Relationship between Ca2+ and cAMP as second messengers in ACTH-induced cortisol production in bovine adrenal fasciculata cells.

In adrenal fasciculata cells stimulated by ACTH, Ca2+ and cAMP play indispensable roles as second messengers in cortisol production. However, whether their second messengers cooperatively or independently participate in steroid production remains unclear. We focused on the roles of Ca2+ and cAMP in cortisol production in bovine adrenal fasciculata cells stimulated by ACTH for a relatively short period (1 h). Incubation of the cells with 100 pM ACTH in Ca2+-containing (normal) medium for 1 h increased cortisol production without affecting cAMP content. In contrast, treatment of the cells with the peptide at a higher concentration (1 nM) significantly augmented both cortisol production and cAMP content. However, ACTH did not increase either of them in the Ca2+-free medium. ACTH rapidly increased the intracellular free Ca2+ concentration ([Ca2+]i) in the normal medium, but did not influence [Ca2+]i in the Ca2+-free medium, indicating that ACTH caused Ca2+ influx into the cells. ACTH-induced Ca2+ influx and cortisol production were suppressed by a voltage-sensitive L-type Ca2+ channel blocker but not by a T-type, N-type, or P-type Ca2+ channel blocker. In contrast, dibutyryl cAMP, a cell-permeable cAMP analog, greatly enhanced cortisol production in the normal or Ca2+-free medium and slowly caused Ca2+ influx into the cells. These results strongly suggest that Ca2+, as a second messenger, is more critical than cAMP for cortisol production. However, both second messengers jointly participate in the production in adrenal fasciculata cells stimulated by ACTH.

Administering xCT Inhibitors Based on Circadian Clock Improves Antitumor Effects.

Clock genes encoding transcription factors that regulate circadian rhythms may inform chronomodulated chemotherapy, where time-dependent dose alterations might affect drug efficacy and reduce side effects. For example, inhibiting the essential cystine transporter xCT with sulfasalazine induces growth arrest in cancer cells. Although the anticancer effects of sulfasalazine have been studied extensively, its effects on transcriptional control of xCT expression have not been studied. Here, we show that sulfasalazine administration during the period of increased xCT expression improves its anticancer effects and that the Clock gene itself induces xCT expression and regulates its circadian rhythm. Our findings highlight the clinical potential of chronomodulated chemotherapy and the importance of xCT-mediated transcriptional regulation in the utility of such strategies. Cancer Res; 77(23); 6603-13. ©2017 AACR.

Impact of Pioglitazone on Macrophage Dynamics in Adipose Tissues of Cecal Ligation and Puncture-Treated Mice.

Pioglitazone improves sepsis-induced organ injury accompanied with anti-inflammatory effects on visceral adipose tissue. However, its action in adipose immune cells remains to be ascertained. We investigated the effects of pioglitazone on visceral adipose macrophage population and polarisation in cecal ligation and puncture (CLP)-induced sepsis mice. Eight-week-old male mice were assigned to 3 groups: 1) sham-operated group, 2) CLP group, or 3) pioglitazone-treated CLP group. Pioglitazone (10 mg/kg) was injected intraperitonally for 7 d and CLP surgery was performed. Visceral adipose tissues were collected 24 h after the surgery. mRNA expression of several macrophage markers (inducible nitric oxide synthase (iNOS) for M1, arginase1 (Arg1) and interleukin (IL)-10 for M2, CD163 and F4/80 for mature macrophages) and inflammatory adipokines (IL-6, monocyte chemoattractant protein-1: MCP-1) was quantified by real-time RT-PCR. Tissue sections were subjected to the immunohistochemical analysis and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. CLP significantly enhanced Arg1, IL-10 and iNOS mRNA expressions as compared with the sham group, and pioglitazone significantly increased the mRNA level of CD163 and F4/80 in CLP mice. Expression of IL-6 and MCP-1 stimulated by CLP was reduced by pioglitazone treatment. Increased CD11b/c- and CD163-positive cells as well as apoptotic cells were observed in the CLP group and the pioglitazone-treated group. The data indicate that M1/M2 macrophage activation of visceral adipose tissues is induced in CLP-induced mice, and the function of macrophages recruited from surrounding organs may be modulated by pioglitazone treatment.

Mechanism Underlying Linezolid-induced Thrombocytopenia in a Chronic Kidney Failure Mouse Model.

OBJECTIVE: To investigate the relationship between renal function and linezolid (LZD)-induced thrombocytopenia and elucidate the underlying mechanism using a chronic renal disease (CRD) mouse model. MATERIALS AND METHODS: CRD was induced in 5-week-old male Institute of Cancer Research (ICR) mice by 5/6 nephrectomy. After this procedure, LZD (25 and 100 mg/kg) was administered intraperitoneally once every day for 28 days. Platelet counts, white blood cell (WBC) counts, and hematocrit (HCT) levels were measured every 7 days. 2-14C-thymidine (0.185 MBq) was administrated intravenously to LZD-administered mice to evaluate the thymidine uptake ability of bone marrow. RESULTS: Platelet counts were significantly lower in the LZD-administered CRD group than in the LZD-nonadministered groups at 14, 21, and 28 days (P < 0.05); however, these changes were not observed in LZD-administered mice with normal renal function, regardless of the duration of LZD administration. No significant changes were observed in WBC counts or HCT levels in any LZD-administered CRD mouse. Moreover, radioactive levels in bone marrow were not significantly different in each group. CONCLUSIONS: These results indicate that LZD-induced decreases in platelet counts were enhanced by renal impairment in vivo, suggesting that LZD-induced thrombocytopenia is not caused by nonimmune-mediated bone marrow suppression.

Pioglitazone attenuates lung injury by modulating adipose inflammation.

BACKGROUND: Pioglitazone modulates adipocyte differentiation and enhances adiponectin promoter activity to increase plasma adiponectin levels. We investigated the effects of pioglitazone on cecal ligation and puncture (CLP)-induced visceral-adipose-tissue inflammation and lung injury in mice. MATERIALS AND METHODS: Eight-wk-old male mice were assigned to three groups: (1) a sham-operated control group, (2) a CLP group, and (3) a pioglitazone-treated CLP group. Pioglitazone (10 mg/kg) was injected intraperitoneally for 7 d. Serum, lung, and visceral adipose tissue were collected 24 h after surgery. Tumor necrosis factor α (TNF-α) levels in peritoneal lavage fluid were measured by an enzyme-linked immunosorbent assay, and TNF-α and interleukin 6 messenger RNA (mRNA) expression levels in visceral adipose tissue were quantified by real-time polymerase chain reaction. Lung tissue specimens were stained with hematoxylin-eosin, and the terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling method was used to evaluate tissue damage. RESULTS: TNF-α levels in peritoneal lavage fluid were significantly higher in the CLP group than in the sham group. TNF-α levels in the pioglitazone-treated CLP group were significantly lower than those in the CLP group. TNF-α and interleukin 6 mRNA expression levels of visceral adipose tissue were significantly higher in the CLP group than in the sham group. Pioglitazone treatment decreased the mRNA expression levels of these cytokines compared with the respective values in the CLP group. Histopathologic analysis of lung tissue revealed significantly increased numbers of terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling-positive cells in the CLP group compared with the sham group. CONCLUSIONS: Pioglitazone effectively prevents lung injury caused by CLP-induced sepsis by maintaining the anti-inflammatory status of visceral adipose tissue.

Evaluation of IGFBP-7 DNA methylation changes and serum protein variation in Swedish subjects with and without type 2 diabetes.

BACKGROUND: Insulin-like growth factor-binding protein 7 (IGFBP-7) is able to interact with insulin-like growth factor 1 (IGF-1) as well as insulin. Previous studies have suggested that serum IGFBP-7 levels may be associated with insulin resistance in type 2 diabetes (T2D). This study aimed to evaluate IGFBP-7 serum protein and IGFBP7 DNA methylation levels in the subjects with and without T2D. RESULTS: A total of 340 Swedish subjects including 100 newly diagnosed T2D patients (50 women/50 men), 100 age-matched nondiabetic control subjects (50/50) and 140 treated T2D patients (54/86) were studied. Serum IGFBP-7 levels were measured with a novel ELISA. IGF1, IGFBP-1, and insulin were determined by in-house radioimmunoassays. DNA methylation levels in the IGFBP7 gene were analyzed with a bisulfite-pyrosequencing technique. Serum IGFBP-7 protein levels were similar among nondiabetic subjects, newly diagnosed, and treated T2D patients and were not correlated with IGFBP7 DNA methylation. However, IGFBP7 DNA methylation was increased in men with newly diagnosed T2D compared with nondiabetic controls (17.6% vs. 12.5%, P < 0.01). Serum IGFBP-7 levels correlated (r = 0.331, P = 0.019) with serum IGFBP-1 levels, a marker of insulin production, in men but not women with newly diagnosed T2D. CONCLUSIONS: This study demonstrates for the first time that IGFBP7 DNA methylation levels are increased in Swedish men with newly diagnosed T2D. The correlation between IGFBP-7 and IGFBP-1 suggests that low IGFBP-7 may be associated with insulin resistance in T2D.

Protease-activated receptor-stimulated interleukin-6 expression in endometriosis-like lesions in an experimental mouse model of endometriosis.

The present study was undertaken to investigate the function of protease-activated receptor (PAR) in endometriotic lesions using a mouse model of endometriosis. Unilateral ovariectomy (uOVX) was performed on female nude mice followed by intraperitoneal transplantation of a suspension mixture of immortalized human endometrial epithelial cells (EM-1) and stromal cells (EtsT-499). Endometriosis-like lesions were observed mostly around the dissection site after transplantation. The expression of interleukin (IL)-6 and cyclooxygenase-2 in the lesions was enhanced in uOVX mice compared to non-uOVX animals. In non-uOVX mice, IL-6 mRNA levels were higher in lesions formed with cells that were pretreated with PAR1/2 agonists (thrombin, 10 U/ml and PAR2-activating peptide, 30 µM) compared to untreated, intact cells. Peritoneal IL-6 concentrations were also increased in the PAR1/2 agonists-treated group. IL-6 expression induced by PAR activation was blocked by the treatment of cells with serine protease inhibitors. In cultured endometrial cells, simultaneous treatment with PAR1 and PAR2 agonists significantly increased the expression of IL-6. These results suggest that peritoneal bleeding may accelerate IL-6 expression in endometriotic lesions in part through the activation of PAR.

Effects of pioglitazone on survival and omental adipocyte function in mice with sepsis induced by cecal ligation and puncture.

BACKGROUND: To examine the effects of pioglitazone, peroxisome proliferator-activated receptor-gamma (PPAR-γ), on mortality and omental adipocyte function in mice with cecal ligation and puncture (CLP). METHODS: Male mice were assigned to receive (1) vehicle/sham-operation, (2) pioglitazone/CLP, or (3) vehicle/CLP. Pioglitazone was injected intraperitoneally for 7 d before operation. Serum and omental tissue were collected before, 24, and 48 h after CLP. Serum levels of adiponectin, cytokine, and chemokine were measured with ELISA. mRNA expressions in omental tissues were determined by RT-PCR. Survival was monitored for 7 d after CLP. RESULTS: Survival after CLP was significantly better in the pioglitazone/CLP than in the vehicle/CLP. Serum adiponectin levels before CLP were higher in the pioglitazone/CLP than in the vehicle/CLP. Treatment with pioglitazone significantly inhibited the increases in the serum interleukin-6 and monocyte chemoattractant protein-1 (MCP-1) levels after CLP and lowered the mRNA expressions of proinflammatory cytokines, interleukin-6, and MCP-1 in omental tissue after CLP. CONCLUSION: The anti-inflammatory effects of pioglitazone on omental adipocyte function appear to be mediated in part by PPAR-γ activation, which down-regulates the production of inflammatory mediators.

Co-expression of somatostatin receptor subtypes and estrogen receptor-α mRNAs by non-functioning pituitary adenomas in young patients.

The expression by non-functioning adenomas (NFoma) of somatostatin receptor (SSTR) subtypes and estrogen receptor (ERα) is poorly understood. Consequently, the mRNAs of SSTR subtypes (SSTR) 1, 2, 3, and 5, dopamine receptor (D2R), and ERα were measured by real-time quantitative RT-PCR in 59 NFomas and 50 functioning adenomas; the latter included 30 GH-secreting adenomas (GHomas) and 20 prolactinomas (PRLomas). NFomas expressed higher levels of SSTR3 than functioning adenomas but had lower levels of SSTR2, SSTR5 and D2R mRNAs than GHomas. Their ERα levels were higher than those of GHomas. The SSTR subtype mRNA levels in NFomas correlated significantly with each other; there was also a good correlation between the SSTR subtypes and ERα in NFomas. These correlations were largely only observed in younger patients (<50 years). The present study describes the differential expression of SSTR subtypes in the largest number of NFoma patients studied thus far, and further proposes possible involvements of SSTR3 and estrogen in the pathophysiology of NFomas.

Knockdown of IGF-binding protein 7 inhibits transformation of the endometrial gland in an in vitro model.

Uterine endometrial glands and their secretory products are critical for the implantation and survival of the peri-implantation embryo, and for the establishment of uterine receptivity. We previously reported that insulin-like growth factor binding protein 7 (IGFBP7) is abundantly expressed in uterine glandular epithelial cells during the secretory phase of the menstrual cycle. In the present study, we used a cultured glandular epithelial cell line of human (EM1) to investigate the significance of IGFBP7 in the function of endometrial glands. EM1 cells formed a mesh-like structure on Matrigel, which was accompanied by elevated levels of intracellular cyclic AMP. However, these morphological changes were blocked by treatment with protein kinase A (PKA) inhibitor (H89). IGFBP7 knockdown using specific short interference RNA (siRNA) inhibited the formation of the mesh-like structure on Matrigel. Cyclic AMP analogs, dibutyryl-cAMP, and N(6)-phenyl-cAMP induced the expression of leukemia inhibitory factor (LIF) which is essential for the onset of implantation. Enhanced LIF expression was suppressed by IGFBP7 siRNA treatment. Western blot analysis revealed that IGFBP7 knockdown results in the aberrant, constitutive expression of the MAPK signaling pathway. These results suggest that IGFBP7 regulates morphological changes of glandular cells by interfering with the normal PKA and MAPK signaling pathways that are associated with the transformation and/or differentiation of endometrial glands.

Insulin-like growth factor binding protein-7 (IGFBP7) blocks vascular endothelial cell growth factor (VEGF)-induced angiogenesis in human vascular endothelial cells.

Insulin-like growth factor binding protein-7 (IGFBP7) and vascular endothelial growth factor (VEGF) are expressed in vascular endothelial cells in several tumor types. In this study, we examined the effect of IGFBP7 on VEGF-induced tube formation in cultured human umbilical vein endothelial cells (HUVECs) and its potential action in the modulation of VEGF signaling in vascular cells. IGFBP7 treatment suppressed VEGF-induced tube formation, proliferation, and the phosphorylation of mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) 1/2 in HUVECs. IGFBP7 attenuated VEGF-enhanced cyclooxygenase (COX)-2 and VEGF mRNA expression, and prostaglandin E(2) secretion. Knocking down endogenous IGFBP7 enhanced COX-2 and VEGF mRNA expression. A significant increase in IGFBP7-induced caspases was not observed in the presence of VEGF. These findings indicate that IGFBP7 can modulate the stimulatory effect of VEGF on angiogenesis by interfering with VEGF expression as well as VEGF signaling and not by inducing apoptosis.

Circulating IGF-binding protein 7 (IGFBP7) levels are elevated in patients with endometriosis or undergoing diabetic hemodialysis.

BACKGROUND: Insulin-like growth factor-binding protein-7 (IGFBP7) is a secretory protein with a molecular mass of approximately 30 kDa. It is abundantly expressed in the uterine endometrium during the secretory phase of the menstrual cycle. Decreased IGFBP7 expression has been observed in some cancers and leiomyomata. METHODS: To determine whether serum IGFBP7 levels reflect changes in uterine IGFBP7 expression in humans during the menstrual cycle, and to examine whether serum IGFBP7 levels are altered in patients with various disorders, we developed a novel, dual-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Firstly, concentrations of IGFBP7 released into the medium were determined in cultured endometrial stromal and glandular cells. Blood samples were collected from women who had normal menstrual cycles and who had been diagnosed with endometriosis. Serum from hemodialysis patients and gastrointestinal cancers was also used to determine the IGFBP7 levels. RESULTS: Using this new ELISA, we demonstrated that cultured uterine cells secrete IGFBP7 into the medium. Patients with endometriosis and those with type II diabetes mellitus undergoing hemodialysis had significantly higher serum concentrations of IGFBP7 than the relevant control subjects. There were no differences in serum IGFBP7 levels in women at different stages of the menstrual cycle. Furthermore, serum IGFBP7 levels in patients with colorectal, esophageal, or endometrial cancer were not different than normal healthy subjects. CONCLUSION: Our observations suggest that IGFBP7 is associated with the pathophysiology of endometriosis and diabetes mellitus, and that serum IGFBP7 levels do not reflect enhanced uterine expression of IGFBP7 mRNA during the menstrual cycle.

[Role of implantation-related factors, stathmin and insulin-like growth factor-binding protein 7 in reproductive endocrinology].

Successful implantation and placentation require that trophoblasts adhere to the uterine epithelium and penetrate the decidualized endometrium. However, the biochemical mechanisms of the establishment of pregnancy including these phenomena have not yet to be definitively elucidated. We have found that stathmin, a cytosolic phosphoprotein that regulates microtubule dynamics, and insulin-like growth factor-binding protein (IGFBP)-related protein 1 (IGFBP-rP1, now called IGF-binding protein 7) were highly expressed in the endometrium around the time of implantation and decidualization. In this article, we review our recent findings of the research regarding the functions of these implantation-associated proteins in endocrine physiology and pharmacology. Analysis of the expression of both factors in rodent and human uterus has revealed that both factors are crucial for the process of endometrial stromal cell differentiation.

Involvement of insulin-like growth factor-binding protein-related protein 1 in decidualization of human endometrial stromal cells.

Uterine decidualization is crucial for successful implantation and the establishment of pregnancy. In the present study, the expression of insulin-like growth factor-binding protein (IGFBP)-related protein 1 (IGFBP-rP1) in the human uterus and endometrial stromal cells (ESCs) and its physiological significance in decidualization were examined. IGFBP-rP1 protein was localized in the glandular epithelium and stromal cells, and blood vessels in the endometrium. Cultured stromal cells expressed IGFBP-rP1 and secreted it into the medium. IGFBP-rP1 was localized mostly in the cytoplasm near the nucleus. Knocking down the endogenous IGFBP-rP1 expression in stromal cells, by a small interfering (si)RNA, diminished the expression of prolactin and IGFBP-1 which serve as decidual markers. These results suggest that IGFBP-rP1 may play a role in decidualization of ESCs.

Effect of insulin-like growth factor-binding protein 7 on steroidogenesis in granulosa cells derived from equine chorionic gonadotropin-primed immature rat ovaries.

Insulin-like growth factor (IGF)-binding protein (IGFBP) 7 is a secreted protein that regulates cellular proliferation, adhesion, and angiogenesis, and has low affinity for IGF compared with that of IGFBP1-IGFBP6. We sought to determine whether IGFBP7 is present in follicular fluid and to elucidate whether IGFBP7 participates in the steroidogenesis of rat mature follicles. Follicular fluid and granulosa cells (GCs) were collected from immature rats 2 days after their treatment with equine chorionic gonadotropin (eCG). IGFBP7 protein was detected in the follicular fluid and the conditioned medium of cultured ovarian GCs by immunoblot analysis. When subconfluent GCs were cultured and treated with FSH and activin, coincubation with FSH and activin markedly increased GC expression of Cyp19a1 (aromatase) mRNA and 17beta-estradiol (E(2)) secretion. The addition of recombinant murine IGFBP7 to these cultures decreased in the activin-enhanced, FSH-stimulated Cyp19a1 mRNA levels in the cells and suppressed the 17beta-E(2) levels in the culture medium. Treatment of GCs with Igfbp7-specific small interfering RNA (siRNA), which knocked down Igfbp7 expression, increased the FSH-stimulated levels of Cyp19a1 but not Cyp11a1 expression. Basal and FSH-stimulated 17beta-E(2) secretion into the culture medium was also enhanced by Igfbp7 siRNA. These results suggest that IGFBP7 suppresses estrogen production in GCs. These observations support the notion that this protein, which is secreted into the follicular fluid, may serve as an intraovarian factor that negatively regulates GC differentiation.

Expression and the biological activities of insulin-like growth factor-binding protein related protein 1 in rat uterus during the periimplantation period.

IGF binding protein-related protein 1 (IGFBP-rP1) is highly expressed in the rat uterus around the time of implantation. In the present study, we determined the periimplantation localization of IGFBP-rP1 mRNA and assessed the effects of recombinant IGFBP-rP1 on the proliferative and prostacyclin (PGI(2))-producing abilities of cultured endometrial cells early in pregnancy. IGFBP-rP1 mRNA was detected at high levels in endometrial stromal cells close to the smooth muscle of interimplantation sites around the time of implantation but absent from decidual zones surrounding the embryo. Differential uterine IGFBP-rP1 expression was also recognized in the delayed implanting pregnant model, but the level of mRNA decreased as decidual tissues formed in the decidualization model. Recombinant IGFBP-rP1 inhibited the proliferation of endometrial stromal cells in vitro and arrested them in the G(1) phase of the cell cycle. Furthermore, IGFBP-rP1 significantly stimulated PGI(2) synthesis and cyclooxygenase II mRNA expression in myometrial cells, both of which are essential molecules for successful implantation. These data suggest that IGFBP-rP1 is an implantation-associated protein and that it modulates the proliferation of rat uterine cells and their production of PGI(2) during the periimplantation period.