Inflammatory Cytokine-Induced HIF-1 Activation Promotes Epithelial-Mesenchymal Transition in Endometrial Epithelial Cells.

The endometrium undergoes repeated proliferation and shedding during the menstrual cycle. Significant changes to this environment include fluctuations in the partial pressure of oxygen, exposure to a high-cytokine environment associated with intrauterine infection, and inflammation. Chronic endometritis is a condition wherein mild inflammation persists in the endometrium and is one of the causes of implantation failure and miscarriage in early pregnancy. It is thought that the invasion of embryos into the endometrium requires epithelial-mesenchymal transition (EMT)-associated changes in the endometrial epithelium. However, the effects of inflammation on the endometrium remain poorly understood. In this study, we investigated the effects of the intrauterine oxygen environment, hypoxia-inducible factor (HIF), and inflammation on the differentiation and function of endometrial epithelial cells. We elucidated the ways in which inflammatory cytokines affect HIF activity and EMT in an immortalized cell line (EM-E6/E7/TERT) derived from endometrial epithelium. Pro-inflammatory cytokines caused significant accumulation of HIF-1α protein, increased HIF-1α mRNA levels, and enhanced hypoxia-induced accumulation of HIF-1α protein. The combined effect of inflammatory cytokines and hypoxia increased the expression of EMT-inducing factors and upregulated cell migration. Our findings indicate that pro-inflammatory factors, including cytokines and LPS, work synergistically with hypoxia to activate HIF-1 and promote EMT in endometrial epithelial cells.

Cigarette Smoke Extract Activates Hypoxia-Inducible Factors in a Reactive Oxygen Species-Dependent Manner in Stroma Cells from Human Endometrium.

Cigarette smoking (CS) is a major contributing factor in the development of a large number of fatal and debilitating disorders, including degenerative diseases and cancers. Smoking and passive smoking also affect the establishment and maintenance of pregnancy. However, to the best of our knowledge, the effects of smoking on the human endometrium remain poorly understood. In this study, we investigated the regulatory mechanism underlying CS-induced hypoxia-inducible factor (HIF)-1α activation using primary human endometrial stromal cells and an immortalized cell line (KC02-44D). We found that the CS extract (CSE) increased reactive oxygen species levels and stimulated HIF-1α protein stabilization in endometrial stromal cells, and that CS-induced HIF-1α-dependent gene expression under non-hypoxic conditions in a concentration- and time-dependent manner. Additionally, we revealed the upregulated expression of a hypoxia-induced gene set following the CSE treatment, even under normoxic conditions. These results indicated that HIF-1α might play an important role in CS-exposure-induced cellular stress, inflammation, and endometrial remodeling.

Exposure to cigarette smoke affects endometrial maturation including angiogenesis and decidualization.

PURPOSE: To elucidate the effects of cigarette smoking on human endometrial maturation for reproductive function, the authors examined the in vitro effects of cigarette smoke extract (CSE) on angiogenesis and decidualization in primary human endometrial stromal cells (ESCs). METHODS: Endometrial stromal cells were cultured with CSE and/or estradiol-17ß (E2) and medroxyprogesterone acetate (MPA). The mRNA, protein levels, and protein secretion of the angiogenic factors and decidual specific factors were assessed using real-time polymerase chain reaction, Western blot analysis, and enzyme-linked immunosorbent assay, respectively. Decidualization was also monitored by the changes in cellular morphology. RESULTS: Endometrial stromal cell proliferation substantially decreased after dose-dependent treatments with CSE at concentrations above 1%, whereas cell death was induced at treatment concentrations above 1% CSE. Treatments above 0.025% CSE led to increased vascular endothelial growth factor mRNA through hypoxia-inducible factor-1α accumulation. CSE concentrations at 0.01% and 0.025% increased the prolactin expression levels after treatment with E2 and MPA, whereas 0.1% and 0.25% CSE concentrations suppressed prolactin. Similar tendencies were observed in cellular morphology and other decidual specific factors. CONCLUSION: These results suggest that exposure to cigarette smoke affects endometrial appropriate maturation including the processes of angiogenesis and decidualization in the reproductive system.

Glucose transporter 1 is important for the glycolytic metabolism of human endometrial stromal cells in hypoxic environment.

AIM: The study aimed to elucidate the glycolytic metabolism of human endometrial stromal cells (hESCs) in hypoxic environment. MAIN METHODS: The hESCs were cultured in hypoxic environment, and their metabolic pathways were analyzed using metabolomics. We assessed glucose uptake using 2-deoxyglucose (2-DG) assay. The expression of glucose transporters (GLUTs) required for glucose uptake was determined using real-time quantitative polymerase chain reaction (qPCR) and western blotting. Furthermore, we knocked down GLUT1 and examined the uptake of 2-DG. KEY FINDINGS: Under hypoxia, glucose-6-phosphate, fructose-6-phosphate, and fructose-1,6-diphosphate were significantly elevated in hESCs (P < 0.05). This finding indicated enhancement in glycolysis. The volume of glucose uptake increased significantly under hypoxia (P < 0.05). Hypoxia simultaneously induced the expression of GLUT1 and GLUT3 mRNA (P < 0.05) and attenuated the expression of GLUT8 (P < 0.05). Glucose uptake was significantly inhibited upon knockdown of GLUT1 (P < 0.0001). SIGNIFICANCE: These results demonstrated a very important role of glucose transport under hypoxia. Also, hESCs utilize glycolysis to adapt to hypoxic conditions that could occur in menstrual and implantation period. These findings pave the way to study implantation failure and tumors originating from the endometrium.

The transcription factor HAND2 up-regulates transcription of the IL15 gene in human endometrial stromal cells.

Cyclic changes of the human endometrium, such as proliferation, secretion, and decidualization, occur during regular menstrual cycles. Heart- and neural crest derivatives-expressed transcript 2 (HAND2) is a key transcription factor in progestin-induced decidualization of human endometrial stromal cells (ESCs). It has been suggested that HAND2 regulates interleukin 15 (IL15), a key immune factor required for the activation and survival of uterine natural killer (uNK) cells. Activated uNK cells can promote spiral artery remodeling and secrete cytokines to induce immunotolerance. To date, no studies have evaluated the transcription factors that regulate IL15 expression in human ESCs. In the present study, we examined whether HAND2 controls IL15 transcriptional regulation in human ESCs. Quantitative RT-PCR and histological analyses revealed that HAND2 and IL15 levels increase considerably in the secretory phase of human endometrium tissues. Results from ChIP-quantitative PCR suggested that HAND2 binds to a putative HAND2 motif, which we identified in the upstream region of the human IL15 gene through in silico analysis. Using a luciferase reporter assay, we found that the upstream region of the human IL15 gene up-regulates reporter gene activities in response to estradiol and a progestin representative (medroxyprogesterone) in ESCs. The upstream region of the human IL15 gene also exhibited increasing responsiveness to transfection with a HAND2 expression vector. Of note, deletion and substitution variants of the putative HAND2 motif in the upstream region of IL15 did not respond to HAND2 transfection. These findings confirm that HAND2 directly up-regulates human IL15 transcription in ESCs.

Resveratrol protects mitochondrial quantity by activating SIRT1/PGC-1α expression during ovarian hypoxia.

PURPOSE: Resveratrol is a well-known potent activator of sirtuin-1 (SIRT1). We investigated the direct effects of hypoxia and resveratrol on SIRT1/ peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) pathways, vascular endothelial growth factor (VEGF), hypoxia-inducible factor (HIF)-1α, and mitochondrial quantity in a steroidogenic human ovarian granulosa-like tumor cell line (KGN) cells. METHODS: KGN cells were cultured with cobalt chloride (CoCl2; a hypoxia-mimicking agent) and/or resveratrol. The mRNA and protein levels, protein secretion, and intracellular localization were assessed by real-time PCR, Western blot analysis, ELISA, and immunofluorescence staining, respectively. Mitochondrial quantity was measured based on the mitochondrial DNA (mtDNA) copy number. RESULTS: CoCl2 simultaneously attenuated the levels of SIRT1 and mtDNA expression, and induced the levels of VEGF protein production. In contrast, resveratrol significantly increased the levels of SIRT1 and mtDNA copy number, but reduced VEGF production in normoxia. Resveratrol could recover CoCl2-suppressed SIRT1 and mtDNA expression and antagonize CoCl2-induced VEGF production. CoCl2 treatment resulted in a downregulation of PGC-1α expression, and this effect was recovered by resveratrol. Resveratrol significantly suppressed the production of the CoCl2-induced HIF-1α and VEGF proteins. CONCLUSION: These results suggest that resveratrol improves mitochondrial quantity by activating the SIRT1/PGC-1α pathway and inhibits VEGF induction through HIF-1α under hypoxic conditions.

Thyroid Hormone Facilitates in vitro Decidualization of Human Endometrial Stromal Cells via Thyroid Hormone Receptors.

Endometrial stromal cells differentiate into decidual cells through the process of decidualization. This differentiation is critical for embryo implantation and the successful establishment of pregnancy. Recent epidemiological studies have suggested that thyroid hormone is important in the endometrium during implantation, and it is commonly believed that thyroid hormone is essential for proper development, differentiation, growth, and metabolism. This study aimed to investigate the impact of thyroid hormone on decidualization in human endometrial stromal cells (hESCs) and define its physiological roles in vitro by gene targeting. To identify the expression patterns of thyroid hormone, we performed gene expression profiling of hESCs during decidualization after treating them with the thyroid hormone levothyroxine (LT4). A major increase in decidual response was observed after combined treatment with ovarian steroid hormones and thyroid hormone. Moreover, LT4 treatment also affected the regulation of many transcription factors important for decidualization. We found that type 3 deiodinase, which is particularly important in fetal and placental tissues, was upregulated during decidualization in the presence of thyroid hormone. Further, it was observed that progesterone receptor, an ovarian steroid hormone receptor, was involved in thyroid hormone-induced decidualization. In the absence of thyroid hormone receptor (TR), due to the simultaneous silencing of TRα and TRß, thyroid hormone expression was unchanged during decidualization. In summary, we demonstrated that thyroid hormone is essential for decidualization in the endometrium. This is the first in vitro study to find impaired decidualization as a possible cause of infertility in subclinical hypothyroidism (SCH) patients.

Concentration of stromal cell-derived factor-1 (SDF-1/CXCL12) in the follicular fluid is associated with blastocyst development.

PURPOSE: To study the association between stromal cell-derived factor-1 (SDF-1/CXCL12) and vascular endothelial growth factor (VEGF) concentrations in individual human ovarian follicles and IVF outcomes. METHODS: Concentrations of SDF-1 and VEGF in 261 follicular fluid samples were measured with enzyme-linked immunosorbent assay. IVF outcome parameters were included in fertilization rate, cleavage rate, embryo morphology on day 3, and blastocyst morphology on day 5. RESULTS: The follicular concentration of SDF-1 and VEGF was not significantly associated with fertilization and cleavage outcome, and embryo morphology. The rates of full blastocysts and good-quality blastocysts were significantly higher in follicles with an SDF-1 concentration of 275-350 pg/mL than in the follicles with SDF-1 concentrations of <200 and ≥350 pg/mL (P < 0.05). The follicular concentration of VEGF was not associated with the blastocyst morphology. CONCLUSION: Our findings showed that follicular concentration of SDF-1, and not VEGF, may be a valuable biochemical marker of blastocyst development.

Progestin-induced heart and neural crest derivatives-expressed transcript 2 inhibits angiopoietin 2 via fibroblast growth factor 9 in human endometrial stromal cells.

Heart and neural crest derivatives-expressed transcript 2 (HAND2) is a key transcription factor in progestin-induced decidualization of human endometrial stromal cells (ESCs). In the mouse, HAND2 plays an important role in uterine receptivity by suppressing several fibroblast growth factors (FGFs). However, the regulation of FGF family members by progestin-induced HAND2 and the role of FGF in vascular regeneration in the endometrium remains poorly understood. To investigate these molecular mechanisms, primary human ESCs were cultured with estradiol (E2), medroxyprogesterone acetate (MPA), progesterone receptor (PR) antagonist RU486, HAND2-specific small interfering RNA (siRNA), and recombinant FGF. The expression levels of FGF family members, HAND2, angiopoietin (ANGPT), and vascular endothelial growth factor (VEGF) were assessed by real-time PCR and ELISA. Out of six FGF genes known to be expressed in the human endometrium, only one, FGF9, was significantly downregulated in human ESCs after 3 days of progestin treatment. E2 + MPA attenuated the mRNA and protein levels of FGF9 during decidualization of ESCs, and this effect was blocked by RU486. Silencing of HAND2 significantly increased FGF9 expression in ESCs treated with E2 + MPA. Moreover, FGF9 activated FGF receptor in human ESCs, triggering ANGPT2 production, which resulted in enhancement of the ANGPT2/ANGPT1 protein ratio. Taken together, progestin-PR signaling and its target HAND2 play an essential role in FGF9 suppression in the human endometrium. In addition, progestin-induced HAND2 inhibits ANGPT2 production by suppressing FGF9 in ESCs. These results suggest that HAND2 may contribute to endometrial vascular maturation by regulating FGF9 during decidualization.

The steroid hormone dydrogesterone inhibits myometrial contraction independently of the progesterone/progesterone receptor pathway.

AIMS: In this study, we aimed to investigate the direct effects of steroid hormones on pregnant myometrial contraction. MAIN METHODS: The effect of steroids on oxytocin-induced contraction was examined in vitro using pregnant rat or human myometrium. Subsequently, we evaluated whether RU486, a potent progesterone antagonist, influenced the effects of progestin on myometrial contraction. Additionally, we evaluated the effects of progestin on high-concentration KCl-induced contraction caused by voltage-dependent calcium channels in order to investigate the mechanisms involved in this process. KEY FINDINGS: Of the natural steroids examined, 17ß-estradiol, progesterone, testosterone, cortisol, and aldosterone did not influence oxytocin-induced contraction at concentrations <10-6 M. Of the tested progestins, medroxyprogesterone acetate, norethisterone, chlormadinone acetate, levonorgesterol, 17α-hydroxyprogesterone capronate, and dienogest had no effect on contraction at <10-6 M. However, dydrogesterone showed rapid and direct inhibition of contraction at 10-6 M, and this inhibitory effect was dependent on dose and time. RU486 did not block the inhibitory effects of dydrogesterone on contraction. High-concentration KCl-induced contraction was also inhibited by dydrogesterone, and the inhibitory effects of dydrogesterone were observed at concentrations as low as 10-7 M. Additionally, oxytocin-induced contraction in pregnant human myometrium was inhibited by 10-6 M dydrogesterone. SIGNIFICANCE: These results suggested that the rapid and direct effects of dydrogesterone on myometrial contraction were caused by a nongenomic pathway and that the progesterone receptor was not required for dydrogesterone action. Additionally, the mechanism of dydrogesterone action may involve voltage-dependent calcium channels.

Importance of transient receptor potential vanilloid 4 (TRPV4) in epidermal barrier function in human skin keratinocytes.

The state of the skin changes drastically depending on the ambient temperature. Skin epidermal keratinocytes express thermosensitive transient receptor potential vanilloid (TRPV) cation channels, TRPV3 and TRPV4. These multimodal receptors are activated by various kinds of chemical and physical stimuli, including warm temperatures (>30°C). It has been suggested that TRPV4 is involved in cell-cell junction maturation; however, the effect of temperature fluctuations on TRPV4-dependent barrier homeostasis is unclear. In the present study, we demonstrated that activation of TRPV4 was crucial for barrier formation and recovery, both of which were critical for the prevention of excess dehydration of human skin keratinocytes. TRPV4 activation by physiological skin temperature (33°C), GSK1016790A or 4α-PDD allowed influx of Ca(2+) from extracellular spaces which promoted cell-cell junction development. These changes resulted in augmentation of intercellular barrier integrity in vitro and ex vivo. TRPV4 disruption reduced the increase in trans-epidermal resistance and increased intercellular permeation after a Ca(2+) switch. Furthermore, barrier recovery after the disruption of the stratum corneum was accelerated by the activation of TRPV4 either by warm temperature or a chemical activator. Our results suggest that physiological skin temperatures play important roles in cell-cell junction and skin barrier homeostasis through TRPV4 activation.

Characteristic effect of an anticancer dinuclear platinum(II) complex on the higher-order structure of DNA.

It is known that a 1,2,3-triazolato-bridged dinuclear platinum(II) complex, [{cis-Pt(NH(3))(2)}(2)(micro-OH)(micro-1,2,3-ta-N (1),N (2))](NO(3))(2) (AMTA), shows high in vitro cytotoxicity against several human tumor cell lines and circumvents cross-resistance to cisplatin. In the present study, we examined a dose- and time-dependent effect of AMTA on the higher-order structure of a large DNA, T4 phage DNA (166 kbp), by adapting single-molecule observation with fluorescence microscopy. It was found that AMTA induces the shrinking of DNA into a compact state with a much higher potency than cisplatin. From a quantitative analysis of the Brownian motion of individual DNA molecules in solution, it became clear that the density of a DNA segment in the compact state is about 2,000 times greater than that in the absence of AMTA. Circular dichroism spectra suggested that AMTA causes a transition from the B to the C form in the secondary structure of DNA, which is characterized by fast and slow processes. Electrophoretic measurements indicated that the binding of AMTA to supercoiled DNA induces unwinding of the double helix. Our results indicate that AMTA acts on DNA through both electrostatic interaction and coordination binding; the former causes a fast change in the secondary structure from the B to the C form, whereas the latter promotes shrinking in the higher-order structure as a relatively slow kinetic process. The shrinking effect of AMTA on DNA is attributable to the possible increase in the number of bridges along a DNA molecule. It is concluded that AMTA interacts with DNA in a manner markedly different from that of cisplatin.

Regulation of tight junction permeability by sodium caprate in human keratinocytes and reconstructed epidermis.

Tight junctions (TJs) restrict paracellular flux of water and solutes in epithelia and endothelia. In epidermis, the physiological role of TJs is not fully understood. In this study, sodium caprate (C10), which dilates intestinal TJs, was applied to cultured human epidermal keratinocytes and reconstructed human epidermis to investigate the effects of C10 on epidermal TJs. C10 treatment decreased transepithelial electrical resistance and increased paracellular permeability, although Western blots showed that the expression of TJ-related transmembrane proteins was not decreased. The effects of C10 were reversible. Immunofluorescence microscopy and immuno-replica electron microscopy showed that the localization of TJ strands were disintegrated, concomitant with the dispersion and/or disappearance of TJ-related molecules from the cell surface. These findings suggest that C10 impairs barrier function by physically disrupting TJ conformation in the epidermis. Furthermore, these results also show that proper localization of the molecules on the cellular membrane is important for TJ barrier function.

Cartilaginous tissue formation from bone marrow cells using rotating wall vessel (RWV) bioreactor.

This is the first successful report of the rapid regeneration of three-dimensional large and homogeneous cartilaginous tissue from rabbit bone marrow cells without a scaffold using a rotating wall vessel (RWV) bioreactor, which simulates a microgravity environment for cells. Bone marrow cells cultured for 3 weeks in DMEM were resuspended and cultured for 4 weeks in the chondrogenic medium within the vessel. Large cylindrical cartilaginous tissue with dimensions of (1.25 +/- 0.06) x (0.60 +/- 0.08) cm (height x diameter) formed. Their cartilage marker expression was confirmed by mRNA expressions of aggrecan, collagen type I and II, and glycosaminoglycan (GAG)/DNA ratio. Their cartilaginous properties were demonstrated by toluidine blue, safranin-O staining, and polarization.