p16INK4A Positively Regulates p21WAF1 Expression by suppressing AUF1-dependent mRNA decay

Background

p16^INK4a and p21^WAF1 are two independent cyclin-dependent kinase inhibitors encoded by the CDKN2A and CDKN1A genes, respectively. p16^INK4a and p21^WAF1 are similarly involved in various anti-cancer processes, including the regulation of the critical G1 to S phase transition of the cell cycle, senescence and apoptosis. Therefore, we sought to elucidate the molecular mechanisms underlying the link between these two important tumor suppressor proteins.

Methodology/Principal Findings

We have shown here that the p16^INK4a protein positively controls the expression of p21^WAF1 in both human and mouse cells. p16^INK4a stabilizes the CDKN1A mRNA through negative regulation of the mRNA decay-promoting AUF1 protein. Immunoprecipitation of AUF1-associated RNAs followed by quantitative RT-PCR indicated that endogenous AUF1 binds to the CDKN1A mRNA in a p16^INK4A-dependent manner. Furthermore, while AUF1 down-regulation increased the expression level of the CDKN1A mRNA, the concurrent knockdown of AUF1 and CDKN2A, using specific silencing RNAs, restored the normal expression of the gene. Moreover, we used EGFP reporter fused to the CDKN2A AU-rich element (ARE) to demonstrate that p16^INK4A regulation of the CDKN1A mRNA is AUF1- and ARE-dependent. Furthermore, ectopic expression of p16^INK4A in p16^INK4A-deficient breast epithelial MCF-10A cells significantly increased the level of p21^WAF1, with no effect on cell proliferation. In addition, we have shown direct correlation between p16^INK4a and p21^WAF1 levels in various cancer cell lines.

Conclusion/Significance

These findings show that p16^INK4a stabilizes the CDKN1A mRNA in an AUF1-dependent manner, and further confirm the presence of a direct link between the 2 important cancer-related pathways, pRB/p16^INK4A and p14^ARF/p53/p21^WAF1.     

The OGF-OGFr axis utilizes the p16INK4a and p21WAF1/CIP1 pathways to restrict normal cell proliferation

Opioid growth factor (OGF) is an endogenous opioid peptide ([Met⁵]enkephalin) that interacts with the OGF receptor (OGFr) and serves as a tonically active negative growth factor in cell proliferation of normal cells. To clarify the mechanism by which OGF inhibits cell replication in normal cells, we investigated the effect of the OGF–OGFr axis on cell cycle activity in human umbilical vein endothelial cells (HUVECs) and human epidermal keratinocytes (NHEKs). OGF markedly depressed cell proliferation of both cell lines by up to 40% of sterile water controls. Peptide treatment induced cyclin-dependent kinase inhibitor (CKI) p16^INK4a protein expression and p21^WAF1/CIP1 protein expression in HUVECs and NHEKs, but had no effect on p15, p18, p19, or p27 protein expression in either cell type. Inhibition of either p16^INK4a or p21^WAF1/CIP1 activation by specific siRNAs blocked OGF inhibitory action. Human dermal fibroblasts and mesenchymal stem cells also showed a similar dependence of OGF action on p16^INK4a and p21^WAF1/CIP1. Collectively, these results indicate that both p16^INK4a and p21^WAF1/CIP1 are required for the OGF–OGFr axis to inhibit cell proliferation in normal cells.     

Chemical DNA damage activates p21 WAF1/CIP1-dependent intra-S checkpoint

When cells progressing in G₁ phase are irradiated with UV light, two damage checkpoint pathways are activated: CHK1-Cdc25A and p53-p21^WAF1/CIP1, both targeting Cdk2 but the latter inducing long lasting inactivation. In similarly irradiated S phase cells, however, p21^WAF1/CIP1-dependent checkpoint is largely inactive. We report here that p21-dependent checkpoint can effectively be activated and induce a prolonged S phase arrest with similarly extended inactivation of Cdk2 by association of p21 if mid-S phase cells are damaged with a base-modifying agent instead of UV light, indicating that the poor utilization of p21-dependent checkpoint is not an innate property of S phase cells.     

The atr protein kinase controls UV-dependent upregulation of p16INK4A through inhibition of Skp2-related polyubiquitination/degradation

The tumor suppressor p16(INK4A), a phosphoprotein that exists in human cells under both phosphorylated and nonphosphorylated forms, plays crucial roles during the cellular response to UV light. However, it is still unclear how this protein is activated in response to this carcinogenic agent. We have shown here that UVC upregulates p16(INK4A) and the phosphorylated form of the protein at the 4 serine sites; Ser-7, Ser-8, Ser-140, and Ser-152. This accumulation of p16(INK4A) occurred through increasing the stability of both forms of the protein. Importantly, phospho-p16(INK4A) showed much higher stability, and UV treatment strongly increased its level in absence of de novo protein synthesis. Furthermore, we have shown that the UV-dependent upregulation of both forms of p16(INK4A) is under the control of the protein kinase Atr, which suppresses their UVC-dependent proteasomal degradation. Interestingly, although this degradation is ubiquitin-related for p16(INK4A) through the Skp2 ubiquitin ligase protein, it is ubiquitin-independent for the phosphorylated form. In addition, we present clear evidence that Skp2 is upregulated in ATR-deficient cells, leading to the downregulation of the p27(Kip1) protein in response to UVC light. Moreover, we have shown a preferential association of endogeneous phospho-p16(INK4A) with Cdk4. This association increased following UV-treatment mainly for p16(INK4A) phosphorylated at Ser-140 and Ser-152. Besides, we have shown that Atr regulates UV-related p16/Cdk4-dependent and -independent phosphorylation of pRB and G1 cell cycle delay. Together, these results indicate that p16(INK4A) and p27(Kip1) are key targets in the Atr-dependent signaling pathway in response to UV damage.     

可逆的组蛋白乙酰化调控p16的表达

p16^INK4a通过抑制CDK4/6的活性而在细胞周期进行中发挥重要的作用,研究发现,组蛋白乙酰转移酶p300能促进p16^INK4a启动子活性,而组蛋白去乙酰化酶HDAC3/4能够逆转由p300介导的p16^INK4a启动子活性的增加,HDAC3/4能够降低p16^INK4a mRNA和蛋白质的水平．染色质免疫沉淀(ChIP)实验结果表明转染p300表达质粒能够逆转由HDAC3/4介导的p16^INK4a启动子组蛋白的低乙酰化状态．此外,免疫荧光实验结果表明HDAC4的核质穿梭起着重要的作用．免疫印迹和染色质免疫沉淀实验证明HDAC的抑制剂丁酸钠盐(NaBu)能通过诱导组蛋白的高乙酰化而促进p16^INK4a的表达．基于这些实验结果,推测出可逆的组蛋白乙酰化参与p16^INK4a基因转录调控的模型．     

p27(Kip1) is an inducer of intestinal epithelial cell differentiation

Constant renewal of the intestinal epitheliumis a highly coordinated process that has been subject to intenseinvestigation, but its regulatory mechanisms are still essentiallyunknown. In this study, we have demonstrated that forced expression ofthe cyclin-dependent kinase inhibitors (CKIs) p27^Kip1 andp21^Cip1/WAF1 in human intestinal epithelial cells led toexpression of differentiation markers at both the mRNA and proteinlevels. Cell differentiation was temporally dissociated from inhibitionof retinoblastoma protein phosphorylation and growth arrest, alreadyestablished 1 day after infection with recombinant adenoviruses.p27^Kip1 proved significantly more efficient thanp21^Cip1/WAF1 in induction of cell differentiation. Incontrast, forced expression of p16^INK4a resulted in growtharrest without induction of differentiation markers. These resultsimplicate both p27^Kip1 and p21^Cip1/WAF1 in thedifferentiation-timing process, but p21^Cip1/WAF1 may actindirectly by increasing p27^Kip1 levels. These results alsosuggest that induction of intestinal epithelial cell differentiation byCKIs is not related to their effects on the cell cycle and may involveinteractions with cellular components other than cyclins andcyclin-dependent kinases.

     

Epstein-Barr Virus Nuclear Antigen 3A Promotes Cellular Proliferation by Repression of the Cyclin-Dependent Kinase Inhibitor p21WAF1/CIP1

Latent infection by Epstein-Barr virus (EBV) is highly associated with the endemic form of Burkitt lymphoma (eBL), which typically limits expression of EBV proteins to EBNA-1 (Latency I). Interestingly, a subset of eBLs maintain a variant program of EBV latency - Wp-restricted latency (Wp-R) - that includes expression of the EBNA-3 proteins (3A, 3B and 3C), in addition to EBNA-1. In xenograft assays, Wp-R BL cell lines were notably more tumorigenic than their counterparts that maintain Latency I, suggesting that the additional latency-associated proteins expressed in Wp-R influence cell proliferation and/or survival. Here, we evaluated the contribution of EBNA-3A. Consistent with the enhanced tumorigenic potential of Wp-R BLs, knockdown of EBNA-3A expression resulted in abrupt cell-cycle arrest in G0/G1 that was concomitant with conversion of retinoblastoma protein (Rb) to its hypophosphorylated state, followed by a loss of Rb protein. Comparable results were seen in EBV-immortalized B lymphoblastoid cell lines (LCLs), consistent with the previous observation that EBNA-3A is essential for sustained growth of these cells. In agreement with the known ability of EBNA-3A and EBNA-3C to cooperatively repress p14^ARF and p16^INK4a expression, knockdown of EBNA-3A in LCLs resulted in rapid elevation of p14^ARF and p16^INK4a. By contrast, p16^INK4a was not detectably expressed in Wp-R BL and the low-level expression of p14^ARF was unchanged by EBNA-3A knockdown. Amongst other G1/S regulatory proteins, only p21^WAF1/CIP1, a potent inducer of G1 arrest, was upregulated following knockdown of EBNA-3A in Wp-R BL Sal cells and LCLs, coincident with hypophosphorylation and destabilization of Rb and growth arrest. Furthermore, knockdown of p21^WAF1/CIP1 expression in Wp-R BL correlated with an increase in cellular proliferation. This novel function of EBNA-3A is distinct from the functions previously described that are shared with EBNA-3C, and likely contributes to the proliferation of Wp-R BL cells and LCLs.     

Cell cycle arrest and cytochrome c-mediated apoptotic induction by MCS-5A is associated with up-regulation of p16INK4a in HL-60 cells

MCS-5A, an analog of sangivamycin, selectively inhibits the cyclin-dependent kinases CDK1 and 4 in HL-60 cells in vitro (IC₅₀: 9.6 and 8.8 μΜ, respectively), while weakly inhibiting other housekeeping protein kinases. MCS-5A effectively induces HL-60 cell cycle arrest at the G₁ and G₂/M phases through direct inhibition of CDK1 and 4 activity. In addition, elevated expression of p16^INK4a and a reduction in the level of hyperphosphorylated pRb showed that 3 μΜ MCS-5A also induces p16^INK4a-mediated cell cycle arrest at the G₁ phase. Furthermore, apoptotic induction in MCS-5A-treated HL-60 cells is associated with the release of cytochrome c from mitochondria, which, in turn, results in the activation of procaspase-8, -9 and -3, and the cleavage of poly(ADP-ribose) polymerase (PARP). In addition, the involvement of p16^INK4a in this apoptotic induction was demonstrated using A549 cells with a homozygous deletion of p16^INK4a. Based on these results, we conclude that MCS-5A is a candidate therapeutic agent for the treatment of human promyelocytic leukemia via the up-regulation of p16^INK4a.     

Indoxyl sulfate, a uremic toxin, promotes cell senescence in aorta of hypertensive rats

We demonstrated that administration of indoxyl sulfate, a uremic toxin, promotes aortic calcification in hypertensive rats. This study aimed to clarify if indoxyl sulfate could contribute to cell senescence in the aorta of hypertensive rats. The rat groups consisted of (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive indoxyl sulfate-administered rats (DN + IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive indoxyl sulfate-administered rats (DH + IS). After 32 weeks, their arcuate aortas were excised for histological and immunohistochemical analysis. Cell senescence was evaluated by immunohistochemistry of senescence-associated β-galactosidase (SA-β-gal), and senescence-related proteins such as p16^INK4a, p21^WAF1/CIP1, p53 and retinoblastoma protein (Rb). Both DH and DH + IS rats showed significantly higher systolic blood pressure than DN and DN + IS rats, respectively. Serum indoxyl sulfate levels were significantly higher in DN + IS and DH + IS rats than in DN and DH rats, respectively. In aorta, DH rats showed significantly increased aortic calcification and wall thickness, and increased expression of SA-β-gal, p16^INK4a, p21^WAF1/CIP1, p53 and Rb in the calcification area of arcuate aorta as compared with DN rats. More notably, DH + IS rats showed significantly increased aortic calcification and wall thickness, and significantly increased expression of SA-β-gal, p16^INK4a, p21^WAF1/CIP1, p53 and Rb in the cells embedded in the calcification area as compared with DH rats. In conclusion, indoxyl sulfate promotes cell senescence with aortic calcification and expression of senescence-related proteins in hypertensive rats.     

ZNF313 is a novel cell cycle activator with an E3 ligase activity inhibiting cellular senescence by destabilizing p21WAF1

ZNF313 encoding a zinc-binding protein is located at chromosome 20q13.13, which exhibits a frequent genomic amplification in multiple human cancers. However, the biological function of ZNF313 remains largely undefined. Here we report that ZNF313 is an ubiquitin E3 ligase that has a critical role in the regulation of cell cycle progression, differentiation and senescence. In this study, ZNF313 is initially identified as a XIAP-associated factor 1 (XAF1)-interacting protein, which upregulates the stability and proapoptotic effect of XAF1. Intriguingly, we found that ZNF313 activates cell cycle progression and suppresses cellular senescence through the RING domain-mediated degradation of p21^WAF1. ZNF313 ubiquitinates p21^WAF1 and also destabilizes p27^KIP1 and p57^KIP2, three members of the CDK-interacting protein (CIP)/kinase inhibitor protein (KIP) family of cyclin-dependent kinase inhibitors, whereas it does not affect the stability of the inhibitor of CDK (INK4) family members, such as p16^INK4A and p15^INK4B. ZNF313 expression is tightly controlled during the cell cycle and its elevation at the late G1 phase is crucial for the G1-to-S phase transition. ZNF313 is induced by mitogenic growth factors and its blockade profoundly delays cell cycle progression and accelerates p21^WAF1-mediated senescence. Both replicative and stress-induced senescence are accompanied with ZNF313 reduction. ZNF313 is downregulated during cellular differentiation process in vitro and in vivo, while it is commonly upregulated in many types of cancer cells. ZNF313 shows both the nuclear and cytoplasmic localization in epithelial cells of normal tissues, but exhibits an intense cytoplasmic distribution in carcinoma cells of tumor tissues. Collectively, ZNF313 is a novel E3 ligase for p21^WAF1, whose alteration might be implicated in the pathogenesis of several human diseases, including cancers.