E2F1-Mediated Upregulation of p19INK4d Determines Its Periodic Expression during Cell Cycle and Regulates Cellular Proliferation

BACKGROUND: A central aspect of development and disease is the control of cell proliferation through regulation of the mitotic cycle. Cell cycle progression and directionality requires an appropriate balance of positive and negative regulators whose expression must fluctuate in a coordinated manner. p19INK4d, a member of the INK4 family of CDK inhibitors, has a unique feature that distinguishes it from the remaining INK4 and makes it a likely candidate for contributing to the directionality of the cell cycle. p19INK4d mRNA and protein levels accumulate periodically during the cell cycle under normal conditions, a feature reminiscent of cyclins. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we demonstrate that p19INK4d is transcriptionally regulated by E2F1 through two response elements present in the p19INK4d promoter. Ablation of this regulation reduced p19 levels and restricted its expression during the cell cycle, reflecting the contribution of a transcriptional effect of E2F1 on p19 periodicity. The induction of p19INK4d is delayed during the cell cycle compared to that of cyclin E, temporally separating the induction of these proliferative and antiproliferative target genes. Specific inhibition of the E2F1-p19INK4d pathway using triplex-forming oligonucleotides that block E2F1 binding on p19 promoter, stimulated cell proliferation and increased the fraction of cells in S phase. CONCLUSIONS/SIGNIFICANCE: The results described here support a model of normal cell cycle progression in which, following phosphorylation of pRb, free E2F induces cyclin E, among other target genes. Once cyclinE/CDK2 takes over as the cell cycle driving kinase activity, the induction of p19 mediated by E2F1 leads to inhibition of the CDK4,6-containing complexes, bringing the G1 phase to an end. This regulatory mechanism constitutes a new negative feedback loop that terminates the G1 phase proliferative signal, contributing to the proper coordination of the cell cycle and provides an additional mechanism to limit E2F activity

CDK2 and PKA Mediated-Sequential Phosphorylation Is Critical for p19INK4d Function in the DNA Damage Response

DNA damage triggers a phosphorylation-based signaling cascade known as the DNA damage response. p19INK4d, a member of the INK4 family of CDK4/6 inhibitors, has been reported to participate in the DNA damage response promoting DNA repair and cell survival. Here, we provide mechanistic insight into the activation mechanism of p19INK4d linked to the response to DNA damage. Results showed that p19INK4d becomes phosphorylated following UV radiation, β-amyloid peptide and cisplatin treatments. ATM-Chk2/ATR-Chk1 signaling pathways were found to be differentially involved in p19INK4d phosphorylation depending on the type of DNA damage. Two sequential phosphorylation events at serine 76 and threonine 141 were identified using p19INK4d single-point mutants in metabolic labeling assays with 32P-orthophosphate. CDK2 and PKA were found to participate in p19INK4d phosphorylation process and that they would mediate serine 76 and threonine 141 modifications respectively. Nuclear translocation of p19INK4d induced by DNA damage was shown to be dependent on serine 76 phosphorylation. Most importantly, both phosphorylation sites were found to be crucial for p19INK4d function in DNA repair and cell survival. In contrast, serine 76 and threonine 141 were dispensable for CDK4/6 inhibition highlighting the independence of p19INK4d functions, in agreement with our previous findings. These results constitute the first description of the activation mechanism of p19INK4d in response to genotoxic stress and demonstrate the functional relevance of this activation following DNA damage

Chromatin relaxation-mediated induction of p19INK4d increases the ability of cells to repair damaged DNA.

The maintenance of genomic integrity is of main importance to the survival and health of organisms which are continuously exposed to genotoxic stress. Cells respond to DNA damage by activating survival pathways consisting of cell cycle checkpoints and repair mechanisms. However, the signal that triggers the DNA damage response is not necessarily a direct detection of the primary DNA lesion. In fact, chromatin defects may serve as initiating signals to activate those mechanisms. If the modulation of chromatin structure could initiate a checkpoint response in a direct manner, this supposes the existence of specific chromatin sensors. p19INK4d, a member of the INK4 cell cycle inhibitors, plays a crucial role in regulating genomic stability and cell viability by enhancing DNA repair. Its expression is induced in cells injured by one of several genotoxic treatments like cis-platin, UV light or neocarzinostatin. Nevertheless, when exogenous DNA damaged molecules are introduced into the cell, this induction is not observed. Here, we show that p19INK4d is enhanced after chromatin relaxation even in the absence of DNA damage. This induction was shown to depend upon ATM/ATR, Chk1/Chk2 and E2F activity, as is the case of p19INK4d induction by endogenous DNA damage. Interestingly, p19INK4d improves DNA repair when the genotoxic damage is caused in a relaxed-chromatin context. These results suggest that changes in chromatin structure, and not DNA damage itself, is the actual trigger of p19INK4d induction. We propose that, in addition to its role as a cell cycle inhibitor, p19INK4d could participate in a signaling network directed to detecting and eventually responding to chromatin anomalies

ATM/ATR signaling pathways are differentially involved in p19 phosphorylation.

<p>(<b>A</b>) Inhibition of p19 phosphorylation by caffeine treatment. WI-38 fibroblasts were incubated with caffeine (5 mM) for 1 hour, then treated with cisplatin (10 µM) or β-amyloid peptide (20 µM) for the indicated times and endogenous p19 phosphorylation analyzed by autoradiography. (<b>B</b>) Evaluation of ATM/ATR involvement in p19 phosphorylation by wortmannin treatment. WI-38 fibroblasts were incubated with the indicated doses of wortmannin for 1 hour, followed by treatment with cisplatin (10 µM) or β-amyloid peptide (20 µM) for 2 hours. (<b>C</b>) Effect of Chk1 and Chk2 inhibitors on p19 phosphorylation. WI-38 fibroblasts were incubated with SB-218078 (SB, 15 nM) or dopamine ß-hidroxylase inhibitor (DBH, 3 µM), both Chk1 inhibitors, or with Chk2 Inhibitor Calbiochem (ICHK2, 20 nM) for 1 hour before treatment with UV light (4 mJ/cm²), cisplatin (10 µM) or β-amyloid peptide (20 µM). After 2 hours, cell extracts were analyzed as in A.</p

Phosphorylation of serine 76 and threonine 141 is required for p19 function linked to the response to DNA damage

<p>(<b>A</b>) DNA repair ability of cells overexpressing p19wt or p19 phosphorylation deficient mutants. WI-38 fibroblasts were transfected with p19wt or the indicated p19 mutants. Cells were maintained in an arginine-free medium containing 1% fetal bovine serum during 48 h, damage with 4 mJ/cm² UV and incubated with [³H]-thymidine. Following 10 h, cell lysates were tested for Unscheduled DNA Synthesis assay (UDS). Bars represent the mean ± s.e.m of three independent experiments performed in triplicate. Student's <i>t</i>-test was used to compare UV-treated control sample (none) with UV-treated p19wt or p19 mutant samples. (*<i>p</i><0,005). Protein expression was analyzed by immunoblot. (<b>B</b>) Similarly as in (<b>A</b>) but overexpressing the phosphomimetic p19 mutants. (<b>C</b>) UV-dependent apoptotic response of cells overexpressing p19wt or phosphorylation deficient mutants of p19. WI-38 fibroblasts were transfected with p19wt or the indicated p19 mutants. Twelve hours following UV irradiation, cell lysates were tested for caspase-3 activity. Results are expressed as percentage of caspase-3 activity with respect to basal activity of cell lysates nontransfected and without UV-treatment, which was set to 100. Bars represent the mean ± s.e.m. of three independent experiments performed in triplicate. Student <i>t</i>-test was used to compare, UV-treated control sample (none) with UV-treated p19wt or p19 mutant samples (^*<i>p</i><0.005). (<b>D</b>) Similarly as in (<b>C)</b> but overexpressing the phosphomimetic p19 mutants.</p

p19 phosphorylation is induced in response to DNA damage.

<p>(<b>A, B</b>) WI-38 fibroblasts were labeled with [³²P]-orthophosphate and treated with β-amyloid peptide (20 µM), cisplatin (10 µM) or UV light (4 mJ/cm²) for the indicated times. Equal amounts of whole cell extracts were subjected to immunoprecipitation with anti-p19 antibody and the immune complexes were analyzed by SDS-PAGE and autoradiography (upper panels; P-p19, phosphorylated p19) or immunoblotting (lower panels; p19). (C; Control, untreated cells).</p

DNA damage induced p19 nuclear translocation is dependent on S76 phosphorylation.

<p>(<b>A</b>) Distribution of phosphorylated p19 in the cytoplasmic and nuclear fractions after DNA damage. <i>In vivo</i> phosphorylation assays were performed in WI-38 fibroblasts. Cells were treated with UV (4 mJ/cm2), collected at the indicated times, and the extracts subjected to a subcellular fractionation protocol. Either the cytoplasmic (C) or nuclear fractions (N) were immunoprecipitated with anti-p19 antibody, and the immunocomplexes analyzed by SDS-PAGE and autoradiography (upper panel). (<b>B</b>) Subcellular distribution of the phosphorylation deficient mutant p19T141A. For <i>in vivo</i> phosphorylation assays, WI-38 cells were transfected with p19wt or p19T141A, treated with UV radiation and collected at the indicated times. After subcellular fractionation, extracts were immunoprecipitated with an anti-V5 antibody and analyzed as in (<b>A</b>). p19wt or p19T141A subcellular distributions were also studied by immunoblot (<b>C</b>) Subcellular localization of endogenous deficiently phosphorylated-p19 after PKA inhibition. For <i>in vivo</i> phosphorylation assays, cells were processed as in (<b>A</b>) but, before UV irradiation, they were incubated with H-89 for 1 hour. Endogenous distribution of p19 was also studied by immunoblot. (<b>D</b>) Subcellular localization of p19S76A mutant following DNA damage. WI-38 cells were transfected with p19S76A and treated with UV radiation. At the indicated times, extracts were prepared by subcellular fractionation and analized by immunoblot with anti V5-antibody.</p

CDK2 and PKA participate in p19 sequential phosphorylation.

<p>(<b>A</b>) CDK and PKA involvement in endogenous p19 phosphorylation. WI-38 fibroblasts were incubated with roscovitine (RSC, 10 µM), or with H-89 (1 µM) for 1 hour before the damaging treatments (4 mJ/cm² UV light, 10 µM cisplatin or 20 µM ß-amyloid peptide). p19 phosphorylation was analyzed by autoradiography. (<b>B, C</b>) Effect of CDK and PKA inhibition on the phosphorylation of T141 mutants. WI-38 cells were transfected with the indicated p19 constructs expression plasmids, incubated with roscovitine or H-89 for 1 hour and then treated with UV light (4 mJ/cm²) or β-amyloid peptide (20 µM) for 2 hours. p19wt or the mutants were immunoprecipitated with anti-V5 antibody and the immunocomplexes were analyzed by autorradiography and immunoblotting. (<b>D</b>) Measurement of CDK1 and CDK2 activities in the phosphorylation process of endogenous p19. WI-38 fibroblasts were incubated for 24 hours with specific CDK1 or CDK2 antisense oligonucleotides before treatment with UV radiation (4 mJ/cm²). After 2 hours, p19 was immunoprecipitated and phosphorylation observed by autoradiography as mentioned before (upper panel). Northern blot results show the efficiency of the antisense oligonucleotides (lower panel).</p

E2F mediates induction of p19 in response to DNA damage or chromatin relaxation.

<p><b>A.</b> HEK-293 cells were transfected with 500 nM E2F decoy oligonucleotide. Twenty four hours later, cells were exposed to 40 J/m² UV or 50 ng/ml NCS and incubated in the presence or in the absence of 100 µM chloroquine. After 4 h, cells were harvested and subjected to northern blot analysis using a ³²P-labelled probe specific for human p19 mRNA and reprobed for E2F1 and β-tubulin mRNA. Figure shows a representative autoradiograph of three independent experiments with similar results. Densitometric analysis of p19 and E2F1 are represented in the lower panel. Bars represent the mean ± S.E of three experiments. Student’s <i>t</i>-test was used to compare treated and non-treated samples (* p<0.05, at least). <b>B.</b> HEK-293 cells transiently cotransfected with 4 µg of p19CAT, or equivalent amount of mutant plasmid, containing the 5′-flanking region of p19 gene and 5 µg pCEFL-β-galactosidase were exposed to 40 J/m² UV or incubated with 50 ng/ml NCS or 100 µM chloroquine or 200 nM TSA or hypotonic medium. After 24 h cells were harvested and CAT activity was determined as described. Results are expressed as relative CAT activity with respect to basal value of p19CAT which was set to 100. Bars represent the mean ± S.D. of three independent experiments performed in quadruplicate. Student’s <i>t</i>-test was used to compare treated with non treated samples (* p<0.01). <b>C.</b> HEK-293 cells, transiently cotransfected with 4 µg of pE2F4XCAT and 5 µg pCEFL-β-galactosidase and, when indicated, 4 µg of a vector expressing E2F1 cDNA, were treated with 100 µM chloroquine, or 200 nM TSA or subjected to hypotonic medium and incubated in the presence or in the absence of 10 µM Ku-55933 or 15 nM SB-218078 or 20 nM Chk2 inhibitor. After 24 h cells were harvested and CAT activity was determined as described. Results are expressed as relative CAT activity with respect to basal value of pE2F4XCAT which was set to 100. Bars represent the mean ± S.D. of three independent experiments performed in quadruplicate. Student’s <i>t</i>-test was used to compare treated with non treated samples (* p<0.01). Decoy E2F oligonucleotide (DecE2F), β-tubulin (β-tub), chloroquine (chlo), hypotonic medium (hypo), neocarzinostatin (NCS), Ku-55933 (Ku), SB-218078 (SB), Chk2 inhibitor (2I).</p