Expression Of Prostate Apoptosis Response-4 Research Articles

Curcumin Sensitizes 4T1 Murine Breast Cancer Cells to Cisplatin Through PAR4 Secretion.

Prostate apoptosis response 4 (PAR4), a tumour-suppressor protein, selectively induces apoptosis of cancer cells without affecting normal cells. Its soluble form is induced by secretagogues (e.g., chloroquine), and it induces apoptosis by interacting with the receptor of glucose-regulated protein 78, which is overexpressed in cancer cells. In this study, curcumin was analyzed as an inducer of PAR4 expression in 4T1 murine breast cancer cell. and its ability to induce PAR4 secretion in Balb/c mice. In addition, the cisplatin sensitizing effect of soluble PAR4 was analyzed. The 4T1 cell line was treated in vitro using different concentrations of curcumin; cell viability was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and PAR4 expression by western blotting. The expression of soluble PAR4 in the serum of mice treated with intraperitoneal curcumin was analyzed using the dot-blot method. Moreover, MTT assay was used to analyze the effects of serum from curcumin-treated mice on cell viability. Tumor size was analyzed in mice treated with curcumin alone and in combination with cisplatin. Curcumin showed a dose- and time-dependent effects on cell viability on 4T1 cells, as well as increasing PAR4 expression. Compared with the control group (phosphate-buffered saline), mice treated with curcumin showed an increase in plasma PAR4. In the Balb/C tumor model, mice treated with curcumin and cisplatin showed greater tumor shrinkage than the control group. These results indicate that curcumin induces expression of soluble PAR4 and sensitizes tumor cells to cisplatin.

Par-4-dependent p53 up-regulation plays a critical role in thymoquinone-induced cellular senescence in human malignant glioma cells

Thymoquinone (TQ), the predominant bioactive constituent present in black cumin (Nigella sativa), exerts tumor suppressive activity against a wide variety of cancer cells. Cellular senescence, characterized by stable and long term loss of proliferative capacity, acts as a potent tumor suppressive mechanism. Here, we provide evidence for the first time that TQ suppresses growth of glioma cells by potentially inducing the expression of prostate apoptosis response-4 (Par-4) tumor suppressor protein. In turn, TQ-induced Par-4 expression triggers cellular senescence, as evidenced by increasing cellular size, β-galactosidase staining, G1 phase arrest, and increased expression of senescence markers such as p53, p21, Rb, and decreased expression of lamin B1, cyclin E and cyclin depended kinase-2 (CDK-2). Further, overexpression of Par-4 significantly increases the expression of p53 and its downstream target p21, and increases β-galactosidase positive cells, while siRNA/shRNA mediated-knockdown of Par-4 reverses the TQ-induced effects. Altogether, we describe a novel mechanism of cross talk between Par-4 and p53, that plays a critical role in TQ-induced senescence in human malignant glioma cells.

Abstract B11: Prostate apoptosis response-4 (PAR4) secretion in breast tumor and normal cell lines cultured in hypoxic conditions

Abstract The prostate apoptosis response-4 (PAR4) gene encodes a 38kDa proapoptotic protein with tumor cell selective action and/or cell sensitization to an additional proapoptotic insult. This selective apoptotic ability is associated with PAR4 nuclear translocation and phosphorylation state. PAR-4 acts through intrinsic and extrinsic apoptotic pathways. PAR-4 can also be secreted by normal/immortalized and cancer cells via the classical ER stress pathway mediated by GRP78. The previous study from our group provided evidence that MCF10A cells cultured in three-dimensional conditions display an increased expression of PAR4 in the center of the acini, where the cells are dying to lumen formation and where the O2 levels are expected to be lower. In the present study, we sought to evaluate the effects of hypoxia on PAR4 secretion in mammary epithelial cells and breast cancer cells. MCF10A (immortalized mammary epithelial cells) and the breast cancer cells MCF7, MDA-MB-231, and SKBR3 were cultured in normoxic and hypoxic (1% O2) conditions. The conditioned medium (CM) was concentrated using Amicon Ultra 10kDA filters and resolved by SDS-PAGE gel electrophoresis in parallel to cell lysate analysis. Cells maintained under hypoxic conditions for 6 h, 24 h, and 48 h showed no alteration in intracellular PAR4 protein levels in relation to cells maintained in normoxia. After 72 h we observed a decrease in intracellular PAR4 protein expression. The GRP78 protein expression did not change in the same time periods. However, Caspase-7 activation could be seen in 24 h, 48 h, and 72 h. Furthermore, we observed an increase (2.7 fold) in PAR4 protein expression in the CM after 72 h under hypoxia. Our results demonstrated for the first time that PAR4 secretion is modulated by hypoxia in normal and tumor breast cells. Further experimental and clinical studies are needed to determine the mechanism and role played by PAR4 secretion in cells' sensitivity to standard chemotherapeutics agents. Supported by FAPESP and CNPq. Citation Format: Simone Aparecida de Bessa Garcia, Irina Gueroldovna Brobovnitchaia, Flavia Rotea Mangone, Maria Aparecida Nagai. Prostate apoptosis response-4 (PAR4) secretion in breast tumor and normal cell lines cultured in hypoxic conditions [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B11.

TNF-α and IFN-γ Together Up-Regulates Par-4 Expression and Induce Apoptosis in Human Neuroblastomas.

The objective of this study was to examine the combined effect of Interferon-gamma (IFN-γ) and Tumor Necrosis factor-alpha (TNF-α) on cytotoxicity and expression of prostate apoptosis response-4 (Par-4) and Par-4 interacting proteins B-cell lymphoma (Bcl-2), nuclear factor kappa-light-chain-enhancer of activated B cells/p65 subunit (NF-κB/p65), Ak mouse strain thymoma (Akt) in human neuroblastoma (NB) cells. Materials and methods included human neuroblastoma cell lines-SK-N-MC, SK-N-SH, and SH-SY5Y, which were treated with IFN-γ and TNF-α individually, or in combination, and were assessed for viability by tetrazolium (MTT) assay. Apoptosis was monitored by hypodiploid population (by flow cytometry), DNA fragmentation, Poly (ADP-ribose) polymerase (PARP) cleavage, and caspase-8 activity. Transcript level of Par-4 was measured by RT-PCR. Protein levels of Par-4 and suppressor of cytokine signaling 3 (SOCS-3) were assessed by immunoblotting. Cellular localization of Par-4 and p65 was examined by immunofluorescence. Unbiased transcript analysis for IFN-γ, TNF-α, and Par-4 were analyzed from three independent clinical datasets from neuroblastoma patients. In terms of results, SK-N-MC cells treated with a combination of, but not individually with, IFN-γ and TNF-α induced apoptosis characterized by hypodiploidy, DNA fragmentation, PARP cleavage, and increased caspase-8 activity. Apoptosis was associated with up-regulation of Par-4 mRNA and protein expression. Immunofluorescence studies revealed that Par-4 was localized exclusively in cytoplasm in SK-N-MC cells cultured for 24 h. but showed nuclear localization at 48 h. Treatment with IFN-γ and TNF-α together enhanced the intensity of nuclear Par-4. In gene expression, data from human neuroblastoma patients, levels of IFN-γ, and TNF-α have strong synergy with Par-4 expression and provide good survival advantage. The findings also demonstrated that apoptosis was associated with reduced level of pro-survival proteins–Bcl-2 and Akt and NF-κB/p65. Furthermore, the apoptotic effect induced by IFN-γ-induced Signal Transducer and Activator of Transcription-1(STAT-1), and could be due to down-regulation of suppressor of cytokine signaling-3 (SOCS3). The study concludes that a combinatorial approach using IFN-γ and TNF-α can be explored to maximize the effect in chemotherapy in neuroblastoma, and implies a role for Par-4 in the process.

Abstract 3803: Transcriptome analysis of MCF7 breast cancer cells with different expression profile of PAR-4 (prostate apoptosis response-4) in the presence and absence of docetaxel

Abstract The ability of cancer cells to evade apoptosis is one of the essential alterations in cell physiology that dictate tumor growth and impact chemo- and radio-resistance. Experimental evidence indicates that protein kinase C apoptosis WT1 regulator (PAWR) gene, also known as prostate apoptosis response-4 (PAR-4), plays an important role in tumor cell survival, and may be considered a candidate for tumor cell selective therapy. This selective ability of Par-4 to directly cause apoptosis in most cancer cells is associated with its nuclear translocation and its phosphorylation state. PAR-4 acts in both intrinsic and extrinsic apoptotic pathways. In the present study, we sought to evaluate the effects of PAR-4 on the expression profile of MCF7 breast cancer cells in the presence and absence of docetaxel. MCF7 breast cancer cells were transfected with the expression vector pCMV6-PAR-4 or pCMV6-Neo. Real time PCR, western blot and immunofluorescence were used to characterize the clones over-expressing PAR-4. Using cDNA microarray analysis, we evaluated the transcriptome changes in the comparison between MCF7 cells with increased expression of PAR-4 and the control cells before and after docetaxel treatment. Several differentially expressed genes were identified as potentially involved in PAR-4-mediated chemosensitivity to docetaxel. Using the Ingenuity program, several gene networks were generated from the differentially expressed genes, including many genes direct and indirectly involved in the canonical WNT (Wingless-Type MMTV Integration 1) signaling pathway. The Wnt Signaling Pathway Human RT2 Profiler PCR Array was used to validate the differentially expressed genes involved in the Wnt pathway identified in MCF-7 cells with differences of Par-4 expression before and after exposure to docetaxel. The validation results confirm the data observed by cDNA microarray showing that the increase in PAR-4 expression modulates the expression of many genes involved in the Wnt signaling pathway in MCF7 breast cells. The results generated could be of particular interest for the understanding the action of PAR-4 in chemosensitivity, especially to increase the specificity and effectiveness of drug treatment and overcoming resistance to chemotherapy. Supported by FAPESP and CNPq. Citation Format: Simone A. de Bessa Garcia, Ana C. Pavanelli, Natalia C. Melo, Maria A. Nagai. Transcriptome analysis of MCF7 breast cancer cells with different expression profile of PAR-4 (prostate apoptosis response-4) in the presence and absence of docetaxel. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3803. doi:10.1158/1538-7445.AM2015-3803

The Expression of Prostate Apoptosis Response-4 in Striatum of A Rat Model of Depression Induced by Chronic Stress

Chronic stress may induce depression, but the mechanism is unclear. Previous studies suggested that dopamine system, especially dopamine receptor D2 (DRD2) may be involved in, and prostate apoptosis response-4 (par-4) may be the link point between DRD2 and depression. The aims of this research are to detect the expression of par-4 in striatum of a rat model of depression induced by chronic stress, and to explore whether DNA methylaion is involved in the regulatory mechanism of par-4 expression. Newborn rats were randomly divided into two groups, when they grew up to ten weeks, the experimental group rats (n = 17) accepted chronic mild stress for 3 weeks, while the control group rats (n = 17) received no experimental handle. Their depressive level was assessed with forced swimming test and sucrose consumption test; the mRNA expression of par-4 and DRD2 in rats’ striatum was detected by Real-Time PCR; the protein expression of par-4 was detected by western blot; and the DNA methylation level of par-4 was investigated by bisulfated DNA sequencing. The result showed that the float time of experimental rats was longer than the control rats (t = 4.01, p 0.05), and the sucrose preference rate of experimental rats was reduced compare with the control rats (t = -2.93, p 0.05). The par-4 and DRD2 mRNA expression of experimental rats were lower than the control rats (t values were -3.56 and -2.507 respectively, p 0.05), and the fold changes of par-4 and DRD2 mRNA expression in experimental rats compared to control rats were 0.33 and 0.33 respectively. The par-4 protein expression of experimental rats was decreased compared with the control rats (t = -4.92, p 0.05). However, there was no significant difference between two groups in methylation level of par-4 promoter region (t = -0.26, p 0.05). These results suggest that chronic mild stress could induce the depressive behaviors of rats and suppress the expression of par-4, but DNA methylation seems to be not involved in the regulatory mechanism.

Abstract 1815: CRM-1 as a potential therapeutic target in pancreatic cancer

Abstract CRM-1 exportin protein encoded by the XPO-1 gene maintains cancer cells through nuclear export signal (NES)-dependent protein exclusion of tumor suppressor proteins (TSPs). High CRM-1 expression has been correlated with poor prognosis in pancreatic cancer making it an attractive therapeutic target. Numerous attempts have been made in the past to develop specific CRM-1 inhibitors; however, earlier inhibitors such as Leptomycin B failed in the clinic due to acute off target toxicity. Using structure based drug design; we have identified new class of selective inhibitors of nuclear export (SINE) that irreversibly inhibits CRM1 and lock TSPs in cancer cell nucleus leading to cancer cell selective apoptosis. The inhibitors have excellent pharmacokinetic parameters and minimal toxicity to normal tissue. Here, we tested SINEs against pancreatic cancer, a disease that urgently needs novel and effective therapies. CRM-1 inhibitors [(KPT-185 (most potent analog), KPT-127, KPT-205, KPT-227 as well as Leptomycin B (as positive control) and not its inactive analog KPT-Tran (as negative control)] induced growth inhibition (MTT) and apoptosis (Annexin V and Histone DNA ELISA) in five cell lines (IC50 ∼150 nM). KPT-SINEs did not have any effect on normal HPDE cells (IC50>5 µM). Mechanistically, KPT-185 mediated apoptosis was found to be through activation, phosphorylation and nuclear localization of a TSP prostate apoptosis response 4 (PAR-4). PAR-4 has earlier been established by our laboratory as a therapeutic target in pancreatic cancer. Co-immunoprecipitation studies demonstrated that KPT-185 dissociated CRM-1-PAR-4 and CRM-1-p-PAR-4 interaction. Further, PAR-4 siRNA knockdown abrogated KPT-SINEs ability to induce growth inhibition or apoptosis. In normal cells PAR-4 localization or phosphorylation was not observed and this was associated with low basal expression of PAR-4 and its phosphorylation inducer protein kinase A protein. Role of other TSPs such as p53 and p27 was also explored, however, our investigations directly point to a PAR-4 specific mechanism. KPT-251 (structurally very similar to KPT-185 with improved pharmacokinetics properties) was well tolerated in SCID mice upto 150 mg/kg, p.o. Oral administration of analog KPT-251 at 25 and 150 mg/kg daily for two weeks reduced pancreatic tumors significantly (p<0.01). Histological examination of remnant tumors showed enhanced staining of PAR-4 and reduced Ki67. Tumor tissue lysates were probed for protein expression using western blotting that revealed activation of PAR-4 signaling and co-immunoprecipitation assay demonstrated dissociation of CRM-1-PAR-4 in tumors as well. In conclusion, discovery of our novel non-toxic KPTs can significantly impact the advancement of the field of CRM-1 inhibitors and could become effective anti-cancer therapeutics for pancreatic cancer as well as other solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1815. doi:1538-7445.AM2012-1815

Vitamin E forms inhibit IL-13/STAT6-induced eotaxin-3 secretion by up-regulation of PAR4, an endogenous inhibitor of atypical PKC in human lung epithelial cells

Eotaxin-3 (CCL-26), a potent chemokine for eosinophil recruitment and contributing significantly to the pathogenesis of asthma, is secreted by lung epithelial cells in response to T helper 2 cytokines including interleukin 13 (IL-13). Here we showed that vitamin E forms, but not their metabolites, differentially inhibited IL-13-stimulated generation of eotaxin-3 in human lung epithelial A549 cells. The relative inhibitory potency was γ-tocotrienol (γ-TE) (IC50 ∼15 μM)>γ-tocopherol, δ-tocopherol (IC50 ∼25–50 μM)>α-tocopherol. Consistent with suppression of eotaxin, γ-TE treatment impaired IL-13-induced phosphorylation of STAT6, the key transcription factor for activation of eotaxin expression, and consequently blocked IL-13-stimulated DNA-binding activity of STAT6. In search of the upstream target of γTE by using inhibitor and siRNA approaches, we discovered that the atypical protein kinase C (aPKC) signaling, instead of classical PKC, p38 MAPK, JNK or ERK, played a critical role in IL-13-stimulated eotaxin generation and STAT6 activation. While showing no obvious effect on aPKC expression or phosphorylation, γ-TE treatment resulted in increased expression of prostate-apoptosis-response 4 (PAR4), an endogenous negative regulator of aPKCs. Importantly, γ-TE treatment led to enhanced formation of aPKC/PAR4 complex that is known to reduce aPKC activity via protein–protein crosstalk. Our study demonstrated that γ-TE inhibited IL-13/STAT6-activated eotaxin secretion via up-regulation of PAR4 expression and enhancement of aPKC–PAR4 complex formation. These results support the notion that specific vitamin E forms may be useful anti-asthmatic agents.

Insulin-like growth factor-1 and 17β-estradiol down-regulate prostate apoptosis response-4 expression in MCF-7 breast cancer cells

The PKC apoptosis WT1 regulator gene, also named prostate apoptosis response-4 (PAR-4), encodes a pro-apoptotic protein that sensitizes cells to numerous apoptotic stimuli. Insulin-like growth factor-1 (IGF-1) and 17β-estradiol (E2), two important factors for breast cancer development and progression, have been shown to down-regulate PAR-4 expression and inhibit apoptosis induced by PAR-4 in neuronal cells. In this study, we sought to investigate the mechanisms of regulation of PAR-4 gene expression in MCF-7 cells treated with E2 or IGF-1. E2 (10 nM) and IGF-1 (12.5 nM) each down-regulated PAR-4 expression in MCF-7 cells after 24 h of treatment. The effect of E2 was dependent on ER activation, as demonstrated by an increase in PAR-4 expression when cells were pretreated for 1 h with 1 µM ICI-182,780 (ICI) before receiving E2 plus ICI. The effect of IGF-1 was abolished by pre-treatment for 1 h with 30 µM LY294002 (a specific PI3-K inhibitor), and significantly inhibited by 30 µM SB202190 (a specific p38MAPK inhibitor). We also demonstrated that E2 acts synergistically with IGF-1, resulting in greater down-regulation of PAR-4 mRNA expression compared with E2 or IGF-1 alone. Our results show for the first time that E2 and IGF-1 inhibit PAR-4 gene expression in MCF-7 cells, suggesting that this down-regulation may provide a selective advantage for breast cancer cell survival.

Neuroprotective Effect of Ghrelin Is Associated with Decreased Expression of Prostate Apoptosis Response-4

Ghrelin is known to promote neuronal defense and survival against ischemic injury by inhibiting apoptotic processes. In the present study, we investigated the role of prostate apoptosis response-4 (Par-4), a proapoptotic gene the expression of which is increased after ischemic injury, in ghrelin-mediated neuroprotection during middle cerebral artery occlusion (MCAO). Both ghrelin and des-acyl ghrelin protected cortical neurons from ischemic injury. Ghrelin receptor specific antagonist abolished the protective effects of ghrelin, whereas those of des-acyl ghrelin were preserved, suggesting the involvement of a receptor that is distinct from GHS-R1a. The expression of Par-4 was increased by MCAO, which was attenuated by ghrelin and des-acyl ghrelin treatments. Both ghrelin and des-acyl ghrelin increased the Bcl-2/Bax ratio, prevented cytochrome c release, and inhibited caspase-3 activation. Our data indicate that des-acyl ghrelin, as well as ghrelin, protect cortical neurons against ischemic injury through the inhibition of Par-4 expression and apoptotic molecules in mitochondrial pathway.

Critical role of prostate apoptosis response-4 in determining the sensitivity of pancreatic cancer cells to small-molecule inhibitor-induced apoptosis

Role of prostate apoptosis response-4 (PAR-4) has been well described in prostate cancer. However, its significance in other cancers has not been fully elucidated. For the current study, we selected four pancreatic cancer cell lines (BxPC-3, Colo-357, L3.6pl, and HPAC) that showed differential endogenous expression of PAR-4. We found that nonpeptidic small-molecule inhibitors (SMI) of Bcl-2 family proteins (apogossypolone and TW-37; 250 nmol/L and 1 micromol/L, respectively) could induce PAR-4-dependent inhibition of cell growth and induction of apoptosis. Sensitivity to apoptosis was directly related to the expression levels of PAR-4 (R = 0.92 and R2 = 0.95). Conversely, small interfering RNA against PAR-4 blocked apoptosis, confirming that PAR-4 is a key player in the apoptotic process. PAR-4 nuclear localization is considered a prerequisite for cells to undergo apoptosis, and we found that the treatment of Colo-357 and L3.6pl cells with 250 nmol/L SMI leads to nuclear localization of PAR-4 as confirmed by 4',6-diamidino-2-phenylindole staining. In combination studies with gemcitabine, pretreatment with SMI leads to sensitization of Colo-357 cells to the growth-inhibitory and apoptotic action of a therapeutic drug, gemcitabine. In an in vivo setting, the maximum tolerated dose of TW-37 in xenograft of severe combined immunodeficient mice (40 mg/kg for three i.v. injections) led to significant tumor inhibition. Our results suggest that the observed antitumor activity of SMIs is mediated through a novel pathway involving induction of PAR-4. To our knowledge, this is the first study reporting SMI-mediated apoptosis involving PAR-4 in pancreatic cancer.

Long‐chain ceramide is elevated in presenilin 1 (PS1M146V) mouse brain and induces apoptosis in PS1 astrocytes

The pro-apoptotic sphingolipid ceramide plays an emergent role in the etiology of Alzheimer's disease (AD), although its function for neurodegeneration is not known. We determined the concentration and composition of ceramide in hippocampal tissue from newborn presenilin 1 (PS1) knock-in (PS1M146V) mice, a mouse model for early-onset familial AD. We found that PS1 tissue contains 3.1 (+/-0.5)-fold more total ceramide than wild-type tissue. In particular, the proportion of C20 and C24 ceramide is increased by 4.0- or 8.5-fold, respectively. The ceramide elevation in PS1 brain is consistent with a 3.7 (+/-0.5)-fold increase of the protein level of the neurotrophin receptor p75NTR, which has been suggested to stimulate the hydrolysis of sphingomyelin to generate ceramide. The predominance of C20 and C24 ceramide is concurrent with the elevated gene expression of lass 2 and lass 4, two isoforms of ceramide synthase that generate dihydroceramide with long-chain fatty acid. Our study indicates that primary cultured astrocytes but not neurons from PS1 mice undergo apoptosis when incubated with C20 ceramide. In contrast, wild-type astrocytes remain unaffected. The sensitivity of PS1 astrocytes is most likely due to the 9.5 (+/-0.4)-fold elevated expression of PAR-4 (prostate apoptosis response-4), a protein that inhibits atypical PKC zeta/lambda in the presence of ceramide. Our results suggest that astroglial death due to ceramide/PAR-4-induced apoptosis may critically contribute to the etiology of AD.

IGF-I inhibition of apoptosis is associated with decreased expression of prostate apoptosis response-4

The neuronal damage caused by ischemic brain injury is associated with increased apoptosis. IGF-I exposure promotes neuronal defense and survival against ischemic insult by inhibiting apoptotic processes. We investigated the role of prostate apoptosis response-4 (Par-4), a proapoptotic gene the expression of which is increased after ischemic injury, in IGF-I-mediated inhibition of apoptosis using PC12 cells exposed to oxygen-glucose deprivation (OGD). The OGD insult resulted in significant increases in apoptotic cell death and Par-4 expression, which were prevented by the treatment of cells with an antisense oligonucleotide of Par-4. IGF-I treatment prior to OGD insult significantly reduced the number of apoptotic cells and the OGD-induced increase in Par-4 expression. OGD-induced nuclear translocation of Par-4 was also attenuated by IGF-I treatment. In addition, we demonstrated that the anti-apoptotic effect of IGF-I was blocked by chemical inhibition of a mitogen activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), or protein kinase A (PKA), but not by a protein kinase C inhibitor. Finally, pretreatment of cells with a MAPK or PI3K inhibitor attenuated IGF-I-induced inhibition of Par-4 expression, suggesting that the MAPK and PI3K pathways contribute to IGF-I-induced Par-4 suppression. In contrast, a PKA inhibitor failed to alter the inhibitory effect of IGF-I on Par-4. These findings indicate that in PC12 cells exposed to OGD insult, IGF-I protects cells from apoptosis, at least in part through the inhibition of Par-4 expression.

A novel isoform of prostate apoptosis response 4 (PAR-4) that co-distributes with F-actin and prevents apoptosis in neural stem cells

The elevated expression of prostate apoptosis response-4 (PAR-4) induces apoptosis in differentiating mouse embryonic stem (ES) cells. In embryoid body (EB) cells and the E15.5 stage of embryonic mouse brain, PAR-4 is expressed as two isoforms (38 and 33 kDa). Using mouse EB-derived RNA as a template we have cloned and characterized a novel isoform of PAR-4 (PAR-4/p33) that lacks exon 3 and shows a bona fide splice junction of exons 2 and 4. The molecular mass for PAR-4/p33 is estimated to be 33 kDa, corresponding to the short form found in the EB cells and E15.5 mouse brain. The fluorescent fusion protein of PAR-4/p33 is mainly found in the cytosol and is co-distributed with F-actin filaments, while that of the 38 kDa full length PAR-4/p38 is predominantly translocated to the nucleus. In contrast to the full length PAR-4 (PAR-4/p38), ectopic expression of PAR-4/p33 does not result in the activation of caspase 3 and the induction of apoptosis. PAR-4/p33 forms a complex with PAR-4/p38, which inhibits its nuclear translocation and the induction of apoptosis. PAR-4/p33 is suggested to be a dominant negative isoform of PAR-4/p38 and may regulate PAR-4-dependent apoptosis.

Direct Binding to Ceramide Activates Protein Kinase Cζ before the Formation of a Pro-apoptotic Complex with PAR-4 in Differentiating Stem Cells

We have reported that ceramide mediates binding of atypical protein kinase C (PKC) ζ to its inhibitor protein, PAR-4 (prostate apoptosis response-4), thereby inducing apoptosis in differentiating embryonic stem cells. Using a novel method of lipid vesicle-mediated affinity chromatography, we showed here that endogenous ceramide binds directly to the PKCζ·PAR-4 complex. Ceramide and its analogs activated PKCζ prior to binding to PAR-4, as determined by increased levels of phosphorylated PKCζ and glycogen synthase kinase-3β and emergence of a PAR-4-to-phosphorylated PKCζ fluorescence resonance energy transfer signal that co-localizes with ceramide. Elevated expression and activation of PKCζ increased cell survival, whereas expression of PAR-4 promoted apoptosis. This suggests that PKCζ counteracts apoptosis, unless its ceramide-induced activation is compromised by binding to PAR-4. A luciferase reporter assay showed that ceramide analogs activate nuclear factor (NF)-κB unless PAR-4-dependent inhibition of PKCζ suppresses NF-κB activation. Taken together, our results show that direct physical association with ceramide and PAR-4 regulates the activity of PKCζ. They also indicate that this interaction regulates the activity of glycogen synthase kinase-3β and NF-κB.

PAR-4 Is Involved in Regulation of β-Secretase Cleavage of the Alzheimer Amyloid Precursor Protein

Mounting evidence indicates that aberrant production and aggregation of amyloid beta-peptide (Abeta)-(1-42) play a central role in the pathogenesis of Alzheimer disease (AD). Abeta is produced when amyloid precursor protein (APP) is cleaved by beta- and gamma-secretases at the N and C termini of the Abeta domain, respectively. The beta-secretase is membrane-bound aspartyl protease, most commonly known as BACE1. Because BACE1 cleaves APP at the N terminus of the Abeta domain, it catalyzes the first step in Abeta generation. PAR-4 (prostate apoptosis response-4) is a leucine zipper protein that was initially identified to be associated with neuronal degeneration and aberrant Abeta production in models of AD. We now report that the C-terminal domain of PAR-4 is necessary for forming a complex with the cytosolic tail of BACE1 in co-immunoprecipitation assays and in vitro pull-down experiments. Overexpression of PAR-4 significantly increased, whereas silencing of PAR-4 expression by RNA interference significantly decreased, beta-secretase cleavage of APP. These results suggest that PAR-4 may be directly involved in regulating the APP cleavage activity of BACE1. Because the increased BACE1 activity observed in AD patients does not seem to arise from genetic mutations or polymorphisms in BACE1, the identification of PAR-4 as an endogenous regulator of BACE1 activity may have significant implications for developing novel therapeutic strategies for AD.

Absence of prostate apoptosis response-4 protein in substantia nigra of Parkinson's disease autopsies.

The morphological evidence for apoptosis of dopaminergic neurons in Parkinson's disease (PD) remains a conflicting issue. The present study examined autopsy material containing substantia nigra and putamen of PD ( n=7) and control subjects ( n=5) for the expression of prostate apoptosis response-4 (Par-4) protein, a protein expressed by apoptotic cells. Par-4 was not detected in the substantia nigra pars compacta. By contrast, duodenal enterocytes, which served as positive controls and are known to be apoptotic, profoundly expressed Par-4. The present study is in agreement with previous studies of the substantia nigra pars compacta in PD, which failed to detect molecules expressed by apoptotic cells. The absence of Par-4 immunoreactivity suggests that death of dopaminergic neurons in PD follows a degenerative pathway that circumvents the induction of Par-4.

Regulation of Apoptosis during Neuronal Differentiation by Ceramide and b-Series Complex Gangliosides

Lipid analysis of gestational day E14.5 mouse brain revealed elevation of ceramide to a tissue concentration that induced apoptosis when added to the medium of neuroprogenitor cells grown in cell culture. Elevation of ceramide was coincident with the first appearance of b-series complex gangliosides (BCGs). Expression of BCGs by stable transfection of murine neuroblastoma (F-11) cells with sialyltransferase-II (ST2) resulted in a 70% reduction of ceramide-induced apoptosis. This was most likely due to an 80% reduced expression of prostate apoptosis response-4 (PAR-4). PAR-4 expression and apoptosis were restored by preincubation of ST2-transfected cells with N-butyl deoxinojirimycin (NB-DNJ) or PD98059, two inhibitors of ganglioside biosynthesis or p42/44 mitogen-activated protein (MAPK) kinase, respectively. In sections of day E14.5 mouse brain, the intermediate zone showed intensive staining for complex gangliosides, but only low staining for apoptosis (TUNEL) and PAR-4. Apoptosis and PAR-4 expression, however, were elevated in the ventricular zone which only weakly stained for complex gangliosides. Whole cell patch clamping revealed a 2-fold increased calcium influx in ST2-transfected cells, the blocking of which with nifedipine restored apoptosis to the level of untransfected cells. In serum-free culture, supplementation of the medium with IGF-1 was required to maintain MAPK phosphorylation and the anti-apoptotic effect of BCG expression. BCG-enhanced calcium influx and the presence of insulin-like growth factor-1 may thus activate a cell survival mechanism that selectively protects developing neurons against ceramide-induced apoptosis by up-regulation of MAPK and reduction of PAR-4 expression.