Jeong-Jae Ko

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.

Effective delivery of anti-miRNA DNA oligonucleotides by functionalized gold nanoparticles

MicroRNAs (miRNAs) are gaining recognition as essential regulators involved in many biological processes, and they are emerging as therapeutic targets for treating disease. Here, we introduce a method for effective delivery of anti-miRNA oligonucleotides (AMOs) using functionalized gold nanoparticles (AuNPs). To demonstrate the ability of AMOs to silence miRNA, we selected miR-29b, which is known to downregulate myeloid cell leukemia-1 (MCL-1), a factor responsible for promoting cell survival. We first generated AuNPs coated with cargo DNA, which was then coupled to complementary DNA linked to an antisense miR-29b sequence. When the AuNPs were delivered into HeLa cells, MCL-1 protein and mRNA levels were increased significantly. Furthermore, apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was inhibited, proving that AMOs targeting miR-29b were effectively delivered by our innovative AuNP. In addition, we provided evidence that AuNP could deliver other AMOs against miR-21 into two independent cell lines, KGN and 293T, suggesting that the AuNP conjugates can be versatile for any AMO and cell type.

MCL-1ES, a novel variant of MCL-1, associates with MCL-1L and induces mitochondrial cell death

MINT‐7255756:MCL1‐ES (uniprotkb:Q07820‐2) physically interacts (MI:0914) with COX IV (uniprotkb:P13073) by anti tag coimmunoprecipitation (MI:0007)

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.

PI3K/Akt/mTOR activation by suppression of ELK3 mediates chemosensitivity of MDA-MB-231 cells to doxorubicin by inhibiting autophagy

Drug resistance in breast cancer remains a major obstacle of clinical therapy. We found that suppression of ELK3 in the triple negative breast cancer cell line MDA-MB-231 impaired autophagy and led to a hypersensitive response to doxorubicin treatment. In ELK3-knockdown MDA-MB-231 cells (ELK3 KD), autophagy was not activated under starvation conditions, which is a major stimulus of autophagy activation. We revealed that activation of the PI3K/Akt pathway was the main cause of impaired autophagy in ELK3 KD. Our results suggest that targeting ELK3 may be a potential approach to overcome doxorubicin resistance in breast cancer therapeutics.

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

Ell3 stabilizes p53 following CDDP treatment via its effects on ubiquitin-dependent and -independent proteasomal degradation pathways in breast cancer cells

The tumor suppressor protein p53 is unstable in quiescent cells and undergoes proteosomal degradation. Under conditions of cellular stress, p53 is rapidly stabilized by post-translational modification, thereby escaping degradation and translocating to the nucleus where it activates genes related to cell cycle arrest or apoptosis. Here, we report that the transcription elongation factor Ell3 sensitizes luminal type-cancer cell line, MCF7, which have wild-type p53, to the chemotherapeutic agent cis-diamminedichloroplatinum(II) (CDDP) by stabilizing p53. Overexpression of Ell3 in MCF7 cells suppressed the MDM2-mediated ubiquitin-dependent degradation pathway. In addition, Ell3 promoted binding of p53 to NADH quinone oxidoreductase 1, which is linked to the ubiquitin-independent degradation of p53. We found that Ell3 activates interleukin-20 (IL20) expression, which is linked to the ERK1/2 signaling pathway. Chemical inhibition of ERK1/2 signaling or molecular suppression of IL20 revealed that the ERK1/2 signaling pathway and IL20 are the main causes of p53 stabilization in Ell3-overexpressing MCF7 cells. These findings suggest that the ERK1/2 pathway can be targeted in the rational development of therapies to induce chemosensitization of breast cancer cells.

Novel Function of Sprouty4 as a Regulator of Stemness and Differentiation of Embryonic Stem Cells.

Sprouty (Spry) genes encode inhibitors of the receptor tyrosine kinase signaling cascade, which plays important roles in stem cells. However, the role of Spry4 in the stemness of embryonic stem cells has not been fully elucidated. Here, we used mouse embryonic stem cells (mESCs) as a model system to investigate the role of Spry4 in the stem cells. Suppression of Spry4 expression results in the decreases of cell proliferation, EB formation and stemness marker expression, suggesting that Spry4 activity is associated with stemness of mESCs. Teratoma assay showed that the cartilage maturation was facilitated in Spry4 knocked down mESCs. Our results suggest that Spry4 is an important regulator of the stemness and differentiation of mESCs.

Rad51 Regulates Reprogramming Efficiency through DNA Repair Pathway

Rad51 is a key component of homologous recombination (HR) to repair DNA double-strand breaks and it forms Rad51 recombinase filaments of broken single-stranded DNA to promote HR. In addition to its role in DNA repair and cell cycle progression, Rad51 contributes to the reprogramming process during the generation of induced pluripotent stem cells. In light of this, we performed reprogramming experiments to examine the effect of co-expression of Rad51 and four reprogramming factors, Oct4, Sox2, Klf4, and c-Myc, on the reprogramming efficiency. Co-expression of Rad51 significantly increased the numbers of alkaline phosphatase-positive colonies and embryonic stem cell-like colonies during the process of reprogramming. Co-expression ofRad51 significantly increased the expression of epithelial markers at an early stage of reprogramming compared with control cells. Phosphorylated histone H2AX (γH2AX), which initiates the DNA double-strand break repair system, was highly accumulated in reprogramming intermediates upon co-expression of Rad51. This study identified a novel role of Rad51 in enhancing the reprogramming efficiency, possibly by facilitating mesenchymal-to-epithelial transition and by regulating a DNA damage repair pathway during the early phase of the reprogramming process.