The differential molecular mechanisms underlying proenkephalin mRNA expression induced by forskolin and phorbol-12-myristic-13-acetate in primary cultured astrocytes

Abstract Cortisol stimulates the synthesis and secretion of oxytocin (OT) from bovine granulosa and luteal cells, but the molecular mechanisms of cortisol action remain unknown. In this study, granulosa cells or luteal cells from days 1-5 and 11-15 of the oestrous cycle were incubated for 4 or 8 h with cortisol (1x10-5, 1x10-7 M). After testing cell viability and hormone secretion (OT, progesterone, estradiol), we studied the effect of cortisol on mRNA expression for precursor of OT (NP-I/OT) and peptidyl glycine-α-amidating mono-oxygenase (PGA). The influence of RU 486 (1x10-5 M), a progesterone receptor blocker and inhibitor of the glucocorticosteroid receptor (GR), on the expression for both genes was tested. Cortisol increased the mRNA expression for NP-I/OT and PGA in granulosa cells and stimulated the expression for NP-I/OT mRNA in luteal cells obtained from days 1-5 and days 11-15 of the oestrous cycle. Expression for PGA mRNA was increased only in luteal cells from days 11-15 of the oestrous cycle. In addition, RU 486 blocked the cortisol-stimulated mRNA expression for NP-I/OT and PGA in both types of cells. These data suggest that cortisol affects OT synthesis and secretion in bovine ovarian cells, by acting on the expression of key genes, that may impair ovary function.

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Role of glucocorticoid receptor and CCAAT/enhancer-binding protein α in the feed-forward induction of 11β-hydroxysteroid dehydrogenase type 1 expression by cortisol in human amnion fibroblasts

Journal of Endocrinology ◽

10.1677/joe-07-0303 ◽

2007 ◽

Vol 195 (2) ◽

pp. 241-253 ◽

Cited By ~ 54

Author(s):

Zhen Yang ◽

Chunming Guo ◽

Ping Zhu ◽

Wenjiao Li ◽

Leslie Myatt ◽

...

Keyword(s):

Glucocorticoid Receptor ◽

Mrna Expression ◽

Promoter Region ◽

Molecular Mechanisms ◽

Hydroxysteroid Dehydrogenase ◽

Dominant Negative ◽

Forward Induction ◽

Human Amnion ◽

Feed Forward

The amount of cortisol available to its receptors is increased by the pre-receptor enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) which converts cortisone to cortisol. We examined the molecular mechanisms of the feedback effect of cortisol on 11β-HSD1 mRNA expression in human amnion fibroblasts. Our data showed that cortisol-induced 11β-HSD1 mRNA expression dose dependently in amnion fibroblasts, which could be completely blocked both by the mRNA transcription inhibitor 5,6-dichlorobenzimidazole riboside and by the glucocorticoid receptor (GR) antagonist RU486, and partially blocked by global inhibition of CCAAT/enhancer-binding proteins (C/EBPs) with transfection of C/EBP-specific dominant-negative expression CMV500 plasmid (AC/EBP) into the cells. Likewise, the induction of the promoter activity by cortisol could also be completely blocked by RU486 and partially by AC/EBP transfection. Progressive 5′ deletion of the 11β-HSD1promoter located the region responsible for cortisol’s induction within −204 bp upstream to the transcription start site. Specific nucleotide mutations of the putative glucocorticoid responsive element or CCAAT in this promoter region attenuated the induction by cortisol. Moreover, chromatin immunoprecipitation assay and electrophoretic mobility shift assay showed that GR and C/EBPα but not C/EBPβ could bind this promoter region upon cortisol stimulation of amnion fibroblasts. In conclusion, we demonstrated that GR and C/EBPα were involved in cortisol-induced 11β-HSD1 mRNA expression via binding to 11β-HSD1 promoter in amnion fibroblasts, which may cast a feed-forward production of cortisol in the fetal membranes at the end of gestation.

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microRNA-4458 Regulates Insulin Resistance in Hepatic Cells by Targeting the Insulin-Like Growth Factor 1 Receptor

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2021.2597 ◽

2021 ◽

Vol 11 (9) ◽

pp. 1812-1817

Author(s):

Jingjing Zhou ◽

Wenjuan Zhu ◽

Zheng Mao ◽

Zhen Li ◽

Xiaoqin Li ◽

...

Keyword(s):

Insulin Resistance ◽

Mrna Expression ◽

Glucose Uptake ◽

Hepg2 Cell ◽

Molecular Mechanisms ◽

Cell Model ◽

Serum Insulin ◽

Luciferase Reporter ◽

Control Group ◽

Hepatic Cells

Background: The objective of the research was to investigate the roles of miR-4458 in the regulation of insulin resistance in hepatic cells and to explore the underlying molecular mechanisms. Methods: The blood samples were collected from the T2D patients and the health controls, and the liver tissues were collected from the DM and control rats. The relationship between IGF1R and miR-4458 was predicted by TargetScan and verified by the dual luciferase reporter gene system. qRT-PCR was used to measure the mRNA expression of miR-4458, IGF1R, G6Pase and PEPCK. The protein expression of IGF1R, p-AKT and AKT were measured by Western blot analysis. The rat insulin ELISA Kit and glucose Uptake Colorimteric Assay Kit were used to determine the level of serum insulin and the glucose uptake. Results: miR-4458 was high expressed in T2D patients. We predicted and verified that IGF1R was a direct target of miR-4458, and the mRNA expression of IGF1R was reduced in type 2 diabetes patients. We established the diabetes model (DM) and IR HepG2 cell model, and found that the blood glucose and serum insulin levels were significantly elevated in the DM group. miR-4458 expression was up-regulated, while the expression of IGF1R and p-AKT, and p-AKT/AKT ratio were reduced in the DM group and IR HepG2 cell model. miR-4458 inhibitor and IGF1R-siRNA significantly decreased the expression of miR-4458 and IGF1R respectively. In comparison with IR+inhibitor control group, miR-4458 inhibitor increased 2-DG6P content, IGF1R expression, p-AKT expression and p-AKT/AKT ratio, reduced the expression of G6Pase and PEPCK, and all the effects were reversed by down-regulating IGF1R. Conclusion: miR-4458 regulated the insulin resistance in hepatic cells by regulating the IGF1R/PI3K/AKT signal pathway, which will be a potential target for the treatment of diabetes.

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Effects of lipotoxicity on a novel insulin-secreting human pancreatic β-cell line, 1.1B4

Biological Chemistry ◽

10.1515/hsz-2013-0115 ◽

2013 ◽

Vol 394 (7) ◽

pp. 909-918 ◽

Cited By ~ 19

Author(s):

Srividya Vasu ◽

Neville H. McClenaghan ◽

Jane T. McCluskey ◽

Peter R. Flatt

Keyword(s):

Mrna Expression ◽

Cell Line ◽

Molecular Mechanisms ◽

Endoplasmic Reticulum Stress Response ◽

Future Research ◽

The Novel ◽

Pancreatic Β Cell ◽

Β Cell ◽

Expression Of Genes ◽

Cellular Ca

Abstract The novel insulin-secreting human pancreatic β-cell line, 1.1B4, demonstrates stability in culture and many of the secretory functional attributes of human pancreatic β-cells. This study investigated the cellular responses of 1.1B4 cells to lipotoxicity. Chronic 18-h exposure of 1.1B4 cells to 0.5 mm palmitate resulted in decreased cell viability and insulin content. Secretory responses to classical insulinotropic agents and cellular Ca2+ handling were also impaired. Palmitate decreased glucokinase activity and mRNA expression of genes involved in secretory function but up-regulated mRNA expression of HSPA5, EIF2A, and EIF2AK3, implicating activation of the endoplasmic reticulum stress response. Palmitate also induced DNA damage and apoptosis of 1.1B4 cells. These responses were accompanied by increased gene expression of the antioxidant enzymes SOD1, SOD2, CAT and GPX1. This study details molecular mechanisms underlying lipotoxicity in 1.1B4 cells and indicates the potential value of the novel β-cell line for future research.

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Gene expression in primary cultured astrocytes affected by aluminum: alteration of chaperons involved in protein folding

Environmental Health and Preventive Medicine ◽

10.1007/s12199-010-0161-2 ◽

2010 ◽

Vol 16 (1) ◽

pp. 16-24 ◽

Cited By ~ 8

Author(s):

David A. Aremu ◽

Ojeiru F. Ezomo ◽

Shunsuke Meshitsuka

Keyword(s):

Gene Expression ◽

Protein Folding ◽

Cultured Astrocytes ◽

Primary Cultured Astrocytes

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Effect of mechanical loading on insulin-like growth factor-I gene expression in rat tibia

Journal of Endocrinology ◽

10.1677/joe.1.06880 ◽

2007 ◽

Vol 192 (1) ◽

pp. 131-140 ◽

Cited By ~ 68

Author(s):

Christianne M A Reijnders ◽

Nathalie Bravenboer ◽

Annechien M Tromp ◽

Marinus A Blankenstein ◽

Paul Lips

Keyword(s):

Bone Formation ◽

Mrna Expression ◽

Mechanical Loading ◽

Mechanical Stimulation ◽

Molecular Mechanisms ◽

Bone Marrow Cells ◽

Mechanical Stimuli ◽

Igf I ◽

Female Wistar Rats ◽

Tibia Shaft

Mechanical loading plays an essential role in maintaining skeletal integrity. Mechanical stimulation leads to increased bone formation. However, the cellular and molecular mechanisms that are involved in the translation of mechanical stimuli into bone formation, are not completely understood. Growth factors and osteocytes, which act as mechanosensors, play a key role during the bone formation after mechanical stimulation. The aim of this study was to characterize the role of IGF-I in the translation of mechanical stimuli into bone formation locally in rat tibiae. Fifteen female Wistar rats were randomly assigned to three groups (n = 5): load, sham-loaded, and control. The four-point bending model of Forwood and Turner was used to induce a single period of mechanical loading on the tibia shaft. The effects of mechanical loading on IGF-I mRNA expression were determined with non-radioactive in situ hybridization on decalcified tibiae sections, 6 h after the loading session. Endogenous IGF-I mRNA was expressed in trabecular and cortical osteoblasts, some trabecular and sub-endocortical osteocytes, intracortical endothelial cells of blood vessels, and periosteum. Megakaryocytes, macrophages, and myeloid cells also expressed IGF-I mRNA. In the growth plate, IGF-I mRNA was located in proliferative and hypertrophic chondrocytes. Mechanical loading did not affect the IGF-I mRNA expression in osteoblasts, bone marrow cells, and chondrocytes, but the osteocytes at the endosteal side of the shaft showed a twofold increase of IGF-I mRNA expression. The proportion of IGF-I mRNA positive osteocytes in loaded tibiae was 29.3 ± 12.9% (mean ± s.d.; n = 5), whereas sham-loaded and contra-lateral control tibiae exhibited 16.7 ± 4.4% (n = 5) and 14.7 ± 4.2% (n = 10) respectively (P < 0.05). Lamellar bone formation after a single mechanical loading session was observed at the endosteal side of the shaft. In conclusion, a single loading session results in a twofold up-regulation of IGF-I mRNA synthesis in osteocytes which are present in multiple layers extending into the cortical bone of mechanically stimulated tibia shaft 6 h after loading. This supports the hypothesis that IGF-I, which is located in osteocytes, is involved in the translation of mechanical stimuli into bone formation.

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