Abstract
p53 protein turnover through the ubiquitination pathway is a vital mechanism in the regulation of its transcriptional activity; however, little is known about p53 turnover through proteasome-independent pathway(s). The digestive organ expansion factor (Def) protein is essential for the development of digestive organs. In zebrafish, loss of function of def selectively upregulates the expression of p53 response genes, which raises a question as to what is the relationship between Def and p53. We report here that Def is a nucleolar protein and that loss of function of def leads to the upregulation of p53 protein, which surprisingly accumulates in the nucleoli. Our extensive studies have demonstrated that Def can mediate the degradation of p53 protein and that this process is independent of the proteasome pathway, but dependent on the activity of Calpain3, a cysteine protease. Our findings define a novel nucleolar pathway that regulates the turnover function of p53, which will advance our understanding of p53's role in organogenesis and tumorigenesis.
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Introduction
p53 is under exquisitely fine regulation and acts as a transcription factor that regulates the expression of thousands of genes that control apoptosis, cell cycle arrest, senescence, metabolism, fertility, aging and autophagy1. Regulation of p53 turnover is essential to keep p53 activity under control in a normal cell. Multiple E3 ligases that mediate p53 degradation through the ubiquitination-26S proteasome pathway have been identified, including the key E3 ligase Mdm2 and others such as Pirh2, COP1, Topors, Arf-BP1, Synoviolin, CARP1/2, E6-AP and Trim24 (reviewed in2). p53 can also be degraded through the MG132-inhibitable but ubiquitination-independent pathway3. However, very little is known to date about p53 turnover through proteasome-independent pathway(s), although some reports have shown that certain cytoplasmically abundant calpains might contribute to the degradation of p534,5,6,7.
The nucleolus is also involved in regulating the function of p53. Disruption of the nucleolus in a cell in response to either internal or external stress normally activates the p53 pathway. In most cases, the activation of p53 is due to the binding of Mdm2 by factors such as ARF8, PML9, RPL510 or RPL1111, which results in physical separation of Mdm2 from p53, thus preventing p53 from degradation by the ubiquitination pathway12.
Def was first identified as a pan-endodermal-enriched factor that is essential for the growth of digestive organs in zebrafish13. Def belongs to a novel protein family that is evolutionally conserved from yeasts to humans and is a component of the ribosomal small subunit (SSU) processome in the nucleolus14,15,16. Human Δ133p53 and its zebrafish Δ113p53 counterpart are N-terminally truncated p53 isoforms whose transcriptional expression is initiated by an alternative p53 promoter that is totally p53 dependent13,17,18,19. Both Δ133p53 and Δ113p53 have been found to modulate p53 activity17,19 and Δ133p53 is upregulated in certain human cancers18,20,21. In the zebrafish def null mutant (defhi429), the transcriptional expression of p53 response genes, including Δ113p53, mdm2 and p21, is selectively upregulated. However, the level of full-length p53 transcripts was not obviously altered in the defhi429 mutant13, which raises the question of whether p53 protein is stabilized or overactivated to upregulate the expression of Δ113p53 in the defhi429 mutant.
In this report, we analyzed the effect of Def on p53 in both zebrafish and human cells and found that Def triggers the degradation of p53 and its isoform Δ133p53/Δ113p53. More importantly, Def-mediated degradation of p53 is dependent on the activity of a specific cysteine proteinase, Calpain 3 (CAPN3), rather than acting through the 26S proteasome pathway. Our results demonstrated that both zebrafish and humans share a conserved common nucleolar pathway that mediates p53 degradation.
Results
Both zebrafish and human Def are localized in the nucleolus
Def homologues in yeast (Upt25p)14,15 and Arabidopsis (NOF1)16 are nucleolar proteins. Zebrafish Def contains a putative nucleolar localization signal (NoLS)22 (Supplementary information, Figure S1A). Co-immunostaining of Def and the nucleolar marker, Fibrillarin (Fib)23 showed that Def was colocalized with Fib in the nucleoli in the intestinal epithelia of the wild-type fish at 3.5 days post-fertilization (dpf) but not in those of the defhi429 mutant (Supplementary information, Figure S1B and S1C). The human def gene (hu-def) is located in 1q32.2 and is called c1orf107. We found that hu-Def was also localized in the nucleolus in MCF-7 cells (Supplementary information, Figure S1D and S1E).
p53 is upregulated and specifically accumulated in the nucleolus in the defhi429 mutant
We demonstrated previously that the transcriptional expression of Δ113p53 is totally p53 dependent and that the transcript level of Δ113p53 was greatly elevated in the defhi429 mutant13,17. Interestingly, the transcript level of p53 was not obviously affected in the defhi429 mutant, which prompted us to speculate that p53 protein might be stabilized or become more active in the mutant to activate the expression of Δ113p53. Western blot using a monoclonal antibody that detects both p53 and Δ113p53 of zebrafish showed that both proteins were upregulated in the defhi429 mutant at 5 dpf (Figure 1A). The def-specific morpholino (def-MO) could knock down Def protein expression in zebrafish by targeting the def gene at its splicing junction of exon 2 and intron 213. Western blot showed that p53 and Δ113p53 were upregulated in the def-MO morphants at 3 dpf (Supplementary information, Figure S1F and S1G). Immunostaining using a polyclonal antibody that recognizes both p53 and Δ113p53 of zebrafish revealed a clear and strong positive signal colocalized with Fib-labeled nucleoli in the cells of the intestinal epithelium of 4 dpf defhi429 mutants but not in those of wild-type zebrafish (Figure 1B; Supplementary information, Figure S2A and S2B). Knockdown of Δ113p53 by its specific morpholino Δ113p53-MO17 had no obvious effect, while knockdown of p53 by its specific morpholino, p53-MOATG, almost abolished the nucleolar staining pattern of p53 in defhi429 mutants (Figure 1B). Therefore, the loss of function of def upregulated p53 protein expression, and p53 protein accumulated in the nucleoli in the defhi429 mutant cells. As Δ113p53 can form a complex with p5317, we speculated that the upregulated Δ113p53 protein probably accumulates, together with p53, in the nucleoli of the mutant cells, although further concrete evidence is needed to prove this hypothesis.
Def selectively triggers the degradation of p53 and Δ113p53 protein
The above results suggested that Def regulates the stability of p53. Indeed, we found that co-injection of Myc-def but not of Myc-defstop (a def mutant that harbors a premature stop codon at codon 55 created by site-directed mutagenesis)13 mRNA drastically reduced the level of p53 protein (Figure 1C, protein panels) but not that of p53 mRNA (Figure 1C, RNA panels) at 6 h post-injection (hpi). In fact, Def reduced the level of p53 as early as 1 hpi (Supplementary information, Figure S2C). To our surprise, overexpression of Def also reduced the level of HA-Δ113p53 protein (Figure 1D, protein panels) but not that of mRNA (Figure 1D, RNA panels) at 6 hpi. To determine whether Def reduced the level of p53 selectively, we replaced p53 mRNA with egfp (enhanced green fluorescent protein) or rcl1 (encoding a nucleolar protein) mRNA and found that Myc-def did not affect the protein levels of EGFP (Figure 1E) or Rcl124 (Supplementary information, Figure S2D). Interestingly, Def failed to reduce the protein levels of two p53 mutants, R143H and R250W (Figure 3D). As R250W and R143H differ from p53 only by an A748 to T748 single nucleotide change and by an R143 codon AGA to H143 codon CAC change in the p53 coding region, respectively, which are relatively distant from the translation start codon ATG, we therefore concluded that Def overexpression selectively promotes the degradation of p53 and Δ113p53 protein without affecting their mRNA stability or protein translation in zebrafish.
Eight def deletion constructs (Supplementary information, Figure S3A) were generated and each in vitro synthesized mRNA was co-injected with HA-p53 mRNA. The level of HA-p53 was then analyzed at 6 hpi. Deletion of the first 189 (construct D1) or 377 (construct D2) amino acids (AAs) from the N-terminus, which contains the NoLS, almost totally abolished the ability of Def to promote HA-p53 degradation. In contrast, deletion of the last 376 AAs (constructs D3, D4 and D5) from the C-terminus did not affect Def-mediated p53 degradation (Supplementary information, Figure S3B). Surprisingly, removal of the last 64 AAs (construct D6) inactivated the Def protein, whereas deletion of AAs 566-627 (construct D7) caused a moderate loss of Def activity. We also deleted the internal region between AAs 628 and 689 (construct D8) and found that the resultant protein was more effective in promoting HA-p53 degradation than the full-length Def (Supplementary information, Figure S3C). Thus, we concluded that the NoLS is essential for the activity of Def on p53. Meanwhile, we identified two promoting regions (AAs 1-189 and 566-627) and one repressing region (AAs 628-689) in the Def protein function, suggesting a highly regulated and complex mechanism of action in Def-mediated p53 degradation.
Def protects the survival of embryos by antagonizing p53 apoptotic activity
The zebrafish mutant tp53M214K carries a M214 to K214 substitution in the p53 DNA-binding domain that creates a loss-of-function allele of p5325. Injection of the normal p53 mRNA alone caused high embryo mortality (∼70%) at 12 hpi in the tp53M214K mutant background. Co-injection of def and p53 mRNAs greatly reduced the embryo mortality seen following injection of p53 mRNA alone (Figure 1F; Supplementary information, Figure S2E and Table S1). The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay showed that injection of the p53 mRNA alone caused massive cell apoptosis, and that injection of def but not of defstop mRNA strongly inhibited p53-induced apoptosis in the co-injected embryos at 10 hpi (Figures 1G; Supplementary information, Figure S2F and Table S2). Concomitantly, compared with their expression in embryos injected with the p53 mRNA alone, the expression of all p53 target genes, including Δ113p53, mdm2, p21 and bax, was downregulated by the injection of def mRNA in the co-injected embryos at 6 hpi (Figure 1H and Supplementary information, Table S3).
Def-mediated degradation of p53 and Δ113p53 is independent of Mdm2
Δ113p53 lacks the motif essential for Mdm2 binding26. Mdm2 binds to the N-terminus of p53 and targets the C-terminus of p53 for ubiquitination, a prerequisite for p53 degradation through the 26S proteasome2. We therefore predicted that Δ113p53 would be resistant to Mdm2-mediated protein degradation. We co-injected Myc-def or Myc-mdm2 mRNA with HA-Δ113p53 or HA-p53_P5 mRNA (similarly to Δ113p53, P5 lacks the Mdm2-binding motif) and found that Def but not Mdm2 promoted the degradation of both Δ113p53 and p53_P5 at 6 hpi (Figure 2A). In addition, knockdown of Mdm2 by an mdm2-specific morpholino (mdm2-MO) did not prevent the degradation of p53 by Def in the injected embryos, and vice versa (Supplementary information, Figure S4). Mdm2 is the key E3 ligase that mediates the degradation of p53 through the 26S proteasome pathway, and its depletion causes embryo lethality in zebrafish27. We used mdm2-MO to knock down Mdm2 protein expression and found that the level of endogenous p53 protein was greatly elevated in the mdm2-MO morphants (Figure 2B). We co-injected def mRNA and mdm2-MO and found that Def reduced the level of p53 protein in the mdm2-MO morphants at both 10 and 24 hpi (Figure 2B), concomitant with a reduction in their apoptosis at 24 hpi (Figure 2C). These results demonstrated that Def promotes the degradation of both p53 and Δ113p53 in an Mdm2-independent manner.
Def targets the p53 DNA-binding domain for degradation
Six p53 deletion constructs (P1-P6) (Figure 3A) were generated for the identification of the Def-targeting domain(s). Def only promoted the degradation of those deletion products that contained the p53 DNA-binding domain (P2, P4 and P5) at 6 hpi (Figure 3B). Noteworthy, Mdm2 triggered the degradation of the P6 product only, but had no or only a weak effect on the products that were truncated from the N- (P2, P3, and P5) or C-terminus (P2 and P4) of p53 (Figure 3C). This is consistent with earlier studies that showed that both the N-terminal Mdm2-binding domain and the C-terminal ubiquitin target sites of p53 are requisite for the Mdm2-driven degradation of p5326.
Three zebrafish p53 mutants were created, each with a point mutation that alters a crucial functional residue, including R143H, R250W and R313C that are counterparts of human hu-p53-R175H, -R282W and -R337C, respectively. hu-p53-R175H and -R282W are known to lose DNA-binding capacity28 whereas hu-p53-R337C displayed the highest DNA-binding activity29. We found that although Mdm2 triggered the degradation of all these three p53 mutant proteins, Def was unable to promote the degradation of zebrafish zf-p53-R143H and -R250W but promoted zf-p53-R313C degradation at 6 hpi (Figure 3D). Therefore, Def-induced p53 degradation is dependent on the molecular status of the p53 protein that requires a wild-type DNA-binding domain.
Def-mediated p53 degradation is independent of the ubiquitination pathway
In addition to Mdm2, many other E3 ligases have been found to trigger the degradation of p53 through the ubiquitination pathway2. We injected one-cell stage zebrafish embryos with HA-ubiquitin mRNA and found that this promoted degradation of p53 in a dosage-dependent manner at 6 hpi (Figure 3E). Multiple lysine residues in the p53 protein are known to serve as the sites for ubiquitination26. We selected nine lysine (K) residues (K292, K293, K295, K345, K347, K348, K360, K361, and K365) and mutated them to arginine (R) (construct zf-p53-9KR). We co-injected zf-p53-9KR with Myc-mdm2 or def mRNA together with HA-ubiquitin mRNA. The results showed that zf-p53-9KR was totally stable after the co-injection of Mdm2 with 400 pg HA-ubiquitin mRNA (Figure 3F) but was degraded by Def with or without HA-ubiquitin at 6 hpi (Figure 3G). Thus, Def-mediated p53 degradation occurs via an ubiquitination-independent pathway.
Two naturally occurring single-nucleotide polymorphisms (SNPs) in hu-def alter hu-Def activity on p53
Before studying hu-Def, we sequenced hu-Def cDNA cloned from different human cell lines including MCF7, HCT116, U2OS, HepG2, SiHa, Ovcar-8/TR, PC-3 and Saos-2 and searched the dbSNP database (http://www.ncbi.nlm.nih.gov/snp/). We identified two SNPs (G/C at position ORF199, converting E67 to Q67, and A/G at position ORF332, converting D111 to G111) in its coding region (Figure 4A). We found that overexpression of the products encoded by all four combinations of these two SNPs in MCF7 cells downregulated the levels of hu-p53 (Figure 4B) and Δ133p53 (Figure 4C) at 24 h post-transfection (hpt). Interestingly, the products of the combination of G199G332 (hu-Def_EG) and C199A332 (hu-Def_QD) exhibited much higher activity than those of G199A332 (hu-Def_ED) and C199G332 (hu-Def_QG) in the degradation of p53 (Figure 4B) and Δ133p53 (Figure 4C). Examination of the p53 target genes showed that p21 but not Bax was downregulated in the hu-def and p53 co-transfected cells (Figure 4B). In contrast, we found that the knockdown of hu-Def by its specific small interfering RNA (siRNA) upregulated the level of endogenous p53 protein in MCF-7, HepG2 and Ovcar-8/TR cells (both MCF-7 and HepG2 cells carry wild-type p53 whereas Ovcar-8/TR has a mutated p53 with an in-frame deletion of 126-132 AAs) at 24 hpt (Figure 4D).
Def-mediated p53 degradation is dependent on Calpain 3
We then tested the effects of various protein degradation inhibitors on Def-mediated p53 degradation. Only cysteine protease inhibitors (leupeptin, pepstatin A and E64) and EDTA, but not inhibitors of the 26S proteasome (MG132), lysosomes (NH4Cl and chloroquine) or autophagy (3-methyladenine, data not shown), inhibited Def-mediated p53 degradation (Figure 4E). In contrast, although MG132 showed a strong inhibitory effect, leupeptin showed only a negligible effect on Hdm2-mediated p53 degradation (Figure 4F). This result also ruled out the possibility of the involvement of free 20S proteasome in Def-mediated degradation of p53 because MG132 was also an effective inhibitor of this process. Interestingly, hu-p53-R175H, which is poorly sensitive to Calpain (a type of cysteine proteases)4, was more resistant to Def-mediated degradation (Supplementary information, Figure S5).
The above results suggested that Def and Calpain might act together to mediate p53 degradation. Humans have 15 known calpain (capn) genes30. We designed specific siRNAs against these 15 human capn genes (Supplementary information, Table S4) and used them to knockdown the expression of their corresponding CAPNs. We then tested the effect of hu-Def on p53 in cells treated with each siRNA. We found that only the knockdown of CAPN3 expression, but not that of CAPN6, -7 or -8 (Figure 5A and 5B) or others (Supplementary information, Figure S6A), blocked Def-mediated p53 degradation. In contrast, the knockdown of CAPN3 expression did not block Hdm2-mediated p53 degradation (Figure 5C). We then determined the effect of the knockdown of CAPN3 on p53 and found that it significantly increased the level of endogenous p53 protein at 24 hpt in MCF7 and HepG2 cells (Figure 5D). Therefore, Def-mediated p53 degradation was CAPN3 dependent. More importantly, because CAPN3 is the only CAPNs known to date to be localized in the nucleolus31, the above results strongly suggested that the Def- and CAPN3-mediated p53 degradation pathway operates in the nucleolus.
hu-Def and CAPN3 form a complex
As shown above, CAPN3 knockdown blocked hu-Def-mediated p53 degradation (Figure 5A-5C). To determine whether hu-Def knockdown could block CAPN3-mediated p53 degradation, MCF7 cells were cotransfected with CAPN3 overexpression plasmid and hu-Def siRNA, and p53 protein was analyzed. Western blot results showed that hu-Def knockdown clearly partially blocked the effect of CAPN3 on p53 (Figure 5E). Apparently, Def and CAPN3 are mutually dependent for the maximization of their effect on p53. We next overexpressed Myc-tagged hu-Def and HA-tagged CAPN3 together in H1299 (p53-null) and MCF7 (p53 wild type) cells and performed coimmuoprecipitation (Co-IP) using the anti-HA antibody. Western blot of Co-IP products using the anti-Myc antibody showed that hu-Def was successfully coprecipitated with CAPN3 (Figure 5F; Supplementary information, Figure S6B). This result demonstrated that Def and CAPN3 can form a complex even in the absence of p53.
The Def-CAPN3 pathway is independent of the RPL5/RPL11 pathway
Previous studies have shown that the ribosomal proteins, RPL5 and RPL11, stabilize and activate p53 by sequestrating MDM2 to the nucleolus under conditions of nucleolar stress10,11. Knockdown of RPL5 or RPL11 would leave MDM2 free in the nucleoplasm, which would trigger the degradation of p53 even under nucleolar stress12. Our extensive data shown above demonstrated that Def-mediated p53 degradation is independent of the Mdm2 pathway. To further verify this conclusion, we tested the effect of the combined knockdown of Def and RPL5, Def and RPL11, CAPN3 and RPL5, and CAPN3 and RPL11 by their respective siRNAs. Clearly, the elevated levels of p53 induced by Def or CAPN3 knockdown were not affected by the concomitant knockdown of either RPL5 or RPL11 (Figure 6A and 6B).
Knockdown of CAPN3 arrests cells at the G1 phase and promotes cell apoptosis
We then performed qPCR to examine the expression of p53 reponse genes, including hdm2, p21, bax and bcl2 in HepG2 (p53 wild type) and H1299 cells after treatment with CAPN3 siRNA at 24 h. We found that hdm2 and bax were significantly upregulated in HepG2 cells but not in H1299 cells after CAPN3 knockdown (Figure 6C). We also analyzed the ratio of cells in the G1, S and G2/M phases after CAPN3 siRNA treatment. The flow cytometry analysis (FACS) result showed that CAPN3 knockdown in HepG2 cells arrested cells at the G1 phase (an average of 74.7% G1 cells in control siRNA samples versus 83.5% in CAPN3 siRNA samples) (Figure 6D). FACS analysis of apoptotic cells after Annexin V staining showed that CAPN3 siRNA-treated HepG2 cells exhibited a much higher level of apoptosis than those treated with control siRNA (Figure 6E).
Capn3b acts together with Def to mediate p53 degradation in zebrafish
A database search in the zebrafish genome assembly Zv9 identified two capn3 genes, located on chromosomes 17 (named capn3a) and 20 (named capn3b). Amino acid sequence alignment revealed that human CAPN3 shares 62% and 57% identity with zebrafish CAPN3a and CAPN3b, respectively (Supplementary information, Figure S7). However, only CAPN3b contains the presumed NoLS KKKxKP (Supplementary information, Figure S8A). We first examined the expression patterns of capn3a and capn3b in zebrafish embryos at 2 and 4 dpf, respectively. Whole-mount in situ hybridization (WISH) results showed that capn3a is mainly expressed in the lens at 2 dpf as recorded in the database (database for Gene Expression, http://zfin.org), and is then enriched in the brain region between the eyes (Figure 7A). In contrast, capn3b expression is enriched in the digestive organs at both 2 and 4 dpf and in the head region at 4 dpf (Figure 7A), and displays a pattern resembling that observed for def13. To determine whether Def-mediated p53 degradation in zebrafish is also CAPN3 dependent, we co-injected def and p53 mRNA with a capn3a-specific (capn3a-MO) or capn3b-specific (capn3b-MO) morpholino (Supplementary information, Figure S8B and S8C) and examined p53 protein levels in the injected embryos. The results showed that capn3b-MO but not capn3a-MO effectively blocked Def-mediated p53 degradation at 6 hpi (Figure 7B). Furthermore, we found that capn3b-MO but not capn3a-MO also increased the level of the endogenous p53 protein as def-MO and mdm2-MO at 3 dpi (Figure 7C). The elevated endogenous p53 protein level could be downregulated by co-injection with Myc-capn3b-5mu mRNA that lacks the target sequence for capn3b-MO (Figure 7D). In contrast, mutating the active site Cys120 (corresponding to Cys129 at the active site in human Capn3)32 to a serine (Capn3b-C120S) abolished the ability of the mutant Capn3 to downregulate the p53 level induced by the injection of capn3b-MO (Figure 7D). This result suggested that Capn3b catalytic activity is essential for Def-mediated p53 degradation and that Capn3b itself does not simply provide a platform for p53 degradation by an alternative mechanism. In humans, CAPN3-C129S was found to be more stable than the wild-type CAPN332. We found that, as expected, the overexpressed zebrafish Capn3b-C120S is more stable than the wild-type Capn3b (Figure 7D).
As shown in Figure 1A, the level of p53 protein was increased in the zebrafish defhi429 mutant. To determine whether capn3a and capn3b were expressed normally in the defhi429 mutant, we measured their expression levels in zebrafish. The qPCR results showed that capn3b but not capn3a was expressed at a higher level in the mutant than in the wild-type control (Figure 7E), demonstrating that Capn3b-mediated p53 degradation is dependent on Def. As shown in Supplementary information, Figure S1F, the level of p53 protein was elevated in the def-MO morphants. We co-injected def-MO with capn3a or capn3b mRNA and found that the overexpression of CAPN3b but not that of CAPN3a partially downregulated the p53 protein level (Figure 7F). It should be noted that the extent of p53 degradation induced by capn3b mRNA was less than that induced by def mRNA, demonstrating that Capn3b-mediated p53 degradation is dependent on Def, as has been observed in human cells. Therefore, the Def- and Capn3b-mediated p53 degradation pathway is also functional in zebrafish.
Discussion
In this study, we demonstrated that Def, a novel nucleolar factor, can induce degradation of the p53 protein in both human and zebrafish, and that this process is independent of the proteasome pathway but is dependent on a specific nucleolus localized cysteine protease, CAPN3. Hence, we concluded that Def and CAPN3 together define a conserved nucleolar protein degradation pathway that negatively regulates the function of p53 by mediating its turnover (Figure 7G).
The regulation of p53 turnover is essential to maintain a low level of p53 in normal cells. Many E3 ligases have been found to mediate p53 degradation through the ubiquitination pathway2. The role of the nucleolus in regulating the p53 function is through the binding of Mdm2 by some nucleolar factors, such as p14ARF, RPL5 and RPL11, so that Mdm2 is physically separated from the nucleoplasmic p53. In this way, p53 degradation through the ubiquitination pathway is blocked12,23. We found (1) that the upregulated p53 and possibly Δ113p53 clearly accumulated in the nucleolus in the defhi429 mutant, and (2) that RPL5 or RPL11 knockdown failed to compromise the elevated level of p53 induced by CAPN3 knockdown. These results demonstrated that the Def-CAPN3-p53 pathway differs from the p14ARF-, PML- or RPL11-mediated nucleolar pathways8,12,23 (Figure 7G).
Calpains belong to the family of Ca2+-dependent cysteine proteases. Although CAPN1 and CAPN2 are considered to be ubiquitous, CAPN3 is mainly expressed in the muscle33. A study of a Calpain3-null knockout mouse revealed that this model exhibited a phenotype that resembles the human limb girdle muscular dystrophy type 2A (LGMD2A) due to aberrant myofibrillogenesis. CAPN3 exists in many alternatively spliced isoforms. The main isoform in muscle is p94, whereas the main isoforms in human melanoma cells are p62, hMp78 and hMp84 (it is proposed that p62 is derived from hMp78 and hMp84). Surprisingly, in human melanoma cells, p62 and hMp78 were located in both the cytoplasm and nucleoli31.
It has been reported that Calpains can regulate p53 stability, however, this was attributed to cytoplasmic abundant calpains4,5,6,7. The nature of the nucleolar localization of CAPN3 explains why Def-mediated p53 degradation depends on the activity of CAPN3 but not that of other Calpains. Importantly, the catalytic activity of Capn3b is indispensible for p53 degradation. Furthermore, we found that Def and CAPN3 can form a complex, which is consistent with the finding that Def and CAPN3 are mutually dependent for the mediation of p53 degradation in the nucleolus. We tried different means to study the potential protein-protein interaction between Def and p53, however, we did not observe any interaction between them. It would be interesting in the future to determine whether the Def-CAPN3-p53 pathway contributes to LGMD2A in the Calpain3-null mouse model.
It appeared that Def knockdown was more effective than that of Capn3b in stabilizing p53. We speculated that an additional nucleolar factor(s) might be required for the formation of a fully functional Def-Capn3 complex. Def might serve as a scaffold protein to recruit the other factor(s). In this scenario, Def knockdown would be more effective in disrupting the complex, whereas the complex could still form after Capn3b knockdown and the residual Capn3b could still induce p53 degradation. This hypothesis might explain why p53 and Δ113p53 are highly stabilized despite that capn3 is highly expressed in the defhi429 mutant or overexpressed in the def-MO morphants. Certainly, the possibility of the existence of other Def effector(s) cannot be excluded. The creation of a capn3b knockout mutant by the transcription activation-like effector nuclease (TALEN) method will help to resolve this query in the future.
Previous studies showed Δ113p53 and Δ133p53 are p53 target genes and function to antagonize p53 apoptotic activity17,19. Our study also demonstrated that Δ113p53/Δ133p53 was degraded by the Def-CAPN3 pathway when overexpressed. This is the first pathway that was found to regulate the turnover of Δ113p53/Δ133p53, although further efforts are needed to prove that this pathway also operates in vivo. The fact that Def acts on the wild-type p53, but not on point mutants p53-R143H and -R250W, and also acts on Δ113p53/Δ133p53 implies that Δ113p53/Δ133p53 protein adopts much of the native structure in its truncated DNA-binding domain. Our findings might provide an explanation of why tumorigenesis occurs in certain cells/tissues even when they harbor wild-type p53. Therefore, the downregulation of hu-Def or CAPN3 in such cancer cells might be a new approach for cancer therapy.
Clearly, the Def-Capn3-p53 pathway has an important role in protecting embryos from the p53-induced reduction in survival. We hypothesized that during early embryogenesis, the Def-Capn3b-p53-Δ113p53 pathway may serve as a check point to monitor environmental changes that would allow the organism to adjust its physiological response to certain cellular stresses and thus to safeguard the proceeding of normal development. However, many questions remain unanswered. For example, how does the Def-CAPN3 pathway control the genesis of the digestive organs? Are other proteins targeted by the Def-CAPN3 pathway? Are the two SNPs in hu-def related to any disease(s)? The resolution of these questions will undoubtedly help to further our understanding of the function of the nucleolus.
Materials and Methods
Zebrafish lines and maintenance
Zebrafish were raised and maintained according to the standard procedure. The defhi429 mutant line was provided by Prof Nancy Hopkins. Two pairs of primers derived from lacZ and def were used to genotype the defhi429 mutant13. The p53-/- mutant allele tp53M214K line25 was provided by Professor Thomas Look.
Cell lines
MCF-7-1, MCF-7-3, HepG2 and SiHa cells were maintained in the DMEM medium (high glucose, GIBCO), MCF-7-2 in DMEM/F12 (1:1) medium (GIBCO), HCT-116, U2OS and Saos-2 in McCoy's 5A (modified) medium, H-1299 and OVCAR-8/TR in RPMI medium 1640 (GIBCO), and PC-3 in Ham's F12. All media were supplemented with 10% newborn calf serum (NBCS, GIBCO) with the exception of 15% NBCS for the Saos-2. Plasmids were transfected into the cell by FuGENE HD (Roche) and siRNAs with Lipofectamine 2000 (GIBCO) transfection reagent according to the manufacturer's recommendations.
Phylogenetic analysis
Human and zebrafish CAPN3 amino acid sequences were retrieved from the GenBank and Ensembl Genome Browser and aligned using the Clustal W program. The phylogenetic tree was built using the MEGA 4 program34.
Plasmid construction
Each def and p53 deletion construct was derived from pCS2+-def-D0 and pCS2+-p53-P0, respectively. The corresponding primer pairs used to generate all relevant plasmids are listed in Supplementary information, Table S5. The method to generate the internal deletion constructs MYC-def_D7, MYC-def_D8, defstop_pCS2+ and the internal deletion construct HA-p53_P6-pCS2+ has been described previously17. Zebrafish mdm2 was cloned by PCR from cDNA of 1 dpf embryos, capn3a and capn3b from cDNA of 5 dpf embryos, using the primer pairs listed in Supplementary information, Table S5. Capn3 and hu-def_ED cDNAs were obtained from MCF7 and hu-def_QG cDNA from RKO cells, and all cDNAs were cloned into the pcDNA3.1+ vector. The Δ133p53 construct was subcloned from hu-p53 plasmid. The hdm2 and ubiquitin constructs were provided by Dr Shuyong Lin and Dr Huizhe Huang, respectively. zf-p53 9KR, R143H, R250W, R313C, hu-def_EG, hu-def_QD, hu-p53 R175H, Myc-capn3b-5mu-pCS2+ and Myc-capn3bC120S-5mu-pCS2+ were constructed through site-directed mutagenesis using the primer pairs listed in Supplementary information, Table S5. The 5′-untranslated regions (5′UTR) of capn3a and capn3b were cloned upstream of the egfp reporter gene to create cmv:capn3a_5′UTR-egfp and cmv:capn3b_5′UTR-egfp constructs in pCS2+, respectively. HA or MYC tags were designed into the primers to generate the tagged constructs. For mRNA injection, mRNAs were synthesized in vitro using the mMESSAGE mMACHINE Kit (Ambion) according to the manufacturer's instructions. 1 nl mRNA or morpholinos or plasmid containing phenol red dye was injected into zebrafish embryos at the one-cell stage.
DNA, RNA and protein analysis
The primer pair 5′-AACCTGTGGCCTAATGCCTGGGA-3′ and 5′-CTGGCAGTGTTCAACCACTCTGCC-3′ was used to amplify the genomic DNA fragment harboring hu-def from different human cells and the DNA fragment was sequenced with the primer 5′-TTAAAAGTGGGGCAGTAGCAGT-3′.
Total RNA was extracted from different samples using TRIzol reagent (Invitrogen). Probes were labeled with Digoxigenin (DIG) and northern blot hybridization was performed according to the manufacturer's instructions (Roche Diagnostics). The p53-P1 probe and egfp probe have been described previously13. The qPCR was performed with a CFX96 Real-Time System (Bio-Rad) using SsoFast EvaGreen Supermix (Bio-Rad). The primer pairs and detailed PCR conditions for amplifying zebrafish Δ113p53, mdm2, p21 and bax have been described previously13. The primer pairs for amplifying zebrafish capn3a, capn3b, def and human hdm2, p21, bax and bcl2 are listed in Supplementary information, Table S6.
The methods used for protein extraction from zebrafish embryos and from cultured cells have been described previously17,18. Protein electrophoresis and western blot were performed according to the instructions provided by the manufacturers.
Protease inhibitors
Protease inhibitors MG132, chloroquine and pepstatin A were purchased from Sigma, Complete Protease Inhibitor Cocktail Tablet (EDTA-free) from Roche, and NH4Cl, leupeptin, PMSF (phenylmethanesulfonyl fluoride), E64 and EDTA from Sangon (Shanghai, China).
Morpholinos and siRNAs
Morpholinos were purchased from Gene Tools (Philomath, USA). def-MO, Δ113p53-MO, p53-MOATG, mdm2-MO, and the human β-globin antisense morpholino (st-MO) were used as described previously13,17,27, capn3a-MO (5′-AACCCAGACGGCGTATAGGGCATCT-3′) and capn3b-MO (5′-CTGAAAACTTCTGTTCTTCTGCCAT-3′) were designed to target the start codon ATG of the corresponding genes. All morpholinos (0.25 pmol) were injected into one-cell stage embryos.
Stealth C1orf107 (hu-def) siRNAs and Negative Control Duplex (control siRNA) were obtained from Invitrogen (Carlsbad, USA). The hu-def siRNA pool containing three duplex oligonucleotides (hu-def-siRNA-1, hu-def-siRNA-2 and hu-def-siRNA-3), CAPN1 siRNA35, CAPN2 siRNA36, CAPN3 siRNA, CAPN5 to CAPN16 siRNAs, RPL5 and RPL11 siRNAs12, and the negative control duplex (control siRNA) were obtained from GenePharma (Shanghai, China). All information on the siRNA sequences is listed in Supplementary information, Table S4.
Antibodies
The zf-p53 monoclonal antibody A7-C10 and polyclonal antibody against the 113-373 aa region of p53 were raised in mouse and rabbit, respectively. Polyclonal antibody against the 34-174 aa region of hu-Def was raised in rabbit. The zf-Def rabbit polyclonal antibody has been described previously13. Anti-Myc mouse monoclonal (9E10) antibody was purchased from Clontech (#631206), anti-HA mouse monoclonal (HA-7) antibody from Sigma (H3663), mouse monoclonal (38F3) antibody against human Fibrillarin (ab4566) and rabbit polyclonal antibodies against GFP (ab6556) and CAPN3 (ab38963) from Abcam. Rabbit monoclonal antibody (EPR1977Y) against GAPDH was from Epitomics (#2251-1), and rabbit polyclonal antibody against β-actin (#4967) was from Cell Signaling Technology. Mouse monoclonal antibodies against human p53 (DO-1, sc-126), Hdm2 (SMP14, sc-965), p21 (F-5, sc-6246), C23 (H-6, sc-55486) and rabbit polyclonal antibody against human Bax (N-20, sc-493) were from Santa Cruz Biotechnology. Polyclonal antibody against the 1-89 aa region of Capn3b was raised in rabbit by HuaAn Biotechnology (Hangzhou, China).
WISH, cryo-sectioning and immunostaining
Probe labeling and WISH were performed as described previously13. The 3′-UTRs of capn3a and capn3b were cloned (Supplementary information, Table S5) and used as probes.
Cryosectioning and immunofluorescence staining were performed as described previously17. For immunofluorescence staining of the cultured cells, cells were plated on coverslips in a six-well plate. Primary antibodies were used at a 1:100 dilution. Immunofluorescence was detected after staining with Alexa Fluor conjugated secondary antibody (Invitrogen) (1:400 dilution). DAPI was used to stain the nuclei. All immunofluorescence staining images were taken under a Leica TCS SP5 confocal microscope.
Coimmunoprecipitation (Co-IP)
Human cell lines transfected with various plasmids were harvested with Tris-HCl protein lysis buffer for total protein extraction. Co-IP was performed using an HA Tag IP/Co-IP Kit (Pierce) according to the manufacturer's instructions. The input and precipitated protein samples were subjected to western blot analysis with corresponding antibodies as stated.
TUNEL assay and embryo viability counting
The TUNEL assay was performed using the in situ Cell Death Detection Kit, TMR red (Roche) as described previously13. For viability counting, dead embryos were characterized by the presence of large, dark debris in the embryos.
Flow cytometry analysis (FACS)
Human cells were digested with trypsin and precipitated at 500× g, 4 °C. After washing in PBS, the cells were stained with PI (Beyotime) or costained with Annexin V-FITC and PI according to the manufacturer's instructions, and subjected to FACS analysis of the cell cycle and apoptosis using a Beckman Coulter FC 500 MCL and a BD FACSCalibur flow cytometer, respectively.
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Acknowledgements
We thank Prof Tuanlao Wang (Xiameng University) for providing the MCF-7-1 cell strain, Prof Zhengping Xu (Zhejiang University) for the MCF-7-2 cell strain and HepG2 cell line, Prof Xing Xie (Zhejiang University) for the SiHa cell line, and Prof Cong Liu (Sichuan University) for the OVCAR-8/TR cell line. We thank Yun Feng, Qian Lang, Xin Ye and Menglian Zhang (Zhejiang University) for their help in maintaining the zebrafish facility. This work is financially supported by the National Natural Science Foundation of China (30825025) and National Basic Research Program of China (2012CB944500).
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( Supplementary information is linked to the online version of the paper on the Cell Research website.)
Supplementary information
Supplementary information, Figure S1
Def is a nucleolar protein both in zebrafish and human. (PDF 173 kb)
Supplementary information, Figure S2
Up-regulated p53/Δ113p53 in defhi429 was enriched in the nucleolus and Def functions as a pro-survival factor by antagonizing p53 apoptotic activity. (PDF 311 kb)
Supplementary information, Figure S3
The NoLS was essential for the Def-mediated degradation of p53. (PDF 165 kb)
Supplementary information, Figure S4
Def-mediated p53 degradation was independent of Mdm2. (PDF 154 kb)
Supplementary information, Figure S5
hu-p53-R175H mutant protein was resistant to Def-mediated but not to Hdm2-mediated protein degradation. (PDF 130 kb)
Supplementary information, Figure S6
Def-mediated p53 degradation depends on the activity of CAPN3, and Def and CAPN3 formed a complex. (PDF 174 kb)
Supplementary information, Figure S7
Comparison of CAPN3 amino acid sequences from humans and zebrafish. (PDF 771 kb)
Supplementary information, Figure S8
Only CAPN3b contained the nucleolar localization motif and capn3a-MO and capn3b-MO knocked down the expression of their targets effectively. (PDF 382 kb)
Supplementary information, Table S1
Summary of survived versus total embryos injected with different mRNAs or buffer as showed in Figure 1F and Supplementary information, Figure S2E. (PDF 146 kb)
Supplementary information, Table S2
Summary of apoptotic events for different treatments as showed in Figure 1G and Supplementary information, Figure S2F. (PDF 166 kb)
Supplementary information, Table S4
siRNA sequences targeting hu-def, 15 calpain genes, rpl5 and rpl11. (PDF 192 kb)
Supplementary information, Table S5
PCR primers used for various cloning and site-directed mutagenesis. (PDF 293 kb)
Supplementary information, Table S6
PCR primers used for qPCR analysis. (PDF 216 kb)
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Tao, T., Shi, H., Guan, Y. et al. Def defines a conserved nucleolar pathway that leads p53 to proteasome-independent degradation. Cell Res 23, 620–634 (2013). https://doi.org/10.1038/cr.2013.16
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DOI: https://doi.org/10.1038/cr.2013.16
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