenous Id1 and E47 in 2BS cells. The proteins of young (Y: PD26), middle-aged (M: PD42), and senescent (S: PD60) cells were immunoprecipitated with a polyclonal anti-Id1 antiserum or with normal rabbit immunoglobulin (Ig) as a negative control. The immunoprecipitate was subjected to 15% SDS-PAGE, and proteins were transferred to a polyvinylidene difluoride filter. Crude cell extract from 2BS cells (PD26) was run in a parallel lane as a positive control (PC). Immunodetection was done with polyclonal anti-E47 or anti-Id1 antiserum, as indicated.

 
E47 Bound to E-Box containing Region of the Endogenous p16INK4a Promoter—Two E-boxes have been identified in the p16INK4a promoter sequence, which locate, respectively, at positions –354 (E1) and –620 (E2) relative to the translation initiation site (31). To seek evidence that E47 can bind to and influence the endogenous p16INK4a promoter, ChIP assays were performed to detect the occupancy of E47 in the two E-boxes in the p16INK4a promoter. The chromatin was isolated in young (PD26) and senescent (PD60) 2BS cells and broken into small fragments via sonication after formaldehyde treatment of the cell cultures (Fig. 3C). An antibody specific for E47 was used to immunoprecipitate E47-cross-linked chromatin fragments. Following reversal of the cross-links and purification of the genomic DNA fragments, the DNA sequences were analyzed by PCR with specific pairs of primers flanking the E1 or E2 boxes (Fig. 3A). The binding of E47 was observed to both E1 and E2 boxes in senescent cells, compared with a weak amplification in young cells (Fig. 3B). The irrelevant antibody control ({beta}-actin) and no antibody control had no amplification products, which suggested the binding of E47 to E1 and E2 boxes was specific. These results identified the binding of E47 to p16INK4a promoter in vivo and implicated the role of E47 in p16INK4a transcriptional regulation.



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  		FIG. 3.
E47 bound to the E-boxes containing region in the p16 gene promoter in 2BS cells. A, schematic diagram of the p16 gene promoter. Solid boxes depict putative E47-binding sites (E-boxes), and arrows indicate primer pairs used for amplifying the E-box-containing region. The numbers are the positions upstream to the p16 gene translation initiation site. B, ChIP assays of young (Y: PD26) and senescent (S: PD60) 2BS cells using antibody against E47. As negative controls, the No Ab sample is an immunoprecipitation that did not contain antibody, and antibody against {beta}-actin was used as irrelevant antibody control. The input sample (Input) contained 0.5% of the total starting chromatin. C, gel electrophoresis of the sonicated DNA after reversion of cross-links. M, 100-bp DNA ladders; Y, young cell DNA; S, senescent cell DNA.

 
Silencing of the Id1 Gene by RNA Interference (RNAi) Induced p16INK4a Expression and Premature Senescence, Whereas Silencing of the E47 Gene Inhibited p16INK4a Expression and Extended the Lifespan of 2BS Cells—Normal human diploid fibroblast 2BS cells enter a senescence state at about PD55–60. To determine the effects of Id1 and E47 expression levels on cellular senescence, we used the pSilencer vector system to stably suppress the expression of Id1 or E47 genes. Early passage of 2BS cells (PD26) were transfected with the pSilencer-Id1 (siId1), pSilencer-E47 (siE47), or pSilencer NC vector, and transformants were selected with G418. The siRNA-transfected cells were analyzed for the relative senescence markers compared with the untransfected 2BS cell control, all of which came from the same batch of early passage cells.

The Efficiency and Specificity of Id1- or E47-specific RNAi and the Impact on p16INK4a Expression—To ensure the specificity of Id1 and E47 siRNA, we compared their potential sequences with the human genome data base using BLAST, and eliminated the sequences with more than 16–17 bp of homology to other coding sequences. We compared the mRNA sequences of the Id family, from Id1 through Id4, at the target domain of siId1 and the surrounding region, and discovered that the target sequence of siId1 had 11 bp of homology to Id2 mRNA, 9 bp of homology to Id3 mRNA, and 7 bp of homology to Id4 mRNA (Fig. 4A), which were all lower than 16–17 bp. Simultaneously, the sequences comparison of E proteins indicated that the target sequence of siE47 had 13 bp of homology to E12 mRNA and 11 bp of homology to E2-2 and HEB mRNAs (Fig. 5A). Hence the used sequences of siId1 and siE47 had less homology to the related proteins, which aided to avoid the impact on the expression of these proteins.



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  		FIG. 4.
Efficiency and specificity of Id1-specific RNAi and the induction of endogenous p16 expression. A, comparison of the mRNA sequences of Id1, Id2, Id3, and Id4 at the target domain of siId1 and the surrounding region. B, Western blot analysis of Id1, Id2, Id3, Id4, and p16 expression in siId1-transfected cells compared with NC cells. Western blotting was performed using specific antibodies against Id1, Id2, Id3, Id4, and p16 as indicated. C, ratios of Id1, Id2, Id3, Id4, and p16 message to {beta}-actin message. The intensity of bands in B was determined by densitometric scanning.

 



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  		FIG. 5.
Efficiency and specificity of E47 specific RNAi and the inhibition of endogenous p16 expression. A, comparison of the mRNA sequences of E47, E12, E2-2, and HEB at the target domain of siE47 and the surrounding region. B, Western blot analysis of E47, E12, E2-2, HEB, and p16 expression in siE47-transfected cells compared with NC cells. As the lifespan of siE47-transfected cells was prolonged, the cells at PD42–45 (M) and PD65–70 (S) were both used to detect the stable knockdown of E47 expression during extended cell growth. NC cells were used at the same PDs. Western blotting was performed using specific antibodies against E47, E12, E2-2, HEB, and p16 as indicated. C, ratios of E47, E12, E2-2, HEB, and p16 message to the {beta}-actin message. The intensity of bands in B was determined by densitometric scanning.

 
To further determine the specificity and efficiency of siId1 and siE47, we detected the expression of related proteins in siId1 and siE47 transfected cells by Western blotting. Because the siId1 cells ceased cell division at PD42–45 (Table I), these pools of resistant clones to G418 were used (Fig. 4B). While the lifespan of siE47-transfected cells was prolonged (Table I), the cells both at PD42–45 and PD65–70 were used to detect the stable knockdown of E47 expression during extended cell growth (Fig. 5B). NC cells were used at same PDs. We found that in siId1-transfected cells the Id1 level reduced 75% compared with the NC cells (Fig. 4, B and C), establishing the efficiency of the Id1 siRNA. However, the levels of Id2, Id3, and Id4 lessened 22%, 8%, and 6%, respectively, which were extremely trifling compared with the level of Id1 knockdown (Fig. 4, B and C). Besides, E47 level decreased 73% in siE47 transfected cells at PD42–45, compared with NC cells at same PD (Fig. 5, B and C). The knockdown of E47 sustained during the extended lifespan and even had a minimal rise in cells at PD65–70. The levels of E12, E2-2, and HEB depressed 13%, 9%, and 12%, respectively, in siE47-transfected cells at PD42–45, but the losses of expression seemed to be minimal and temporary, because in cells at PD65–70, their expressions were restored. Moreover, E12 and HEB levels advanced a little compared with NC cells at same PD (Fig. 5, B and C). These data identified the efficiency and specificity of siId1 and siE47.


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  		TABLE I
Cumulative population doublings of siE47, siId1, and control cells

 
In addition, Western blot assays revealed an increased p16INK4a expression in the siId1-transfected cells compared with the NC cells (Fig. 4, B and C), which provided the in vivo significance of Id1 repression of p16INK4a. The expression of p16INK4a in siE47-transfected cells markedly reduced at PD42–45 (Fig. 5, B and C), however, when cells entered irreversible growth arrest at PD65–70, the p16INK4a level increased to some extent, although the knockdown of E47 still kept stable. The results identified E47 as a latent activator of p16INK4a, yet there may be some other mechanisms stimulated when E47 expression was inhibited.

siE47 Extended the Proliferative Lifespan of 2BS Cells, but siId1 Shortened It—The replicative senescence of normal human diploid fibroblasts is directly correlated to their number of PD rather than to the growth and metabolic time (38–40). After completing a finite number of divisions, cells enter permanent growth arrest. To determine the effects of Id1 and E47 gene-specific silencing on the lifespan of 2BS cells, the number of PDs for siRNA transfected and untransfected cells from the same batch of early passage cells was counted. The lifespan of siE47 cells (PD65–70) was about 10–15 PDs greater than the NC control and normal 2BS cells (PD55–60) (Table I). In contrast, siId1-transfected cells ceased cell division of 13–15 PDs (PD42–45) earlier than the control cells, keeping in the subconfluent stage even after 2 months under normal culture conditions.

siE47 Promoted Cell Growth, Whereas siId1 Led to Growth Inhibition—To observe the impact of Id1 and E47 gene-specific silencing on the cell proliferation, the growth curves for siRNA transfected and untransfected 2BS cells were compared. The curve of siId1-transfected cells was approaching that of senescent cells, showing nearly complete growth inhibition (Fig. 6A); however, the growth curve of siE47 advanced quickly, displaying its strong proliferation potential similar to young cells (Fig. 6B). As a control, NC cells had almost the same growth rate or growth potential as middle-aged cells without transfection.



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  		FIG. 6.
Growth curves of siId1 (A), siE47 (B), and NC cells compared with young (PD26), middle-aged (PD42), and senescent (PD60) 2BS cells. 2000 cells per well were plated onto 96-well plates. At the indicated time points, cells were stained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and the cell number was obtained from an optical density at 570 nm. Each experiment was performed at least twice.

 
siId1 Brought G1 Cell Cycle Arrest, and siE47 Postponed It—To clarify the mechanisms of growth rate inhibition described above, the cell cycle profile of siRNA transfected and untransfected 2BS cells was analyzed by flow cytometry. Each experiment was performed at least three times, and representative data are shown in Fig. 7A. In contrast to the irreversible G1 arrest imposed by siId1, siE47 postponed the irreversible growth arrest. As a control, NC cells had a parallel percentage of cells in G1 phase to middle-aged 2BS cells (Fig. 7B). Thus, silencing the Id1 expression led to premature senescence by initiating the G1 arrest, whereas silencing the E47 expression efficiently delayed the onset of cellular senescence by postponing it.



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  		FIG. 7.
Flow cytometry analysis of siId1, siE47, and NC cells compared with young (PD26), middle-aged (PD42), and senescent (PD60) 2BS cells. Each experiment was performed at least three times. A showed the representative data. B, the graph depicted data from three independent experiments (means ± S.E.).

 
SA-{beta}-galactosidase Staining—A common marker for cellular senescence, SA-{beta}-galactosidase staining was also checked with siRNA transfected and untransfected 2BS cells. Nearly all of the siId1-transfected cells were strongly stained blue, with gross enlargement and flattened morphology as with senescent cells (Fig. 8). siE47 cells and young cells retained a refractive cytoplasm with thin and long projections, and there were a few dispersed cells that were SA-{beta}-Gal-stained. For NC cells, the positive ratio was comparable with middle-aged cells (Fig. 8).



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  		FIG. 8.
Morphology and SA-{beta}-gal staining for siRNA-transfected and untransfected cells. A, NC cells; B, siE47 cells; C, siId1 cells; D, young cells (PD26); E, middle-aged cells (PD42); F, senescent cells (PD60). Photographs are at x400 magnification.

 
All these outcomes manifested that RNAi-mediated silencing of Id1 gene could induce premature senescence accompanied with increased p16INK4a levels, however, doing the same thing to E47 could cut down the p16INK4a expression, extend the proliferative lifespan, and finally delay the onset of senescence of 2BS cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
p16INK4a, the inhibitor of D-type CDK4 and CDK6, is implicated in cell immortalization and senescence (31). Previous studies have demonstrated that it is the accumulation of p16INK4a that triggers the onset of cellular senescence (34). So how is p16INK4a expression raised during cellular senescence? p16INK4a expression is more likely to be ruled by physiological stress than by telomere shortening, because in rodent senescent cells where telomere shortening can't be detected the accumulation of p16INK4a is also observed. We have been studying p16INK4a transcription modulation for years and found a novel negative regulatory element, the INK4a transcription silence element, in the p16INK4a promoter (35). Moreover, the GC-rich region of the p16INK4a promoter is a positive transcription regulatory element (35). Based on these findings we propose that p16INK4a transcription control may deal with a number of known and unknown nuclear factors, such as members of the Sp family.

To find clues of the transcriptional regulators we searched the p16INK4a promoter sequence and identified two E-boxes at the positions –354 (E1) and –620 (E2) upstream to the translation initiation site. In addition, one recent report showed that Id1-null primary mouse embryo fibroblasts underwent premature senescence and accompanied increased p16INK4a expression (29). There is a view that the primary mode through which Id proteins function is the antagonism of basic HLH transcriptional regulators. Subtle changes in the equilibrium of heterodimeric interactions between basic HLH factors and Id proteins cause dramatic cellular effects (41). The mammalian Id proteins (Id1 through Id4) preferentially target the ubiquitously expressed E proteins, which belong to the Class I basic HLH proteins (E47, E12, E2-2, and HEB) (42). As a member of the E proteins, E47 has the DNA-binding specificity that is limited to the consensus DNA sequence CANNTG, known as E-box (43). E47 may bind to the E-box as homodimers or heterodimers with MyoD. The two E-boxes in the p16INK4a promoter with a consensus sequence of CAGGTG belong to the Group I E-boxes with high affinity to E47 homodimers (7). It has been reported recently that E47 transcriptionally activates p16INK4a promoter in an E-box-dependent manner (26). On the basis of these findings, we reason that Id1 may participate in cellular senescence by blocking E47-mediated transcriptional activation of the key senescence effector, p16INK4a.

In the present study, we detected endogenous expression patterns of p16INK4a and Id1 during the lifespan of human diploid fibroblasts, setting up the relationship between p16INK4a and Id1 expression as well as the correlation between Id1 expression and the age of cells. The high levels in young cells and the strikingly decrease in senescent cells of Id1 expression were consistent with the postulated role of Id1 in the regulation of cell senescence (Fig. 1). The function of constitutively expressed E47 protein may be modulated by altering the level of Id1 protein at a definite stage of cell growth. In general, quiescent cells express low or absent levels of Id genes, whereas the expression of Id is high in proliferating cells (44, 45). Id1 expression changes in diverse phases of the cell cycle, which is rapidly induced following the stimulation of quiescent fibroblasts with serum. After an initial decline, Id1 expression is further up-regulated as cells progress through G1 and enter S phase of the cell cycle (22). To ensure that the expression variations originated from the senescence process other than cell cycle, we performed Western blot assays with synchronized 2BS cells.

Id1 belongs to Class V HLH proteins that lack a basic region, thus the heterodimers can't bind to DNA when Id1 combines to E proteins, therefore, blocking the transcriptional activation of E proteins to target genes. There are many evidences for the interaction of Id1 and E47. For example, in vitro translated Id1 protein coimmunoprecipitated with E47 protein and inhibited DNA binding by E47 very efficiently (9). Sun et al. (12) have shown that Id1 can form complexes with E47 by glutathione S-transferase-pull down, thus provide a molecular basis for inhibition of DNA binding by Id proteins through formation of heterodimers. Moreover, dimerization of Id1 protein with E47 was tested in a quantitative yeast two-hybrid assay, finding that Id1 bound with high affinity to E47 protein (46). These observations were confirmed by co-immunoprecipitation and mammalian two-hybrid analyses (46). We suppose Id1 could act on p16INK4a gene by sequestering the E47 protein. To test this hypothesis, the endogenous interaction of Id1 and E47 was examined by immunoprecipitation. We identified the presence of Id1 and E47 protein complexes in 2BS cells and further ascertained that binding strength of the two proteins changed following the population divisions, which provided evidence for the mediator role of E47 (Fig. 2). The presence of endogenous complex is not able to sufficiently prove that Id1 and E47 dimerize with each other, because the surroundings in vivo are so complex that they contain multiple protein factors. Nevertheless, due to the evidence for direct dimerization described above, it is extremely possible that Id1 and E47 interact directly even though they are both part of a large complex.

Peverali et al. (24) have found E2A can cause growth arrest in NIH 3T3 cells, which occurs before the G1 to S transition in the cell cycle. Overexpression of E47 can activate the expression of p21 gene, which encodes an inhibitor of the CDK and is essential for all phases of cell cycle transition (25). In addition, E47 has the latent capacity to activate transcription of p16INK4a promoter through elements known as the E-box and to suppress the cell colony-forming efficiency of several cell lines (26). These observations lead us to propose that E47 may promote cellular senescence through the transcriptional activation of negative regulators of cell cycle progression, such as p16INK4a gene. There are a number of examples for in vitro binding of E47 to E-box containing sequences by gel mobility shift analysis (EMSA) (1, 7), and the existence of two E-boxes in p16INK4a promoter provides the possibility of DNA binding to E47 protein. Although EMSA is a fast and easy method for identifying which nucleotides are required for protein-DNA association under in vitro conditions, in vivo binding conditions are often difficult to recreate in vitro. To test the supposition described above, we performed ChIP assays in human diploid fibroblasts to further ascertain the combination of E47 to p16INK4a promoter under unperturbed physiological conditions. The results showed that E47 indeed bound to the E-box-containing region in the p16INK4a promoter, and the DNA-binding activity is stronger in senescent cells, where p16INK4a is highly expressed, than in young cells, offering support for the promotion role of E47 to p16INK4a transcription (Fig. 3). The strong DNA-binding activity of E47 in senescent cells may be due to the presence of E47 not sequestered by Id1. The occupancy of E47 to both E1 and E2 boxes is coincident with the redundant role of these two elements in p16INK4a transcription activation. Although recent work proposed a model in which up-regulation of p16INK4a depended on the augmented expression of Ets1 in senescent cells, where the interference by Id1 was absent (47), this view didn't contradict our conclusion that Id1 regulated p16INK4a through the interaction with E47. The p16INK4a promoter is subject to multiple levels of control (31, 35), thus, not all aspects of p16INK4a regulation can be explained by one isolated event. There would be the hierarchy and interplay of these regulatory pathways related to additional proteins that connect with DNA either directly via its DNA binding domain or indirectly as a coactivator or corepressor. E47 and Ets1 may participate in p16INK4a regulation together, and previous data cannot exclude the likelihood that these two proteins may contact with each other in vivo. There are four probable patterns for proteins bonding to DNA as follows. (a) A transcription factor directly contacts with either a consensus or nonconsensus binding site. (b) Two or more factors bind to separated sites that are drawn near by looping of the DNA by way of protein-protein interactions. (c) A protein factor binding to a low affinity site is stabilized by contact with another protein that is recruited by a factor bound to a different site. (d) Transcription factors are brought to sites through interaction with other DNA-binding proteins. According to the first pattern, E47 and Ets1 may both function through specific binding sites in the p16INK4a promoter, and they also can interact directly through the second way. The precise mechanism by which E47 and Ets1 function is currently under investigation in our laboratory.

It was reported that ectopic expression of Id1 immortalized primary human keratinocytes and activated telomerase activity (27). Not long after, Nickoloff et al. (28) observed that Id1 extended the lifespan of human keratinocytes but could not immortalize them. A key unresolved issue is the cellular effects of Id1 on human diploid fibroblasts. Our studies try to address this question through specific silencing of Id1 or E47 expression by RNAi in 2BS cells. To ensure the specificity of RNAi, we compared the mRNA sequences of members of Id family and E proteins, respectively, then selected the target sequences of siId1 and siE47 out of the HLH domain in which the sequences are highly homologous among the members of HLH proteins. The target sequence of siE47 locates N-terminal to the basic region of E47, designated as the A region (7), which is strikingly different from that of E12. The chosen siId1 and siE47 sequences have less homology to related proteins.

Introduction of siId1 or siE47 efficiently inhibited the expression of target genes, because the level of Id1 was significantly reduced by 75% in siId1 cells (Fig. 4, B and C), and E47 expression was reduced by 73% in siE47 cells (Fig. 5, B and C). As expected, the introduction of siId1 could barely impact on the expressions of Id2, Id3, and Id4, which have only minimal changes compared with those in NC cells. There were analogous effects on the expression of E12, E2-2, and HEB in siE47-transfected cells at PD42–45, implying the nonspecific inhibition of siE47 was insignificant. When siE47 cells were cultured to senescence, the knockdown of E47 was still maintained stable, whereas the expression of E12, E2-2, and HEB was restored to the levels of NC cells. It seems that certain factors in vivo arouse these genes to return the normal levels at senescence, thereby compensating for the lost function of E47. The expression patters of p16INK4a in siE47 cells also confirmed this view. In siE47 cells at PD42–45, the p16INK4a level dropped off remarkably. However, it rose at PD65–70 and exceeded the level in siE47 cells at PD42–45, although the absolute value was lower than that of senescent NC cells. On the basis of these data, we conclude that E47 gene silencing decreased the expression of p16INK4a, yet there are other pathways through which p16INK4a is motivated for the senescence progression. The members of E proteins other than E47 may be involved in the process.

siE47 cells have a finite elongation in lifespan and a considerable delay in onset of senescence. In contrast, the siId1-transfected cells exhibited elevated p16INK4a expression, accompanied with irreversible growth arrest and several other cell senescent features as well as a shortened lifespan. These data are consistent with previous reports and give direct evidence for the function of endogenous Id1 and E47. The growth curve showed that siId1-transfected cells were growth-inhibited with the trend to approaching senescent cells, however, having distinction with senescent cells. This phenomenon may be due to the incomplete inhibition of Id1 and p16INK4a expression, because other members of the Id family may also be involved in cellular senescence by their influence on the p16INK4a/Rb pathway. For example, Id2 has the capacity to bind to Rb and potentiate S phase progression by attenuating the growth-suppressing activity of Rb protein (18, 48).

In summary, Id1 is one of the critical factors in the regulation of cell senescence. We conclude that the high level of Id1 in young cells represses p16INK4a expression by sequestering E47 protein and, therefore, maintains the proliferation state of 2BS cells. The shrinking Id1 expression may be one of the reasons for the raised p16INK4a expression in senescent 2BS cells, which in turn contributes to the onset of cellular senescence. Although there are several independent pathways that control the process of replicative senescence in human cells, the inactivation of the Id1 pathway would result in momentous cellular effects that incite senescent phenotype prematurely. A growing body of evidence suggests that cellular senescence is a barrier to cancer in vivo, so the induction of premature senescence of tumor cells might be a tool for cancer therapy. With this goal in mind, members of the Id family of proteins may be effective candidates.


   FOOTNOTES
 
* This work was supported by grants from the Special Funds for Major State Basis Research of China (G2000057001) and National Natural Science Foundation of China (39930170 and 30271432). 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. Back

To whom correspondence should be addressed. Tel.: 8610-82801454; Fax: 8610-82802931; E-mail: ttj{at}bjmu.edu.cn.

1 The abbreviations used are: HLH, helix-loop-helix; CDK, cyclin-dependent kinase; Rb, retinoblastoma; PD, population doubling; PBS, phosphate-buffered saline; Ig, rabbit immunoglobulin; siRNA, small interfering RNA; SA, senescence-associated; EMSA, electrophoretic mobility shift analysis; RNAi, RNA interference; siId1, pSilencer-Id1; siE47, pSilencer-E47; ChIP, chromosomal immunoprecipitation. Back


   ACKNOWLEDGMENTS
 
We are very grateful to Prof. Yong-Feng Shang for the kind gift of pSilencer NC plasmid.



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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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