FLT3 signaling augments macrophage production from human pluripotent stem cells.

Recent advances in cell engineering technologies enable immune cells to be utilized for adoptive cell transfer (ACT) immunotherapy against cancers. Macrophages have the potential to directly and indirectly exterminate cancers and are therefore an attractive option for therapies. To develop new ACT therapies using macrophages, a great number of macrophages are required. Human induced pluripotent stem cells (iPSCs) are expected to be a source of macrophages; therefore, a system to efficiently produce macrophages from human iPSCs is needed. Here, we demonstrated that human iPSCs were robustly differentiated into macrophages by enforced FMS-like tyrosine kinase-3 (FLT3) signaling via the introduction of exogenous FLT3 into iPSCs and the addition of its ligand FLT3L to the macrophage induction culture. These iPSC-derived macrophages were identical to those obtained by standard differentiation induction methods. Thus, our novel system enables the preparation of scalable macrophages from human iPSCs. We believe that this system will be useful to develop a novel ACT therapy using macrophages.

Circadian protection against bacterial skin infection by epidermal CXCL14-mediated innate immunity.

The epidermis is the outermost layer of the skin and the body's primary barrier to external pathogens; however, the early epidermal immune response remains to be mechanistically understood. We show that the chemokine CXCL14, produced by epidermal keratinocytes, exhibits robust circadian fluctuations and initiates innate immunity. Clearance of the skin pathogen Staphylococcus aureus in nocturnal mice was associated with CXCL14 expression, which was high during subjective daytime and low at night. In contrast, in marmosets, a diurnal primate, circadian CXCL14 expression was reversed. Rhythmically expressed CXCL14 binds to S. aureus DNA and induces inflammatory cytokine production by activating Toll-like receptor (TLR)9-dependent innate pathways in dendritic cells and macrophages underneath the epidermis. CXCL14 also promoted phagocytosis by macrophages in a TLR9-independent manner. These data indicate that circadian production of the epidermal chemokine CXCL14 rhythmically suppresses skin bacterial proliferation in mammals by activating the innate immune system.

Identification of compounds that preferentially suppress the growth of T-cell acute lymphoblastic leukemia-derived cells.

T-cell acute lymphoblastic leukemia (T-ALL) is one of the most frequently occurring cancers in children and is associated with a poor prognosis. Here, we performed large-scale screening of natural compound libraries to identify potential drugs against T-ALL. We identified three low-molecular-weight compounds (auxarconjugatin-B, rumbrin, and lavendamycin) that inhibited the proliferation of the T-ALL cell line CCRF-CEM, but not that of the B lymphoma cell line Raji in a low concentration range. Among them, auxarconjugatin-B and rumbrin commonly contained a polyenyl 3-chloropyrrol in their chemical structure, therefore we chose auxarconjugatin-B for further analyses. Auxarconjugatin-B suppressed the in vitro growth of five human T-ALL cell lines and two T-ALL patient-derived cells, but not that of adult T-cell leukemia patient-derived cells. Cultured normal T cells were several-fold resistant to auxarconjugatin-B. Auxarconjugatin-B and its synthetic analogue Ra#37 depolarized the mitochondrial membrane potential of CCRF-CEM cells within 3 h of treatment. These compounds are promising seeds for developing novel anti-T-ALL drugs.

An Interferon-γ/FLT3 Axis Positively Regulates Hematopoietic Progenitor Cell Expansion from Human Pluripotent Stem Cells.

Since it became possible to differentiate human pluripotent stem cells (hPSCs) into hematopoietic cells in vitro, great efforts have been made to obtain highly potent hematopoietic stem/progenitor cells (HSPCs) from hPSCs. Immunophenotypical HSPCs can be obtained from hPSCs, but their repopulating potential in vivo is low. Here, we developed a novel hematopoietic differentiation method for human-induced pluripotent stem cells (hiPSCs) to determine why the existing hPSC differentiation systems are inadequate. hiPSC-derived CD45+CD34+ cells in our system were mostly CD38- immunophenotypical HSPCs. The vast majority of human CD45+CD34+ cells in umbilical cord blood, fetal liver, and bone marrow are CD38+ hematopoietic progenitor cells (HPCs); therefore, the poor production of CD38+ HPCs was indicative of a systematic problem. hiPSC-derived CD45+CD34+ cells did not express FLT3, a receptor tyrosine kinase. Exogenous FLT3 activity significantly enhanced the production of CD38+ HPCs from hiPSCs. Thus, poor production of CD38+ HPCs was due to a lack of FLT3 expression. Interferon-γ upregulated expression of FLT3 and increased the number of CD38+ HPCs among hiPSC-derived CD45+CD34+ cells. These results suggest that the poor production of CD38+ HPCs with hPSC differentiation systems is due to a lack of FLT3 expression, and that the addition of interferon-γ can solve this problem.

Synthesis and anti-tumor activities in human leukemia-derived cells of polyenylpyrroles with a methyl group at the conjugated polyene terminus.

To develop novel drugs for treating T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML) which are highly malignant hematological tumors, a series of analogs having a polyenylpyrrole structure of natural compounds (rumbrin and auxarconjugatin B) were synthesized and investigated their structure-activity relationships (SAR) of in vitro anti-T-ALL and anti-AML activities. We obtained three findings: (1) introduction of a methyl group at the conjugated polyene terminus enhanced anti-T-ALL activity, (2) analogs with a 3-chloropyrrole moiety had even higher selectivity for T-ALL cells, and (3) some analogs were effective against AML-derived cells. Among the studied compounds, 3-chloro-2-(8-ethoxycarbonylnona-1,3,5,7-tetraenyl) pyrrole 4e was the most promising candidate of T-ALL- and AML-treating drug. This study provides useful structural information for designing novel drugs treating T-ALL and AML.

Identification of Functional Domains of CXCL14 Involved in High-Affinity Binding and Intracellular Transport of CpG DNA.

Some CXC chemokines, including CXCL14, transport CpG oligodeoxynucleotides (ODNs) into dendritic cells (DCs), thereby activating TLR9. The molecular basis of this noncanonical function of CXC chemokines is not well understood. In this study, we investigated the CpG ODN binding and intracellular transport activities of various CXC chemokines and partial peptides of CXCL14 in mouse bone marrow-derived dendritic cells. CXCL14, CXCL4, and CXCL12 specifically bound CpG ODN, but CXCL12 failed to transport it into cells at low dose. CXCL14 N-terminal peptides 1-47, but not 1-40, was capable of transporting CpG ODN into the cell, resulting in an increase in cytokine production. However, both the 1-47 and 1-40 peptides bound CpG ODN. By contrast, CXCL14 peptides 13-50 did not possess CpG ODN binding capacity or transport activity. The chimeric peptides CXCL12 (1-22)-CXCL14 (13-47) bound CpG ODN but failed to transport it. These results suggest that amino acids 1-12 and 41-47 of CXCL14 are required for binding and intracellular transport of CpG ODN, respectively. We found that an anti-CXCL14 Ab blocked cell-surface binding and internalization of the CpG ODN/CXCL14 complex. On the basis of these findings, we propose that CXCL14 has two functional domains, one involved in DNA recognition and the other in internalization of CXCL14-CpG DNA complex via an unidentified CXCL14 receptor, which together are responsible for eliciting the CXCL14/CpG ODN-mediated TLR9 activation. These domains could play roles in CXCL14-related diseases such as arthritis, obesity-induced diabetes, and various types of carcinoma.

A novel all-in-one conditional knockout system uncovered an essential role of DDX1 in ribosomal RNA processing.

Generation of conditional knockout (cKO) and various gene-modified cells is laborious and time-consuming. Here, we established an all-in-one cKO system, which enables highly efficient generation of cKO cells and simultaneous gene modifications, including epitope tagging and reporter gene knock-in. We applied this system to mouse embryonic stem cells (ESCs) and generated RNA helicase Ddx1 cKO ESCs. The targeted cells displayed endogenous promoter-driven EGFP and FLAG-tagged DDX1 expression, and they were converted to Ddx1 KO via FLP recombinase. We further established TetFE ESCs, which carried a reverse tetracycline transactivator (rtTA) expression cassette and a tetracycline response element (TRE)-regulated FLPERT2 cassette in the Gt(ROSA26)Sor locus for instant and tightly regulated induction of gene KO. By utilizing TetFE Ddx1F/F ESCs, we isolated highly pure Ddx1F/F and Ddx1-/- ESCs and found that loss of Ddx1 caused rRNA processing defects, thereby activating the ribosome stress-p53 pathway. We also demonstrated cKO of various genes in ESCs and homologous recombination-non-proficient human HT1080 cells. The frequency of cKO clones was remarkably high for both cell types and reached up to 96% when EGFP-positive clones were analyzed. This all-in-one cKO system will be a powerful tool for rapid and precise analyses of gene functions.

[Validation of Method for Nitrite Determination in Foods].

In general, nitrite in food is extracted under slightly alkaline conditions, deproteinized, and analyzed by a colorimetric method using color development by diazotization. However, depending on the sample, the sample solution may become cloudy and difficult to filter by the deproteinization treatment of the analytical method. Recently, an improved analytical method that solves these problems has been reported. Therefore, a validation study was performed on the improved analytical method was performed. The concentrations of sodium nitrite added to cod roe, fish sausage, and ham, which were not labeled with sodium nitrite, were set at the upper limits of the standards for use. We set the target values of 70-120% for trueness, less than 15% for intralaboratory reproducibility, and less than intralaboratory reproducibility for repeatability. As a result, the target values were met for the three samples verified: 88-92% for trueness, 2.0-3.0% for repeatability, and 3.2-4.3% for intralaboratory reproducibility. In addition, an interlaboratory study was conducted by eight institutes on the improved analytical method for nitrite. At each institution, sodium nitrite was added to the same three samples as in the validation study, at concentrations equivalent to twice the lower limit of quantification and the upper limit of the standards for use and analyzed in triplicate. The estimated trueness from the obtained analyses ranged from 82 to 95%, the repeatability ranged from 2.3 to 5.8%, and the inter-room reproducibility ranged from 3.5 to 11%. Thus, the improved analytical method could be useful for determining nitrite in foods.

Synthesis of polyenylpyrrole derivatives with selective growth inhibitory activity against T-cell acute lymphoblastic leukemia cells.

T-cell acute lymphoblastic leukemia (T-ALL) is a hardly curable disease with a high relapse rate. 20 analogs were synthesized based on the structures of two kinds of fungi-derived polyenylpyrrole products (rumbrin (1) and auxarconjugatin-B (2)) to suppress the growth of T-ALL-derived cell line CCRF-CEM and tested for growth-inhibiting activity. The octatetraenylpyrrole analog gave an IC50 of 0.27 µM in CCRF-CEM cells, while it did not affect Burkitt lymphoma-derived cell line Raji and the cervical cancer cell line HeLa, or the oral cancer cell line HSC-3 (IC50 > 10 µM). This compound will be a promising compound for developing T-ALL-specific drugs.

Current advances in microcell-mediated chromosome transfer technology and its applications.

Chromosomes and chromosomal gene delivery vectors, human/mouse artificial chromosomes (HACs/MACs), can introduce megabase-order DNA sequences into target cells and are used for applications including gene mapping, gene expression control, gene/cell therapy, and the development of humanized animals and animal models of human disease. Microcell-mediated chromosome transfer (MMCT), which enables chromosome transfer from donor cells to target cells, is a key technology for these applications. In this review, we summarize the principles of gene transfer with HACs/MACs; their engineering, characteristics, and utility; and recent advances in the chromosome transfer technology.

Inhibitory action of an ERK1/2 inhibitor on primitive endoderm cell differentiation from mouse embryonic stem cells.

A combination of extracellular signal-regulated kinase 1/2 (ERK1/2) and glycogen synthase kinase 3ß (GSK3ß) inhibitors, called 2i, is widely used for maintaining the pluripotency of mouse embryonic stem cells (ESCs) in vitro. Without 2i, a few mouse ESCs spontaneously gives rise to primitive endoderm (PrE) cells, whereas 2i completely blocks this PrE cell differentiation. However, the molecular mechanisms underlying the inhibitory action of 2i on PrE cell differentiation remain unclear. Robust PrE cell induction is achieved by enforced expression of the transcription factor Gata4. Here, we analyzed how 2i inhibits the PrE cell differentiation using mouse ESCs carrying an inducible Gata4 expression cassette. We found that 2i effectively inhibited the Gata4-induced PrE cell differentiation and the ERK1/2 inhibitor was responsible for this effect. We further revealed that the transcriptional activation ability of Gata4 was necessary for PrE cell induction and its disruption by the ERK1/2 inhibitor. The phosphorylation of Ser105, Ser266, and Ser411 of the Gata4 protein was not involved in the PrE cell induction. Overexpression of Klf4, an ERK1/2 substrate, inhibited the Gata4-mediated transcriptional activation. Our data indicated that ERK1/2 supported the PrE cell induction via the indirect transcriptional activation of Gata4.

In vitro differentiation of mouse T cell-derived hybrid cells obtained through cell fusion with embryonic stem cells.

Nuclear reprogramming is an innovative advance in cell biology. An important research initiative in this field is cell fusion-mediated nuclear reprogramming, wherein the nuclei of somatic cells, such as thymocytes, are initialized through cell fusion with embryonic stem cells (ESCs). However, hybrid cells obtained through cell fusion between ESCs and thymocytes failed to contribute to the embryo proper when injected into blastocysts, which suggested that there are fundamental defects in such hybrid cells. Here, we performed side-by-side comparative analyses of the in vitro growth and differentiation capacities of ESCs and ESC-T hybrid cells. We found that the hybrid cells were larger and proliferated more slowly than the ESCs in 2i/LIF medium. Upon in vitro induction of differentiation, hybrid cells gave rise to cells of the three germ layers. Under culture conditions for hematopoietic differentiation, hybrid cells successively differentiated into lateral mesodermal cells, hemogenic endothelial cells, and various types of hematopoietic cells, including erythroid, myeloid, and lymphoid cells, although T cell maturation in the CD4/CD8 double-negative fraction was delayed. These results verified the multi-lineage differentiation capacity of ESC-T hybrid cells. The minimal contribution of hybrid cells to chimeric embryos may be due to their slow growth.

Domain-specific biological functions of the transcription factor Gata2 on hematopoietic differentiation of mouse embryonic stem cells.

The generation of mouse hematopoietic stem cells from hemogenic endothelial cells (HECs) in the aorta/gonad/mesonephros region of developing embryos requires a zinc finger transcription factor Gata2. In the previous study, an enforced expression of Gata2 in vitro promoted the production of HECs from mesodermal cells differentiated from mouse embryonic stem cells (ESCs). Our research group has previously demonstrated that the enforced expression of Gata2 in ESC-derived HECs enhances erythroid and megakaryocyte differentiation and inhibits macrophage differentiation. However, the manner in which the multiple functions of Gata2 are regulated remains unclear. Mouse ESCs differentiate into various types of hematopoietic cells when cocultured with OP9 stromal cells (OP9 system). Using this system and the inducible gene cassette exchange system, which facilitates the establishment of ESCs carrying inducible transgenes under an identical gene expression regulatory unit, the domain-specific functions of Gata2 were systematically dissected in this study. We determined that the N-terminal (amino acid 1-110) region of Gata2 was an erythroid-inducing region, both the middle (amino acid 111-200) and C-terminal (amino acid 413-480) regions were megakaryocyte-inducing regions. Furthermore, the present data strongly suggest that intramolecular antagonistic interactions between each of these regions fine-tune the biological functions of Gata2.

DEAD box protein DDX1 promotes colorectal tumorigenesis through transcriptional activation of the LGR5 gene.

DDX1, a member of the DEAD box RNA helicase family, plays a critical role in testicular tumors. However, it remains to be clarified whether DDX1 is involved in other types of malignant tumors such as colorectal cancer. We disrupted the DDX1 gene in a human colorectal cancer cell line LoVo using the CRISPR/Cas9-mediated gene-targeting system. DDX1-KO LoVo cells exhibited a much slower growth rate, produced fewer colonies in soft agar medium, and generated smaller solid tumors in nude mice than parental LoVo cells. Such phenotypes of the DDX1-KO cells were mostly reversed by exogenous expression of DDX1. These results indicate that DDX1 is required for tumorigenicity of colorectal cancer cells. In the DDX1-KO cells, the cancer stem cell marker genes LGR5, CD133, ALDH1 and SOX2 were markedly suppressed. Among them, expression of LGR5, which is essential for tumorigenicity of colorectal cancer cells, was restored in the DDX1-transfected DDX1-KO cells. Consistently, the DDX1-KO cells lost sphere-forming capacity in a DDX1-dependent fashion. Reporter and chromatin immunoprecipitation assays revealed that DDX1 directly bound to the -1837 to -1662 region of the enhancer/promoter region of the human LGR5 gene and enhanced its transcription in LoVo cells. Repression of LGR5 by DDX1 knockdown was observed in 2 other human colorectal cancer cell lines, Colo320 and SW837. These results suggest that LGR5 is a critical effector of DDX1 in colorectal cancer cells. The DDX1-LGR5 axis could be a new drug target for this type of malignant cancer.

Overexpression of Lhx2 suppresses proliferation of human T cell acute lymphoblastic leukemia-derived cells, partly by reducing LMO2 protein levels.

T cell acute lymphoblastic leukemia (T-ALL) is a malignant cancer with poor prognosis. The transcriptional co-factor LIM domain only 2 (LMO2) and its target gene HHEX are essential for self-renewal of T cell precursors and T-ALL etiology. LMO2 directly associates with LDB1 in a large DNA-containing nuclear complex and controls the transcription of T-ALL-related genes. Recently, we reported that overexpression of the LIM-homeodomain transcription factor, Lhx2, results in liberation of the Lmo2 protein from the Lmo2-Ldb1 complex, followed by ubiquitin proteasome mediated degradation. Here, we found that proliferation of five human T-ALL-derived cell lines, including CCRF-CEM, was significantly suppressed by retroviral overexpression of Lhx2. The majority of Lhx2-transduced CCRF-CEM cells arrested in G0 phase and subsequently underwent apoptosis. Expression of LMO2 protein as well as HHEX, ERG, HES1 and MYC genes was repressed in CCRF-CEM cells by transduction with Lhx2. Lhx2-mediated growth inhibition was partially rescued by simultaneous overexpression of Lmo2; however, both the C-terminal LIM domain and the homeodomain of Lhx2 were required for its growth-suppressive activity. These data indicate that Lhx2 is capable of blocking proliferation of T-ALL-derived cells by both LMO2-dependent and -independent means. We propose Lhx2 as a new molecular tool for anti-T-ALL drug development.

Epigenetic regulation of the glucose transporter gene Slc2a1 by ß-hydroxybutyrate underlies preferential glucose supply to the brain of fasted mice.

We carried out liquid chromatography-tandem mass spectrometry analysis of metabolites in mice. Those metabolome data showed that hepatic glucose content is reduced, but that brain glucose content is unaffected, during fasting, consistent with the priority given to brain glucose consumption during fasting. The molecular mechanisms for this preferential glucose supply to the brain are not fully understood. We also showed that the fasting-induced production of the ketone body ß-hydroxybutyrate (ß-OHB) enhances expression of the glucose transporter gene Slc2a1 (Glut1) via histone modification. Upon ß-OHB treatment, Slc2a1 expression was up-regulated, with a concomitant increase in H3K9 acetylation at the critical cis-regulatory region of the Slc2a1 gene in brain microvascular endothelial cells and NB2a neuronal cells, shown by quantitative PCR analysis and chromatin immunoprecipitation assay. CRISPR/Cas9-mediated disruption of the Hdac2 gene increased Slc2a1 expression, suggesting that it is one of the responsible histone deacetylases (HDACs). These results confirm that ß-OHB is a HDAC inhibitor and show that ß-OHB plays an important role in fasting-induced epigenetic activation of a glucose transporter gene in the brain.

The Efficacy of Trimodal Chemoradiotherapy with Cisplatin as a Bladder-Preserving Strategy for the Treatment of Muscle-Invasive Bladder Cancer.

INTRODUCTION: Open radical cystectomy (ORC) is currently the standard treatment for muscle-invasive bladder cancer (MIBC) without metastasis, while many patients with MIBC are not always appropriate candidates due to multiple comorbidities. To evaluate the bladder-preservation strategy, we compared the results with those obtained by ORC. PATIENTS AND METHODS: We retrospectively analyzed the data of 50 patients with MIBC treated by trimodal chemoradiotherapy with cisplatin (CDDP-radiation [CDDP-R]). Transurethral resection of the bladder tumor (TURBT) was performed before treatment to confirm pathological stage ≥T2. Extensive TURBT was performed after chemoradiotherapy to evaluate the pathological response to treatment. We compared the survival outcomes of our CDDP-R with those of ORC (retrospective cohort, n = 205) by propensity score matching analysis. RESULTS: The 2- and 5-year progression-free survival, bladder-intact survival, cancer-specific survival, and overall survival (OS) rates after treatment were 70.8 and 63.9%, 64.0 and 49.8%, 86.7 and 71.8%, and 84.3 and 64.8%, respectively. The 2- and 5-year OS rates after CDDP-R were 90.5 and 74.3%, respectively, and those after ORC were 71.8 and 59.9%, respectively, indicating a significant survival advantage conferred by CDDP-R over ORC (p < 0.05, HR 0.45, 95% CI 0.21-0.94). CONCLUSIONS: In selected patients, CDDP-R for MIBC may provide comparative oncological outcomes as ORC.

Efficient production of platelets from mouse embryonic stem cells by enforced expression of Gata2 in late hemogenic endothelial cells.

Platelets are essential for blood circulation and coagulation. Previous study indicated that overexpression of Gata2 in differentiated mouse embryonic stem cells (ESCs) resulted in robust induction of megakaryocytes (Mks). To evaluate platelet production capacity of the Gata2-induced ESC-derived Mks, we generated iGata2-ESC line carrying the doxycycline-inducible Gata2 expression cassette. When doxycycline was added to day 5 hemogenic endothelial cells in the in vitro differentiation culture of iGata2-ESCs, c-Kit(-)Tie2(-)CD41(+) Mks were predominantly generated. These iGata2-ESC-derived Mks efficiently produced CD41(+)CD42b(+)CD61(+) platelets and adhered to fibrinogen-coated glass coverslips in response to thrombin stimulation. Transmission electron microscopy analysis demonstrated that the iGata2-ESC-derived platelets were discoid-shaped with α-granules and an open canalicular system, but were larger than peripheral blood platelets in size. These results demonstrated that an enforced expression of Gata2 in late HECs of differentiated ESCs efficiently promotes megakaryopoiesis followed by platelet production. This study provides valuable information for ex vivo platelet production from human pluripotent stem cells in future.

Efficient production of T cells from mouse pluripotent stem cells by controlled expression of Lhx2.

LIM-homeobox transcription factor Lhx2 induces ex vivo amplification of adult hematopoietic stem cells (HSCs) in mice. We previously showed that engraftable HSC-like cells are generated from mouse embryonic stem cells (ESCs) and induced pluripotent stem cells by enforced expression of Lhx2. However, when these HSC-like cells were transplanted into irradiated congenic mice, donor-derived T cells were barely detectable, whereas other lineages of hematopoietic cells were continuously produced. Here we investigated T-cell differentiation potential of the Lhx2-induced HSC-like cells using ESCs carrying doxycycline (dox)-inducible Lhx2 expression cassette. Dox-mediated over-expression of Lhx2 conferred a self-renewing activity to ESC-derived c-Kit(+) CD41(+) embryonic hematopoietic progenitor cells (HPCs), thereby converting them to HSC-like cells. When these HSC-like cells were transplanted into irradiated immunodeficient mice and they were supplied with a dox-containing water, CD4/8 double negative T cells were detected in their thymi. Once the Lhx2 expression was terminated, differentiation of CD4/8 double positive and single positive T cells was initiated in the thymi of transplanted mice and mature T cells were released in the peripheral blood. These results showed that engraftable HSC-like cells with full hematopoietic potential can be obtained from ESCs by the conditional expression of Lhx2.

A positive feedback loop between progesterone and microsomal prostaglandin E synthase-1-mediated PGE2 promotes production of both in mouse granulosa cells.

Microsomal prostaglandin E synthase-1 (mPGES-1) is primarily expressed in granulosa cells (GCs) in the preovulatory follicle. Both prostaglandin E2 (PGE2) and progesterone (P4) are implicated in various reproductive functions. Here, we demonstrate that mPges-1 may be a direct downstream target gene of the P4 receptor and P4-stimulated PGE2 secretion can stimulate P4 production in a newly generated mouse GC line (GtsT). Treatment of GtsT cells with a P4 receptor agonist, norgestrel, markedly increased mPGES-1 expression detected by RT-PCR analysis. PGE2 secretion measured by an enzyme-linked immunosorbent assay was enhanced by P4 treatment. Luciferase assays revealed that the proximal promoter region of the mPges-1 gene was responsible for the effects of P4 treatment. Conversely, PGE2 treatment stimulated P4 secretion, which coordinated with mRNA expression of steroidogenic acute regulatory protein. Taken together, P4 may regulate mPGES-1 expression to increase PGE2 secretion and in turn P4 production. An autocrine loop between P4 and PGE2 might function to maintain the increased levels of both in GCs.