Intrinsic Apoptosis Signaling Pathways Research Articles

Depletion of the receptor for advanced glycation end products (RAGE) sensitizes towards apoptosis via p53 and p73 posttranslational regulation

The receptor for advanced glycation endproduct (RAGE) is involved in diabetic complications and chronic inflammation, conditions known to affect the sensitivity towards apoptosis. Here, we studied the effect of genetically depleting RAGE on the susceptibility towards apoptosis. In murine osteoblastic cells, RAGE knockout increased both spontaneous and induced apoptosis. Decreased levels of B-cell lymphoma 2 protein and increased intrinsic apoptosis were observed in Rage(-/-) cells. Furthermore, loss of RAGE increased expression of the death receptor CD95 (Fas, Apo-1), CD95-dependent caspase activation and extrinsic apoptosis, whereas NF-kB-p65 nuclear translocation was diminished. Importantly, depletion of RAGE reduced the ubiquitination and degradation of p53 and p73 and increased their nuclear translocation. The increase of p53 and p73 transactivational activity was essential for the RAGE-dependent regulation of apoptosis, because knockdown of p53 and p73 significantly decreased apoptosis in RAGE-deficient but not in RAGE-expressing cells. Thus, the RAGE-mediated posttranslational regulation of p53 and p73 orchestrates a sequence of events culminating in control of intrinsic and extrinsic apoptosis signaling pathways.

Identification of Chemosensitivity Nodes for Vinblastine through Small Interfering RNA High-Throughput Screens

Discovering chemosensitivity pathways or nodes is an attractive strategy for formulating new drug combinations for cancer. Microtubules are among the most successful anticancer drug targets. Therefore, we implemented a small interfering RNA (siRNA) synthetic lethal screen targeting 5520 unique druggable genes to identify novel chemosensitivity nodes for vinblastine, a microtubule-destabilizing agent used clinically. We transiently transfected human glioblastoma cells with siRNAs for 48 h and then treated cells with a sublethal concentration of vinblastine. Forty-eight hours later, we analyzed cell viability and, using a series of statistical methods, identified 65 gene products that, when suppressed, sensitized glioblastoma cells to vinblastine. After completion of the secondary assays, we focused on one siRNA, B-cell lymphoma extra large (BCL-xL), because of its role in the intrinsic apoptosis signaling pathway as well as the availability of pharmacological inhibitors. We found that nontoxic concentrations of 4-[4-[[2-(4-chlorophenyl)-5,5-dimethylcyclohexen-1-yl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-morpholin-4-yl-1-phenylsulfanylbutan-2-yl]amino]-3-(trifluoromethylsulfonyl)phenyl]sulfonylbenzamide (ABT-263), an inhibitor of the BCL-2 family members (BCL-2, BCL-xL, and BCL-w), sensitized glioblastoma and non-small-cell lung cancer cells to vinblastine and induced apoptosis through the intrinsic cell death pathway. These results illustrate the usefulness of unbiased siRNA screens as a method for identifying potential novel anticancer therapeutic combinations.

Upregulation of Cellular Bcl-2 by the KSHV Encoded RTA Promotes Virion Production

Apoptosis of virus infected cells can restrict or dampen full blown virus propagation and this can serve as a protective mechanism against virus infection. Consequently, viruses can also delay programmed cell death by enhancing the expression of anti-apoptotic proteins. Human Bcl-2 is expressed on the surface of the mitochondrial membrane and functions as the regulator of the delicate balance between cell survival and apoptosis. In this report, we showed that the replication and transcription activator (RTA) encoded by KSHV ORF 50, a key regulator for KSHV reactivation from latent to lytic infection, upregulates the mRNA and protein levels of Bcl-2 in 293 cells, and TPA-induced KSHV-infected cells. Further analysis revealed that upregulation of the cellular Bcl-2 promoter by RTA is dose-dependent and acts through targeting of the CCN9GG motifs within the Bcl-2 promoter. The Bcl-2 P2 but not the P1 promoter is primarily responsive to RTA. The results of ChIP confirmed the direct interaction of RTA protein with the CCN9GG motifs. Knockdown of cellular Bcl-2 by lentivirus-delivered small hairpin RNA (shRNA) resulted in increased cell apoptosis and decreased virion production in KSHV-infected cells. These findings provide an insight into another mechanism by which KSHV utilizes the intrinsic apoptosis signaling pathways for prolonging the survival of lytically infected host cells to allow for maximum production of virus progeny.

Protein protein interaction of p53 and p73 with YY1 in human hepatocellular carcinoma

Introduction: We have previously described a relevant role of the p53 family members in tumorigenesis, therapeutic response and prognosis of hepatocellular carcinoma (HCC). Since protein interaction networks play a key role in signaling pathways of different tumor entities we used the LUMIER-assay (LUminescence-based Mammalian IntERactome mapping) which is a high-throughput technology to identify protein protein interactions of the p53 family in HCC. Methods and results: First we established the LUMIER-assay in Hep3B cells to be able to map protein interaction networks in liver cells and apply it to the p53 and p73 pathway. A set of relevant proteins from extrinsic and intrinsic apoptosis signaling pathways, cell cycle and proliferation control was selected using the Ingenuity Pathway Analysis System and the BIOGRID database. Selected proteins were transiently expressed in Hep3B cells using GATEWAY-compatible ORF clones. Positive interactions were further analysed and verified by co-immunoprecipitations.Among the protein protein interactions newly identified, the interaction of p53 with YY1 (Ying Yang 1) and the interaction of p73 with YY1 was most notable. YY1 is known as a transcription factor and as a negative regulator of p53. YY1 overexpression stimulates p53 ubiquitination and degradation through direct physical interaction with p53 and Hdm2. The direct interaction of YY1 and p73 has not been described so far and may be relevant for hepatocarcinogenesis. We are investigating the influence of YY1 on p73 levels and induction of apoptosis of HCC cells. Conclusion: We have adapted LUMIER technology to map protein protein interactions of p53 and p73 in hepatocellular carcinoma cell lines. First results have revealed so far undescribed direct interaction of YY1 and p73 which plays a relevant role in hepatocarcinogenesis.

8주간 운동이 생쥐의 gastrocnemius에서 Bcl-2, Bax, caspase-8, caspase-3와 HSP70에 미치는 영향

The aim of this study was to investigate the effects of exercise on intrinsic and extrinsic apoptosis signaling pathways in skeletal muscle. ICR-type white male mice were divided into a control group (CON: n=10) and an exercise training group (EX: n=10) after a 1 week adaptation period. EX per-formed treadmill running at 16.4 m/min with a 4% incline, 40 min/day and 5 days/week for 8 weeks. Cervical dislocation was performed at 48 hours after the last bout of exercise, after which gas-trocnemius skeletal muscles were immediately collected. The results of verifying the intrinsic apopto-sis pathway showed that there were no significant differences in Bcl-2, Bax, or the ratio of Bax/Bcl-2 proteins between EX and CON. On the other hand, the results of verifying the extrinsic apoptosis pathway showed that caspase-8 proteins were significantly lower in EX than in CON (p<0.05). Apoptosis suppressing protein HSP70 was higher in EX than in CON. In addition, caspase-3, which is the final factor for apoptosis, was not activated. These results indicate that apoptosis did not devel-op since caspase-3 is non-cleaved by the effects of caspase-8 and HSP70 extrinsic pathways rather than Bcl-2 and Bax intrinsic pathways among signal pathways for apoptosis.Keywords : Exercise, Bcl-2, Bax, caspase-8, caspase-3, HSP70

Differentiation and neural integration of hippocampal neuronal progenitors: Signaling pathways sequentially involved

In the context of their potential implication in regenerative strategies, we characterized cell mechanisms underlying the fate of embryonic rat hippocampal H19-7 progenitors in culture upon induction of their differentiation, and tested their capacities to integrate into a neuronal network in vitro. Without addition of growth factors, nearly 100% of cells expressed various neuronal markers, with a progressive rise of the expression of Synapsin I and II, suggesting that cells developed as mature neurons with synaptogenic capacities. Fully differentiated neurons were identified as glutamatergic and expressed the receptor-associated protein PSD-95. Quantification of ATP showed that 60% of cells died within 24 h after differentiation. Cell death was shown to imply Erk1/2-dependent intrinsic mitochondrial apoptosis signaling pathway, with activation of caspase-9 and -3, finally leading to single-strand DNA. Surviving neurons displayed high levels of Akt, phospho-Akt, and antiapoptotic proteins such as Bcl-2 and Bcl-XL, with decreased caspase activation. In the absence of trophic support, the proapoptotic death-associated protein (DAP) kinase was dramatically stimulated by 24 h postdifferentiation, along with increased levels of p38 and phospho-p38, and caspase reactivation. These findings show that different signaling pathways are sequentially triggered by differentiation, and highlight that ultimate cell death would involve p38 and DAP kinase activation. This was supported by the improvement of cell survival at 24-h postdifferentiation when cells were treated by PD169316, a specific inhibitor of p38. Finally, when seeded on rat hippocampal primary cultured neurons, a significant number of differentiated H19-7 cells were able to survive and to develop cell-cell communication.

ΔNp73β is oncogenic in hepatocellular carcinoma by blocking apoptosis signaling via death receptors and mitochondria

Background & Aims: p73 belongs to the p53 family of transcription factors known to regulate cell cycle and apoptosis. The Trp73 gene has two promoters that drive the expression of two major p73 isoform subfamilies: TA and ΔN. In general, TAp73 isoforms show proapoptotic activities, whereas members of the N-terminally truncated (ΔN) p73 subfamily that lack the transactivation domain show antiapoptotic functions. We found that upregulation of ΔNp73 in hepatocellular carcinoma (HCC) correlated with reduced survival. Here, we investigated the molecular mechanisms accounting for the oncogenic role of ΔNp73 in HCC. Results: ΔNp73ß can directly interfere with the transcriptional activation function of the TA (containing the transactivation domain) isoforms of the p53 family and consequently inhibit transactivation of proapoptotic target genes. Interference of ΔNp73ß with apoptosis-/chemosensitivity takes place at several levels of apoptosis signaling. ΔNp73ß negatively regulates the genes encoding for the death receptors CD95, TNF-R1, TRAIL-R2 and TNFRSF18. Furthermore, ΔNp73β represses the genes encoding caspase-2, -3, -6, -8 and -9. Concomitantly, ΔNp73ß inhibits apoptosis emanating from mitochondria. Conclusions: Thus, ΔNp73 expression in HCC selects against both the death receptor and the mitochondrial apoptosis activity of the TA isoforms. Our data suggest that ΔNp73 isoforms repress apoptosis-related genes of the extrinsic and intrinsic apoptosis signaling pathways thereby contributing to chemoresistance. The clinical importance of these data is evidenced by our finding that the ΔNp73ß target gene signature can predict the prognosis of patients suffering from HCC.

Dominant negative (ΔN) p63α induces drug resistance in hepatocellular carcinoma by interference with apoptosis signaling pathways

p63 belongs to the family of p53-related transcription factors expressing a variety of isoforms. The Trp63 gene has two promoters that drive the expression of two major p63 isoform subfamilies. Isoforms of the TAp63 subfamily show pro-apoptotic activities, whereas members of the N-terminally truncated (ΔN) p63 subfamily have anti-apoptotic functions. We have previously shown an important role for TAp63α in the induction of apoptosis and chemosensitivity of hepatocellular carcinoma (HCC). Here, we investigated the molecular mechanisms accounting for the oncogenic role of ΔNp63α in HCC. ΔNp63α can directly interfere with the transcriptional activation function of the TA (containing the transactivation domain) isoforms of the p53 family and consequently inhibit transactivation of pro-apoptotic target genes. ΔNp63α negatively regulates the genes encoding for the death receptor CD95 and the pro-apoptotic Bcl-2 family member BAX. Thus, ΔNp63α expression in HCC interferes with both the death receptor and the mitochondrial apoptosis activity of the TA isoforms. In addition and of clinical relevance, ΔNp63α inhibits activation of p53 family target genes and apoptosis induced by chemotherapeutic drugs. Chemotherapeutic treatment induces expression of Bax, Bim, Noxa, Puma and Perp; this is antagonized by ΔNp63α. Our data suggest that the ΔNp63α isoform represses apoptosis-related genes of the extrinsic and intrinsic apoptosis signaling pathways, thereby contributing to chemoresistance of HCC.

Activation of the receptor for parathyroid hormone and parathyroid hormone related protein induces apoptosis via the extrinsic and intrinsic signaling pathway

Parathyroid hormone (PTH) is the primary regulator of serum calcium homeostasis and bone metabolism. PTH acts primarily by binding to its receptor, PTH1R, in the bone and kidney. In addition to PTH, PTH1R also recognizes PTH-related peptide (PTHrP), a paracrine/autocrine factor originally described as the hormone responsible for hypercalcemia of malignancy. PTHrP is developmentally regulated and expressed, and it has been shown to play a physiological role in development, differentiation, cell proliferation and survival. We investigated the effects of PTH1R activation on the apoptosis signaling programs of human embryonic kidney (HEK) cells. Stimulation experiments of the CD95, TNF-R and TRAIL-R death receptor systems revealed that activation of PTH1R in HEK cells triggers signaling via each of these death receptors. Furthermore, our findings demonstrate a link between activation of PTH1R and the mitochondrial apoptosis pathway. PTHR1R overexpression led to an alteration of the mitochondrial membrane potential and activation of the intrinsic apoptosis signaling pathway. Our data indicate that activation of PTH1R engages major apoptosis signaling pathways by inducing signaling via death receptors and mitochondria in HEK cells. Thus, beyond its importance in development and differentiation, we describe an important role for the PTH/PTHrP receptor system in apoptosis of differentiating/embryonic cells.

Siva-1 Promotes K-48 Polyubiquitination of TRAF2 and Inhibits TCR-Mediated Activation of NF-kappaB

The proapoptotic protein Siva-1 plays an important role in some of the extrinsic and intrinsic apoptosis signaling pathways in cancer cells. Previously, we showed that Siva-1 inhibited the activity of the prosurvival transcription factor NF-kappaB. In the present study, upon TCR cross-linking of Jurkat T leukemia cells, we demonstrated that the inhibitory target of Siva-1 is upstream of the IKK complex in the NF-kappaB signaling pathway. Additionally, Siva-1 also suppressed the activity of another crucial transcription factor AP-1, and a common mediator of both these pathways is the adaptor protein TRAF2. Further, we observed that Siva-1 indeed interacted with TRAF2 and negatively regulated its activity by promoting K48-hnked polyubiquitination. Siva-1 specifically interacted with the ring finger domain of TRAF2, which is essential for its E3 hgase activity and its ability to subsequently activate NF-kappaB. TCR cross-linking of Jurkat T cells that lacked Siva-1 revealed significantly lowered K48- but elevated K63-ubiquitinated TRAF2 levels upon TCR cross-linking, suggesting that the differential pattern of ubiquitination in these cells essentially contributed to a robust and sustained activation of NF-kappaB. The above results demonstrated an important role for endogenous Siva-1 in negatively regulating NF-kappaB activation by targeting TRAF2.