Regulation of rDNA Transcription by Proto-Oncogene PELP1

Proline-, glutamic acid-, and leucine-rich protein (PELP1) is a novel nuclear receptor coregulator with a multitude of functions. PELP1 serves as a scaffolding protein that couples various signaling complexes with nuclear receptors and participates as a transcriptional coregulator. Recent data suggest that PELP1 expression is deregulated in hormonal cancers, and that PELP1 functions as a proto-oncogene; however, the mechanism by which PELP1 promotes oncogenesis remains elusive.Using pharmacological inhibitors, confocal microscopy and biochemical assays, we demonstrated that PELP1 is localized in the nucleolus and that PELP1 is associated with the active ribosomal RNA transcription. Cell synchronization studies showed that PELP1 nucleolar localization varies and the greatest amount of nucleolar localization was observed during S and G2 phases. Using pharmacological compounds and CDK site mutants of PELP1, we found that CDK's activity plays an important role on PELP1 nucleolar localization. Depletion of PELP1 by siRNA decreased the expression of pre-rRNA. Reporter gene assays using ribosomal DNA (pHrD) luc-reporter revealed that PELP1WT but not PELP1MT enhanced the expression of reporter. Deletion of nucleolar domains abolished PELP1-mediated activation of the pHrD reporter. ChIP analysis revealed that PELP1 is recruited to the promoter regions of rDNA and is needed for optimal transcription of ribosomal RNA.Collectively, our results suggest that proto-oncogene PELP1 plays a vital role in rDNA transcription. PELP1 modulation of rRNA transcription, a key step in ribosomal biogenesis may have implications in PELP1-mediated oncogenic functions

Reticulocalbin 3 Is a Novel Mediator of Glioblastoma Progression

Glioblastoma is the most common malignant primary brain tumor. Molecular mechanisms underlying the pathobiology of glioblastoma are incompletely understood, emphasizing an unmet need for the identification of new therapeutic candidates. Reticulocalbin 3 (RCN3), an ER lumen-residing Ca2+ binding protein, plays an essential role in protein biosynthesis processes via the secretory pathway. Emerging studies demonstrated that RCN3 is a target for therapeutic intervention in various diseases. However, a knowledge gap exists about whether RCN3 plays a role in glioblastoma. Publicly available datasets suggest RCN3 is overexpressed in glioblastoma and portends poor survival rates. The knockdown or knockout of RCN3 using shRNA or CRISPR/Cas9 gRNA, respectively, significantly reduced proliferation, neurosphere formation, and self-renewal of GSCs. The RNA-seq studies showed downregulation of genes related to translation, ribosome, and cytokine signaling and upregulation of genes related to immune response, stem cell differentiation, and extracellular matrix (ECM) in RCN3 knockdown cells. Mechanistic studies using qRT-PCR showed decreased expression of ribosomal and increased expression of ER stress genes. Further, in silico analysis of glioblastoma patient datasets showed RCN3 expression correlated with the ribosome, ECM, and immune response pathway genes. Importantly, the knockdown of RCN3 using shRNA significantly enhanced the survival of tumor-bearing mice in orthotopic glioblastoma models. Our study suggests that RCN3 could be a potential target for the development of a therapeutic intervention in glioblastoma

Development and Characterization of Inducible Astrocyte-Specific Aromatase Knockout Mice

17β-estradiol (E2) is produced in the brain as a neurosteroid, in addition to being an endocrine signal in the periphery. The current animal models for studying brain-derived E2 include global and conditional non-inducible knockout mouse models. The aim of this study was to develop a tamoxifen (TMX)-inducible astrocyte-specific aromatase knockout mouse line (GFAP-ARO-iKO mice) to specifically deplete the E2 synthesis enzymes and aromatase in astrocytes after their development in adult mice. The characterization of the GFAP-ARO-iKO mice revealed a specific and robust depletion in the aromatase expressions of their astrocytes and a significant decrease in their hippocampal E2 levels after a GCI. The GFAP-ARO-iKO animals were alive and fertile and had a normal general brain anatomy, with a normal astrocyte shape, intensity, and distribution. In the hippocampus, after a GCI, the GFAP-ARO-iKO animals showed a major deficiency in their reactive astrogliosis, a dramatically increased neuronal loss, and increased microglial activation. These findings indicate that astrocyte-derived E2 (ADE2) regulates the ischemic induction of reactive astrogliosis and microglial activation and is neuroprotective in the ischemic brain. The GFAP-ARO-iKO mouse models thus provide an important new model to help elucidate the roles and functions of ADE2 in the brain

Brain-Derived Estrogen and Neurological Disorders

Astrocytes and neurons in the male and female brains produce the neurosteroid brain-derived 17β-estradiol (BDE2) from androgen precursors. In this review, we discuss evidence that suggest BDE2 has a role in a number of neurological conditions, such as focal and global cerebral ischemia, traumatic brain injury, excitotoxicity, epilepsy, Alzheimer’s disease, and Parkinson’s disease. Much of what we have learned about BDE2 in neurological disorders has come from use of aromatase inhibitors and global aromatase knockout mice. Recently, our group developed astrocyte- and neuron-specific aromatase knockout mice, which have helped to clarify the precise functions of astrocyte-derived 17β-estradiol (ADE2) and neuron-derived 17β-estradiol (NDE2) in the brain. The available evidence to date suggests a primarily beneficial role of BDE2 in facilitating neuroprotection, synaptic and cognitive preservation, regulation of reactive astrocyte and microglia activation, and anti-inflammatory effects. Most of these beneficial effects appear to be due to ADE2, which is induced in most neurological disorders, but there is also recent evidence that NDE2 exerts similar beneficial effects. Furthermore, in certain situations, BDE2 may also have deleterious effects, as recent evidence suggests its overproduction in epilepsy contributes to seizure induction. In this review, we examine the current state of this quickly developing topic, as well as possible future studies that may be required to provide continuing growth in the field

Neuron-Derived Estrogen—A Key Neuromodulator in Synaptic Function and Memory

In addition to being a steroid hormone, 17β-estradiol (E2) is also a neurosteroid produced in neurons in various regions of the brain of many species, including humans. Neuron-derived E2 (NDE2) is synthesized from androgen precursors via the action of the biosynthetic enzyme aromatase, which is located at synapses and in presynaptic terminals in neurons in both the male and female brain. In this review, we discuss evidence supporting a key role for NDE2 as a neuromodulator that regulates synaptic plasticity and memory. Evidence supporting an important neuromodulatory role of NDE2 in the brain has come from studies using aromatase inhibitors, aromatase overexpression in neurons, global aromatase knockout mice, and the recent development of conditional forebrain neuron-specific knockout mice. Collectively, these studies demonstrate a key role of NDE2 in the regulation of synapse and spine density, efficacy of excitatory synaptic transmission and long-term potentiation, and regulation of hippocampal-dependent recognition memory, spatial reference memory, and contextual fear memory. NDE2 is suggested to achieve these effects through estrogen receptor-mediated regulation of rapid kinase signaling and CREB-BDNF signaling pathways, which regulate actin remodeling, as well as transcription, translation, and transport of synaptic proteins critical for synaptic plasticity and function

Global Genomic and Proteomic Analysis Identified Critical Pathways Modulated by Proto-Oncogene PELP1 in TNBC

The PELP1 oncogene is commonly overexpressed in many cancers, including triple negative breast cancer (TNBC). However, the mechanisms by which PELP1 contributes to TNBC progression are not well understood. To elucidate these mechanisms, we generated CRISPR-Cas9 mediated PELP1 knockout TNBC cell lines, and alterations in the proteome were examined using global data-independent acquisition mass spectrometry (DIA-MS). Further mechanistic studies utilized shRNA knockdown, Western blotting, and RNA-seq approaches. TCGA data sets were utilized for determining the status of PELP1 in TNBC patient tumors and for examining its correlation with ribosomal proteins. Global DIA-MS studies revealed that 127 proteins are upregulated while 220 proteins are downregulated upon PELP1-KO. Bioinformatic analyses suggested that the oncogenic activities of PELP1 involve regulation of expression of ribosomal proteins and ribosomal complexes. RNA-seq studies further suggested PELP1 modulates the functions of transcription factor c-Myc in TNBC. TCGA data confirmed PELP1 has high expression in TNBC patient tumors, and this high expression pattern correlates with c-Myc, a regulator of ribosomal proteins. Collectively, our global approach studies suggest that PELP1 contributes to TNBC progression by modulation of cell cycle, apoptosis, and ribosome biogenesis pathways

Brain-derived estrogen and neural function

Brain-derived estrogen (BDE2) produced by the enzyme aromatase has several important functions in the brain. Work using aromatase inhibitors, aromatase knockdown models, and conditional forebrain neuron-specific and astrocyte-specific aromatase knockout mouse models have provided evidence that BDE2 has a critical role in regulating synaptic function and plasticity, cognitive function, sexual differentiation and reproduction, socio-sexual behavior, and neuroprotection. [Display omitted] •Aromatase is localized at synaptic and non-synaptic locations in the brain.•Neuron-derived E2 functions as a neuromodulator to regulate plasticity, memory and behavior.•Neuron-derived E2 is also neuroprotective and anti-inflammatory.•Following brain injury or ischemia, astrocytes also produce E2.•Astrocyte-derived E2 regulates gliosis, neuroinflammation, and is neuroprotective. Although classically known as an endocrine signal produced by the ovary, 17β-estradiol (E2) is also a neurosteroid produced in neurons and astrocytes in the brain of many different species. In this review, we provide a comprehensive overview of the localization, regulation, sex differences, and physiological/pathological roles of brain-derived E2 (BDE2). Much of what we know regarding the functional roles of BDE2 has come from studies using specific inhibitors of the E2 synthesis enzyme, aromatase, as well as the recent development of conditional forebrain neuron-specific and astrocyte-specific aromatase knockout mouse models. The evidence from these studies support a critical role for neuron-derived E2 (NDE2) in the regulation of synaptic plasticity, memory, socio-sexual behavior, sexual differentiation, reproduction, injury-induced reactive gliosis, and neuroprotection. Furthermore, we review evidence that astrocyte-derived E2 (ADE2) is induced following brain injury/ischemia, and plays a key role in reactive gliosis, neuroprotection, and cognitive preservation. Finally, we conclude by discussing the key controversies and challenges in this area, as well as potential future directions for the field

CDKs regulate PELP1 nucleolar localization.

<p>(<b>A</b>) ZR-75 cells at sub confluence were fixed and the localization of ^phos991PELP1was analyzed by using confocal microscopy. (<b>B</b>) Localization of GST-tagged PELP1 or PELP1 S991AMT or PELP1 S991EMT were visualized by using confocal microscopy with the antibody against the GST epitope in ZR-75 cells. (<b>C</b>) PELP1 was visualized in ZR-75 cells after serum starved for two days and released with 10% serum in absence or presence of roscovitine (10 µM) by using confocal microscopy. (<b>D</b>) 293T cells were transfected with pHrD luciferase reporter vector along with PELP1 WT or PELP1 S991AMT or PELP1 S991EMT expressing vectors. Cells were serum starved for 24 h and then stimulated with 10% serum for 24 h and the reporter gene activity was measured. Results are the average of 3 independent experiments. *<i>p-value </i><0.05.</p