Biology subjects

Ganeshkumar, S.

Publications and source records attributed to Ganeshkumar, S..

5 recordsLinked to original sources

bioRxiv2025

Regulatory Role for Tumor Suppressor REST on Estrogen Receptor (ESR1) Expression and Leiomyoma Pathophysiology

Uterine fibroids, benign tumors of the smooth muscle layer of the uterus, plague approximately 80% of the female population by age 50. While there have been efforts to understand the mechanism behind this pathophysiology, it largely remains unclear. Lack of preclinical animal models that recapitulate aberrant steroid hormone pathways in UL has significantly hampered the development of long-term hormonal therapies for uterine fibroids. In addition, cultured myometrial as well as leiomyoma smooth muscle cells rapidly silence both estrogen receptor alpha (ESR1) and progesterone receptor (PGR) expression through unknown mechanisms, further limiting in vitro mechanistic studies of UL. Previous work by our lab has determined the loss of REST, a master regulator of epigenetic gene silencing, in leiomyoma results in the upregulation of ESR1 targets and therefore estrogen signaling. Using ChIP-PCR, we find REST is directly associated with ESR1 genomic locus, playing a role in its epigenetic regulation. ChIP-seq analysis of Rest cKO mouse uterus samples reveals a global role for REST in the regulation of progesterone receptor target genes and highlights alterations in PGR binding within the Esr1 locus. Additionally, we find REST inhibition of ESR1 expression is regulated through upstream WNT planar cell polarity molecule, PRICKLE1. Based on role of REST in silencing ESR1 expression in cultured myometrial cells, our results support the development of a potential cell culture method to maintain ESR1 expression through REST modulation. Finally, we establish a broad role for REST in epigenetic regulation relevant to leiomyoma pathophysiology.

cell biology↗

bioRxiv2024

Loss of PRICKLE1 in the myometrium leads to reduced fertility, abnormal myometrial architecture, and aberrant extracellular matrix deposition in mice.

Uterine leiomyomas (UL) are the most prevalent benign tumors of the female reproductive tract, originating from the myometrium and affecting over 75% of reproductive-age women. Symptoms of UL include pelvic pain, pressure, dysmenorrhea, menorrhagia, anemia, and reproductive dysfunction. Currently, there is no effective long-term pharmacotherapy for UL, making them the leading cause of hysterectomies in the United States. The lack of treatment options is attributed to the absence of accurate animal models and a limited understanding of UL pathogenesis. Previous research has shown the loss of repressor of element 1 silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) within the myometrium promotes UL pathogenesis. In addition, deletion of Rest in the mouse myometrium leads to a UL phenotype. PRICKLE1, also known as Rest-interacting LIM-domain Protein (RILP), is required for nuclear localization of REST and Wnt/planar cell polarity (PCP) signaling, making it a critical target for UL studies. In the context of PCP, smooth muscle cells in UL show abnormal organization, aberrant ECM structure, and expression levels, potentially influenced by PRICKLE1 loss. The exact role of PRICKLE1 and Wnt/PCP in UL pathogenesis remains unclear. To explore PRICKLE1s role in UL, we deleted Prickle1 using our myometrial-specific icre. Our findings demonstrate that Prickle1 loss in the myometrium results in a UL phenotype characterized by altered collagen expression, excessive extracellular matrix (ECM) deposition, aberrant smooth muscle cell organization, increased Esr1 and Pgr expression, and dysregulated Wnt/PCP signaling. This novel mouse model serves as a valuable preclinical tool for understanding UL pathogenesis and developing future pharmacotherapies.

physiology↗

bioRxiv2024

Loss of PRICKLE1 leads to abnormal endometrial epithelial architecture, decreased embryo implantation, and reduced fertility in mice.

Successful embryo implantation requires coordinated changes in the uterine luminal epithelium, including structural adaptations, apical-basal polarity shifts, intrauterine fluid resorption, and cellular communication. Planar cell polarity (PCP) proteins, essential for cell organization, are understudied in the context of uterine physiology and implantation. PRICKLE proteins, components of PCP, are suggested to play critical roles in epithelial polarization and tissue morphogenesis. However, their function in the polarized unicellular layer of endometrial epithelium, which supports embryo implantation, is unknown. We developed an endometrial epithelial-specific knockout (cKO) of mouse Prickle1 using Lactoferrin-iCre to investigate itss role in uterine physiology. Prickle1 ablation in the endometrial epithelium of mice resulted in decreased embryo implantation by gestational day 4.5 leading to lower fertility. Three-dimensional imaging of the uterus revealed abnormal luminal folding, impaired luminal closure, and altered glandular length in mutant uteri. Additionally, we observed decreased aquaporin-2 expression, disrupted cellular architecture, and altered E-Cadherin expression and localization in the mutant uterine epithelium. Evidence of epithelial-mesenchymal transition (EMT) was found within luminal epithelial cells, further linking PRICKLE1 loss to uterine pathologies. Furthermore, altered polarity of cell division leading to incomplete cytokinesis and increase in binuclear or multinucleated cells suggests a crucial role for PRICKLE1 in the maintenance of epithelial architecture. Our findings highlight PRICKLE1s critical role in the PCP pathway within the uterus, revealing its importance in the molecular and cellular responses essential for successful pregnancy and fertility. Significance StatementConservative cell division is essential to maintain apical-basal polarity and proper epithelial function in the uterus. Wnt/ Planar cell polarity signaling molecules are hypothesized to provide the spatial cues to organize unicellular, 2-dimensional sheet of epithelium in a plane orthogonal to the apical-basal polarity. Conditional ablation of Prickle1, a crucial Wnt/ PCP gene, in mouse uterine epithelium results in aberrant expression of epithelial cadherin, altered plane of cell division, incomplete cytokinesis leading to binucleated/ multinucleated cells, epithelial - mesenchymal transition, and defective implantation. Role of Prickle1 in maintaining symmetric uterine epithelial cell division and tissue architecture is unique among Wnt/PCP genes, including previously described mouse models for Vangl2, Ror2, and Wnt5a. Classification: Biological Sciences (Major) Cell Biology (Minor), Physiology (Minor) Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/605120v2_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@cfd16corg.highwire.dtl.DTLVardef@1be29aborg.highwire.dtl.DTLVardef@1a1b938org.highwire.dtl.DTLVardef@87138c_HPS_FORMAT_FIGEXP M_FIG Conditional ablation of Prickle1, a crucial Wnt/ PCP gene, in mouse uterine epithelium results in altered plane of cell division and incomplete cytokinesis leading to binucleated/multinucleated cells, epithelial - mesenchymal transition, altered gland length, and defective implantation. Some images adapted from BioRender.com (2024). C_FIG

physiology↗

bioRxiv2022

Development of allosteric, selective cyclin-dependent kinase 2 (CDK2) inhibitors that are negatively cooperative with cyclin binding and show potential as contraceptive agents

Compared to most ATP-site kinase inhibitors, small molecules that target an allosteric pocket have the potential for improved selectivity due to the often observed lower structural similarity at these distal sites. Despite their promise, relatively few examples of structurally confirmed, high-affinity allosteric kinase inhibitors exist. Cyclin-dependent kinase 2 (CDK2) is a target for many therapeutic indications, including non-hormonal contraception.1 However, an inhibitor against this kinase with exquisite selectivity has not reached the market because of the structural similarity between CDKs.1-2 In this paper, we describe the development and mechanism of action of new type III inhibitors that bind CDK2 with nanomolar affinity, making them the highest affinity, structurally confirmed allosteric CDK inhibitors reported. Notably, these anthranilic acid inhibitors exhibit a strong negative cooperative relationship with cyclin binding, which remains an underexplored mechanism for CDK2 inhibition. Furthermore, the binding profile of these compounds in both biophysical and cellular assays demonstrate the promise of this series for further development into a therapeutic selective for CDK2 over highly similar kinases like CDK1. The potential of these inhibitors as efficacious contraceptive agents is seen by incubation with mouse testicular explants, where they recapitulate Cdk2-/- and Spdya-/- phenotypes.

pharmacology and toxicology↗

bioRxiv2022

Loss of the repressor REST affects progesterone receptor function and promotes uterine leiomyoma pathogenesis

Uterine Leiomyomas (UL) are benign tumors that arise in the myometrial layer of the uterus. The standard treatment option for UL is hysterectomy, although hormonal therapies such as selective progesterone receptor modulators are often used as temporary treatment options to reduce symptoms or slow the growth of tumors. However, since the pathogenesis of UL is poorly understood and most hormonal therapies are not based on UL-specific, divergent hormone signaling pathways, hallmarks that predict long-term efficacy and safety of pharmacotherapies remain largely undefined. In a previous study, we reported aberrant expression of REST/NRSF target genes activate UL growth due to the near ubiquitous loss of REST. Here, we show that ablation of the Rest gene in mouse uterus leads to UL phenotype and gene expression patterns analogous to UL, including altered estrogen and progesterone signaling pathways. We demonstrate that many of the genes dysregulated in UL harbor cis-regulatory elements bound by REST and progesterone receptor (PGR) adjacent to each other. Crucially, we identify an interaction between REST and PGR in healthy myometrium and present a putative mechanism for the dysregulation of progesterone responsive genes in UL ensuing the loss of REST. Using three Rest conditional knockout mouse lines, we provide a comprehensive picture of the impact loss of REST has in UL pathogenesis and in altering the response of UL to steroid hormones. Significance statementAblation of Rest gene in the mouse uterus, modelling the loss REST in uterine fibroids, results in tumor formation and gene expression patterns analogous to human UL, including altered estrogen and progesterone receptor (PGR) pathways. The current study provides a putative mechanism for the aberrant function of PGR in UL.

physiology↗