REGγ Mediated Regulation of p21^Waf/Cip1, p16^INK4a and p14^ARF/p19^ARF <i>in Vivo</i>

p21^Waf/Cip1, p16^INK4a and p14^ARF (p19^ARF in mice) have been demonstrated to be degraded by REGγ-proteasome pathway in an ATP- and ubiquitin-independent manner in vitro. However, the in vivo roles of REGγ mediated-degradation of p21^Waf/Cip1, p16^INK4a and p14^ARF remain unclear. In this study, we showed enhanced expression of p21^Waf/Cip1, p16^INK4a and p19^ARF in multiple tissues from REGg^–/– mice compared to REGg^+/+ mice. Furthermore, we examined the expression of p21^Waf/Cip1, p16^INK4a and p14^ARF in different cancer tissues and observed that the REGγ protein levels were highly expressed in different human cancers while the level of p21^Waf/Cip1, p16^INK4a and p14^ARF appears to be inversely correlated. These results demonstrate that REGγ may exert its function in physiological and pathological conditions through degradation of p21^Waf/Cip1, p16^INK4a and p14^ARF in vivo.

• REGγ; p21^Waf/Cip1;p16^INK4a; p14^ARF; p19^ARF; Cancer

1. Introduction

The INK4a/ARF locus encodes two proteins, p16^INK4a and p14^ARF (equivalent to p19^ARF in mice) [1,2]. Both proteins demonstrate tumor suppressor activity and play an important role in cell proliferation, cell progression and cell growth arrest. Mice lacking either p19^ARF or p16^INK4a are more prone to tumorigenesis compared to wild-type littermates [3,4]. P16^INK4a is a well characterized tumor suppressor and was shown to inhibit the kinase activities of the cyclin D-dependent kinases CDK4 and CDK6 [5,6]. P16^INK4a inactivation has been implicated in the deregulation of the cell cycle in the malignant melanoma [7,8].

P14^ARF and p19^ARF proteins are present in normal cells at low levels but accumulate in response to oncogene activation [9-12]. High p14^ARF and 19^ARF protein levels promote cell cycle arrest, senescence, or apoptosis by binding directly to the p53-degrading ubiquitin ligase Mdm2 and protecting p53 from Mdm2-mediated degradation [12-14]. The biological properties of p16^INK4a, p14^ARF and p19^ARF have been studied extensively, but the regulation of these proteins remains unknown.

REGγ (also known as PA28g or PSME3) is a member of the 11S proteasomes which binds and activates the 20S proteasome to promote ATPand ubiquitin-independent protein degradation. Recent observations show that REGg could enhance proteasomal degradation of some proteins, such as steroid receptor coactivator-3, p21^Waf/Cip1 and smurf1 [15-17] and REGg was also involved in p16^INK4a and p19^ARF degradation in vitro [16]. Furthermore, there is some evidence suggesting that REGg is involved in cancer progression and REGg has been reported to be overexpressed in colorectal cancer, thyroid cancer, liver cancer and lung cancer [18-22]. However, there is no evidence about the degradation of p21^Waf/Cip1, p16^INK4a, p14^ARFand p19^ARF by REGg in vivo.

In this study, we demonstrate that REGγ mediated the degradation of p21^Waf/Cip1, p16^INK4a and p14^ARF/p19^ARF in multiple mouse tissues and in human cancer tissues. In addition, the mRNA levels of p21^Waf/Cip1, p16^INK4a and p19^ARF were not affected by REGg in mouse tissues. Our results provide the first evidence that REGγ plays an important role in the degradation of p21^Waf/Cip1, p16^INK4a and p14^ARF/p19^ARF in vivo.

2. Materials and Methods

2.1. Animals

The REGg^–/– mice with C57BL/6 genetic background were acquired from Dr. John J. Monaco and bred in the Animal Core. To generate the mice required in this study, we kept REGg^+/– mice intercrosses between males and females. Genotyping of REGg^+/+ and REGg^–/– mice was carried out by PCR analysis of genomic DNA as described.

The REGg^+/+ and REG^–/– mice were born at Mendelian frequency, grew normally and used in the subsequent experiments.

2.2. Antibody and Cell Culture

Antibodies were purchased from Invitrogen (REGg/PA 28g), BD Pharmingen (p21^Cip/WAF1), Sigma (β-actin, p14), Santa Cruz (p16, p19). The HeLa stable cell lines were generated by retroviral shRNA vectors specific for REGg or a control vector from OriGene (Rockville, MD). These two cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (Gibco).

2.3. RNA Analyses

Tissues were homogenized in 1 ml RNAisoTM Plus (TAKARA). Total RNA was extracted and 2 μg RNA was reversely transcribed into cDNA with M-MLV reverse transcriptase (Invitrogen) following the manufacturer’s instruction. The mouse gene-specific primers are as follows:

REGg sense primer, 5-TCCTCACCAATAGCCACG-3;

REGg antisense primer, 5-CTCGATCAGCAGCCGAAT-3;

p16 sense primer, 5-GCTGCAGACAGACTGGCCA-3;

p16 antisense primer, 5-GTCCTCGCAGTTCGAATCTG-3;

p19 sense primer, 5-CGCAGGTTCTTGGTCACTGT-3;

p19 antisense primer, 5-TGTTCACGAAAGCCAGAGCG-3;

p21 sense primer, 5-CCTGGTGATGTCCGACCTG-3;

p21 antisense primer, 5-CCATGAGCGCATCGCAATC-3.

2.4. Western Blot Analysis

Tissues were homogenized in liquid nitrogen and lysed using RIPA buffer (1.0 mM EDTA 150 mM 1% Triton X-100 50 mM Tris-HCl (pH 7.4) NaCl 0.1% SDS 1% sodium deoxyholate) for 30 min. Then the homogenized tissues were centrifuged at 12,000 g for 15 min and the supernatants were collected. Equivalent amount of protein from each sample was analyzed by using primary antibodies specific for p16^INK4a, p21^Waf/Cip1, p19^ARF and REGg overnight at 4˚C. After incubation with fluorescent labeled secondary antibody, specific signals for proteins were visualized by LI-COR Odyssey Infrared Imaging System.

2.5. Immunohistochemistry (IHC) and H&E Staining

Tissues were fixed with 4% paraformaldehyde for 48 hours. Then the samples were dehydrated through a graded series of ethanol, embedded in paraffin and sectioned at 4 μm.

Immunohistochemistry (IHC) and H&E staining were performed as described [23]. Antibodies against p16^INK4a, p21^Waf/Cip1, p19^ARF and REGg were used at 1:500, 1:600, 1:600, and 1:400 dilutions, respectively.

2.6. Data Collection and Statistical Analysis The statistical data was got by GraphPad Prism 5.0 software. The results were expressed as the mean ± s.d. Statistical analysis was performed using the two tailed, paired Student’s t test. A p value of less than 0.05 was considered statistically significant.

3. Results

3.1. The p21 Waf/Cip1 , p16 INK4a , and p19 ARF Expression is Higher in REG( –/– Mouse Tissues

Numerous REGγ targets proteins such as p21^Waf/Cip1, p16^INK4a, and p14^ARF/p19^ARF (p19^ARF in the mouse) of REGγ-proteasome, have been identified in vitro [16, 17]. However, the molecular details and in vivo biological significance of REGγ, p21^Waf/Cip1, p16^INK4a and p19^ARF interplay remain elusive. We investigated cell specific expression patterns of REGγ, p21^Waf/Cip1, p16^INK4a and p19^ARF by IHC analysis in cerebellum (Figure 1(a)), kidney (Figure 1(b)), stomach (Figure 1(c)) and liver (Figure 1(d)) from REGg^+/+ and REGg^–/– littermate mice. The staining of REGγ was negative in the tissues from REGg^–/– mice. Intriguingly, there was an increased expression of p21^Waf/Cip1, p16^INK4a and p19^ARF in REGg depleted tissues, suggesting that REGg may regulate the expression p21^Waf/Cip1, p16^INK4a and p19^ARF in vivo.

3.2. Comparative Analysis of REG( Protein and mRNA Expression

To verify the REGγ expression observed by IHC, p21^Waf/Cip1, p16^INK4a and p19^ARF protein and mRNA levels were examined using extracts from different mouse tissues. Firstly, the relative protein levels were normalized to β-actin and the results showed that p21^Waf/Cip1, p16^INK4a and p19^ARF protein levels were higher in REGg deficient cerebellum (Figure 2(a)), stomach (Figure 2(b)) and liver (Figure 2(c)). The comparison of relative mRNA and protein levels in different tissues may provide intact information about tissue-specific roles for a gene. Next, we consistently observed a higher mRNA expression of p16^INK4a and p19^ARF in multiple tissues, indicating that REGg regulated p16^INK4a and p19^ARF in protein level (Figure 2(d)). To further demonstrate that REGg is involved in p21^Waf/Cip1, p16^INK4a and p19^ARF degradation, we generated stable HeLa cell line constitutively expressing either a control non-specific shRNA (shN) or a REGg-specific shRNA (shR). We monitored the p21^Waf/Cip1, p16^INK4a and p14^ARF protein levels in Hela stable cells. As expected, the p21^Waf/Cip1, p16^INK4a and p14ARF protein levels were higher in Hela-ShR cells compared with Hela-shN cells (Figure 2(e)). The same phenomenon was also found in Hela cells described previously [16].

3.3. Highly Expressed REG( and Reduced Expression of p21 Waf/Cip1 , p16 INK4a and p14 ARF are Observed in Human Cancers

It is known that REGg is high expressed in lungs, liver, thyroid and colon cancer [20,24]. However, the biological links between REGg and its correlated genes such as p21^Waf/Cip1, p16^INK4a and p14^ARF in cancers remain unknown. Immunohistochemistry (IHC) analysis of REGg regulated proteins revealed that the REGg level was high expressed in human cancers such as kidney (Figure 3(a)), lungs (Figure 3(b)) and brain (Figure 3(c)) while the protein level of p21^Waf/Cip1, p16^INK4a and p14^ARF was decreased. However, the protein level of p21^Waf/Cip1, p16^INK4a and p14^ARF was increased when REGg level was lower in stomach cancer (Figure 3(d)). These results suggest that REGγ mediated degradation of p21^Waf/Cip1, p16^INK4a and p14^ARF is one of the important mechanisms by which REGγ mediates its physiological function in different cancers.

4. Discussion

p21^Waf/Cip1, p19^ARF and p16^INK4a are important tumor-suppressor proteins. p21^Waf/Cip1 is encoded by CDKN1A gene, which can bind to and inhibit the activity of cyclin-CDK2 or -CDK4 complexes, and thus functioning as a regulator of cell cycle progression at G1. p16^INK4a inhibits Cdk4 and Cdk6 activity leading to activating RBp19^ARF stabilizes p53 protein levels by binding to Mdm2 and promoting its degradation [25]. Mice lacking p21^Waf/Cip1, p16^INK4a or p19^ARF are prone to form tumors induced by carcinogens. Currently, several studies showed that REGγ can promote the degradation of p21^Waf/Cip1 and p53 in vitro. However, the detailed link between REGγ and the cyclin-dependent kinase inhibitors is still unclear. This study systematically demonstrated that REGγ can promote the degradation of

p21^Waf/Cip1, p16^INK4a and p19^ARF in vivo.

REGγ-deficient mice [23,26] have been demonstrated growth retardation and cell-specific mitotic defects, in-

dicating a role of REGγ in regulation of cell cycle and cell proliferation. It is well known that p16^INK4a, p19^ARF, smARF and p21^Waf/Cip1 are important CDK inhibitors. Therefore, we used the REGγ-knockout mice to detect the effects of REGγ on p16^INK4a/p19^ARF/p21^{Waf/Cip1 in several normal tissues. As expected, our IHC and blot results showed that the levels of p16INK4a}/p19^ARF/ p21^Waf/Cip1 were markedly increased in cerebellum, stomach, kidney and liver when REGγ was deleted. Our result demonstrated that REGγ can promote the degradation of CDK inhibitors in normal tissues. These results indicated that REGγ plays an important role in regulating the dynamic balance of body such as organizational self-renewal and tissue remodeling. These results also provide the first time to study the effects of REGγ on CDK inhibitors in vivo. Recent studies demonstrate that REGγ can promote proteasome-dependent degradation of cyclin-dependent kinase inhibitors p21^Waf/Cip1, p16^INK4a and p19^ARF, in a ubiquitinand ATP-independent manner [16,17]. However, their conclusion is mainly limited to in vitro studies. Thus, our finding is consistent with previous suggestion that REGγ is involved in the regulation of cell cycle and cell proliferation.

Besides as the CDK inhibitors, p16^INK4a/p19^ARF/ p21^Waf/Cip1 are well known as the tumor suppressor. The first genetic evidence supporting a tumor suppressor activity for p21^Waf/Cip1 came from the discovery that CDKN1A deficient mice developed spontaneous tumours. Many human cancers such as colorectal, cervical, head and neck, and small-cell lung cancers are associated with reduced p21^Waf/Cip1 expression. The frequent mutations and deletions of p16^INK4a and p14^ARF in many types of human cancers cancer cell lines suggested an important role for p16^INK4a and p14^ARF in carcinogenesis. Thus, the effects of REGγ on p16^INK4a/p19^ARF/p21^Waf/Cip1 were investigated in several human cancer tissues. In cancer tissues of stomach, lung, kidney and brain, we found that REGγ has different expression patterns. REGγ’s expression was higher in the kidney and lung cancer compared with stomach cancer. These results indicated that REGγ showed its specificity for participating in carcinogensis. Consistently, previous studies revealed that REGγ can degrade not only tumor suppressor but also tumor activator, which suggests its dual function in cancer. Thus, we assumed that the mechanism of REGγ involved in cancer is complicated. Interestingly, we found the levels of p16^INK4a/p19^ARF/p21^Waf/Cip1 are firmly regulated by REGγ both in normal tissues and in cancer tissues. When REGγ levels are high in the kidney and lung cancer, the expressions of p16^INK4a/p19^ARF/p21^Waf/Cip1 are low. On the contrary, the high expression of p16^INK4a/p19^ARF/ p21^Waf/Cip1 was observed in the stomach cancer which showed low expression of REGγ. Our results demonstrated that REGγ can promote the degradation of p16^INK4a/p19^ARF/p21^Waf/Cip1 not only in normal tissues but also in cancer tissues. REGγ has been reported to be involved in some types of cancer [22,27]. Therefore, we assumed that REGγ maybe play its role in the formation and development of cancer by altering the levels of CDK inhibitors under some conditions.

Recently, REGγ was shown to facilitate the turnover of tumor suppressor p53 by promoting MDM2-mediated p53 ubiquitination [28]. P53 is known as “guardian of the genome” because it shuts down cell division in response to DNA damage. Previous research had linked the function of p14^ARF to p53. It is also reported that p53 mediates the DNA damage-induced checkpoint through transactivation of p21^Waf/Cip1. Thus, the relations between REGγ and p53 in vivo are also required to be systemically studied in the future. The members in the CDK inhibitor family also included p15 (INK4B), p18 (INK4C) in INK4 subfamily and p27 (KIP1), p57 (KIP2) in the KIP subfamily. It is still unclear if REGγ can promote the degradation of p15, p18, p27 and p57. Furthermore, the effects of REGγ on p16^INK4a/p19^ARF/p21^{Waf/Cip1 in the other tissues of mice or in the other types of cancer are needed to be clarified.}

In summary, this study is the first to systematically study the effects of REGγ on p16^INK4a/p19^ARF/p21^Waf/Cip1 in vivo. We found that REGγ can promote the degradation of CDK inhibitor both in normal tissue of mice and in human caner tissues. The data suggest that inhibition of REGγ may be of potential benefit in preventing and treating some types of human cancer.

5. Acknowledgements

This work was supported by National Institutes of Health (1R01CA131914, P30 CA125123), Norman Hackerman Advanced Research Program (1082318401; PN00 4949- 0012-2009), and supported in part by the Pilot/Feasibility Program of the Diabetes & Endocrinology Research Center (P30-DK079638) at Baylor College of Medicine. This manuscript was also funded in part by the National Natural Science Foundation of China (81261120555, 81071657, 81272943, 31100946, 31200878); the Science and Technology Commission of Shanghai Municipality (11DZ2260300, 10JC1404200, 11ZR1410000, 12ZR14 09300); the National Basic Research Program (2009 CB918402, 2011CB504200); and the Fundamental Research Funds for the Central Universities (78210101 and 78210139).

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