Sushil Devkota

Pierce1, a novel p53 target gene contributing to the ultraviolet-induced DNA damage response.

Retinoblastoma (Rb) and p53 genes are mutated or inactivated in most human cancers and mutually regulate each other. Recently, we reported that expression of diverse genes was altered in Rb-deficient mouse embryonic fibroblasts (MEF). In this study, we found that Pierce1, a novel transcript upregulated in Rb-deficient MEFs, is a transcriptional target of p53. Although Pierce1 promoter did not respond to the ectopic expression of E2F1, it was strongly activated by p53 via 2 cis-elements. Consistently, the expression of Pierce1 was induced by genotoxic stresses that activate p53 but was not detected in p53-deficient MEFs. Pierce1 was posttranslationally stabilized by ultraviolet C (UVC) irradiation, and UVC-activated ATR (ataxia telangiectasia-mutated and Rad3-related) signaling suppressed proteosomal degradation of Pierce1 protein. Furthermore, knockdown of Pierce1 compromised the checkpoint response of wild-type MEFs to UVC irradiation, accompanying the diminished expression of p53 target genes. Together, our data suggest that Pierce1 is an important p53 target gene contributing to normal DNA damage response and may play crucial roles in maintaining genomic integrity against genotoxic stresses, including UVC irradiation.

Ei24-deficiency attenuates protein kinase Cα signaling and skin carcinogenesis in mice

Etoposide-induced gene 24 (Ei24) is a p53 target gene that inhibits growth, induces apoptosis and autophagy, as well as suppresses breast cancer. To evaluate the role of Ei24 in in vivo tumorigenesis, we generated an Ei24-deficient mouse model. Here, we report that, although Ei24 homozygous knockout mice are embryonic lethal, Ei24 heterozygous null mice are attenuated to DMBA/TPA-induced carcinogenesis with regard to the number and size of tumors but not the incidence. Ei24 contains a functional consensus motif, named as an R motif that is highly analogous to amino acids 105-110 of RINCK1, an E3 ligase for protein kinase C (PKC) proteins. We found that Ei24 stabilizes PKCαvia RINCK degradation and competition with RINCK for binding with the C1a domain of PKCα. We also found that Ei24 contributes to PKCα-mediated transactivation of EGFR by promoting PKCα membrane localization and interaction with EGFR. Finally, using Oncomine database we show that Ei24 and EGFR are upregulated in some subsets of human HNSCC. These results suggest that Ei24 is a regulator of the RINCK1-PKCα-EGFR signaling pathway in the development of skin-cancer.

Functional characterization of EI24-induced autophagy in the degradation of RING-domain E3 ligases

ABSTRACT Historically, the ubiquitin-proteasome system (UPS) and autophagy pathways were believed to be independent; however, recent data indicate that these pathways engage in crosstalk. To date, the players mediating this crosstalk have been elusive. Here, we show experimentally that EI24 (EI24, autophagy associated transmembrane protein), a key component of basal macroautophagy/autophagy, degrades 14 physiologically important E3 ligases with a RING (really interesting new gene) domain, whereas 5 other ligases were not degraded. Based on the degradation results, we built a statistical model that predicts the RING E3 ligases targeted by EI24 using partial least squares discriminant analysis. Of 381 RING E3 ligases examined computationally, our model predicted 161 EI24 targets. Those targets are primarily involved in transcription, proteolysis, cellular bioenergetics, and apoptosis and regulated by TP53 and MTOR signaling. Collectively, our work demonstrates that EI24 is an essential player in UPS-autophagy crosstalk via degradation of RING E3 ligases. These results indicate a paradigm shift regarding the fate of E3 ligases.

Ei24, a Novel E2F Target Gene, Affects p53-independent Cell Death upon Ultraviolet C Irradiation

Background: Rb loss deregulates E2F and thus induces the expressions of E2F target genes. Results: Ei24 is an E2F target up-regulated in Rb−/− MEFs and affects susceptibility of p53-deficient MEFs against UVC. Conclusion: E2F1 can provide a p53-independent modulation of cellular sensitivity against UVC via Ei24. Significance: EI24 may be a potential therapeutic target for treating p53-deficient tumors. The deficiency of retinoblastoma (Rb) gene deregulates E2F transcription factors and thus induces E2F target genes directly or p53 target genes indirectly via mouse p19Arf (or p14ARF in humans), an E2F target gene. Here, we identified that etoposide-induced 2.4 mRNA (Ei24)/p53-induced gene 8 (Pig8), a p53 target gene involved in apoptosis and autophagy, was up-regulated in Rb−/− mouse embryonic fibroblasts (MEFs). The Ei24 promoter was activated by E2F1 via multiple E2F-responsive elements, independently of the previously reported p53-responsive element. Chromatin immunoprecipitation assays revealed that E2F1 directly acts on the mouse Ei24 promoter. We observed that Ei24 expression was suppressed in p53−/− MEFs upon UVC irradiation, which was exacerbated in p53−/− E2f1−/− MEFs, supporting the positive role of E2F1 on Ei24 transcription. Furthermore, Ei24 knockdown sensitized p53−/− MEFs against UVC irradiation. Together, our data indicate that Ei24 is a novel E2F target gene contributing to the survival of p53-deficient cells upon UVC irradiation and thus may have a potential significance as a therapeutic target of certain chemotherapy for treating p53-deficient tumors.

Reactivation of Tert in the medial prefrontal cortex and hippocampus rescues aggression and depression of Tert(-/-) mice

The role of telomerase reverse transcriptase (TERT) has been extensively investigated in the contexts of aging and cancer. Interestingly, Tert−/− mice exhibit additional but unexpected aggressive and depressive behaviors, implying the potential involvement of TERT function in mood control. Our conditional rescue experiments revealed that the depressive and aggressive behaviors of Tert−/− mice originate from Tert deficiency in two distinct brain structures. Reactivation of Tert in the hippocampus was sufficient to normalize the depressive but not the aggressive behaviors of Tert−/− mice. Conversely, re-expression of Tert in the medial prefrontal cortex (mPFC) reversed the aggressive but not the depressive behavior of Tert−/− mice. Mechanistically, decreased serotonergic signaling and increased nitric oxide (NO) transmission in the hippocampus transduced Tert deficiency into depression as evidenced by our observation that the infusion of a pharmacological agonist for serotonin receptor 1a (5-HTR1A) and a selective antagonist for neuronal NO synthase into the hippocampus successfully normalized the depressive behavior of Tert−/− mice. In addition, increased serotonergic transmission by the 5-HTR1A agonist in the mPFC was sufficient to rescue the aggressive behavior of Tert−/− mice. Thus, our studies revealed a novel function of TERT in the pathology of depression and aggression in a brain structure-specific manner, providing direct evidence for the contribution of TERT to emotional control.

Telomerase reverse transcriptase induces basal and amino acid starvation-induced autophagy through mTORC1

Telomerase is a reverse transcriptase that consists of the telomerase RNA component (TERC) and the reverse transcriptase catalytic subunit (TERT) and specializes in the elongation of telomere ends. New evidence suggests that beyond classical telomere maintenance, TERT also possesses telomere length-independent functions that are executed via interaction with other binding proteins. One such reported TERT-interacting proteins is mTOR, a master nutrient sensor that is upregulated in several cancers; however, the physiological implications of the TERT-mTOR interaction in normal cellular processes as well as in tumorigenesis are poorly understood. Here, we report that TERT inhibits the kinase activity of mTOR complex 1 (mTORC1) in multiple cell lines, resulting in the activation of autophagy under both basal and amino acid-deprived conditions. Furthermore, TERT-deficient cells display the inability to properly execute the autophagy flux. Functionally, TERT-induced autophagy provides a survival advantage to cells in nutrient-deprived conditions. Collectively, these findings support a model in which gain of TERT function modulates mTORC1 activity and induces autophagy, which is required for metabolic rewiring to scavenge the nutrients necessary for fueling cancer cell growth in challenging tumor microenvironments.

Hippocampal TERT Regulates Spatial Memory Formation through Modulation of Neural Development

Summary The molecular mechanism of memory formation remains a mystery. Here, we show that TERT, the catalytic subunit of telomerase, gene knockout (Tert−/−) causes extremely poor ability in spatial memory formation. Knockdown of TERT in the dentate gyrus of adult hippocampus impairs spatial memory processes, while overexpression facilitates it. We find that TERT plays a critical role in neural development including dendritic development and neuritogenesis of hippocampal newborn neurons. A monosynaptic pseudotyped rabies virus retrograde tracing method shows that TERT is required for neural circuit integration of hippocampal newborn neurons. Interestingly, TERT regulated neural development and spatial memory formation in a reverse transcription activity-independent manner. Using X-ray irradiation, we find that hippocampal newborn neurons mediate the modulation of spatial memory processes by TERT. These observations reveal an important function of TERT through a non-canonical pathway and encourage the development of a TERT-based strategy to treat neurological disease-associated memory impairment.