p16INK4A Positively Regulates Cyclin D1 and E2F1 through Negative Control of AUF1

Abstract

BackgroundThe cyclin-D/CDK4,6/p16INK4a/pRB/E2F pathway, a key regulator of the critical G1 to S phase transition of the cell cycle, is universally disrupted in human cancer. However, the precise function of the different members of this pathway and their functional interplay are still not well defined.Methodology/Principal FindingsWe have shown here that the tumor suppressor p16INK4a protein positively controls the expression of cyclin D1 and E2F1 in both human and mouse cells. p16INK4a stabilizes the mRNAs of the corresponding genes through negative regulation of the mRNA decay-promoting AUF1 protein. Immunoprecipitation of AUF1-associated RNAs followed by RT-PCR indicated that endogenous AUF1 binds to the cyclin D1 and E2F1 mRNAs. Furthermore, AUF1 down-regulation increased the expression levels of these genes, while concurrent silencing of AUF1 and p16INK4a, using specific siRNAs, restored normal expression of both cyclinD1 and E2F1. Besides, we have shown the presence of functional AU-rich elements in the E2F1 3′UTR, which contributed to p16/AUF1-mediated regulation of E2F1 post-transcriptional events in vivo. Importantly, genome-wide gene expression microarray analysis revealed the presence of a large number of genes differentially expressed in a p16INK4a -dependent manner, and several of these genes are also members of the AUF1 and E2F1 regulons. We also present evidence that E2F1 mediates p16-dependent regulation of several pro- and anti-apoptotic proteins, and the consequent induction of spontaneous as well as doxorubicin-induced apoptosis.Conclusion/SignificanceThese findings show that the cyclin-dependent kinase inhibitor p16 INK4a is also a modulator of transcription and apoptosis through controlling the expression of two major transcription regulators, AUF1 and E2F1.

The Cytokine IL-6 Reactivates Breast Stromal Fibroblasts through Transcription Factor STAT3-dependent Up-regulation of the RNA-binding Protein AUF1

“…9). Indeed, p16 also negatively regulates AUF1 post-transcriptionally (46), which could explain the key role of p16 in the activation of stromal fibroblasts. Therefore, either AUF1 up-regulation or p16 down-regulation will both lead to stromal fibroblast transactivation and the consequent promotion of metastasis.…”

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

“…Apoptosis Analysis by Annexin V/Flow Cytometry-Cells were either not treated or challenged with UV light (10 Jm Ϫ2 ), then harvested after 72 h, centrifuged, and stained by propidium iodide and Alexa Fluor 488 annexin V (Molecular Probes, Eugene, OR) as described previously (18).…”

“…Additionally, several lines of evidence indicate that p16 is overexpressed at both protein and mRNA levels in certain tumors, and this has been shown to be associated with poor prognosis (13,14). Furthermore, it became clear that p16 interacts with several proteins (15,16), and it also regulates the expression of different genes involved in various cellular processes (17,18). We have recently shown that p16 negatively regulates the expression of AUF1 through activating the turnover of its mRNA (18).…”

“…Furthermore, it became clear that p16 interacts with several proteins (15,16), and it also regulates the expression of different genes involved in various cellular processes (17,18). We have recently shown that p16 negatively regulates the expression of AUF1 through activating the turnover of its mRNA (18). However, the molecular mechanism(s) underlying this process were not delineated.…”

“…To determine whether p16 influence is global or specific for some Sp1 target genes, the effect of p16 on the binding of Sp1 to the promoter of the FGF21 and IHPK2 genes, which are positively regulated by Sp1 (30) but are not p16 targets (18), was investigated by the ChIP assay. Fig.…”