Sang Jun Han

A new isoform of steroid receptor coactivator-1 is crucial for pathogenic progression of endometriosis

Endometriosis is considered to be an estrogen-dependent inflammatory disease, but its etiology is unclear. Thus far, a mechanistic role for steroid receptor coactivators (SRCs) in the progression of endometriosis has not been elucidated. An SRC-1–null mouse model reveals that the mouse SRC-1 gene has an essential role in endometriosis progression. Notably, a previously unidentified 70-kDa SRC-1 proteolytic isoform is highly elevated both in the endometriotic tissue of mice with surgically induced endometriosis and in endometriotic stromal cells biopsied from patients with endometriosis compared to normal endometrium. Tnf−/− and Mmp9−/− mice with surgically induced endometriosis showed that activation of tumor necrosis factor α (TNF-α)–induced matrix metallopeptidase 9 (MMP9) activity mediates formation of the 70-kDa SRC-1 C-terminal isoform in endometriotic mouse tissue. In contrast to full-length SRC-1, the endometriotic 70-kDa SRC-1 C-terminal fragment prevents TNF-α–mediated apoptosis in human endometrial epithelial cells and causes the epithelial-mesenchymal transition and the invasion of human endometrial cells that are hallmarks of progressive endometriosis. Collectively, the newly identified TNF-α–MMP9–SRC-1 isoform functional axis promotes pathogenic progression of endometriosis.

Multi-modulation of nuclear receptor coactivators through posttranslational modifications.

Nuclear receptor (NR) coactivators are recruited to DNA by NRs, potentiating NR-dependent gene transcription. To obtain the complexity of NR-mediated gene regulation with a finite number of coactivators, the molecular properties of coactivators are dynamically modulated by posttranslational modifications (PTMs) in response to external stimuli. PTMs can regulate the molecular interactions of coactivators with transcription factors and other coactivators, in addition to their cellular location, protein stability, conformation and enzymatic activity. Therefore, dynamic regulation of the molecular properties of coactivators by PTMs allows for the complexity of NR-dependent gene expression and influences the regulation of NR-mediated physiological processes. This review focuses on recent progress in our understanding of how coactivator PTMs influence NR-mediated gene transcription and addresses their biological relevance.

Estrogen Receptor β Modulates Apoptosis Complexes and the Inflammasome to Drive the Pathogenesis of Endometriosis

Alterations in estrogen-mediated cellular signaling play an essential role in the pathogenesis of endometriosis. In addition to higher estrogen receptor (ER) β levels, enhanced ERβ activity was detected in endometriotic tissues, and the inhibition of enhanced ERβ activity by an ERβ-selective antagonist suppressed mouse ectopic lesion growth. Notably, gain of ERβ function stimulated the progression of endometriosis. As a mechanism to evade endogenous immune surveillance for cell survival, ERβ interacts with cellular apoptotic machinery in the cytoplasm to inhibit TNF-α-induced apoptosis. ERβ also interacts with components of the cytoplasmic inflammasome to increase interleukin-1β and thus enhance its cellular adhesion and proliferation properties. Furthermore, this gain of ERβ function enhances epithelial-mesenchymal transition signaling, thereby increasing the invasion activity of endometriotic tissues for establishment of ectopic lesions. Collectively, we reveal how endometrial tissue generated by retrograde menstruation can escape immune surveillance and develop into sustained ectopic lesions via gain of ERβ function.

Dynamic Cell Type Specificity of SRC-1 Coactivator in Modulating Uterine Progesterone Receptor Function in Mice

ABSTRACT Regulation of gene transcription by the progesterone receptor (PR) in cooperation with coactivator/corepressor complexes coordinates crucial processes in female reproduction. To investigate functional relationships between PR and steroid receptor coactivators (SRCs) in distinct cell types of uterine tissue during gene transcription, we generated a new transgenic mouse model utilizing a Progesterone Receptor Activity Indicator (PRAI) system that could monitor PR activity in vivo. The PRAI system consists of a modified PR bacterial artificial chromosome (BAC) clone in which the DNA binding domain of the PR was replaced with the yeast Gal4 DNA binding domain. A humanized green fluorescent protein (hrGFP) reporter controlled by the Upstream Activating Sequences for the Gal4 gene (UASG) was inserted in tandem with the modified PR gene. Expression of hrGFP in the uterus demonstrated that the PRAI animal model faithfully replicated PR signaling under various endocrine states. Bigenic PRAI-SRC-1−/− mice revealed that SRC-1 modulates PR activity in the uterus in a cell-specific fashion and is involved in PR gene activation in stroma and myometrium of the uterus in response to estrogen and progesterone. In contrast, SRC-1 was involved in the down-regulation of PR target gene expression in the luminal and glandular epithelial compartments of the uterus after chronic progesterone treatment. Finally, we dissected the means by which SRC-1 dynamically regulates PR activity in each uterine cell compartment and demonstrated that it involves the differential ability of SRC-1 to modulate expression levels of distinct coactivators, corepressors, and PR in a cell-specific fashion.

Cooperative Activation of Gene Expression by Agonists and Antagonists Mediated by Estrogen Receptor Heteroligand Dimer Complexes

Estrogen receptor (ER) antagonists are generally thought to inhibit estrogen action through competitive inhibition, resulting in receptor binding to antagonist rather than agonist. However, microarray analyses reveal a group of genes for which ER agonist and antagonist cooperatively regulate expression, suggesting additional models of combined agonist/antagonist action must exist. In conjunction with a chimeric reporter gene and two modified ERs, one [ERα(GSCKV)] with a mutation in the DNA-binding domain and the other (ERα-G521R) with a ligand-binding specificity mutation, we herein demonstrate that ER agonist and antagonist cooperatively activate gene expression through an ER heteroligand dimer complex (ER-HLD) consisting of one subunit of the receptor dimer bound to agonist and another occupied by antagonist. Coimmunoprecipitation experiments confirmed interaction between the agonist-bound and antagonist-bound receptors. This cooperative activation of gene expression was enhanced by steroid receptor coactivator 3 coactivator, and required each ligand-bound subunit of the dimer to bind to DNA, as well as both activation function 1 domains for maximal transcriptional activity. Ligand combinations able to induce ER-HLD transcriptional activity include the agonists 17β-estradiol or conjugated estrogens with the antagonists tamoxifen, raloxifene, bazedoxifene, or fulvestrant. Moreover, ER-HLD can activate transcription in the context of a natural promoter. Taken together, these findings broaden our understanding of the complex relationship between ER agonist and antagonist, and suggest a novel model by which cell and tissue selective effects of antiestrogens may be achieved.

The Dual Estrogen Receptor α Inhibitory Effects of the Tissue-Selective Estrogen Complex for Endometrial and Breast Safety

The conjugated estrogen/bazedoxifene tissue-selective estrogen complex (TSEC) is designed to minimize the undesirable effects of estrogen in the uterus and breast tissues and to allow the beneficial effects of estrogen in other estrogen-target tissues, such as the bone and brain. However, the molecular mechanism underlying endometrial and breast safety during TSEC use is not fully understood. Estrogen receptor α (ERα)–estrogen response element (ERE)–DNA pull-down assays using HeLa nuclear extracts followed by mass spectrometry–immunoblotting analyses revealed that, upon TSEC treatment, ERα interacted with transcriptional repressors rather than coactivators. Therefore, the TSEC-mediated recruitment of transcriptional repressors suppresses ERα-mediated transcription in the breast and uterus. In addition, TSEC treatment also degraded ERα protein in uterine tissue and breast cancer cells, but not in bone cells. Interestingly, ERα-ERE-DNA pull-down assays also revealed that, upon TSEC treatment, ERα interacted with the F-box protein 45 (FBXO45) E3 ubiquitin ligase. The loss-of- and gain-of-FBXO45 function analyses indicated that FBXO45 is involved in TSEC-mediated degradation of the ERα protein in endometrial and breast cells. In preclinical studies, these synergistic effects of TSEC on ERα inhibition also suppressed the estrogen-dependent progression of endometriosis. Therefore, the endometrial and breast safety effects of TSEC are associated with synergy between the selective recruitment of transcriptional repressors to ERα and FBXO45-mediated degradation of the ERα protein.

Targeting CreER T2 expression to keratin 8-expressing murine simple epithelia using bacterial artificial chromosome transgenesis

Keratin 8 (K8) is a type II keratin that is associated with the type I keratins K18 or K19 in single layered epithelia. We generated a bacterial artificial chromosome (BAC) transgenic mouse line that expresses the tamoxifen inducible CreERT2 inserted into the endogenous murine K8 gene. The transgenic mouse line contains two copies of the BAC transgene. To determine the expression specificity and inducibility of CreERT2, the K8–CreERT2 mice were bred with a Gt(ROSA26)ACTB–tdTomato–EGFP fluorescent protein-based reporter transgenic mouse line. We demonstrated that CreERT2 and the endogenous K8 gene share the same patterns of expression and that the enzymatic activity of CreERT2 can be efficiently induced by tamoxifen in all K8-expressing tissues. This mouse line will be useful for studying gene function in development and homeostasis of simple epithelia, and investigating both tissue lineage hierarchy and the identity of the cells of origin for epithelial cancers.

Dynamic Regulation of Progesterone Receptor Activity in Female Reproductive Tissues

The progesterone receptor (PR) in cooperation with coregulator complexes coordinates crucial processes in female reproduction. To investigate the dynamic regulation of PR activity in vivo, a new transgenic mouse model utilizing a PR activity indicator (PRAI) system was generated. Studies utilizing the PRAI mouse have revealed that progesterone temporally regulates PR activity in female reproductive tissues. Specifically, progesterone rapidly enhances PR activity immediately after administration. However, chronic progesterone stimulation represses PR activity in female reproductive organs. Like progesterone, RU486 also temporally regulates PR activity in female reproductive organs. However, the temporal regulation of PR activity by RU486 is the inverse of progesterones activity. RU486 acutely represses PR activity after injection but increases PR activity after chronic treatment in female reproductive tissues. Treatment with a mixed antagonist/agonist of PR, when compared to natural hormone, results in dramatically different tissue-specific patterns of intracellular PR activity, coregulator levels, and kinase activity. Transcriptional regulation of gene expression by PR is facilitated by coordinate interactions with the steroid receptor coactivators (SRCs). Bigenic PRAI-SRC knockout mouse models enabled us to draw a tissue-specific coactivator atlas for PR activity in vivo. Based on this atlas, we conclude that the endogenous physiological function of PR in distinct tissues is modulated by different SRCs. SRC-3 is the primary coactivator for PR in the breast and SRC-1 is the primary coactivator for PR in the uterus.

A scoring system for the follow up study of nuclear receptor coactivator complexes

We have systematically isolated a variety of coactivator complexes from HeLa S3 cells using proteomic approaches. In the present report, we have evaluated twelve coactivator complexes involved in nuclear receptor-dependent gene transcription that have been purified by using an immunoprecipitation method. The twelve purified coactivator complexes are SRC-1, SRC-2, SRC-3, CBP, p300, CAPER, E6-AP, ASC-1, CoREST, CRSP3, CRSP2, and CDK7 containing complexes. We have identified 153 protein components associated with these coactivator complexes using mass spectrometry. In order to systematically characterize the functional roles for these components in nuclear receptor-dependent gene transcription and their investigative potential, we have developed a scoring system. This scoring system is comprised of biological and experimental parameters. The biological evaluation considers aspects such as intrinsic enzymatic activity of a protein component, cellular signaling processes in which protein components may be involved, associations with human disease, specific protein motifs, and the known biological roles of other interacting partners of the identified protein. In the experimental evaluation, we include parameters, such as the availability of research materials for the functional study of the identified protein component; such as full-length cDNA clones, antibodies, and commercially available knock-out embryonic stem (ES) cells. Each scoring parameter has been assigned an arbitrary number of points according to perceived relative importance. On the basis of this scoring system, we prioritized each of the protein components in terms of the likelihood of their importance for coactivator complex networking in nuclear receptor-dependent gene transcription.

An estrogen receptor alpha activity indicator model in mice.

Estrogen receptor alpha (ERα), plays essential roles in the female reproduction. To investigate the dynamic changes in ERα activity in vivo, we have developed an ER Alpha Activity Indicator (ERAAI) mouse. This ERAAI mouse harbors both a modified ERα Bacterial Artificial Chromosome (BAC) clone and a reporter gene which is regulated specifically by the modified receptor. The ERα modification (Gal4‐ERα) consists replacing the DNA binding domain (DBD) of ERα with the DBD of yeast Gal4 transcription factor. This reporter transgene consisting of a humanized renilla Green Fluorescent Protein (hrGFP) sequence controlled by the Upstream Activating Sequences for the Gal4 gene (UASG) was inserted into the modified ERα BAC clone. Expression of Gal4‐ERα and hrGFP reliably recapitulates endogenous ERα expression and activity in the estrogen target tissues in response to estrogen stimulation. Therefore, the ERAAI mouse represents a novel animal model to investigate dynamic ERα activity in vivo. genesis 47:815–824, 2009.