Mira Park

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.

Modulation of biological processes in the nucleus by delivery of DNA oligonucleotides conjugated with gold nanoparticles.

The development of a method that can efficiently deliver nucleic acids into the nucleus of living systems remains one of the key challenges for experimental and therapeutic use of nonbiological gene delivery agents. In the current study, we demonstrate a functionalized gold nanoparticle (AuNP) that can serve as a universal carrier for the delivery of DNA oligonucleotides (oligos) into the nucleus. We designed various types of DNA oligos to redirect alternative splicing of pre-mRNAs, such as MCL-1 and BCL-6, and to sequester transcriptional factors, including estrogen receptor α and p53. We successfully delivered the oligos into the nucleus, resulting in the targeted effects. In addition, injection of the antisense DNAs into a xenograft tumor in a mouse model system resulted in inhibited development of the tumor by redirecting the alternative splicing of the pre-mRNA. Our findings show that these nanoconjugates efficiently load and deliver antisense DNAs to redirect gene splicing or double-stranded DNAs to decoy gene transcription by transcriptional factors into mammalian cells and in vivo animals. Therefore, our lego-like AuNP gene delivery system can be used universally to control different biological processes by modulating nuclear gene expression events in living systems.

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.

Inhibition of xenograft tumor growth in mice by gold nanoparticle-assisted delivery of short hairpin RNAs against Mcl-1L

A prerequisite for the therapeutic use of small RNAs is the development of a method that can deliver them into animals. Previous studies have shown the capability of functionalized gold nanoparticles to serve as a general platform for loading and delivering DNA oligonucleotides and short hairpin RNAs (shRNAs) into cultured human cells. Here, we report the ability of the gold nanoparticle-assisted gene delivery system (AuNP-GDS) to deliver shRNA to a xenograft tumor in a mouse model. AuNP-GDS delivery of in vitro synthesized shRNA targeted to the Mcl-1L gene knocked down levels of Mcl-1L mRNA and protein by ~36% and ~26%, respectively, which were sufficient to induce apoptosis of the xenograft tumor cells and consequently inhibited the development of the tumor. We demonstrated that our lego-like AuNP-GDS, which can easily load and deliver shRNAs targeted to any gene of interest into living systems, can deliver shRNAs into xenograft tumors, leading to antitumor activity in an animal model.

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

PLGA nanoparticles with multiple modes are a biologically safe nanocarrier for mammalian development and their offspring

Nano-sized particles (NPs) of various materials have been extensively used as therapeutic and diagnostic agents, drug delivery systems, and biomedical devices. However, the biological impacts of NP exposure during early embryogenesis on following development and next generations have not been investigated. Here, we demonstrated that polylactic-co-glycolic acid (PLGA)-NPs were not toxic and did not perturb development of preimplantation mouse embryos in vitro. Moreover, subsequent fetal development in vivo after embryo transfer proceeded normally and healthy pups were born without any genetic aberrations, suggesting biosafety of PLGA-NPs during developmental processes. TRITC-labeled PLGA-NPs, named TRITC nano-tracer (TnT) were used to visualize the successful delivery of the NPs into sperms, oocytes and early embryos. Various molecular markers for early embryogenesis demonstrated that TnT treatment at various developmental stages did not compromise embryo development to the blastocyst. mRNA-Seq analyses reinforced that TnT treatment did not significantly affect mRNA landscapes of blastocysts which undergo embryo implantation critical for following developmental processes. Moreover, when 2-cell embryos exposed to TnT were transferred into pseudopregnant recipients, healthy offspring were born without any distinct morphologic and chromosomal abnormalities. TnT treatment did not affect the sex ratio of the exposed embryos after birth. When mated with male mice, female mice that were exposed to TnT during early embryogenesis produced a comparable number of pups as control females. Furthermore, the phenotypes of the offspring of mice experienced TnT at their early life clearly demonstrated that TnT did not elicit any negative transgenerational effects on mammalian development.