p53 is required for nerve growth factor-mediated differentiation of PC12 cells via regulation of TrkA levels

p53 is necessary for the elimination of neural cells inappropriately differentiated or in response to stimuli. However, the role of p53 in neuronal differentiation is not certain. Here, we showed that nerve growth factor (NGF)-mediated differentiation in PC12 cells is enhanced by overexpression of wild-type p53 but inhibited by mutant p53 or knockdown of endogenous wild-type p53, the latter of which can be rescued by expression of exogenous wild-type p53. Interestingly, p53 knockdown or overexpression of mutant p53 attenuates NGF-mediated activation of TrkA, the high-affinity receptor for NGF and a tyrosine kinase, and activation of the mitogen-activated protein kinase pathway. In addition, p53 knockdown reduces the constitutive levels of TrkA, which renders PC12 cells inert to NGF. And finally, we showed that both constitutive and stimuli-induced expressions of TrkA are regulated by p53 and that induction of TrkA by activated endogenous p53 enhances NGF-mediated differentiation. Taken together, our data demonstrate that p53 plays a critical role in NGF-mediated neuronal differentiation in PC12 cells at least in part via regulation of TrkA levels.     

p73 induces apoptosis by different mechanisms

p73, like its homologue, the tumor suppressor p53, is able to induce apoptosis in several cell types. This property is important for the involvement of p73 in cancer development and therapy. However, in contrast with p53, the TAp73 gene has two distinct promoters coding for two protein isoforms with opposite effects: while the transactivation proficient TAp73 shows pro-apoptotic effects, the amino-terminal-deleted DeltaNp73 has an anti-apoptotic function. Indeed, the relative expression of these two proteins is related to the prognosis of several cancers. Here we discuss recent developments in the control of p73-induced apoptosis. First, TAp73 induces ER stress via the direct transactivation of Scotin. Second, TAp73 induces the mitochondrial pathway by directly transactivating both Bax and the BH3 only protein PUMA promoters. While the first transactivation is weak, and not sufficient to trigger apoptosis (at least in the in vitro cellular models so far evaluated), the induction of PUMA is strong and lethal. Third, the promoter of the death receptor CD95 contains a p53 responsive element and preliminary experiments suggest that TAp73 also activates the death receptor pathway. In addition, TAp73 is able to transactivate its own second promoter, thus inducing the expression of the anti-apoptotic DeltaNp73 isoform. Therefore, the balance between TAp73 and DeltaNp73 finely regulates cellular sensitivity to death.     

TAp73 Protein Stability Is Controlled by Histone Deacetylase 1 via Regulation of Hsp90 Chaperone Function

Histone deacetylases (HDACs) play important roles in fundamental cellular processes, and HDAC inhibitors are emerging as promising cancer therapeutics. p73, a member of the p53 family, plays a critical role in tumor suppression and neural development. Interestingly, p73 produces two classes of proteins with opposing functions: the full-length TAp73 and the N-terminally truncated ΔNp73. In the current study, we sought to characterize the potential regulation of p73 by HDACs and found that histone deacetylase 1 (HDAC1) is a key regulator of TAp73 protein stability. Specifically, we showed that HDAC1 inhibition by HDAC inhibitors or by siRNA shortened the half-life of TAp73 protein and subsequently decreased TAp73 expression under normal and DNA damage-induced conditions. Mechanistically, we found that HDAC1 knockdown resulted in hyperacetylation and inactivation of heat shock protein 90, which disrupted the interaction between heat shock protein 90 and TAp73 and thus promoted the proteasomal degradation of TAp73. Functionally, we found that down-regulation of TAp73 was required for the enhanced cell migration mediated by HDAC1 knockdown. Together, we uncover a novel regulation of TAp73 protein stability by HDAC1-heat shock protein 90 chaperone complex, and our data suggest that TAp73 is a critical downstream mediator of HDAC1-regulated cell migration.     

RabGEF1/Rabex-5 Regulates TrkA-Mediated Neurite Outgrowth and NMDA-Induced Signaling Activation in NGF-Differentiated PC12 Cells

Nerve growth factor (NGF) binds to its cognate receptor TrkA and induces neuronal differentiation by activating distinct downstream signal transduction events. RabGEF1 (also known as Rabex-5) is a guanine nucleotide exchange factor for Rab5, which regulates early endosome fusion and vesicular trafficking in endocytic pathways. Here, we used the antisense (AS) expression approach to induce an NGF-dependent sustained knockdown of RabGEF1 protein expression in stable PC12 transfectants. We show that RabGEF1 is a negative regulator of NGF-induced neurite outgrowth and modulates other cellular and signaling processes that are activated by the interaction of NGF with TrkA receptors, such as cell cycle progression, cessation of proliferation, and activation of NGF-mediated downstream signaling responses. Moreover, RabGEF1 can bind to Rac1, and the activation of Rac1 upon NGF treatment is significantly enhanced in AS transfectants, suggesting that RabGEF1 is a negative regulator of NGF-induced Rac1 activation in PC12 cells. Furthermore, we show that RabGEF1 can also interact with NMDA receptors by binding to the NR2B subunit and its associated binding partner SynGAP, and negatively regulates activation of nitric oxide synthase activity induced by NMDA receptor stimulation in NGF-differentiated PC12 cells. Our data suggest that RabGEF1 is a negative regulator of TrkA-dependent neuronal differentiation and of NMDA receptor-mediated signaling activation in NGF-differentiated PC12 cells.     

TAp73/Delta Np73 influences apoptotic response, chemosensitivity and prognosis in hepatocellular carcinoma

We investigated the mechanisms by which TAp73 beta and dominant-negative p73 (Delta Np73) regulate apoptosis. TAp73 beta transactivated the CD95 gene via the p53-binding site in the first intron. In addition, TAp73 beta induced expression of proapoptotic Bcl-2 family members and led to apoptosis via the mitochondrial pathway. Endogenous TAp73 was upregulated in response to DNA damage by chemotherapeutic drugs. On the contrary, DeltaNp73 conferred resistance to chemotherapy. Inhibition of CD95 gene transactivation was one mechanism by which DeltaNp73 functionally inactivated the tumor suppressor action of p53 and TAp73 beta. Concomitantly, DeltaNp73 inhibited apoptosis emanating from mitochondria. Thus, DeltaNp73 expression in tumors selects against both the death receptor and the mitochondrial apoptosis activity of TAp73 beta. The importance of these data is evidenced by our finding that upregulation of DeltaNp73 in hepatocellular carcinoma patients correlates with reduced survival. Our data indicate that Delta Np73 is an important gene in hepatocarcinogenesis and a relevant prognostic factor.     

LAMTOR2-Mediated Modulation of NGF/MAPK Activation Kinetics during Differentiation of PC12 Cells

LAMTOR2 (p14), a part of the larger LAMTOR/Ragulator complex, plays a crucial role in EGF-dependent activation of p42/44 mitogen-activated protein kinases (MAPK, ERK1/2). In this study, we investigated the role of LAMTOR2 in nerve growth factor (NGF)-mediated neuronal differentiation. Stimulation of PC12 (rat adrenal pheochromocytoma) cells with NGF is known to activate the MAPK. Pharmacological inhibition of MEK1 as well as siRNA–mediated knockdown of both p42 and p44 MAPK resulted in inhibition of neurite outgrowth. Contrary to expectations, siRNA–mediated knockdown of LAMTOR2 effectively augmented neurite formation and neurite length of PC12 cells. Ectopic expression of a siRNA-resistant LAMTOR2 ortholog reversed this phenotype back to wildtype levels, ruling out nonspecific off-target effects of this LAMTOR2 siRNA approach. Mechanistically, LAMTOR2 siRNA treatment significantly enhanced NGF-dependent MAPK activity, and this effect again was reversed upon expression of the siRNA-resistant LAMTOR2 ortholog. Studies of intracellular trafficking of the NGF receptor TrkA revealed a rapid colocalization with early endosomes, which was modulated by LAMTOR2 siRNA. Inhibition of LAMTOR2 and concomitant destabilization of the remaining members of the LAMTOR complex apparently leads to a faster release of the TrkA/MAPK signaling module and nuclear increase of activated MAPK. These results suggest a modulatory role of the MEK1 adapter protein LAMTOR2 in NGF-mediated MAPK activation required for induction of neurite outgrowth in PC12 cells.     

MicroRNA expression profiling of NGF-treated PC12 cells revealed a critical role for miR-221 in neuronal differentiation

MicroRNAs (miRNAs) are small non-coding RNAs that control protein expression through translational inhibition or mRNA degradation. MiRNAs have been implicated in diverse biological processes such as development, proliferation, apoptosis and differentiation. Upon treatment with nerve growth factor (NGF), rat pheochromocytoma PC12 cells elicit neurite outgrowth and differentiate into neuron-like cells. NGF plays a critical role not only in neuronal differentiation but also in protection against apoptosis. In an attempt to identify NGF-regulated miRNAs in PC12 cells, we performed miRNA microarray analysis using total RNA harvested from cells treated with NGF. In response to NGF treatment, expression of 8 and 12 miRNAs were up- and down-regulated, respectively. Quantitative RT-PCR analysis of 11 out of 20 miRNAs verified increased expression of miR-181a^∗, miR-221 and miR-326, and decreased expression of miR-106b^∗, miR-126, miR-139-3p, miR-143, miR-210 and miR-532-3p after NGF treatment, among which miR-221 was drastically up-regulated. Functional annotation analysis of potential target genes of 7 out of 9 miRNAs excluding the passenger strands (*) revealed that NGF may regulate expression of various genes by controlling miRNA expression, including those whose functions and processes are known to be related to NGF. Overexpression of miR-221 induced neuronal differentiation of PC12 cells in the absence of NGF treatment, and also enhanced neuronal differentiation caused by low-dose NGF. Furthermore, miR-221 potentiated formation of neurite network, which was associated with increased expression of synapsin I, a marker for synapse formation. More importantly, knockdown of miR-221 expression by antagomir attenuated NGF-mediated neuronal differentiation. Finally, miR-221 decreased expression of Foxo3a and Apaf-1, both of which are known to be involved in apoptosis in PC12 cells. Our results suggest that miR-221 plays a critical role in neuronal differentiation as well as protection against apoptosis in PC12 cells.     

TAp63 plays compensatory roles in p53-deficient cancer cells under genotoxic stress

p53, p63, and p73 belong to the p53 family of proteins, which mediate development, differentiation, and various other cellular responses. p53 is involved in many anti-cancer mechanisms, such as cell cycle regulation, apoptosis, and the maintenance of genomic integrity. The p63 gene is controlled by two promoters that direct the expression of two isoforms, one with and one without transactivating properties, known as TAp63 and ΔNp63. In this study, p53-deficient cells (Hep3B and PC-3) and p53-expressing cells (A549 and HepG2) were treated with doxorubicin to examine the possible roles of TAp63 in these cells under genotoxic stress; TAp63 expression was induced in p53-deficient cell lines, but not in p53-expressing cell lines. The ectopic expression of p53 in p53-deficient cells (Hep3B) reduced TAp63 promoter activity, and knockdown of TAp63 attenuated doxorubicin-induced cell growth arrest by promoting cell cycle progression, leading to an increase in the percentage of G(2)/M cells. Moreover, knockdown of TAp63 increased cell sensitivity to doxorubicin-induced genomic damage. Our results suggest that TAp63 may play a compensatory role in cell cycle regulation and DNA damage repair in p53-deficient cancer cells.     

MKK7γ1 reverses nerve growth factor signals: proliferation and cell death instead of neuritogenesis and protection

c-Jun N-terminal kinases (JNKs) are the exclusive downstream substrates of mitogen-activated protein kinase kinase 7 (MKK7). Recently, we have shown that a single MKK7 splice variant, MKK7γ1, substantially changes the functions of JNKs in naïve PC12 cells. Here we provide evidence that MKK7γ1 blocks NGF-mediated differentiation and sustains proliferation by interfering with the NGF-triggered differentiation programme at several levels: (i) down-regulation of the NGF receptors TrkA and p75; (ii) attenuation of the differentiation-promoting pathways ERK1/2 and AKT; (iii) increase of JNK1 and JNK2, especially the JNK2 54 kDa splice variants; (iv) repression of the cyclin-dependent kinase inhibitor p21^WAF1/CIP1, which normally supports NGF-mediated cell cycle arrest; (v) strong induction of the cell cycle promoter CyclinD1, and (vi) profound changes of p53 functions. Moreover, MKK7γ1 substantially changes the responsiveness to stress. Whereas NGF differentiation protects PC12 cells against taxol-induced apoptosis, MKK7γ1 triggers an escape from cell cycle arrest and renders transfected cells sensitive to taxol-induced death. This stress response completely differs from naïve PC12 cells, where MKK7γ1 protects against taxol-induced cell death. These novel aspects on the regulation of JNK signalling emphasise the importance of MKK7γ1 in its ability to reverse basic cellular programmes by simply using JNKs as effectors. Furthermore, our results highlight the necessity for the cells to balance the expression of JNK activators to ensure precise intracellular processes.