Changhwan Ahn

Streptozotocin induces endoplasmic reticulum stress and apoptosis via disruption of calcium homeostasis in mouse pancreas.

Calcium homeostasis refers to the regulation of calcium ion concentration in the body. This concentration is tightly controlled by a stabilizing system consisting of calcium channels and calcium buffering proteins. Calcium homeostasis is crucial for cell survival. Various forms of cell death (e.g., necrosis and apoptosis) also share calcium signaling pathways and molecular effectors. Calcium acts not only as a ubiquitous second messenger involved in apoptosis along with various cell death inducers but also a regulator for the synthesis of enzymes/hormones such as insulin. We hypothesized that streptozotocin disrupts calcium homeostasis and the altered intracellular calcium levels may induce cell death. After streptozotocin administration, blood glucose level was increased while insulin levels decreased. The expression of insulin response markers also decreased relative to the vehicle group. L-type voltage-gated calcium channel expression and sarcoplasmic reticulum Ca(2+) ATPase were increased by streptozotocin. Calcium buffering protein calbindin-D9k and calmodulin family members were also increased. The expression of genes involved in transporting calcium ions to the endoplasmic reticulum (ER) was decrease while the expression of those affecting the removal of calcium from the ER was increased. Depletion of calcium from the ER leads to ER-stress and can induce apoptosis. In the streptozotocin-treatment group, apoptosis markers were increased. Taken together, these results imply that the disruption of calcium homeostasis by streptozotocin induces ER-stress and leads to the apoptosis of pancreatic cells. Additionally, findings from this study suggest that imbalances in calcium homeostasis could promote pancreatic beta cell death and result in type I diabetes.

Alteration of Tight Junction Gene Expression by Calcium- and Vitamin D-Deficient Diet in the Duodenum of Calbindin-Null Mice

Calcium absorption is regulated by both active (transcellular) and passive (paracellular) pathways. Although each pathway has been studied, correlations between the two pathways have not been well elucidated. In previous investigations, the critical transcellular proteins, calbindin-D9k (CaBP-9k) and -D28k (CaBP-28k), were shown to affect other transcellular pathways by buffering intracellular calcium concentrations. The rate of paracellular calcium transport in the duodenum is generally determined by the expression of tight junction genes. In the present study, the effect of dietary calcium and/or vitamin D supplementation on the expression of tight junction genes (occludin, ZO-1 and claudin 2, 10b, 12 and 15) in the duodenum of CaBP-9k- and/or -28k-deficient mice was examined. With a normal diet, the expression of most tight junction genes in the duodenum was significantly increased in CaBP-9k knockout (KO) mice compared to wild-type (WT) animals. With a calcium- and vitamin D-deficient diet, tight junction gene expression was significantly decreased in the duodenum of the CaBP-9k KO mice. These findings suggest that expression of paracellular tight junction genes is regulated by transcellular CaBP proteins, suggesting that active and passive calcium transport pathways may function cooperatively.

Differential expression of calcium transport genes caused by COMT inhibition in the duodenum, kidney and placenta of pregnant mice

Preeclampsia is a pregnancy-specific disease characterized by concurrent development of hypertension, proteinuria, and oxidative stress in the placenta. Preeclampsia-like genetic models were also developed by modification of preeclampsia-related genes, such as catechol-O-methyltranferase (COMT). In this study, we induced COMT inhibition in mice during pregnancy in order to reproduce physiological conditions associated with preeclampsia. Expression of the gene known as hypoxia biomarker, HIF-1α, was highly induced in the placenta of this model. The over-expression of HIF-1α demonstrates that our experimental conditions were similar to those of preeclampsia. We measured the expression of several calcium transport genes (CTGs; TRPV5, TRPV6, PMCA1 and CaBP-9k) in the placenta, duodenum and kidney after COMT inhibition on gestation day 17.5 (GD 17.5). In addition, we evaluated the calcium transporters in the kidney, duodenum of non-pregnant female mice. Placental TRPV5, TRPV6 and PMCA1 expressions were down-regulated by COMT inhibitor (ro41-0960). In addition, the reduced PMCA1 expression in the placenta was reversed by calcium supplementation. Duodenal expressions of TRPV5, TRPV6, and PMCA1 were decreased in COMT-inhibited mice, and recovered slightly after calcium supplementation. Renal expression of TRPV5, TRPV6, and PMCA1 was also decreased by COMT inhibition, while it was reversed by calcium supplementation to the level of control. Duodenal- and renal calcium transporting genes, TRPV5, TPRV6, PMCA1 and CaBP-9k, were down-regulated by COMT treatment in female mice. Taken together, these results indicate that physiological changes observed in COMT inhibition were similar to symptoms of preeclampsia, which may be related to disturbance of calcium metabolism during pregnancy.

2,4,6-Tribromophenol Interferes with the Thyroid Hormone System by Regulating Thyroid Hormones and the Responsible Genes in Mice

2,4,6-Tribromophenol (TBP) is a brominated flame retardant (BFR). Based on its affinity for transthyretin, TBP could compete with endogenous thyroid hormone. In this study, the effects of TBP on the thyroid hormone system were assessed in mice. Briefly, animals were exposed to 40 and 250 mg/kg TBP. Thyroid hormones were also administered with or without TBP. When mice were treated with TBP, deiodinase 1 (Dio1) and thyroid hormone receptor β isoform 2 (Thrβ2) decreased in the pituitary gland. The levels of deiodinase 2 (Dio2) and growth hormone (Gh) mRNA increased in response to 250 mg/kg of TBP, and the relative mRNA level of thyroid stimulating hormone β (Tshβ) increased in the pituitary gland. Dio1 and Thrβ1 expression in the liver were not altered, while Dio1 decreased in response to co-treatment with thyroid hormones. The thyroid gland activity decreased in response to TBP, as did the levels of free triiodothyronine and free thyroxine in serum. Taken together, these findings indicate that TBP can disrupt thyroid hormone homeostasis and the presence of TBP influenced thyroid actions as regulators of gene expression. These data suggest that TBP interferes with thyroid hormone systems

Claudin-1, -2, -4, and -5: comparison of expression levels and distribution in equine tissues

Claudins, which are known as transmembrane proteins play an essential role in tight junctions (TJs) to form physical barriers and regulate paracellular transportation. To understand equine diseases, it is helpful to measure the tissue-specific expression of TJs in horses. Major equine diseases such as colic and West Nile cause damage to TJs. In this study, the expression level and distribution of claudin-1, -2, -4, and -5 in eight tissues were assessed by Western blotting and immunohistochemistry methods. Claudin-1 was primarily identified in the lung, duodenum, and uterus, claudin-2 was evenly observed in equine tissues, claudin-4 was abundantly detected in the liver, kidney and uterus, and claudin-5 was strongly expressed in the lung, duodenum, ovary, and uterus, as determined by Western blotting method. The localization of equine claudins was observed by immunohistochemistry methods. These findings provide knowledge regarding the expression patterns and localization of equine claudins, as well as valuable information to understand tight junction-related diseases according to tissue specificity and function of claudins in horses.

Induction of the Estrogenic Marker Calbindn-D9k by Octamethylcyclotetrasiloxane

Interrupting the hormonal balance of an organism by interfering with hormones and their target receptors gives rise to various problems such as developmental disorders. Collectively, these reagents are known as endocrine disruptors (EDs). Cyclic volatile methyl siloxanes (cVMSs) are a group of silicone polymers that including octamethylcyclotetrasiloxane (D4). In the present study, we examined the estrogenicity of D4 through in vitro and in vivo assays that employed calcium-binding protein 9K (calbindin-D9k; CaBP-9K) as a biomarker. For in vitro investigation, GH3 rat pituitary cells were exposed to vehicle, 17β-estradiol (E2), or D4 with/without ICI 182 780 (ICI). CaBP-9K and progesterone receptor (PR) both were up-regulated by E2 and D4 which were completely blocked by ICI. Transcription of estrogen receptor α (ER α) was decreased by E2 and D4 but increased by ICI. D4 was also administered to immature female rats for an uterotrophic (UT) assay and detection of CaBP-9K. Ethinyl estradiol (EE) or D4 was administered subcutaneously with or without ICI. Although uterine weight was not significant altered by D4, an effect thought to be due to cytochrome P450 (CYP), it induced CaBP-9K and PR gene expression. Based on these results we reveal that D4 has estrogenic potential proven under in vitro and in vivo experimental conditions.

Establishment of a rapid drug screening system based on embryonic stem cells

Embryonic stem (ES) cells have the capacity for self-renewal and differentiation into three germ layers following formation of embryonic bodies (EB). To investigate toxicity of pharmaceutical compounds, five toxic chemicals, indomethacin, dexamethasone, hydroxyurea, 5-fluorouracil, and cytosine arabinoside were applied in mouse ES cells during formation of EBs. Using microscopic evaluation, the size of EBs was reduced in a dose-dependent manner by treatment with pharmaceutical chemicals. While apoptosis-related proteins, cleaved caspase-3 and PARP, were decreased in compound-exposed EBs, necrosis-related protein (Hmgb1) was present in culture media of EBs, indicating that detection of Hmgb1 can result in activation of necrosis by pharmaceutical compounds. While pharmaceutical compounds impaired the differentiation of mES cells linked with spontaneous apoptotic cell death, it was determined that cytotoxic cell damage is necrosis-dependent in mES cells. In addition, an apoptotic transcript (Noxa mRNA) in toxicant-exposed EBs was decreased in parallel with apoptosis-related proteins. Following impairment of apoptosis, differentiation-related markers including un-differentiation (Sox2), endoderm (Hnf4), mesoderm (Bmp4), and ectoderm (Pax6) also fluctuated by treatment with pharmaceutical compounds. Taken together, the data imply that exposure to pharmaceutical compounds results in increased cell death hindering the spontaneous apoptosis of cells to undergo differentiation. Using both characteristics of ES cells like self-renewal or cellular pluripotency and potentials of ES cells for evaluation in toxicity of various compounds, the current study was conducted for establishment of a novel drug screening system beyond hidden virtues of the well-known chemicals.

Effects of Octylphenol and Bisphenol A on the Metal Cation Transporter Channels of Mouse Placentas

Octylphenol (OP) and bisphenol A (BPA) are known as endocrine-disrupting chemicals (EDCs). During pregnancy, the expression of steroid hormone receptors is controlled by maternal and fetal nutrition. To evaluate the impact of EDCs during pregnancy, ethinyl estradiol (EE, 0.2 mg/kg/day), OP (50 mg/kg/day), and BPA (50 mg/kg/day) were administered to pregnant mice. The mRNA levels of TRPV6 (transient receptor potential cation channels in subfamily V, member 6) decreased significantly by EE and OP. The PMCA1 (ATPase, Ca++ transporting, plasma membrane 1) mRNA and protein levels decreased significantly by EE, OP, and BPA. CTR1 (solute carrier family 31, member 1) and ATP7A (ATPase, Cu++ transporting, alpha polypeptide) expression decreased significantly by EE, OP, and BPA. The mRNA levels of IREG1 (iron-regulated transporter, member 1) decreased significantly by EE. Hephaestin (HEPH) mRNA levels decreased significantly by EE, OP, and BPA, and protein levels decreased significantly by BPA. As a result of immunohistochemistry analysis, all cation transporter proteins were found in labyrinth of placenta. To confirm the cytosolic level of cations, levels of cation level in fetal serum were measured. EE, OP, and BPA significantly reduced serum calcium and copper levels, and iron levels were reduced by BPA. Taken together, some EDCs, such as OP and BPA, could modulate the calcium, copper, and iron ion-transporting channels during pregnancy. The fetus relies on the mother for ionic transportation, and, therefore, pregnant women should avoid exposure to cation-channel-disrupting chemicals.

Two faces of the estrogen metabolite 2-methoxyestradiol in vitro and in vivo

2-Methoxyestradiol (2-ME), an endogenous metabolite of 17β-estradiol (E2), interacts with estrogen receptors (ERs) and microtubules, however, 2-ME has a low affinity for ERs. Furthermore, 2‑ME has been identified as a potential novel antitumor agent, combining its anti‑proliferative effects on a variety of tumor cell types with its anti‑angiogenic action. Therefore, 2‑ME is of interest due to its potential anticancer therapeutic effects. In the current study, the estrogenic effect of 2‑ME on CaBP‑9k, ERα, and progesterone receptor (PR) mRNA levels in the absence and presence of E2 and progesterone (P4) in in vivo and in vitro models was examined. In GH3 cells, the mRNA level of CaBP‑9k was induced in the E2 treatment group (concentration, 10‑9 M), and the expression of CaBP‑9k was also upregulated in the 2‑ME‑treated group (concentration, 10‑7 M). Uterine lactoferrin (Ltf) mRNA expression was also increased in the 2‑ME group [dose, 40 mg/kg body weight (BW)], which was comparable to the response with E2 (dose, 40 µg/kg BW) observed in mice. As inhibitors of ER and PR activity, ICI 182,780 and mifepristone (RU486) were observed to reverse the E2 or 2‑ME mediated increase of CaBP‑9k and Ltf mRNA expression. In addition, it was found that 2‑ME significantly decreased the levels of ERα and increased PR transcripts. Consistent with the in vitro results, the mRNA levels revealed decreased ERα and increased PR in in vivo treatment of E2 and 2‑ME. These findings demonstrate that the expression of estrogenic markers, CaBP‑9k and Ltf, is regulated by 2‑ME in in vitro and in vivo models, therefore, estrogenic activi-ties of 2-ME may be increased in females during the estrous cycle via the ER and/or PR-mediated signaling pathway.

Elemol from Chamaecyparis obtusa ameliorates 2,4-dinitrochlorobenzene-induced atopic dermatitis

Chamaecyparis obtusa has been traditionally used as an antibiotic agent and in cosmetics for the prevention of microorganism infection and skin troubles. Atopic dermatitis (AD) is a chronic inflammatory skin disease that encompasses immunologic responses, susceptibility factors and compromised skin-barrier function. Use of plant medicines in therapeutic treatment of AD has recently been suggested as an alternative therapeutic option. The present study examined the effect of elemol, an active component of Chamaecyparis obtusa, on AD using in vivo and in vitro models. RBL-2H3 cells were stimulated with concanavalin A and dinitrophenyl human serum albumin, and atopic dermatitis was induced in BALB/c mice by topical application of 2,4-dinitrochlorobenzene (DNCB) prior to elemol treatment. The mRNA expression was evaluated by reverse transcription quantitative polymerase chain reaction, and the levels of β-hexosaminidase and serum immunoglobulin E (IgE) were examined by ELISA. Histological changes were also performed by microscopy. Elemol attenuated the onset of AD-like skin lesions, reduced serum IgE levels and decreased mast cell infiltration into the dermis and hypodermis. In addition, elemol downregulated the transcriptional expression of several pro-inflammatory cytokines, including TNF-α, IL-1β, IL-6 and IκBα, in the skin of the DNCB-induced animal models of AD. In the RBL-2H3 mast cell line, elemol significantly inhibited the mRNA expression of IL-4 and IL-13, and further attenuated the release of β-hexosaminidase from mast cells. Histological examination revealed that elemol significantly ameliorated the DNCB-induced dermal destruction in mice. The results of the present study suggested that elemol may have therapeutic potential in the treatment of AD due to its immunosuppressive effects.