Calcium (Ca2+) plays an important role as a second messenger, transmitting the message of arrival of stimuli such as hormones and neurotransmitters to the intracellular system that carries out the cellular response to the stimulus. The universality of Ca2+ as an intracellular messenger depends on its enormous versatility. This versatility is exploited to control processes as diverse as fertilization, proliferation, development, learning and memory, contraction and secretion, and must be accomplished within the context of Ca2+ being highly toxic. Ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs) are Ca2+ -release channels located on intracellular membranes of the endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR) that perform essential functions as key targets of hormone/neurotransmitter action to initiate intracellular Ca2+ signals. The purpose of this project was to study the role of RyR2 in Ca2+ signaling in the NG115-401L neuronal cell line. siRNA transfection methods were employed to knockdown RyR2 expression levels in NG115-401L cells. We used reverse transcription and real-time PCR to evaluate RyR2 gene expression in transfected/untransfected cells. We also evaluated cytosolic Ca2+ changes induced by RyR activators or regulators, using fura-2 AM as the Ca2+ indicator. Successful RyR2 gene knockdown allowed us to carry out some initial experiments to characterize the specific roles played by the RyR2 receptor isoform. We examined cell responses to FK-506 under the condition of RyR2 knockdown, finding that RyR2 appears to confer selectivity to this response. Finally, the effects of siRNA transfection and FK-506 treatment on NG115-401L cell growth were evaluated. These experimental results may contribute to future studies of RyR2, and help develop novel treatments for RyR2-base d dysfunctional diseases.

Indiana University-Purdue University Indianapolis (IUPUI) / In response to different environmental stresses, phosphorylation of eIF2 (eIF2P) represses global translation coincident with preferential translation of ATF4. ATF4 is a transcriptional activator of the integrated stress response, a program of gene expression involved in metabolism, nutrient uptake, anti-oxidation, and the activation of additional transcription factors, such as CHOP/GADD153, that can induce apoptosis. Although eIF2P elicits translational control in response to many different stress arrangements, there are selected stresses, such as exposure to UV irradiation, that do not increase ATF4 expression despite robust eIF2P. In this study we addressed the underlying mechanism for variable expression of ATF4 in response to eIF2P during different stress conditions and the biological significance of omission of enhanced ATF4 function. We show that in addition to translational control, ATF4 expression is subject to transcriptional regulation. Stress conditions such as endoplasmic reticulum stress induce both transcription and translation of ATF4, which together enhance expression of ATF4 and its target genes in response to eIF2P. By contrast, UV irradiation represses ATF4 transcription, which diminishes ATF4 mRNA available for translation during eIF2∼P. eIF2P enhances cell survival in response to UV irradiation. However, forced expression of ATF4 and its target gene CHOP leads to increased sensitivity to UV irradiation. In this study, we also show that C/EBPβ is a transcriptional repressor of ATF4 during UV stress. C/EBPβ binds to critical elements in the ATF4 promoter resulting in its transcriptional repression. The LIP isoform of C/EBPβ, but not the LAP version is regulated following UV exposure and directly represses ATF4 transcription. Loss of the LIP isoform results in increased ATF4 mRNA levels in response to UV irradiation, and subsequent recovery of ATF4 translation, leading to enhanced expression of its target genes. Together these results illustrate how eIF2P and translational control, combined with transcription factors regulated by alternative signaling pathways, can direct programs of gene expression that are specifically tailored to each environmental stress.

Indiana University-Purdue University Indianapolis (IUPUI) / Mammalian target of rapamycin (mTOR) is a key regulator of protein synthesis. Dysregulation of mTOR signaling occurs in many human cancers and its inhibition causes arrest at the G1 cell cycle stage. However, mTOR’s impact on mitosis (M-phase) is less clear. Here, suppressing mTOR activity impacted the G2-M transition and reduced levels of M-phase kinase, Aurora A. mTOR inhibitors did not affect Aurora A mRNA levels. However, translational reporter constructs showed that mRNA containing a short, simple 5’-untranslated region (UTR), rather than a complex structure, is more responsive to mTOR inhibition. mTOR inhibitors decreased Aurora A protein amount whereas blocking proteasomal degradation rescues this phenomenon, revealing that mTOR affects Aurora A protein stability. Inhibition of protein phosphatase, PP2A, a known mTOR substrate and Aurora A partner, restored mTOR-mediated Aurora A abundance. Finally, a non-phosphorylatable Aurora A mutant was more sensitive to destruction in the presence of mTOR inhibitor. These data strongly support the notion that mTOR controls Aurora A destruction by inactivating PP2A and elevating the phosphorylation level of Ser51 in the “activation-box” of Aurora A, which dictates its sensitivity to proteasomal degradation. In summary, this study is the first to demonstrate that mTOR signaling regulates Aurora-A protein expression and stability and provides a better understanding of how mTOR regulates mitotic kinase expression and coordinates cell cycle progression. The involvement of mTOR signaling in the regulation of cell migration by its upstream activator, Rheb, was also examined. Knockdown of Rheb was found to promote F-actin reorganization and was associated with Rac1 activation and increased migration of glioma cells. Suppression of Rheb promoted platelet-derived growth factor receptor (PDGFR) expression. Pharmacological inhibition of PDGFR blocked these events. Therefore, Rheb appears to suppress tumor cell migration by inhibiting expression of growth factor receptors that in turn drive Rac1-mediate actin polymerization.