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Molecular and Cellular Biology, May 2007, p. 3868-3880, Vol. 27, No. 10
0270-7306/07/$08.00+0     doi:10.1128/MCB.02112-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Np73 Modulates Nerve Growth Factor-Mediated Neuronal Differentiation through Repression of TrkA

Jin Zhang and Xinbin Chen^*

Center for Comparative Oncology, University of California at Davis, Davis, California 95616, and Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294

Received 10 November 2006/ Returned for modification 8 January 2007/ Accepted 6 March 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
p73, a member of the p53 family, expresses two classes of proteins: the full-length TAp73 and the N-terminally truncated Np73. While TAp73 possesses many p53-like features, Np73 is dominant negative towards TAp73 and p53 and appears to have distinct functions in tumorigenesis and neuronal development. Given its biological importance, we investigated the role of Np73 in nerve growth factor (NGF)-mediated neuronal differentiation in PC12 cells. We show that overexpression of Np73 or Np73ß inhibits NGF-mediated neuronal differentiation in both p53-dependent and -independent manners. In line with this, we showed that the level of endogenous Np73 is progressively diminished in differentiating PC12 cells upon NGF treatment and knockdown of Np73 promotes NGF-mediated neuronal differentiation. Interestingly, we found that the ability of Np73 to suppress NGF-mediated neuronal differentiation is correlated with its ability to regulate the expression of TrkA, the high-affinity NGF receptor. Specifically, we found that Np73 directly binds to the TrkA promoter and transcriptionally represses TrkA expression, which in turn attenuates the NGF-mediated mitogen-activated protein kinase pathway. Conversely, the steady-state level of TrkA is increased upon knockdown of Np73. Furthermore, we found that histone deacetylase 1 (HDAC1) and HDAC2 are recruited by Np73 to the TrkA promoter and act as corepressors to suppress TrkA expression, which can be relieved by trichostatin A, an HDAC inhibitor. Taken together, we conclude that Np73 negatively regulates NGF-mediated neuronal differentiation by transrepressing TrkA.

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p73 belongs to the p53 family, which also includes p53 and p63. These proteins share a significant degree of sequence similarity, especially in the DNA binding domain (20, 26, 27). However, p73 is not a classic Knudson-type tumor suppressor despite its remarkable structural and functional similarities to p53. For example, p73-deficient mice lack a tumor phenotype. Instead, these mice exhibit abnormalities in neuronal development, such as hippocampal dysgenesis, hydrocephalus, loss of Cajal-Retzius neurons, and defects in pheromone sensory pathways (53). In addition, wild-type p73 is frequently up-regulated, but not inactivated, in human primary tumors, suggesting that p73 may promote, rather than suppress, tumor formation (42).

These apparent contradictions of p73 activities might be due to the fact that p73 produces two classes of proteins, which are generated from two separate promoters and have distinct or even opposing functions. One, termed TAp73, is transcribed from the upstream promoter and contains an N-terminal activation domain with homology to that in p53. The other one, denoted Np73, is transcribed from the downstream promoter in intron 3 and N-terminally truncated. Consequently, the TAp73 isoforms contain many p53-like properties, such as transactivation of a subset of p53 target genes necessary for induction of cell cycle arrest and apoptosis (4, 11, 15, 18, 24, 46, 49). In contrast, the Np73 isoforms, because they lack the activation domain conserved in p53, are dominant negative towards TAp73 or p53 and thus are thought to have oncogenic potential (32). Consistent with this notion, Np73 has been reported to be frequently up-regulated in human cancers, including breast cancer (54), lung cancer (45), and neuroblastoma (3). Moreover, overexpression of Np73 promotes immortalization in primary cells and malignant transformation in NIH 3T3 fibroblasts (35, 43). In addition to its oncogenic potential, Np73 plays a crucial role in neuronal survival through both p53-dependent and -independent mechanisms (29, 36, 37, 48). The underlying mechanism of these biological phenomena has been hypothesized as follows: Np73, by forming a heterotetramer with TAp73 or competing with TAp73 or p53 to bind to the p53-responsive element (p53-RE), antagonizes TAp73 or p53 since it was thought not to contain an activation domain. However, we have reported recently that Np73ß, one of the Np73 isoforms, is active in transactivation and suppression of cancer cell growth (30). In addition, Np73, the full-length Np73, has been reported to specifically transactivate BTG2 in a p53-dependent manner in neuroblastoma cells but not in other cell types (4, 13). These findings suggest that Np73 retains transcriptional activities, at least in some settings. On the other hand, these findings point out a possibility that in addition to antagonizing TAp73 and p53, Np73 may regulate its own unique target genes for its biological activities.

Cellular differentiation, a highly coordinated process, is tightly regulated by a group of key transcriptional factors (25, 33). Precise regulation of this process is very important to ensure the production of properly differentiated cells. The transcriptional activity of p53 plays an important role in several types of cellular differentiation, such as epithelial differentiation, myogenic differentiation, and neuronal differentiation (1, 6, 28). Like p53, TAp73 also plays a critical role in cellular differentiation. Overexpression of TAp73 is sufficient to induce differentiation in neuroblastoma cells (5) and platelet-derived growth factor withdrawal- or thyroid hormone-induced differentiation in oligodendrocyte precursor cells (2). In contrast, overexpression of Np73 inhibits differentiation in oligodendrocyte precursor cells (2) and interferes with multiple developmental programs (23). Moreover, Np73 is likely to be responsible for the neuronal defects in p73-deficient mice since it is the most predominant isoform in the developing mouse brain (37). Such observations support a model whereby Np73 might regulate cellular differentiation or development at least in part by antagonizing other p53 family members. However, whether these observations reflect the role of endogenous Np73 remains unclear.

Nerve growth factor (NGF) plays a fundamental role in the neuronal development and maintenance of sympathetic and sensory neurons. The pheochromocytoma PC12 cell line is a well-established model for studying NGF-mediated neuronal differentiation (17). Upon NGF treatment, PC12 cells cease proliferation, exhibit somatic hypertrophy, and acquire neurites (47). The signal is initiated when NGF binds to its high-affinity receptor, TrkA, which is then phosphorylated and dimerized to activate its downstream cascades (22). Thus, the PC12 cell line is an ideal system with which to investigate the role of Np73 in NGF-mediated neuronal differentiation. In this study, we show that overexpression of Np73 or Np73ß inhibits, whereas knockdown of Np73 promotes, NGF-mediated neuronal differentiation. Moreover, we show that endogenous Np73 is gradually diminished in PC12 cells upon NGF treatment. To uncover the underlying mechanism, we show that Np73, through binding to the p53-RE in the TrkA promoter, transrepresses TrkA expression, which then inhibits the NGF-mediated mitogen-activated protein kinase (MAPK) pathway. Conversely, we showed that the steady-state level of TrkA is increased in PC12 cells upon knockdown of Np73. Finally, we showed that Np73, by recruiting histone deacetylase 1 (HDAC1) and HDAC2 to the TrkA promoter, transcriptionally represses TrkA expression, which can be relieved by trichostatin A (TSA), an HDAC inhibitor.

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Reagents. The dual-luciferase assay kit and nerve growth factor were purchased from Promega (Madison, WI). Anti-p21 and anti-TrkA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antihemagglutinin (anti-HA) was purchased from Covance. TSA and antibodies against HDAC1/2, p-MEK1/2, and p-RSK90 were purchased from Upstate-Cell Signaling Biotechnology (Charlottesville, VA). Antiactin, anti-growth-associated protein 43 (anti-GAP-43), proteinase inhibitor cocktail, RNase A, and propidium iodide were purchased from Sigma (St. Louis, MO). p73-Ab1, p73-Ab2, and p73-Ab3 antibodies were purchased from Calbiochem (San Diego, CA). BL906 antibody was purchased from Bethyl Laboratories (Montgomery, TX). An expressed sequence tag (EST) clone (source ID 6826464; GenBank accession no. BC069182) was purchased from Open Biosystems (Huntsville, AL). Scrambled Np73 small interfering RNA (siRNA) (GGCCGAUUGUCAAAUAAUU) was purchased from Dharmacon RNA Technologies. The iScript cDNA synthesis kit was purchased from Bio-Rad Laboratories.

Plasmids. To generate HA-tagged murine Np73, a PCR fragment was amplified using an EST clone (ID 6826464) as a template. The following primers were used: upstream primer, 5'-ACCATGTACCCATACGATGTTCCAGATTACGCTCTTTACGTCGGTGACCCCATGAGA-3'; and downstream primer, 5'-CCGGGTCTCCAGGGTGATGATGACAAGGATGGGCCTCCGATTC-3'. This PCR product, which was then digested with SacII and SfiI, together with another DNA fragment, which was digested from an EST clone (ID 6826464) with SfiI and EcoRI, was cloned into pUHD10-3, a tetracycline-regulated expression vector (16), at SacII and EcoRI sites to generate pUHD10-3-HA-Np73. To generate HA-tagged murine Np73ß, a PCR fragment was amplified using an EST clone (ID 6826464) as a template with an upstream primer (5'-GGCCGCCGGTCTTTCGAGGGTCGCATCTGT-3') and a downstream primer (5'-TTCAGAGCCCCAAGGTCCTGACGAGGCTGGGGT-3'). The fragment was then used to replace the region in pUHD10-3-HA-Np73 between the KpnI and EcoRI sites. To generate a construct expressing siRNA targeting the rat Np73 gene, two oligonucleotides were synthesized, which were annealed and then cloned into the pBabe-U6 expression vector (31) to generate pBabe-U6-Si-Np73. The sequences of the oligonucleotides were as follows (lowercase letters indicate link sequences and restriction enzyme sites): forward, 5'-gatccccGCCACCAGTCCGCCCCTACttcaagagaGTAGGGGCGGACTGGTGGCtttttggaaa-3'; reverse, 5'-agcttttccaaaaaGCCACCAGTCCGCCCCTACtctcttgaaGTAGGGGCGGACTGGTGGCggg-3'. The nucleotides in boldface above represent the siRNA target region. pSuper-Si-p53 vector, which expresses an siRNA targeting the rat p53 coding region, pUHD10-3-HA-p53 vector, which expresses HA-tagged murine p53, and the luciferase reporters (the wild-type and mutant TrkA promoters) were described previously (55).

Cell culture. The PC12 tet-off cells were maintained in DMEM (Dulbecco/Vogt modified Eagle's minimal essential medium) supplemented with 10% horse serum and 5% fetal bovine serum (FBS). H1299-HA-Np73-1 and H1299-HA-Np73ß cells were generated as described previously (29) and cultured in DMEM with 10% FBS. Selection drugs were added to the medium as needed. Cells were grown at 37°C with 5% CO₂. For protein induction, cells were washed three times with DMEM and then cultured in DMEM with 10% tetracycline-free FBS. For NGF treatment, cells were uninduced or induced to express a protein of interest and then switched to DMEM with 2% tetracycline-free FBS plus 100 ng/ml of NGF for parental PC12 cells or 200 ng/ml of NGF for p53 knockdown PC12 cells.

Establishment of stable cell lines. All transfections were achieved using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). To generate stable cell lines that inducibly express murine Np73 or Np73ß, PC12 cells, which are capable of expressing Tet-VP16 for the tetracycline-repressible expression system (purchased from ClonTech), were transfected with a pUHD10-3 vector expressing HA-tagged murine Np73 or Np73ß. To generate a PC12 cell line in which endogenous p53 is knocked down and an exogenous murine Np73 is inducibly expressed, pSuper-Si-p53 and pUHD10-3-HA-Np73 were cotransfected into PC12 cells. To generate Np73 knockdown cell lines, pBabe-U6-Si-Np73 was transfected into PC12 cells. After transfection, cells were selected with 1 µg/ml of puromycin or 1 µg/ml of puromycin and 100 µg/ml of G418 for 4 weeks. Individual clones were screened for inducible expression of Np73 and Np73ß or lack of endogenous p53 or Np73 by Western blot analysis.

Growth rate analysis. To determine the rate of cell growth, 5 x 10⁴ PC12 cells were seeded per 60-mm-diameter plate. Cells were uninduced or induced to express a protein of interest in the presence or absence of tetracycline (2 µg/ml). Culture media were replaced every 72 h. Each day over a 5-day period, three plates for each group were rinsed twice with phosphate-buffered saline (PBS) to remove dead cells. Live cells on the plates were trypsinized, and samples from each plate were counted three times using a Coulter cell counter (Coulter Corporation, Miami, FL). The average number of cells from each group was used for growth rate determination.

DNA histogram analysis. Cells were seeded at 2 x 10⁵ per 90-mm-diameter plate and uninduced or induced to express Np73 or Np73ß in the presence or absence of tetracycline (2 µg/ml). After 48 h of induction, both floating and dead cells were collected and fixed with 10 ml of 75% ethanol overnight and resuspended in 300 µl of PBS solution containing 50 µg each of RNase A and propidium iodide per ml. The stained cells were analyzed in a fluorescence-activated cell sorter (FACS Calibur; Becton Dickinson) within 4 h. The percentage of cells in the sub-G₁, G₁, S, and G₂/M phases was determined by the Cell Quest program.

Neurite outgrowth assay. A total of 5 x 10⁴ cells were plated in each well of a six-well plate. Cells were uninduced or induced as needed. After induction, cells were subject to NGF treatment and the differentiation medium was replaced every 2 days. Cells with at least one neurite longer than two body lengths were counted as neurite positive. At least 500 cells were counted for each group, with experiments performed in triplicate.

Luciferase assay. A total of 1 x 10⁴ PC12 cells were plated in each well of a 24-well plate for 16 h and then transfected with a luciferase reporter along with pUHD10-3 control vector or pUHD10-3 vector that expresses murine Np73, murine Np73ß, or wild-type murine p53. pRL-CMV, a Renilla luciferase assay vector, was also cotransfected as an internal control. Cell extracts were prepared 16 h after transfection and used for the dual-luciferase assay according to the manufacturer's instructions (Promega). The increase in relative luciferase activity is a product of the luciferase activity induced by activator divided by that induced by an empty vector. All experiments were repeated in triplicate.

Immunoprecipitation and Western blot analysis. For immunoprecipitation, cells were lysed in 1% Triton lysis buffer (25 mM Tris [pH 7.4], 25 mM NaCl, 1% Triton X-100) supplemented with the proteinase inhibitor cocktail (100 µg/ml). Cell lysates were clarified by centrifugation at 14,000 rpm for 15 min at 4°C and then incubated with 1 µg antibody overnight. The immunocomplexes were brought down by protein A/G beads and subjected to Western blot analysis. For Western blot analysis, cell lysates were resuspended with 2x sample buffer and boiled for 5 min. Proteins were then resolved in a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred to a nitrocellulose membrane. Membrane blocking, washing, antibody incubation, and detection by enhanced chemiluminescence were performed as previously described (7, 31).

RNA isolation and Northern blot analysis. Total RNA was isolated with Trizol reagent and used for Northern blot analysis as described previously (7). The TrkA probe was made from a 650-bp cDNA fragment amplified by reverse transcription-PCR (RT-PCR) with an upstream primer (5'-ATGAGACCAGCTTCATC-3') and a downstream primer (5'-GCTCCCACTTGAGAATG-3'). The glyceraldehyde-3-phosphate dehydrogenase (GADPH) probe was made from a 1.25-kbp PstI-PstI cDNA fragment of the rat GAPDH cDNA (12).

RT-PCR analysis. RT-PCR was performed with the the iScript cDNA synthesis kit (Bio-Rad Laboratories) according to the manufacturer's instructions. The PCR program used for amplification was (i) 94°C for 5 min, (ii) 94°C for 45 s, (iii) 55°C for actin or 58°C for Np73 for 45 s and (iv) 72°C for 1 min, and (v) 72°C for 10 min. From steps 2 to 4, the cycle was repeated 20 times for actin or 35 times for Np73. The primers used to amplify actin were upstream primer 5'-GTGGGCCGCTCTAGGCACCAA-3' and downstream primer '5-CTCTTTGATGTCACGCACGAT-3'. The primers for Np73 were upstream primer 5'-TCCAACACATCTCCGGGCAA-3' and downstream primer 5'-TGGGACGAGGCATGAGTCTG-3'.

ChIP assay. Cells were seeded at 2 x 10⁷ per 150-mm plate and uninduced or induced to express a protein of interest. After induction, Np73-DNA complexes were cross-linked by formaldehyde and used for chromatin immunoprecipitation (ChIP) assay as described previously (19, 50). The primers used to detect the TrkA promoter were TrkA-RE-5 (5'-CTCCTGGGCTTTCAGACTTC-3') and TrkA-RE-3 (5'-CCCTAGTTGACAGGCTAAATA-3'). The primers used to detect the p21 promoter were p21-RE-5 (5'-GCCTCCTGAGTGCTGGG-3') and p21-RE-3 (5'-AGGGTGGGGGGTGGTAT-3').

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Overexpression of murine Np73ß, but not Np73, suppresses cell proliferation in PC12 cells. To investigate the role of Np73 in neuronal differentiation, we generated stable PC12 cell lines that inducibly express HA-tagged murine Np73 or Np73ß under the control of tetracycline-regulated promoter. The results are presented in Fig. 1A and B. Upon induction, Np73 and Np73ß were detectable by anti-HA (Fig. 1A, Np73; and B, Np73ß), and their levels were comparable. We also examined the induction of p21, a common target of the p53 family. We found that p21 was induced by Np73ß (Fig. 1B, p21; compare lanes 1 and 3 with 2 and 4, respectively) but not by Np73 (Fig. 1A, p21). To characterize if Np73 or Np73ß has an effect on cell proliferation, growth curve analysis was performed over a 5-day period. We found that cell proliferation was markedly inhibited by Np73ß (Fig. 1D) but little if any by Np73 (Fig. 1C). This is consistent with an earlier report (30). To further examine the effect of Np73 on cell proliferation, DNA histogram analysis was performed. We found that overexpression of Np73 had no effect on the cell cycle (Fig. 1E). However, apoptosis was induced by Np73ß as the percentage of cells with sub-G₁ DNA content was increased from 2.85% to 13.37% (Fig. 1F). We would like note that a low level of Np73ß can induce only cell cycle arrest, but not apoptosis (data not shown). Together, we showed that Np73ß, but not Np73, is capable of inhibiting cell proliferation in PC12 cells.

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FIG. 1. Overexpression of Np73ß, but not Np73, suppresses cell proliferation in PC12 cells. (A and B) Establishment of stable PC12 cell lines inducibly expressing HA-tagged murine Np73 (A) or Np73ß (B). PC12 cells were uninduced or induced to express Np73 (A) or Np73ß (B) for 18 h. Cell extracts were collected for Western blot analysis. The levels of HA-tagged Np73 (A) or Np73ß (B), p21, and actin were analyzed by Western blot analysis with antibodies against HA (-HA), p21, and actin, respectively. Actin was used as a loading control. (C and D) Growth rates of cells with or without Np73 (C) or Np73ß (D) expression over a 5-day period. A total of 5 x 104 cells were seeded per 60-mm-diameter plate, which were then uninduced or induced to express Np73 (C) or Np73ß (D). At the time points indicated, live cells were collected and counted by the Coulter cell counter. The average numbers of cells from three plates were used for growth rate determination. (E and F) DNA histogram analysis of PC12 cells in the presence or absence of Np73 (E) or Np73ß (F). PC12 cells were uninduced or induced to express Np73 (E) or Np73ß (F) for 48 h, and the percentage of cells in each phase of the cell cycle was determined by DNA histogram analysis. DNA content was quantified using the data from at least three independent experiments.

Overexpression of murine Np73 or Np73ß inhibits NGF-mediated neuronal differentiation in PC12 cells. To determine if Np73 or Np73ß plays a role in NGF-mediated neuronal differentiation, PC12 cells were uninduced or induced to express Np73 or Np73ß for 18 h followed by treatment with NGF or no NGF treatment for 3 or 6 days. Phase-contrast microscopy was utilized to examine the morphology of PC12 cells. We showed that in the absence of NGF, no neurite outgrowth was observed in PC12 cells, regardless of expression of Np73 (Fig. 2A, a and b) or Np73ß (Fig. 2B, a and b). However, in the presence of NGF, much less and shorter neurite outgrowth was observed in PC12 cells expressing Np73 than in control cells (Fig. 2A, compare c and e with d and f, respectively). Surprisingly, neurites were also found to be fewer and shorter in PC12 cells expressing Np73ß than those in control cells (Fig. 2B, compare c and e with d and f, respectively), given that Np73ß has a strong transcriptional activity (Fig. 1). The neurite outgrowth was then quantified by counting cells with neurites longer than two body lengths. We found that the percentage of cells with neurites was decreased by Np73 from 23% to 9% after 3 days of treatment and from 53% to 24.7% after 6 days of treatment (Fig. 2C). Likewise, Np73ß reduced NGF-mediated neurite outgrowth from 30.9% to 16.9% after 3 days of NGF treatment and from 58.3% to 33.6% after 6 days of NGF treatment (Fig. 2C). To confirm this, the induction of growth-associated protein 43 (GAP-43) and p21 was examined by Western blot analysis. GAP-43 is a neuronal differentiation marker whose expression directly correlates with the extent of differentiation, while p21 is required for the cell cycle arrest mediated by NGF during differentiation (14, 41). We showed that GAP-43 was induced in an NGF-dependent manner (Fig. 2D and E, GAP-43, compare lanes 1 and 2 with 3 and 4). However, the induction of GAP-43 was significantly decreased upon expression of Np73 (Fig. 2D, GAP-43, compare lanes 3 and 4) or Np73ß (Fig. 2E, GAP-43, compare lanes 3 and 4). Interestingly, we found that while Np73 attenuated NGF-mediated activation of p21 (Fig. 2D, p21, compare lanes 3 and 4), Np73ß induced p21 expression regardless of NGF treatment (Fig. 2E, p21, compare lanes 1 and 3 with 2 and 4, respectively). We also determined the level of p53, which was found not to be consistently up- or down-regulated by Np73 or Np73ß regardless of NGF treatment (Fig. 2D and E, p53, compare lanes 1 and 3 with 2 and 4, respectively). Furthermore, we determined if the induction of p21 has an effect on the ability of Np73ß to inhibit NGF-mediated neuronal differentiation. To address this, two clones, PC12-HA-Np73ß-2 and PC12-HA-Np73ß-1, which express various levels of Np73ß (Fig. 2F, Np73ß, compare lanes 2 and 4), were selected for NGF treatment. We found that the extent of p21 induction was higher in PC12-HA-Np73ß-2 cells than that in PC12-HA-Np73ß-1 cells (Fig. 2F, p21, compare lanes 2 and 4). However, NGF-mediated expression of GAP-43 and neuronal differentiation were suppressed by Np73ß in both clones (Fig. 2F, GAP-43, compare lanes 1 and 3 with 2 and 4, respectively) (data not shown). This suggests that p21 induced by Np73ß does not impinge on the ability of Np73ß to suppress NGF-mediated neuronal differentiation. Taken together, our data indicate that Np73 and Np73ß are capable of inhibiting NGF-mediated neuronal differentiation in PC12 cells.

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FIG. 2. Overexpression of Np73 or Np73ß inhibits NGF-mediated neuronal differentiation in PC12 cells. (A and B) Representative microscopy images of PC12 cells with or without Np73 (A) or Np73ß (B) expression in the presence or absence of NGF. PC12 cells were uninduced (a, c, and e) or induced (b, d, and f) to express Np73 (A) or Np73 (B) for 18 h followed by treatment with 100 ng/ml NGF (c, d, e, and f) or no NGF treatment (a and b) for 3 (c and d) or 6 (e and f) days. (C) Quantitative analysis of PC12 cells bearing neurites. Cells were treated as described for panels A and B. Neurite-positive cells are the ones with at least one neurite longer than two body lengths. The percentage of neurite outgrowth is calculated by dividing the neurite-positive cells by the total number of cells counted. Approximately 500 cells were counted for each sample. The error bars represent the standard deviations of triplicate results. (D, E, and F) Overexpression of Np73 (D) or Np73ß (E and F) attenuates the induction of GAP-43. PC12 cells were uninduced or induced to express Np73 (D) or Np73ß (E and F) for 18 h followed by treatment with NGF or no NGF treatment for 3 days. Cell lysates were examined by Western blot analysis with antibodies against HA (-HA), p21, GAP-43, p53, and actin, respectively. Long Exp., long expression; Short Exp., short expression.

Np73 inhibits NGF-mediated neuronal differentiation in p53 knockdown PC12 cells. Previously, we and others have reported that p53 plays an important role in NGF-mediated neuronal differentiation in PC12 cells (9, 55). Since Np73 has been found to be dominant negative towards p53 (37), it is important to determine whether Np73 would inhibit NGF-mediated neuronal differentiation in the absence of p53. To this end, we generated stable PC12 cell lines in which endogenous p53 is targeted by siRNA and an HA-tagged murine Np73 is inducibly expressed under a tetracycline-regulated promoter. Multiple clones were selected, and one representative clone, PC12(p53KD)-HA-Np73, is shown in Fig. 3A. Upon induction, Np73 was detectable by anti-HA (Fig. 3A, Np73). Moreover, upon NGF treatment, endogenous p53 was accumulated in PC12 cells but not in p53 knockdown PC12 cells (Fig. 3A, p53, compare lanes 3 and 4 with lanes 7 and 8), suggesting that p53 is silenced by siRNA in PC12(p53KD) cells.

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FIG. 3. Np73 inhibits NGF-mediated neuronal differentiation in p53 knockdown PC12 cells. (A) PC12(p53KD)-HA-Np73 and PC12-HA-Np73 cells were uninduced or induced to express Np73 for 18 h followed by treatment with NGF or no NGF treatment for 3 days. Cell lysates were collected for Western blot analysis with antibodies against HA, p53, p21, GAP-43, and actin, respectively. (B) Representative microscopy images of p53 knockdown PC12 cells with or without Np73 expression in the presence or absence of NGF. PC12 cells were uninduced (a, c, and e) or induced (b, d, and f) to express Np73 for 18 h followed by treatment with 200 ng/ml NGT (c, d, e, and f) or no NGF treatment (a and b) for 3 (c and d) or 6 (e and f) days. (C) Quantitative analysis of PC12 cells bearing neurites shown in panel B.

Next, to determine if Np73 inhibits NGF-mediated neuronal differentiation in the absence of p53, PC12(p53KD) cells were uninduced or induced to express Np73 for 18 h followed by treatment with NGF or no NGF treatment for 3 or 6 days. It should be mentioned that a higher dosage of NGF (200 ng/ml instead of 100 ng/ml) was used since p53 knockdown renders PC12 cells less sensitive to NGF (55). As shown in Fig. 3B, in the absence of NGF, no morphological change was found in PC12(p53KD) cells regardless of Np73 induction (Fig. 3B, a and b). In the presence of NGF, control PC12(p53KD) cells extended neurites in a time-dependent manner (Fig. 3C, c and e). However, the neurites were much fewer and shorter in Np73-expressing PC12(p53KD) cells than in control cells (Fig. 3B, compare d and f with c and e, respectively), suggesting that Np73 is capable of inhibiting NGF-mediated neurite outgrowth in the absence of p53. By counting cells with neurites, we showed that Np73 decreased NGF-mediated neurite outgrowth in p53 knockdown PC12 cells from 13.9% to 1.32% after a 3-day treatment and from 31.8% to 8.3% after a 6-day treatment (Fig. 3C). To confirm this, induction of GAP-43 and p21 was examined by Western blot analysis. We showed that due to lack of p53, the extent of GAP-43 induction was less in p53 knockdown PC12 cells than that in parental PC12 cells (Fig. 3A, GAP-43, compare lanes 3 and 4 with 7 and 8). Nevertheless, a further reduction was found in p53 knockdown PC12 cells upon Np73 expression (Fig. 3A, GAP-43, compare lanes 3 and 4). In addition, p21 was not induced by NGF in p53 knockdown PC12 cells due to lack of p53 (Fig. 3A, p21, lanes 3 and 4), consistent with a previous report (55). Together, these data suggest that Np73 is capable of inhibiting NGF-mediated neuronal differentiation independent of p53.

Np73 is the predominant form in undifferentiated PC12 cells and down-regulated upon NGF treatment. To determine the role of endogenous Np73 in NGF-mediated neuronal differentiation, we examined whether commercially available anti-p73 antibodies are able to recognize exogenous murine Np73, which shares a high degree of sequence identity with rat Np73. To do this, PC12 cells were uninduced or induced to express Np73 or Np73ß for 18 h. Cell lysates were immunoprecipitated with BL906 antibody (Bethyl Laboratories, catalog no. A300-126A), which was raised against amino acids 1 to 62 of human TAp73 and expected to react with only the TAp73 isoforms, or p73-Ab2 antibody (Calbiochem, catalog no. 0P109), which was raised against amino acids 380 to 495 of human p73 and expected to detect both the and ß isoforms of TA/Np73. The immunoprecipitates were then examined by Western blot analysis with p73-Ab1 and p73-Ab3 antibodies, which preferentially detect (TA/Np73) and ß (TA/Np73ß) isoforms of p73, respectively. We showed that murine Np73 was detectable by p73-Ab1 in p73-Ab2, but not BL906, immunoprecipitates (Fig. 4A, compare lanes 6 and 4). Similarly, murine Np73ß was detected by p73-Ab3 in p73-Ab2, but not BL906, immunoprecipitates (Fig. 4B, compare lanes 6 and 4).

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FIG. 4. Np73 is the predominant isoform in undifferentiated PC12 cells and is down-regulated upon NGF treatment. (A and B) Characterization of p73 antibodies that can recognize exogenous murine Np73 (A) or Np73ß (B). PC12 cells were uninduced or induced to express Np73 (A) or Np73ß (B) for 18 h. Cell lysates were immunoprecipitated (IP) with 1 µg of BL906 and p73Ab-2 antibodies, respectively, followed by Western blot (WB) analysis with p73-Ab1 (A) or p73-Ab3 (B). Approximately 2% of cell lysates was used as input. (C, D, and E) Characterization of endogenous p73 in PC12 and HCT116 cells. Cell lysates were immunoprecipitated with 1 µg of BL906 and p73-Ab2 antibodies, respectively, followed by Western blot analysis with p73-Ab1 (C), p73-Ab3 (D), or BL906 (E). The HA-tagged murine Np73 (C, lane 2), Np73ß (C and D, lane 1), and p73ß (E, lane 2) as well as human TAp73 (E, lane 1) were used as controls. The asterisk in panel E, lane 3, represents a nonspecific protein. (F) Endogenous Np73 is down-regulated upon NGF treatment in PC12 cells. PC12 cells were treated with NGF or not treated with NGF for 0 to 72 h. Cell lysates were collected at each time point and analyzed by Western blot analysis with p73-Ab1, p73-Ab3, GAP-43, and actin antibodies, respectively.

Next, we determined the expression of p73 in PC12 cells. To this end, PC12 cell extracts were immunoprecipitated with BL906 or p73-Ab2 antibody followed by Western blot analysis with p73-Ab1 or p73-Ab3 antibody. We showed that two polypeptides, whose apparent molecular masses are close to that for HA-tagged murine Np73 (Fig. 4C, lane 2), were detected by p73-Ab1 in p73-Ab2, but not BL906, immunoprecipitates (Fig. 4C, compare lanes 4 and 5). We would like to note that HA-tagged Np73 and Np73ß expressed in PC12 cells exist as a doublet (Fig. 4C, lanes 1 and 2). Another polypeptide, whose apparent molecular mass is close to that for HA-tagged Np73ß (Fig. 4D, lane 1), was detected by p73-Ab3 in p73-Ab2, but not BL906, immunoprecipitates (Fig. 4D, compare lanes 3 and 4). Therefore, these polypeptides are likely to be Np73 since they were pulled down by p73-Ab2, an antibody for both TA/Np73 isoforms, but not BL906, an antibody specific for TAp73 isoforms. To further confirm this, PC12 and HCT116 cell lysates were immunoprecipitated with BL906 or p73-Ab2 antibody followed by Western blot analysis with BL906 antibody. HCT116 cells, which express endogenous TAp73 (46), were utilized as a positive control. We showed that a polypeptide whose molecular mass is close to that of human TAp73 (Fig. 4E, lane 1) was detected by BL906 antibody in HCT116 but not PC12 cells (Fig. 4E, compare lanes 4 and 5 with lanes 7 and 8). To rule out the possibility of species variation, we also showed that BL906 was able to detect HA-tagged TAp73ß, a murine TAp73 (Fig. 4E, lane 2). Together, we concluded that these polypeptides detected by p73-Ab1 and p73-Ab3 in PC12 cells are likely to be Np73 and Np73ß. Thus, Np73 is the predominant p73 isoform in undifferentiated PC12 cells.

To determine if expression of endogenous Np73 is altered during NGF-mediated neuronal differentiation, PC12 cells were treated with NGF for 0 to 72 h. Cell lysates were collected at each time point and analyzed by Western blot analysis with antibodies against Np73 (p73-Ab1), Np73ß (p73-Ab3), GAP-43, and actin, respectively. We showed that upon NGF treatment, GAP-43 was accumulated in a time-dependent manner (Fig. 4F, GAP-43). However, unlike GAP-43, the level of Np73 was rapidly diminished in the first 18 h upon NGF treatment and then came back and Np73was maintained at low levels from 24 to 72 h (Fig. 4E, Np73). Similarly, upon NGF treatment, the level of Np73ß was also decreased in the first 18 h and then Np73ß was maintained at low levels in differentiating PC12 cells (Fig. 4E, Np73ß).

Knockdown of endogenous Np73 promotes NGF-mediated neuronal differentiation in PC12 cells. To determine if down-regulation of Np73 in differentiating PC12 cells plays a role in NGF-mediated neuronal differentiation, we generated stable PC12 cell lines in which endogenous Np73 is silenced by siRNA. Two representative clones, PC12-Np73-KD-1 and PC12-Np73-KD-4, are presented in Fig. 5A. Western blot analysis revealed that the levels of endogenous Np73 and Np73ß were significantly decreased in PC12-Np73-KD cells compared to those in parental PC12 cells (Fig. 5A, Np73 and Np73ß, compare lane 1 with 2 and 3). In line with this, RT-PCR analysis also showed that the level of Np73 transcripts was markedly reduced in PC12-Np73-KD cells compared to that in parental PC12 cells (Fig. 5B, Np73, compare lane 1 with 2 and 3). Next, to determine if knockdown of Np73 affects NGF-mediated neuronal differentiation, PC12 and PC12-Np73-KD cells were treated with NGF or without NGF for 3 or 6 days. As shown in Fig. 5C, neither PC12 nor PC12-Np73-KD cells were able to extend neurites in the absence of NGF (Fig. 5C, a to c). However, upon NGF treatment for 3 days, PC12-Np73-KD cells appeared to extend many more and longer neurites than those in parental PC12 cells (Fig. 5C, compare d with e and f). Similar patterns were found when cells were treated with NGF for 6 days (Fig. 5C, compare g with h and i). By counting cells with neurites, we found that knockdown of Np73 in PC12 cells substantially increased NGF-mediated neurite outgrowth from 33.5% to 49.1 to 53.5% after 3 days of treatment and from 61.2% to 77.6 to 90.1% after 6 days of treatment (Fig. 5D). To confirm this, the levels of Np73 and GAP-43 were analyzed by Western blot analysis. We showed that the steady-state levels of Np73 and Np73ß were much lower in PC12-Np73-KD cells than those in PC12 cells (Fig. 5E, Np73, compare lanes 1 to 4 with 5 and 6; and 5F, Np73ß, compare lanes 3 to 6 with 1 and 2). We also noticed that the levels of Np73 in two PC12-KD-Np73 cells were slightly increased upon NGF treatment for 3 days (Fig. 5F, compare lanes 3 and 5 with 4 and 6, respectively), although the levels were much lower than those in the parental PC12 cells (Fig. 5F, compare lane 1 with 4 and 6, respectively). This is likely due to the effect of NGF on Np73 modifications since NGF is known to up-regulate p53 via posttranslational modifications, such as acetylation (51). Nevertheless, we show that upon NGF treatment, the induction of GAP-43 was greatly increased in Np73 knockdown PC12 cells compared to that in parental PC12 cells (Fig. 5F, GAP-43, compare lane 2 with lanes 4 and 6). We would like to note that even in the absence of NGF treatment, GAP-43 was detectable in Np73 knockdown PC12 cells (Fig. 5E, GAP-43 Long Exp, compare lane 1 with lanes 3 and 5). Furthermore, a scrambled siRNA was used as a negative control. We showed that the scrambled siRNA was unable to target Np73 (Fig. 5G, Np73, compare lanes 1 and 3) or enhance GAP-43 expression upon NGF treatment (Fig. 5G, GAP-43, compare lanes 1 and 2 with 3 and 4, respectively). Together, these data suggest that knockdown of Np73 in PC12 cells promotes NGF-mediated neuronal differentiation.

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FIG. 5. Knockdown of Np73 promotes NGF-mediated neuronal differentiation in PC12 cells. (A) Establishment of stable PC12 cell line with Np73 knockdown. PC12 and PC12-Np73-KD cell lysates were collected and examined by Western blot analysis with p73-Ab1, p73-Ab3, and actin antibodies, respectively. (B)Total RNAs from PC12 and PC12-Np73-KD cells were purified and subjected to RT-PCR analysis with primers to amplify Np73 and actin. (C) Representative microscopy images of PC12 cells with or without Np73 knockdown in the absence or presence of NGF. PC12 and PC12-Np73-KD cells were treated without (a to c) or with (d to i) NGF for 3 days (NGF-3D [d to f]) or 6 days (NGF-6D [h and i]). (D) Quantitative analysis of PC12 and PC12-Np73-KD cells with neurites shown in panel C. (E) PC12 and PC12-Np73-KD cells were treated with or without NGF for 3 days followed by Western blot analysis with p73-Ab1 and actin, respectively. (F) The expression of GAP-43 is increased in PC12 cells upon knockdown of Np73. PC12 cells were treated as described in panel E and examined by Western blot analysis with antibodies against Np73ß (p73-Ab3), GAP-43, and actin, respectively. (G) PC12 cells were either mock transfected or transfected with 25 nM scrambled siRNA for 3 days, followed by NGF treatment or no NGF treatment for 3 days. Cell lysates were collected for Western blot analysis with antibodies against Np73ß (p73-Ab3), GAP-43, and actin, respectively.

Np73 transcriptionally suppresses TrkA expression, which attenuates the NGF-mediated MAPK pathway. Previously, we reported that p53 plays an important role in NGF-mediated neuronal differentiation in PC12 cells by up-regulating TrkA, the high-affinity NGF receptor (55). Here, we found that knockdown of Np73 promotes, whereas overexpression of Np73 inhibits, NGF-mediated neuronal differentiation, suggesting that Np73 might be able to inhibit TrkA expression. To test this, PC12-HA-Np73 or PC12-HA-Np73ß cells were uninduced or induced to express Np73 or Np73ß for 12 h followed by NGF treatment for 24 h or no NGF treatment. We found that TrkA expression was increased upon NGF treatment (Fig. 6A and B, TrkA, compare lanes 1 and 3), consistent with an early report (8). Moreover, we showed that Np73 was capable of suppressing TrkA expression regardless of NGF treatment (Fig. 6A, TrkA, compare lanes 1 and 3 with 2 and 4, respectively). Similarly, TrkA expression was suppressed by Np73ß (Fig. 6B, TrkA, compare lanes 1 and 3 with 2 and 4, respectively). Additionally, TrkA expression was suppressed by Np73 in p53 knockdown PC12 cells (data not shown). Next, to determine whether the repression of TrkA by Np73 is preceded by a decrease in TrkA transcript, Northern blot analysis was performed and showed that the level of TrkA mRNA was down-regulated upon induction of Np73 or Np73ß (Fig. 6C, TrkA, compare lanes 1 and 3 with 2 and 4, respectively). To determine if Np73 transcriptionally represses TrkA expression in the absence of p53, p53 knockdown PC12 cells were uninduced or induced to express Np73 in the presence or absence of NGF. We found that the level of TrkA mRNA was decreased upon Np73 induction regardless of NGF treatment (Fig. 6D, TrkA, compare lanes 1 and 3 with 2 and 4, respectively). This suggests that Np73 can repress TrkA expression in a p53-independent manner. Moreover, to determine if endogenous Np73 has an effect on TrkA expression, we analyzed the steady-state level of TrkA in the parental and Np73 knockdown PC12 cells. We showed that the steady-state level of TrkA was increased in Np73 knockdown PC12 cells compared to that in parental PC12 cells (Fig. 6E, TrkA, compare lane 1 with lanes 2 and 3). Finally, to rule out the possibility that the inhibition of TrkA by Np73 may be cell-type specific, H1299 cells that can inducibly express HA-tagged human Np73 or Np73ß were utilized. We showed that upon Np73/ß induction, the expression of TrkA was repressed in H1299 cells (Fig. 6F, TrkA, compare lanes 1 and 3 with 2 and 4, respectively). Next, we determined if Np73-mediated repression of TrkA has an effect on the NGF-mediated MAPK pathway, whose sustained activation is required for NGF-mediated neurite outgrowth in PC12 cells (38). To this end, PC12-HA-Np73ß cells were uninduced or induced to express Np73ß for 12 h followed by NGF treatment for 0, 5, 15, and 30 min. As shown in Fig. 6G, NGF-mediated activation of MEK1/2 and RSK90 was attenuated in PC12 cells upon Np73ß induction compared to that in control cells (Fig. 6G, p-MEK1/2 and p-RSK90, compare lanes 1, 3, 5, and 7 with 2, 4, 6, and 8, respectively).

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FIG. 6. Np73 transcriptionally inhibits TrkA expression, which leads to repression of the NGF-mediated MAPK pathway. (A and B) Overexpression of Np73 (A) or Np73ß (B) represses TrkA expression. PC12 cells were uninduced or induced to express Np73 (A) or Np73ß (B) for 12 h followed by treatment with NGF or no NGF treatment for 24 h. Cell extracts were analyzed by Western blot analysis with antibodies against HA, TrkA, and actin, respectively. (C) The level of TrkA mRNA is down-regulated by Np73 or Np73ß. PC12 cells were uninduced or induced to express Np73 or Np73ß for 18 h. The total RNAs from these cells were isolated and prepared for Northern blots, which were sequentially probed with cDNAs derived from the TrkA and GAPDH genes, respectively. (D) Np73 down-regulates TrkA expression independent of p53. p53 knockdown PC12 cells were uninduced or induced to express Np73 for 12 h followed by NGF treatment for 12 h. Total RNAs were isolated for Northern blot analysis as described in panel C. (E) The steady-state level of TrkA protein is increased in PC12 cells upon knockdown of Np73. Cell lysates were collected for Western blot analysis with antibodies against TrkA and actin, respectively. (F) Overexpression of Np73 and Np73ß suppresses TrkA expression in H1299 cells. H1299 cells were uninduced or induced to express Np73 or Np73ß for 24 h. Cell lysates were collected and analyzed by Western blot analysis with antibodies against HA (-HA), TrkA, and actin, respectively. (G) Np73 attenuates the NGF-mediated MAPK pathway. PC12 cells were uninduced or induced to express Np73ß for 18 h followed by NGF treatment for 0, 5, 15, or 30 min. Cell extracts were collected for Western blot analysis with antibodies against HA, p-MEK1/2, p-RSK90, and actin, respectively.

Np73 directly represses the TrkA promoter. To further investigate the underlying mechanism, we sought to determine if Np73 inhibits the TrkA promoter, which contains a p53-RE (55). To this end, a luciferase reporter under the control of the TrkA promoter with or without p53-RE (Fig. 7A, bottom panel) was cotransfected into PC12 cells with a control vector or a vector that expresses murine p53, Np73, or Np73ß. We show that the TrkA promoter with p53-RE was responsive to p53 (Fig. 7A), consistent with an early report (55). In contrast, this promoter was repressed by Np73 and Np73ß in a dosage-dependent manner (Fig. 7A). However, the TrkA promoter without p53-RE was inert to p53, Np73, and Np73ß (Fig. 7A). Next, to determine if Np73 directly binds to the TrkA promoter in vivo, a ChIP assay was performed. The Np73-DNA complexes were immunoprecipitated by anti-HA and used for identification of Np73-binding regions with primers located in the TrkA and p21 promoters (Fig. 7B). Normal immunoglobulin G (IgG) was used as a control. We found that upon Np73ß induction, the fragments containing the p53-RE in the TrkA or p21 promoters were enriched by anti-HA (Fig. 7C, TrkA-p53-RE and p21-p53-RE, compare lanes 1 and 2). No DNA fragment was enriched by the control IgG (Fig. 7C, TrkA-p53-RE and p21-p53-RE, lanes 3 and 4). Similarly, Np73 was found to bind to the p53-RE in the TrkA and p21 promoters (data not shown). We also show that Np73 directly bound to the TrkA and p21 promoters in the absence of p53 (Fig. 7D, TrkA-p53-RE and p21-p53-RE, compare lanes 1 and 2). Furthermore, to determine if endogenous Np73 is able to bind to the TrkA promoter in vivo, PC12 cells were used for a ChIP assay. P73-Ab1 and -3 antibodies were used to immunoprecipitate Np73-DNA complexes, while normal IgG and BL906 antibodies were used as controls. We showed that the fragment containing the p53-RE was enriched in p73-Ab1 and -3, but not in BL906 or IgG, immunocomplexes (Fig. 7E, TrkA-p53-RE, lanes 1 to 3).

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FIG. 7. Np73 directly represses the TrkA promoter. (A) Wild-type, but not mutant, TrkA promoter is suppressed by Np73. A schematic diagram of wild-type and mutant TrkA promoters is presented in the bottom panel. The dual-luciferase assay was performed as described in Materials and Methods. The increase in relative luciferase activity for each construct was calculated using an empty vector as a control. (B) Schematic presentation of the TrkA and p21 promoters with the locations of the transcriptional start site, p53-RE, and primers used for ChIP assays. (C and D) Np73 binds to the TrkA and p21 promoters in vivo. Cells were uninduced or induced to express Np73ß (C) or Np73 (D) for 18 h followed by the ChIP assay as described in Materials and Methods. Anti-HA antibody (-HA) was used to immunoprecipitate Np73ß- or Np73-DNA complexes. Normal IgG was used as a control. (E) Endogenous Np73 binds to the TrkA promoter under a nonstressed condition in PC12 cells. p73-Ab1 and -3 antibodies were used to immunoprecipitate Np73-DNA complexes. Rabbit IgG and BL906 antibodies were used as a control.

Np73-mediated repression of TrkA requires histone deacetylases. Many studies have shown that gene silencing/repression is a highly regulated process including coordinated histone modifications. Therefore, we examined if HDACs are involved in Np73-mediated repression of TrkA. To this end, trichostatin A, a histone deactylase inhibitor, was utilized. If HDACs are involved, Np73-mediated TrkA repression would be derepressed upon TSA treatment. To test this, PC12-HA-Np73ß or PC12(p53KD)-HA-Np73 cells were uninduced or induced to express Np73ß or Np73 for 12 h and then treated with 0, 15, and 50 ng/ml of TSA for an additional 12 h. We found that in the absence of TSA, the expression of TrkA was repressed by Np73 or Np73ß (Fig. 8A and B, TrkA, compare lanes 1 and 2). However, in the presence of TSA, the repression of TrkA by Np73 or Np73ß was attenuated (Fig. 8A and B, TrkA, compare lanes 3 and 5 with 4 and 6, respectively). Moreover, immunoprecipitation followed by Western blot analysis was performed to determine if Np73 can physically interact with HDACs. Anti-HA was used to immunoprecipitate Np73-containing complexes or to detect Np73 by Western blot analysis. We found that endogenous HDAC1 and HDAC2 were detected in Np73- or Np73ß-containing immunoprecipitates (Fig. 8C and D). Conversely, Np73 was detected in both HDAC1- and HDAC2-containing immunoprecipitates (Fig. 8E). These data suggest that Np73 physically interacts with HDAC1 and HDAC2. Furthermore, we determined if Np73 is able to recruit HDAC1 and -2 to the TrkA promoter. To this end, PC12 cells were uninduced or induced to express Np73ß for 12 h for a ChIP assay. Chromatin was immunoprecipitated with normal IgG, antibody against acetylated histone 3, or a mixture of HDAC1 and -2 antibodies. We showed that upon Np73ß induction, the fragment containing the p53-RE in the TrkA promoter was enriched in HDAC1/2 immunocomplexes (Fig. 8F, compare lanes 7 and 8). In addition, the level of acetylated histone 3 bound to the p53-RE in the TrkA promoter was markedly decreased upon Np73ß expression (Fig. 8F, compare lanes 5 and 6). No fragment was enriched by normal IgG (Fig. 8F, lanes 3 and 4). Taken together, these results suggest that HDAC1 and -2 are recruited to the TrkA promoter by Np73, which leads to diminished acetylation of histones and, subsequently, repression of TrkA.

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FIG. 8. Np73-mediated repression of TrkA requires histone deacetylases. (A and B) Trichostatin A derepresses Np73-mediated repression of TrkA. PC12 cells were uninduced or induced to express Np73ß (A) or Np73 (B) for 12 h and then treated with 0, 15, and 50 ng/ml of TSA for additional 12 h. Cell lysates were collected for Western blot analysis with antibodies against HA, TrkA, and actin, respectively. (C to E) Np73 interacts with endogenous HDACs. PC12 cells were uninduced or induced for 18 h to express Np73 (C and E) or Np73ß (D). Cell lysates were immunoprecipitated (IP) with 1 µg of HA (C and D), HDAC1 (E), or HDAC2 (E) antibodies overnight. The immunocomplexes were examined by Western blot (WB) analysis with a mixture of anti-HDAC1 and -2 (C and D, upper panel) or with anti-HA (E, upper panel). The blots were then stripped and reblotted with anti-HA (C and D, lower panel) or a mixture of anti-HDAC1 and -2 (E, lower panel). (F) HDAC1 and -2 are recruited to the TrkA promoter. PC12-HA-Np73ß cells were uninduced and induced to express Np73ß for ChIP assay. Antiacetylated H3 and anti-HDAC1/2 were used to enrich the fragment containing the p53-RE in the TrkA promoter. Rabbit IgG was used as a control.

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Many studies have shown that Np73 possesses multiple functions distinct from TAp73, the latter of which has many p53-like features (4, 18, 24, 44). Np73 is frequently overexpressed in a variety of human cancers (54). In addition, overexpression of Np73 directly correlates with the poor prognosis of neuroblastoma (3) and lung cancer (45). Furthermore, Np73 plays a crucial role in neuronal development and is a prosurvival protein in the nerve system (37, 53). However, the mechanism by which Np73 exerts these biological activities is not clear. In the present study, we have investigated this and shown that Np73 is a negative regulator of NGF-mediated neuronal differentiation in PC12 cells with the following pieces of evidence: (i) overexpression of Np73 inhibits NGF-mediated neuronal differentiation, (ii) the level of endogenous Np73 is progressively diminished in differentiating PC12 cells upon treatment with NGF, and (iii) knockdown of Np73 promotes NGF-mediated neuronal differentiation. Interestingly, we also found that Np73, by recruiting HDAC corepressors to the TrkA promoter, transcriptionally inhibits TrkA expression and consequently inhibits NGF-mediated neuronal differentiation. In line with this, we have shown that knockdown of Np73 increases the steady-state level of TrkA and leads to enhanced neurite outgrowth in PC12 cells upon NGF treatment. Based on these findings, we present a model for the role of Np73 in NGF-mediated neuronal differentiation (Fig. 9).

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FIG. 9. Model of the role of Np73 in NGF-mediated neuronal differentiation in PC12 cells.

In this study, we found that while Np73 and Np73ß are capable of inhibiting NGF-mediated neuronal differentiation via repression of TrkA, Np73ß exhibits several unique activities. Like its human counterpart, murine Np73ß is a strong transcriptional activator, as demonstrated by its ability to induce p21 and apoptosis (Fig. 1). Remarkably, these properties make Np73ß a p53/TAp73-like protein. However, unlike p53, which transactivates TrkA and promotes NGF-mediated neuronal differentiation (55), Np73ß has an opposing function. One possibility is that the activation domain in Np73, which has no homology to that in p53, functions differently. As a result, p53, but not Np73, can transactivate expression of certain genes, such as TrkA, which are required for neuronal differentiation. The other possibility is that Np73 may regulate its own unique target genes to counter NGF-mediated neuronal differentiation, such as repression of GAP-43 induced by NGF (Fig. 2E). We would like to note that the induction of p21 by Np73ß is not sufficient, although p21 is known to be required, for NGF-mediated neuronal differentiation. This is consistent with a previous report that overexpression of p21 is not sufficient to induce PC12 cells to undergo differentiation (10).

Here, we found that Np73 is the predominant p73 isoform in undifferentiated PC12 cells. Upon NGF treatment, endogenous Np73 is down-regulated and maintained at low levels in differentiated PC12 cells. Similar to our findings, other studies showed that Np73 is preferentially expressed in proliferating nephron precursors, while TAp73 is predominantly expressed in the differentiation domain of renal cortex during nephrogenesis (39, 40). Moreover, Np63, another member of the p53 family, is also preferentially expressed in undifferentiated basal epithelium cells and its expression is repressed in differentiated cells (52). These observations suggest that different p53 family members have opposing functions during differentiation and that inhibition of cellular differentiation by Np73 or Np63 may contribute to tumor formation since lack of proper differentiation and unbalanced proliferation are two of the hallmarks of many types of cancer cells.

The finding that Np73 represses TrkA expression may provide an insight into the mechanism of neuroblastoma pathogenesis. Both up-regulation of Np73 and down-regulation of TrkA have been reported to be associated with the poor prognosis in neuroblastoma patients (3, 21, 34). Therefore, it would be interesting to find out if these two prognosis makers are directly correlated. In addition, we have shown that upon knockdown of Np73, TrkA expression is up-regulated, leading to enhanced NGF-mediated neuronal differentiation. Furthermore, we have shown that upon treatment with TSA, an HDAC inhibitor, Np73-mediated repression of TrkA is at least partially derepressed. These data point out the possibility that Np73 might be explored as a therapeutic target for neuroblastoma and that HDAC inhibitors and others that target Np73 can be used as agents for managing neuroblastoma.

 
This work was supported in part by NIH grants CA081237 and CA102188.

 
* Corresponding author. Mailing address: Center for Comparative Oncology, 2128 Tupper Hall, University of California at Davis, Davis, CA 95616. Phone: (530) 754-8404. Fax: (530) 752-6042. E-mail: xbchen{at}ucdavis.edu

Published ahead of print on 12 March 2007.

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Molecular and Cellular Biology, May 2007, p. 3868-3880, Vol. 27, No. 10
0270-7306/07/$08.00+0     doi:10.1128/MCB.02112-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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