Membrane-Localized Estrogen Receptor α Is Required for Normal Organ Development and Function

SummarySteroid receptors are found in discrete cellular locations, but it is unknown whether extranuclear pools are necessary for normal organ development. To assess this, we developed a point mutant estrogen receptor α (ERα) knockin mouse (C451A) that precludes palmitoylation and membrane trafficking of the steroid receptor in all organs. Homozygous knockin female mice (nuclear-only ERα [NOER]) show loss of rapid signaling that occurs from membrane ERα in wild-type mice. Multiple developmental abnormalities were found, including infertility, relatively hypoplastic uteri, abnormal ovaries, stunted mammary gland ductal development, and abnormal pituitary hormone regulation in NOER mice. These abnormalities were rescued in heterozygous NOER mice that were comparable to wild-type mice. mRNAs implicated in organ development were often poorly stimulated by estrogen only in homozygous NOER mice. We conclude that many organs require membrane ERα and resulting signal transduction to collaborate with nuclear ERα for normal development and function

Just when you thought it was safe to go into the membrane: the growing complexities of extra-nuclear progesterone signaling

The diversity of membrane-initiated progesterone actions has made characterization and establishment of its biological importance a complicated endeavor. A new study by Zuo and colleagues shows that progesterone via endogenous membrane progesterone receptor-α acts as a negative regulator of proliferation and epithelial to mesenchymal transition in a breast cancer cell line. These progesterone-mediated actions appear to be regulated through epidermal growth factor receptor and phosphatidylinositol 3-kinase signaling localized in caveolae. Moreover, the study shows expression of membrane progesterone receptor-α in benign and malignant breast cancer tissues. These data bring forth novel concepts with regard to progesterone actions in the breast; however, further work is warranted to fully characterize the physiologic actions of extra-nuclear progesterone signaling in the breast

A selective cyclic integrin antagonist blocks the integrin receptors α(v)β(3 )and α(v)β(5 )and inhibits retinal pigment epithelium cell attachment, migration and invasion

BACKGROUND: Proliferative vitreoretinopathy (PVR) is a leading cause of blindness after failed retinal reattachment surgery. PVR is characterized by the proliferation, migration and contraction of retinal pigmented epithelial cells (RPE), and these cellular responses are influenced by the expression and function of integrin receptors. The effect of a cyclic integrin antagonist containing the amino acid sequence Arg-Gly-Asp-D-Phe-Val (RGDfV), specific for the integrin receptors α(v)β(3 )and α(v)β(5), was investigated on basic fibroblast growth factor (bFGF), platelet derived growth factor-BB (PDGF-BB), and serum induced human RPE proliferation, migration, invasion and attachment to the extracellular matrix. Furthermore, the effects of bFGF and PDGF-BB regulated expression of integrins α(v)β(3 )and α(v)β(5 )on RPE cells was examined. METHODS: The effect of a cyclic integrin antagonist and a control peptide (0.01 μg/ml to 300 μg/ml) was investigated on serum or cytokine (bFGF or PDGF-BB pretreatment) induced human fetal RPE cell proliferation by H(3)-thymidine uptake. The effect of the cyclic integrin antagonist on RPE cell attachment onto different extracellular matrices (laminin, collagen IV, fibronectin), RPE cell invasion stimulated by PDGF-BB or serum, and migration stimulated by PDGF-BB, vascular endothelial growth factor (VEGF) or serum was explored. PDGF-BB and bFGF modulation of the integrin receptors α(v)β(3 )and α(v)β(5 )was evaluated by flow cytometry. RESULTS: The integrin antagonist did not inhibit DNA synthesis stimulated by serum, bFGF, or PDGF-BB treatment. RPE attachment onto fibronectin was inhibited in a concentration range of 1–10 μg/ml (p < 0.05). Attachment of the RPE cells onto collagen IV and laminin was inhibited in a range of 3–10 μg/ml (p < 0.05). Serum and PDGF-BB stimulated migration was inhibited by the cyclic integrin antagonist in a concentration range of 1–10 μg/ml (p < 0.05). Furthermore, the cyclic integrin antagonist inhibited PDGF-BB stimulated RPE cell invasion through fibronectin (3μg/ml: 66% inhibition, p < 0.001). In each of these experiments, the control peptides had no significant effects. PDGF-BB and bFGF pretreatment of RPE cells increased the expression of integrin receptors α(v)β(3 )(bFGF: 1.9 fold, PDGF-BB: 2.3 fold) and α(v)β(5 )(bFGF: 2.9 fold, PDGF-BB: 1.5 fold). CONCLUSION: A selective inhibition of the integrin receptors α(v)β(3 )and α(v)β(5 )through a cyclic integrin antagonist is able to inhibit RPE cell attachment, migration and invasion. Since these steps are of importance for the progression of PVR, a cyclic integrin antagonist should be further evaluated for the treatment of this disease

Software for the frontiers of quantum chemistry:An overview of developments in the Q-Chem 5 package

This article summarizes technical advances contained in the fifth major release of the Q-Chem quantum chemistry program package, covering developments since 2015. A comprehensive library of exchange–correlation functionals, along with a suite of correlated many-body methods, continues to be a hallmark of the Q-Chem software. The many-body methods include novel variants of both coupled-cluster and configuration-interaction approaches along with methods based on the algebraic diagrammatic construction and variational reduced density-matrix methods. Methods highlighted in Q-Chem 5 include a suite of tools for modeling core-level spectroscopy, methods for describing metastable resonances, methods for computing vibronic spectra, the nuclear–electronic orbital method, and several different energy decomposition analysis techniques. High-performance capabilities including multithreaded parallelism and support for calculations on graphics processing units are described. Q-Chem boasts a community of well over 100 active academic developers, and the continuing evolution of the software is supported by an “open teamware” model and an increasingly modular design

Nuclear receptors outside the nucleus: extranuclear signalling by steroid receptors

Steroid hormone receptors mediate numerous crucial biological processes and are classically thought to function as transcriptional regulators in the nucleus. However, it has been known for more than 50 years that steroids evoke rapid responses in many organs that cannot be explained by gene regulation. Mounting evidence indicates that most steroid receptors in fact exist in extranuclear cellular pools, including at the plasma membrane. This latter pool, when engaged by a steroid ligand, rapidly activates signals that affect various aspects of cellular biology. Research into the mechanisms of signalling instigated by extranuclear steroid receptor pools and how this extranuclear signalling is integrated with responses elicited by nuclear receptor pools provides novel understanding of steroid hormone signalling and its roles in health and disease

mTOR-mediated Leiomyoma Growth in Uterine-specific Tsc2-null Mice is Exclusively Dependent on Estradiol Signaling - Implications in the Treatment of Lymphangioleiomyomatosis (LAM)

Thesis (Ph. D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Pharmacology and Physiology, 2015.Lymphangioleiomyomatosis (LAM) is a devastating rare lung disease in which adenomas consisting of abnormal smooth-muscle cells grow within the lungs and progressively lead to loss of pulmonary function. The true origin of the LAM cell is unknown; however, important features provide us with clues. First, LAM cells contain inactivating mutations in genes encoding tuberous sclerosis complex (TSC) 1 or TSC2, proteins that limit cell growth through mammalian target of rapamycin complex1 (mTORC1) activity. Second, LAM cells appear to be metastatic. Third, LAM cells express estrogen and progesterone receptors as well as melanocytic-differentiation markers. Finally, LAM is found almost exclusively in women. Interestingly, LAM shares many features with uterine leiomyomas, benign tumors of myometrial cells. To test the hypothesis that LAM cells originate from uterine leiomyomas containing Tsc mutations, we developed a mouse model for leiomyoma and LAM by targeting uterine-specific Tsc2 deletion. 100% of uteri from uterine-specific Tsc2-null mice developed LAM-like leiomyomas by 18-24 weeks and approximately 50% of 28-34 week old mice developed smooth-muscle Tsc2-null tumors in the lungs. Uniquely, uterine Tsc2-null cells expressed melanocytic markers premelanosomal protein (PMEL) and Melan-A (MLANA), both considered hallmarks of LAM. Ten-weeks after eliminating estrogen production through oophorectomy or by aromatase inhibition using letrozole, Tsc2-null uteri had reduced mTORC1 activity and decreased myometrial proliferation, resulting in tumor regression and reversal of the uterine phenotype. Thus, loss of TSC2 is not sufficient to promote mTORC1-mediated leiomyoma growth without a continuous positive proliferative signal from estradiol. Mechanistically, we find that matrix metalloproteinases (MMP)-2 activity and MMP-9 expression are dependent on estrogen in the absence of TSC2. In-vivo fluorescent imaging using an MMP-sensitive optical biomarker showed higher signal specifically in leiomyomas, suggesting that MMP activity is primarily in Tsc2-null myometrial cells. Finally, we identified another melanocytic-related marker, glycoprotein NMB (GPNMB) that was over-expressed in Tsc2-null myometrial cells as well as in human LAM. Thus, GPNMB may be useful as a biomarker for LAM. Together, we suggest that lung LAM-like myometrial lesions may indeed originate from the uterus and that anti-estrogen therapy may be an effective means of treating not only leiomyoma, but LAM as well

Neutrophil Elastase and SERPINB1 are Critical Regulators of Prostate Cancer Progression

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pathology and Laboratory Medicine, 2018.Tissue infiltration and elevated peripheral circulation of granulocytic myeloid-derived cells is associated with poor outcomes in prostate cancer and other malignancies. Although myeloid-derived cells have the ability to suppress T-cell function, little is known about the direct impact of these innate cells on prostate tumor growth. Here, we report that granulocytic myeloid-derived suppressor cells (MDSC) are the predominant tumor-infiltrating cells in prostate cancer xenografts established in athymic nude mice. MDSCs significantly increase in number in the peripheral circulation as a function of xenograft growth and are successfully depleted in vivo by Gr-1 antibody treatment. Importantly, MDSC depletion significantly decreases xenograft growth. We hypothesized that MDSCs might exert their pro-tumorigenic actions in part through neutrophil elastase (NE). Indeed, we demonstrate that NE is expressed by infiltrating immune cells and enzymatically active in prostate cancer xenografts and tumors of prostate-specific Ptennull mice. Importantly, treatment with sivelestat, a small-molecule inhibitor specific for NE, significantly decreases xenograft and Pten-null tumor growth. Mechanistically, NE activates MAPK signaling and induces MAPK-dependent transcription of the proliferative gene cFOS in prostate cancer cells. Functionally, NE stimulates proliferation, migration, and invasion of prostate cancer cells in vitro. Immunohistochemistry on human prostate cancer biopsies reveals expression of NE by infiltrating CD33+ MDSCs, which predict poor patient prognosis. Interestingly, reduced expression of SERPINB1, an endogenous NE inhibitor, is also correlated with diminished survival in several cancers. Accordingly, examining gene expression datasets of human prostate cancer, we find reduced SERPINB1 in metastatic and locally advanced disease compared to normal tissue, with low expression predicting reduced progression-free survival. Moreover, SERPINB1 is down-regulated in Pten-null prostate tumors compared to wild type prostates. Therefore, we sought to characterize the role of SERPINB1 in prostate cancer. We demonstrate that SERPINB1 expression and secretion is high in the human non-malignant prostatic epithelial cell line RWPE-1 but low in human prostate cancer cell lines. Knockdown of SERPINB1 in RWPE-1 cells increases proliferation, decreases apoptosis, and stimulates expression of epithelialmesenchymal transition (EMT) markers, including MMP9, TWIST1, and SNAI1. In contrast, stable SERPINB1 overexpression in C4-2 prostate cancer cells that lack endogenous expression results in reduced xenograft growth and NE activity in vivo. Finally, we uncover a novel epigenetic mechanism for SERPINB1 repression in prostate cancer that involves EZH2-mediated histone (H3K27me3) methylation and DNMTmediated DNA methylation. Using TCGA data, we confirm that the SERPINB1 promoter is significantly hypermethylated in prostate cancer compared to normal tissue and strongly correlates with reduced expression. Our findings suggest that the balance between NE and SERPINB1 is physiologically important within the prostate and may serve as a biomarker and therapeutic target in prostate cancer

Overlapping nongenomic and genomic actions of thyroid hormone and steroids

The genomic actions of thyroid hormone and steroids depend upon primary interactions of the hormones with their specific nuclear receptor proteins. Formation of nuclear co-activator or co-repressor complexes involving the liganded receptors subsequently result in transcriptional events—either activation or suppression—at genes that are specific targets of thyroid hormone or steroids. Nongenomic actions of thyroid hormone and steroids are in contrast initiated at binding sites on the plasma membrane or in cytoplasm or organelles and do not primarily require formation of intranuclear receptor protein-hormone complexes. Importantly, hormonal actions that begin nongenomically outside the nucleus often culminate in changes in nuclear transcriptional events that are regulated by both traditional intranuclear receptors as well as other nuclear transcription factors. In the case of thyroid hormone, the extranuclear receptor can be the classical “nuclear” thyroid receptor (TR), a TR isoform, or integrin αvβ3. In the case of steroid hormones, the membrane receptor is usually, but not always, the classical “nuclear” steroid receptor. This concept defines the paradigm of overlapping nongenomic and genomic hormone mechanisms of action. Here we review some examples of how extranuclear signaling by thyroid hormone and by estrogens and androgens modulates intranuclear hormone signaling to regulate a number of vital biological processes both in normal physiology and in cancer progression. We also point out that nongenomic actions of thyroid hormone may mimic effects of estrogen in certain tumors