The cyclin-dependent kinase inhibitor p16INK4a physically interacts with transcription factor Sp1 and cyclin-dependent kinase 4 to transactivate microRNA-141 and microRNA-146b-5p spontaneously and in response to ultraviolet light-induced DNA damage

Abstract

p16(INK4a) is a tumor suppressor protein involved in several stress-related cellular responses, including apoptosis. Recent lines of evidence indicate that p16(INK4a) is also a modulator of gene expression. However, the molecular mechanisms underlying this novel function are still obscure. Here, we present clear evidence that p16(INK4a) modulates the levels of various microRNAs, with marked positive effect on miR-141 and miR-146b-5p. This effect is mediated through the formation of the p16-CDK4-Sp1 heterocomplex, which binds to Sp1 consensus-binding motifs present in the promoters of miR-141 and miR-146b-5p, and it enables their transcription. In addition, we have shown that p16(INK4a) interacts with Sp1 through the fourth ankyrin repeat, which is crucial for Sp1 binding to the miR-141 and miR-146b-5p promoters and their transcriptional activation. The physiological importance of this association was revealed by the inability of cancer-related p16(INK4a) mutants to interact with Sp1. Moreover, we have shown p16-CDK4-Sp1-dependent up-regulation of miR-141 and miR-146b-5p following UV light-induced DNA damage and the role of these two microRNAs in mediating p16-related induction of apoptosis in response to this genotoxic stress. Together, these results indicate that p16(INK4a) associates with CDK4 not only to inhibit the cell cycle but also to enable the transcription of two important onco-microRNAs, which act as downstream effectors.

Keywords: Protein Complexes; Protein-DNA Interaction; Signaling; Tumor suppressor gene; shRNA.

FIGURE 1.

p16 down-regulation does not affect cell proliferation in primary skin fibroblasts. A, HFSN1 cells expressing either control-shRNA (HFSN1C) or CDKN2A-shRNA (HFSN1p16sh) were seeded in E-16 plates and reincubated for 48 h. The proliferation rate was measured using the RTCA-DP xCelligence system. B, cell cycle analysis using flow cytometry. C, immunostaining using Ki-67 antibody. Scale bars, 50 μm. Labeling index for Ki-67 staining was determined for at least 500 cells per data point and expressed as mean ± S.D. of triplicate determinations. D, whole cell lysates were prepared from the indicated cells and used for immunoblotting using the indicated antibodies. The numbers below the bands indicate protein expression relative to total pRB and GAPDH.

FIGURE 2.

p16-dependent differential expression of miRNAs. A, total RNA samples were prepared from the indicated cells and were used to perform microRNA array. B and C, qRT-PCR using primers corresponding to the indicated genes. The relative transcript values were normalized to GAPDH transcript levels. Error bars represent means ± S.D. D, total RNA was extracted from the indicated cells utilizing the RNeasy mini kit (Qiagen), and then was used for Northern blot analysis.

FIGURE 3.

p16 modulates the expression of miR-141 and miR-146b-5p in a cell cycle-independent manner. A, confluent cells were starved for 24 h in serum-free media, and then the cell cycle was analyzed by flow cytometry. The numbers in the boxes indicate the proportion of cells in each phase of the cell cycle. PI, propidium iodide. B, total RNA was purified from the indicated cells and was amplified by qRT-PCR using specific primers for the indicated genes. Error bars represent means ± S.D.

FIGURE 4.

p16 positively regulates miR-141 and miR-146b-5p at the transcriptional level. A, Cells were treated with actinomycin D for the indicated periods of time. Total RNA was extracted, and the remaining levels of miR-141 and miR-146b-5p were assessed by RT-PCR following electrophoresis on 2% ethidium bromide-stained agarose gels. B, schematic diagrams of miR-141 and miR-146b-5p genomic loci and their putative promoters. Chr, chromosome; EST, expressed sequence tag; TSS, transcription start site. C, miR-141 and miR-146b-5p promoters were inserted into SmaI/HindIII sites in the pGL3 luciferase reporter plasmid. The pGL3 vector was used to transfect U2OS and EH1 cells together with Renilla luciferase phRLTK vector to normalize for variation in transfection efficiency, and the firefly (FL) and Renilla (RL) luciferase activities were measured 24 h post-transfection using bio-imaging apparatus and software (BD Biosciences). Histogram shows the activity of luciferase under miR-141 and miR-146b-5p promoters. Data are presented as means ± S.E. of three independent experiments and each is measured in triplicate (*, p < 0.05).

FIGURE 5.

p16 positively regulates the expression of miR-141 and miR-146b-5p through the Sp1 transcription factor. A, nucleotide sequences of the miR-141 and miR-146b-5p promoters, −39 to +9 nucleotides and −93 to −26 nucleotides, respectively, relative to the putative transcription start site. Highlighted are the various overlapped Sp1-binding sites in the miR-141 and miR-146b-5p promoters. B and D, whole cell lysates were prepared from the indicated cells and used for immunoblotting using the indicated antibodies. C and E, EMSAs were performed using nuclear extracts from the indicated cells and biotin-labeled Sp1 consensus binding site of the miR-146b-5p promoter. Oligos, oligonucleotides.

FIGURE 6.

Sp1 binds the miR-141 and miR-146b-5p promoters and activates their transcription in a p16-dependent manner. A, ChIP assay. Chromatin was purified from HFSN1C and HFSN1p16sh, and then immunoprecipitated (IP) using anti-Sp1 antibody. miR-141 and miR-146b-5p promoters were amplified by PCR (upper panel) and qPCR (lower panel) using specific primers, and their abundances were plotted relative to the input, and GAPDH (unlinked locus) was used as negative control. These experiments were performed in triplicate (*, p < 0.05). B, ChIP assay. The promoters of the indicated genes were amplified using qPCR. C, total RNA was extracted from HFSN1 cells expressing either Sp1-shRNA (scrambled sequence was used as control) or Sp1-ORF (empty vector was used as control) and was utilized to assess the expression level of the indicated genes by qRT-PCR. Error bars represent means ± S.D.

FIGURE 7.

p16, Sp1, and CDK4 proteins form a heterocomplex that binds the miR-141 and miR-146b-5p promoters. A, whole cell extracts were prepared from HFSN1 cells and immunoprecipitated (IP) with anti-p16, anti-Sp1, or anti-CDK4 antibodies (mouse IgG was used as control), and immunoblotting was performed using the indicated antibodies. B, EMSA. Supershift assay was performed using nuclear extracts from the indicated cells that were preincubated with the indicated antibodies (1 μg) for an hour before adding the biotin-labeled oligonucleotides of the Sp1 consensus-binding site of the miR-146b-5p promoter. C, EMSA. Pure proteins were used in the indicated combinations and biotin-labeled oligonucleotides of the Sp1 consensus binding sites of the miR-141 and miR-146b-5p promoters. The numbers indicate the amount of proteins in nanograms. D, ChIP assay. Chromatin was purified from the indicated cells and then immunoprecipitated using anti-p16 or anti-CDK4 antibodies. miR-141 and miR-146b-5p promoters were amplified by PCR (upper panel) and qPCR (lower panel), and their abundances were plotted relative to the input. E, total proteins and RNA were prepared from HFSN1 cells expressing either control-shRNA or CDK4-shRNA and were used to assess the level of CDK4 by immunoblotting and the level of miR-141 and miR-146b-5p by qRT-PCR. Error bars represent means ± S.D.

FIGURE 8.

p16 interacts with Sp1 through the fourth ankyrin repeat. A, p16-negative U2OS cells were transfected with the indicated constructs, and then whole cell extracts were prepared and used for immunoprecipitation (IP) with anti-p16 antibody (mouse IgG was used as control), and immunoblotting was performed utilizing the indicated antibodies. B, whole cell extracts were prepared from U2OS expressing the indicated constructs, and immunoblotting was performed for the indicated antibodies. C, ChIP assay. Chromatin was purified from the indicated cells and then immunoprecipitated using anti-Sp1 antibody. miR-141 and miR-146b-5p promoters were amplified by qPCR using specific primers. D, total RNA was prepared from U2OS expressing the indicated constructs and used to assess the level of the indicated genes by qRT-PCR using GAPDH as internal control. Error bars represent means ± S.D. E, U2OS cells were transfected with constructs bearing the indicated tumor-associated point mutations in the fourth ankyrin repeat of human p16. Whole cell extracts were prepared and used for immunoprecipitation with anti-p16 antibody (mouse IgG was used as control), and immunoblotting was performed utilizing the indicated antibodies.

FIGURE 9.

p16 modulates the expression of different Sp1 targets. A and C, total RNA was prepared from the indicated cells, and quantitative RT-PCR was performed using specific primers for the indicated genes. B, EMSA was performed using nuclear extracts from the indicated cells and biotin-labeled oligonucleotides corresponding to three different Sp1 consensus-binding sites of the miR-365 promoter. Error bars represent means ± S.D. Oligos, oligonucleotides.

FIGURE 10.

p16-CDK4-Sp1-dependent up-regulation of miR-141 and miR-146b-5p and their role in apoptosis following UV damage. A, cells were either mock-treated or challenged with UV light (10 Jm⁻²) and then re-incubated for the indicated periods of time. Total RNA was then isolated and used for RT-PCR using specific primers for the indicated genes. The numbers below the bands indicate the corresponding expression levels following loading correction against GAPDH. B, qRT-PCR on mRNAs corresponding to 2 h of post-irradiation relative to time 0. These experiments were performed in triplicate (*, p < 0.05). C, cells were either mock-treated or challenged with UV light (10 Jm⁻²) and incubated for 72 h. Apoptosis was analyzed by annexin V/propidium iodide flow cytometry. D, the histogram shows the proportions of spontaneous and induced apoptosis (early + late). The error bars represent standard deviations of at least three different experiments. E, HFSN1C and HFSN1p16sh cells expressing the control plasmid, pre-miR-141 or pre-miR-146b-5p, were treated as above and collected after 48 and 72 h. Whole cell lysates were prepared and used for immunoblotting analysis. Error bars represent means ± S.D.