Miae Won

FOXL2 Interacts with Steroidogenic Factor-1 (SF-1) and Represses SF-1-Induced CYP17 Transcription in Granulosa Cells

Mutations in FOXL2 are responsible for blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) type I, in which affected women exhibit premature ovarian failure. FOXL2-null mice showed defects in granulosa cell development during folliculogenesis. We screened a rat ovarian yeast two-hybrid cDNA library to identify FOXL2-interacting proteins and found steroidogenic factor-1 (SF-1). Here, we show that human FOXL2 and SF-1 proteins interact in human granulosa cells and that FOXL2 negatively regulates the transcriptional activation of a steroidogenic enzyme, CYP17, by SF-1. Furthermore, FOXL2 mutants found in blepharophimosis-ptosis-epicanthus inversus syndrome type I patients lost the ability to repress CYP17 induction mediated by SF-1. Chromatin immunoprecipitation and EMSA results further revealed that FOXL2 inhibited the binding of SF-1 to the CYP17 promoter, whereas the FOXL2 mutants failed to block this interaction. Therefore, this study identifies a novel regulatory role for FOXL2 on a key steroidogenic enzyme and provides a possible mechanism by which mutations in FOXL2 disrupt normal ovarian follicle development.

Gold nanoparticle–DNA aptamer composites as a universal carrier for in vivo delivery of biologically functional proteins

Although the delivery of biologically functional protein(s) into mammalian cells could be of tremendous value to biomedical research, the development of such technology has been hindered by the lack of a safe and effective delivery method. Here, we present a simple, efficient, and versatile gold nanoparticle-DNA aptamer conjugate (AuNP-Apt)-based system, with nanoblock-like properties, that allows any recombinant protein to be loaded without additional modifications and delivered into mammalian living systems. AuNP-Apt-based protein delivery system was able to deliver various proteins into variety of cell types in vitro without showing cytotoxicity. This AuNP-Apt system was also effective for the local and systemic targeted delivery of proteins in vivo. A local injection of the AuNP-Apt loaded with the apoptosis-inducing BIM protein efficiently inhibited the growth of xenograft tumors in mice. Furthermore, an intravenous injection of AuNP-Apt loaded with both epidermal growth factor (EGF) and BIM resulted in the targeted delivery of BIM into a xenograft tumor derived from EGF receptor-overexpressing cancer cells with no detectable systemic toxicity. Our findings show that this system can serve as an innovative platform for the development of protein-based biomedical applications.

The apolipoprotein A-I level is downregulated in the granulosa cells of patients with polycystic ovary syndrome and affects steroidogenesis.

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder found in women. The etiology of PCOS is still not clear, and there are no available studies on the proteome analysis of granulosa cells (GCs) in PCOS patients. To identify the pathogenic mechanisms and potential diagnostic markers for PCOS, we conducted proteomic profiling of GCs in PCOS patients by two-dimensional gel electrophoresis and liquid chromatography coupled with mass spectrometry (LC-MS/MS) analyses. The proteomic analysis yielded eight downregulated and 12 upregulated proteins in PCOS patients, among which apolipoprotein A-I (ApoA-I) showed significant downregulation in PCOS patients as confirmed by Western blotting. Knockdown of ApoA-I decreased the number of transcripts of steroidogenic enzymes in a granulosa cell line (KGN), while its overexpression generally increased the level of expression of these enzymes. Furthermore, modulation of the expression level of ApoA-I in the granulosa cells altered progesterone production. Therefore, this study suggests that ApoA-I can be useful as a granulosa cell biomarker of PCOS patients and that downregulated ApoA-I may be related to the disturbed production of steroid hormones in PCOS patients.

HIP1R interacts with a member of Bcl-2 family, BCL2L10, and induces BAK-dependent cell death.

The Bcl-2 family members are evolutionally conserved and crucial regulators of apoptosis. BCL2L10 (human Diva or BCL-B) is a member of the Bcl-2 family that has contradictory functions in apoptosis. In the present study, we identified the Huntington-interacting protein 1-related (HIP1R) protein following a search for Diva-interacting proteins using the yeast two-hybrid system. HIP1R is a multi-domain protein that regulates the clathrin-mediated endocytic machinery and actin assembly in cells. Interaction of endogenous proteins of BCL2L10 and HIP1R in 293T cells was determined by immunoprecipitation, and their direct association was confirmed by the Far-Western analysis. The deletion of both the AP180-homology (ANTH) and F-actin-binding the talin-HIP1/R/Sla2p actin-tethering C-terminal homology (THATCH) domains of HIP1R greatly compromised its binding ability to BCL2L10. Ectopic expression of HIP1R resulted in moderate cell death of 293T cells in conjunction with the dissipation of mitochondrial membrane potential and caspase 9 activation. A member of proapoptotic Bcl-2 family, BAK, was required for HIP1R to induce cell death, while BAX was dispensable. In addition, BCL2L10 was associated with endogenous caspase 9, and their binding was augmented by HIP1R overexpression. Thus, this study provided the previously unknown function of HIP1R involved in the intrinsic cell death pathway and further explored possible mechanisms by which HIP1R induces cell death.

Inhibition of Xenograft Tumor Growth by Gold Nanoparticle-DNA Oligonucleotide Conjugates-Assisted Delivery of BAX mRNA

Use of non-biological agents for mRNA delivery into living systems in order to induce heterologous expression of functional proteins may provide more advantages than the use of DNA and/or biological vectors for delivery. However, the low efficiency of mRNA delivery into live animals, using non-biological systems, has hampered the use of mRNA as a therapeutic molecule. Here, we show that gold nanoparticle-DNA oligonucleotide (AuNP-DNA) conjugates can serve as universal vehicles for more efficient delivery of mRNA into human cells, as well as into xenograft tumors generated in mice. Injections of BAX mRNA loaded on AuNP-DNA conjugates into xenograft tumors resulted in highly efficient mRNA delivery. The delivered mRNA directed the efficient production of biologically functional BAX protein, a pro-apoptotic factor, consequently inhibiting tumor growth. These results demonstrate that mRNA delivery by AuNP-DNA conjugates can serve as a new platform for the development of safe and efficient gene therapy.

Identification of differentially expressed microRNAs in endometrial cancer cells after progesterone treatment.

Introduction The main methods of treatment of endometrial carcinoma are hysterectomy and bilateral oophorectomy with lymphadenectomy. However, another option for hormonal treatment of endometrial carcinoma, the use of progesterone in young patients to preserve childbearing capacity, has been reported, and a high remission rate has been described in well-selected stage I, grade 1 endometrial cancer. Although it is intriguing that hormonal therapy alone can treat endometrial carcinoma without surgery or cytotoxic chemotherapy, the molecular basis for the effects of progesterone on endometrial carcinoma is not clearly known. MicroRNAs (miRNAs) are a class of naturally occurring small, noncoding RNA molecules that control cellular function and are known to function as both tumor suppressors and oncogenes. Thus, in the present study, changes in the miRNA profile of endometrial carcinoma cells on progesterone treatment were studied. Method To elucidate the mechanism of hormonal treatment in endometrial carcinoma cells, we studied the changes in miRNA expression in endometrial carcinoma cells on treatment with progesterone using the Hec1A endometrial carcinoma cell line as a model system. Hec1A cells were treated with medroxyprogesterone acetate, and total RNA was extracted. The changes in the miRNA profile after progesterone treatment were determined using a microarray containing 868 miRNAs. Results Of 868 miRNAs, the expression levels of miR-625*, -21, -142-5p, and 146b-5p were increased by more than 400%, whereas the expression levels of miR-633, -29c, -29*, and -193b were decreased by 50%. To validate the array results, quantitative real-time polymerase chain reactions were performed. Conclusions MiRNA expression was modulated by progesterone treatment in endometrial carcinoma cells, implying a possible critical role of miRNAs in regulating posttranscriptional gene expression on progesterone treatment. Further studies are required to clarify the mechanism involved in the hormonal treatment of endometrial carcinoma.

IEX-1-induced cell death requires BIM and is modulated by MCL-1.

MCL-1 (myeloid cell leukemia-1) is a distinguished and pivotal member of the pro-survival BCL-2 family of proteins, and we isolated IEX-1 (immediate early response gene X-1) as a MCL-1-interacting protein using the yeast two-hybrid system and confirmed their endogenous association in human cells. The underlying mechanisms by which IEX-1 affects cell survival and death are largely unknown. Ectopic expression of IEX-1-induced caspase-dependent apoptosis in 293T cells, and the response was significantly modulated by changes in the MCL-1 expression level in cells. Forced expression of IEX-1 was unable to induce cell death or to perturb mitochondrial membrane potential in BIM-depleted cells. Additionally, knockouts of NOXA or PUMA did not affect the activities of IEX-1, indicating that the pro-death action of IEX-1 specifically requires BIM. Our findings provide insight into a new regulatory circuit that controls cell death and survival by the coordinated action of MCL-1, IEX-1, and BIM.

Increased expression of the testicular estrogen receptor alpha in adult mice exposed to low doses of methiocarb.

Methiocarb is a widely used carbamate pesticide and a suspected endocrine disrupter. The objective of this study was to examine the in vivo effects of methiocarb at low doses on testicular expression of steroid receptors, spermatogenesis and sperm quality in adult mice. Eighteen‐week‐old DBA/2 males were treated with daily intraperitoneal injection of methiocarb (0, 0.03, 0.3, 1.0 or 3.0 µg kg−1 of body weight) for 20 days. Kidney and liver weights were significantly increased in the 1.0 or 3.0 µg kg−1 treatment groups (P < 0.05). The testicular expression of estrogen receptor α (ERα) was significantly increased in mice treated with methiocarb as confirmed by Western blot analysis. The sperm production and sperm quality of the methiocarb‐exposed mice were not significantly altered as determined using a computer‐assisted sperm analysis system. Therefore, these results demonstrate, that although the exposure to methiocarb at low doses alters testicular ERα expression in adult mice, both sperm production and quality remain unaffected. Copyright

Identification of amino acid residues in the catalytic domain of RNase E essential for survival of Escherichia coli: functional analysis of DNase I subdomain.

RNase E is an essential Escherichia coli endoribonuclease that plays a major role in the decay and processing of a large fraction of RNAs in the cell. To better understand the molecular mechanisms of RNase E action, we performed a genetic screen for amino acid substitutions in the catalytic domain of the protein (N-Rne) that knock down the ability of RNase E to support survival of E. coli. Comparative phylogenetic analysis of RNase E homologs shows that wild-type residues at these mutated positions are nearly invariably conserved. Cells conditionally expressing these N-Rne mutants in the absence of wild-type RNase E show a decrease in copy number of plasmids regulated by the RNase E substrate RNA I, and accumulation of 5S ribosomal RNA, M1 RNA, and tRNAAsn precursors, as has been found in Rne-depleted cells, suggesting that the inability of these mutants to support cellular growth results from loss of ribonucleolytic activity. Purified mutant proteins containing an amino acid substitution in the DNase I subdomain, which is spatially distant from the catalytic site posited from crystallographic studies, showed defective binding to an RNase E substrate, p23 RNA, but still retained RNA cleavage activity—implicating a previously unidentified structural motif in the DNase I subdomain in the binding of RNase E to targeted RNA molecules, demonstrating the role of the DNase I domain in RNase E activity.

Absence of a FOXL2 mutation (402C→G) in the blood of adult-type granulosa cell tumor patients possessing the FOXL2 mutation.

Recently, a new mutation in FOXL2, c.402C→G leading to a p.C134W change, was reported to be found in 97% of adult-type ovarian granulosa cell tumors (GCTs) tested. In the current study, we compared the FOXL2 sequences of genomic DNA isolated from both GCT and blood. Although the GCTs of patients possessed the FOXL2 mutation, their FOXL2 nucleotide sequences of genomic DNA isolated from matching blood samples lacked the 402C→G mutation. Therefore, we confirmed that the nucleotide alteration of FOXL2 is due to a somatic mutation and demonstrated that sequencing of blood DNA for the detection of the FOXL2 mutation is not a useful method for the diagnosis of GCT.