Cytoplasmic Accumulation of Incompletely Glycosylated SHBG Enhances Androgen Action in Proximal Tubule Epithelial Cells

Abstract Human sex hormone-binding globulin (SHBG) accumulates within the cytoplasm of epithelial cells lining the proximal convoluted tubules of mice expressing human SHBG transgenes. The main ligands of SHBG, testosterone and its metabolite, 5α-dihydrotestosterone (DHT), alter expression of androgen-responsive genes in the kidney. To determine how intracellular SHBG might influence androgen action, we used a mouse proximal convoluted tubule (PCT) cell line with characteristics of S1/S2 epithelial cells in which human SHBG accumulates. Western blotting revealed that SHBG extracted from PCT cells expressing a human SHBG cDNA (PCT-SHBG) is 5–8 kDa smaller than the SHBG secreted by these cells, due to incomplete N-glycosylation and absence of O-linked oligosaccharides. PCT-SHBG cells sequester [3H]DHT more effectively from culture medium than parental PCT cells, and the presence of SHBG accentuates androgen-dependent activation of a luciferase reporter gene, as well as the endogenous kidney androgen-regulated protein (Kap) gene. After androgen withdrawal, androgen-induced Kap mRNA levels in PCT-SHBG cells are maintained for more than 2 wk vs 2 d in parental PCT cells. Transcriptome profiling after testosterone or DHT pretreatments, followed by 3 d of steroid withdrawal, also demonstrated that intracellular SHBG enhances androgen-dependent stimulation (e.g.Adh7, Vcam1, Areg, Tnfaip2) or repression (e.g.Cldn2 and Osr2) of many other genes in PCT cells. In addition, nuclear localization of the androgen receptor is enhanced and retained longer after steroid withdrawal in PCT cells containing functional SHBG. Thus, intracellular SHBG accentuates the uptake of androgens and sustains androgens access to the androgen receptor, especially under conditions of limited androgen supply.

Download Full-text

Leukotriene B4 mediates histamine induction of NF-κB and IL-8 in human bronchial epithelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1998.274.6.l1030 ◽

1998 ◽

Vol 274 (6) ◽

pp. L1030-L1039 ◽

Cited By ~ 11

Author(s):

Yosuke Aoki ◽

Daoming Qiu ◽

Guo Hua Zhao ◽

Peter N. Kao

Keyword(s):

Epithelial Cells ◽

Protein Secretion ◽

Bronchial Epithelial Cells ◽

Luciferase Reporter ◽

Mrna Levels ◽

Human Bronchial Epithelial Cells ◽

Lipoxygenase Inhibitor ◽

Luciferase Reporter Gene ◽

Bronchial Epithelial ◽

Histamine Stimulation

In 16HBE transformed human bronchial epithelial cells, histamine stimulated interleukin (IL)-8 mRNA and protein secretion, and this histamine stimulation was inhibited by the H1-receptor antagonist diphenhydramine (DPH), by the inhibitor of 5-lipoxygenase-activating protein (FLAP) MK-886, by the 5-lipoxygenase inhibitor Zileuton, and by dexamethasone. Histamine stimulated bronchial epithelial cell production of leukotriene B4(LTB4), and this production was inhibited by FLAP inhibitors MK-886 and L-655,238 and Zileuton. Histamine stimulated IL-8 luciferase reporter gene activity that was inhibited with DPH, dexamethasone, MK-886 and L-655,238, and Zileuton. The inhibition of IL-8 transcription and protein secretion by FLAP inhibitors and Zileuton was reversed with exogenous LTB4. There was increased IL-8 nuclear factor-κB (NF-κB) DNA-binding activity after histamine stimulation, and this was inhibited by DPH and MK-886. Cytoplasmic phospholipase A2 mRNA levels were also potently induced by histamine. Thus histamine stimulation of bronchial epithelial cells involves binding at H1receptors, production of LTB4, activation of NF-κB and increased expression of IL-8.

Download Full-text

Suppression of IL-8 production in gastric epithelial cells by MUC1 mucin and peroxisome proliferator-associated receptor-γ

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00023.2012 ◽

2012 ◽

Vol 303 (6) ◽

pp. G765-G774 ◽

Cited By ~ 9

Author(s):

Yong Sung Park ◽

Wei Guang ◽

Thomas G. Blanchard ◽

K. Chul Kim ◽

Erik P. Lillehoj

Keyword(s):

Epithelial Cells ◽

Negative Control ◽

Luciferase Reporter ◽

Peroxisome Proliferator ◽

Apical Surface ◽

Gastric Epithelial Cells ◽

Luciferase Reporter Gene ◽

Ags Cells ◽

Pparγ Agonist ◽

H Pylori

MUC1 is a membrane-tethered mucin expressed on the apical surface of epithelial cells. Our previous report (Guang W, Ding H, Czinn SJ, Kim KC, Blanchard TG, Lillehoj EP. J Biol Chem 285: 20547–20557, 2010) demonstrated that expression of MUC1 in AGS gastric epithelial cells limits Helicobacter pylori infection and reduces bacterial-driven IL-8 production. In this study, we identified the peroxisome proliferator-associated receptor-γ (PPARγ) upstream of MUC1 in the anti-inflammatory pathway suppressing H. pylori- and phorbol 12-myristate 13-acetate (PMA)-stimulated IL-8 production. Treatment of AGS cells with H. pylori or PMA increased IL-8 levels in cell culture supernatants compared with cells treated with the respective vehicle controls. Prior small interfering (si)RNA-induced MUC1 silencing further increased H. pylori - and PMA-stimulated IL-8 levels compared with a negative control siRNA. MUC1-expressing AGS cells pretreated with the PPARγ agonist troglitazone (TGN) had reduced H. pylori - and PMA-stimulated IL-8 levels compared with cells treated with H. pylori or PMA alone. However, following MUC1 siRNA knockdown, no differences in IL-8 levels were seen between TGN/ H. pylori and H. pylori -only cells or between TGN/PMA and PMA-only cells. Finally, TGN-treated AGS cells had increased Muc1 promoter activity, as measured using a Muc1-luciferase reporter gene, and greater MUC1 protein levels by Western blot analysis, compared with vehicle controls. These results support the hypothesis that PPARγ stimulates MUC1 expression by AGS cells, thereby attenuating H. pylori - and PMA-induced IL-8 production.

Download Full-text

PPARγ inhibits airway epithelial cell inflammatory response through a MUC1-dependent mechanism

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00360.2011 ◽

2012 ◽

Vol 302 (7) ◽

pp. L679-L687 ◽

Cited By ~ 11

Author(s):

Yong Sung Park ◽

Erik P. Lillehoj ◽

Kosuke Kato ◽

Choon Sik Park ◽

Kwang Chul Kim

Keyword(s):

Epithelial Cells ◽

Culture Media ◽

A549 Cells ◽

Luciferase Reporter ◽

Muc1 Expression ◽

Mrna Levels ◽

Airway Epithelial ◽

Pparγ Agonist ◽

Tnf Α ◽

Primary Mouse

This study was conducted to examine the relationship between the peroxisome proliferator-associated receptor-γ (PPARγ) and MUC1 mucin, two anti-inflammatory molecules expressed in the airways. Treatment of A549 lung epithelial cells or primary mouse tracheal surface epithelial (MTSE) cells with phorbol 12-myristate 13-acetate (PMA) increased the levels of tumor necrosis factor (TNF)-α in cell culture media compared with cells treated with vehicle alone. Overexpression of MUC1 in A549 cells decreased PMA-stimulated TNF-α levels, whereas deficiency of Muc1 expression in MTSE cells from Muc1 null mice increased PMA-induced TNF-α levels. Treatment of A549 or MTSE cells with the PPARγ agonist troglitazone (TGN) blocked the ability of PMA to stimulate TNF-α levels. However, the effect of TGN required the presence of MUC1/Muc1, since no differences in TNF-α levels were seen between PMA and PMA plus TGN in MUC1/Muc1-deficient cells. Similarly, whereas TGN decreased interleukin-8 (IL-8) levels in culture media of MUC1-expressing A549 cells treated with Pseudomonas aeruginosa strain K (PAK), no differences in IL-8 levels were seen between PAK and PAK plus TGN in MUC1-nonexpressing cells. EMSA confirmed the presence of a PPARγ-binding element in the MUC1 gene promoter. Finally, TGN treatment of A549 cells increased MUC1 promoter activity measured using a MUC1-luciferase reporter gene, augmented MUC1 mRNA levels by quantitative RT-PCR, and enhanced MUC1 protein expression by Western blot analysis. These combined data are consistent with the hypothesis that PPARγ stimulates MUC1/Muc1 expression, thereby blocking PMA/PAK-induced TNF-α/IL-8 production by airway epithelial cells.

Download Full-text

Cyclin G2 Regulates Adipogenesis through PPARγ Coactivation

Endocrinology ◽

10.1210/en.2010-0461 ◽

2010 ◽

Vol 151 (11) ◽

pp. 5247-5254 ◽

Cited By ~ 32

Author(s):

Victor Aguilar ◽

Jean-Sébastien Annicotte ◽

Xavier Escote ◽

Joan Vendrell ◽

Dominique Langin ◽

...

Keyword(s):

Adipocyte Differentiation ◽

Luciferase Reporter ◽

Peroxisome Proliferator ◽

Mrna Levels ◽

Cell Cycle Regulators ◽

Luciferase Reporter Gene ◽

Peroxisome Proliferator Activated Receptor ◽

Cyclin G2 ◽

Regulatory Effects

Cell cycle regulators such as cyclins, cyclin-dependent kinases, or retinoblastoma protein play important roles in the differentiation of adipocytes. In the present paper, we investigated the role of cyclin G2 as a positive regulator of adipogenesis. Cyclin G2 is an unconventional cyclin which expression is up-regulated during growth inhibition or apoptosis. Using the 3T3-F442A cell line, we observed an up-regulation of cyclin G2 expression at protein and mRNA levels throughout the process of cell differentiation, with a further induction of adipogenesis when the protein is transiently overexpressed. We show here that the positive regulatory effects of cyclin G2 in adipocyte differentiation are mediated by direct binding of cyclin G2 to peroxisome proliferator-activated receptor γ (PPARγ), the key regulator of adipocyte differentiation. The role of cyclin G2 as a novel PPARγ coactivator was further demonstrated by chromatin immunoprecipitation assays, which showed that the protein is present in the PPARγ-responsive element of the promoter of aP2, which is a PPARγ target gene. Luciferase reporter gene assays, showed that cyclin G2 positively regulates the transcriptional activity of PPARγ. The role of cyclin G2 in adipogenesis is further underscored by its increased expression in mice fed a high-fat diet. Taken together, our results demonstrate a novel role for cyclin G2 in the regulation of adipogenesis.

Download Full-text

A distal activation domain is critical in the regulation of the rat androgen receptor gene promoter

Biochemical Journal ◽

10.1042/bj2940779 ◽

1993 ◽

Vol 294 (3) ◽

pp. 779-784 ◽

Cited By ~ 19

Author(s):

C S Song ◽

S Her ◽

M Slomczynska ◽

S J Choi ◽

M H Jung ◽

...

Keyword(s):

Androgen Receptor ◽

Nucleotide Sequence ◽

Receptor Gene ◽

Critical Role ◽

Androgen Receptor Gene ◽

Luciferase Reporter ◽

Upstream Region ◽

Luciferase Reporter Gene ◽

Mobility Shift ◽

Cis Element

The far upstream region of the rat androgen receptor (AR) gene has been cloned, and the nucleotide sequence up to -2656 bp established. Nested deletion mutants of rat AR 5′ flanking sequences were ligated to the luciferase reporter gene, and their promoter activities were examined in transfected COS1 cells. Results show a critical cis-acting domain located between positions -960 and -940. Deletion of this cis element resulted in a greater than 90% decrease in the promoter activity. A nuclear protein that specifically binds to this 21-nucleotide sequence was identified by gel mobility shift analysis. The -960/-940 cis element has no identify to the binding sequence of any known transcription factor. Furthermore, the cognate binding protein is present in both rat and human (HeLa) cell nuclear extracts. We conclude that a novel trans-activator interacting at the -960/-940 region plays a critical role in the regulation of AR gene expression.

Download Full-text

Downregulation of PU.1 Leads to Decreased Expression of Dectin-1 in Alveolar Macrophages during Pneumocystis Pneumonia

Infection and Immunity ◽

10.1128/iai.01141-09 ◽

2010 ◽

Vol 78 (3) ◽

pp. 1058-1065 ◽

Cited By ~ 16

Author(s):

Chen Zhang ◽

Shao-Hung Wang ◽

Chung-Ping Liao ◽

Shoujin Shao ◽

Mark E. Lasbury ◽

...

Keyword(s):

Alveolar Macrophages ◽

Mrna Level ◽

Gene Promoter ◽

Pneumocystis Pneumonia ◽

Luciferase Reporter ◽

Mrna Levels ◽

Dependent Manner ◽

Luciferase Reporter Gene ◽

Mobility Shift ◽

Mobility Shift Assay

ABSTRACT Dectin-1 is an important macrophage phagocytic receptor recognizing fungal β-glucans. In this study, the mRNA levels of the Dectin-1 gene were found to be decreased by 61% in alveolar macrophages (AMs) from Pneumocystis-infected mice. The expression of Dectin-1 protein on the surface of these cells was also significantly decreased. By fluorescence in situ hybridization, mRNA expression levels of the transcription factor PU.1 were also found to be significantly reduced in AMs from Pneumocystis-infected mice. Electrophoretic mobility shift assay showed that PU.1 protein bound Dectin-1 gene promoter. With a luciferase reporter gene driven by the Dectin-1 gene promoter, the expression of the PU.1 gene in NIH 3T3 cells was found to enhance the luciferase activity in a dose-dependent manner. PU.1 expression knockdown by small interfering RNA (siRNA) caused a 63% decrease in Dectin-1 mRNA level and 40% decrease in protein level in AMs. Results of this study indicate that downregulation of PU.1 during Pneumocystis pneumonia leads to decreased expression of Dectin-1 in AMs.

Download Full-text

Tumor necrosis factor-α inhibits peroxisome proliferator-activated receptor γ activity at a posttranslational level in hepatic stellate cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00411.2003 ◽

2004 ◽

Vol 286 (5) ◽

pp. G722-G729 ◽

Cited By ~ 49

Author(s):

Chin K. Sung ◽

Hongyun She ◽

Shigang Xiong ◽

Hidekazu Tsukamoto

Keyword(s):

Hepatic Stellate Cells ◽

Stellate Cells ◽

Luciferase Reporter ◽

Critical Event ◽

Peroxisome Proliferator ◽

Mrna Levels ◽

Luciferase Reporter Gene ◽

Mobility Shift ◽

Peroxisome Proliferator Activated Receptor ◽

Tnf Α

Diminished activity of peroxisome proliferator-activated receptor γ (PPARγ) is implicated in activation of hepatic stellate cells (HSC), a critical event in the development of liver fibrosis. In the present study, we investigated PPARγ regulation by TNF-α in an HSC line designated as BSC. In BSC, TNF-α decreased both basal and ligand (GW1929)-induced PPARγ mRNA levels without changing its protein expression. Nuclear extracts from BSC treated with TNF-α showed decreased binding of PPARγ to PPAR-responsive element (PPRE) as determined by electrophoretic mobility shift assay. In BSC transiently transfected with a PPARγ1 expression vector and a PPRE-luciferase reporter gene, TNF-α decreased both basal and GW1929-induced transactivation of the PPRE promoter. TNF-α increased activation of ERK1/2 and JNK, previously implicated in phosphorylation of Ser82 of PPARγ1 and resultant negative regulation of PPARγ transactivity. In fact, TNF-α failed to inhibit transactivity of a Ser82Ala PPARγ1 mutant in BSC. TNF-α-mediated inhibition of PPARγ transactivity was not blocked with a Ser32Ala/Ser36Ala mutant of inhibitory NF-κBα (IκBα). These results suggest that TNF-α inhibits PPARγ transactivity in cultured HSC, at least in part, by diminished PPARγ-PPRE (DNA) binding and ERK1/2-mediated phosphorylation of Ser82 of PPARγ1, but not via the NF-κB pathway.

Download Full-text

MicroRNA-182-5p protects human lens epithelial cells against oxidative stress-induced apoptosis by inhibiting NOX4 and p38 MAPK signalling

BMC Ophthalmology ◽

10.1186/s12886-020-01489-8 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Zhao-Na Li ◽

Ming-Xu Ge ◽

Zhong-Fang Yuan

Keyword(s):

Epithelial Cells ◽

P38 Mapk ◽

Mapk Signaling ◽

Luciferase Reporter ◽

Human Lens ◽

Lens Epithelial Cells ◽

Luciferase Reporter Gene ◽

Age Related ◽

Induced Apoptosis

Abstract Background MicroRNAs (miRNAs) are abnormally expressed in various ocular diseases, including age-related cataract. However, the role of miR-182-5p in the progression of age-related cataract remains unclear. Methods The expression of miR-182-5p in HLE-B3 cells was detected by qRT-PCR. HLE-B3 cells were transfected with miR-182-5p mimics. CCK-8, EdU, flow cytometry, 2′,7′-dichlorodihydrofluorescein diacetate, JC-1 kit, and western blot were used to assess the cell viability, proliferation, apoptosis, reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP), and protein expression, respectively, in vitro. The relationship between miR-182-5p and NOX4 was confirmed using the dual-luciferase reporter gene analysis. Results We found that miR-182-5p expression was significantly decreased by the H2O2 exposure. Overexpression of miR-182-5p promoted cell proliferation and inhibited ROS production and apoptosis in H2O2-induced HLE-B3 cells. Moreover, p-p-38, p-ERK, and p-JNK were up-regulated in H2O2-treated HLE-B3 cells, and overexpression of miR-182-5p reversed the effects of H2O2 on HLE-B3 cells. In addition, dual-luciferase reporter assay substantiated that NOX4 was a direct target and downregulated by miR-182-5p. Conclusions We concluded that miR-182-5p inhibited lens epithelial cells apoptosis through regulating NOX4 and p38 MAPK signaling, providing a novel biomarker for treatment of age-related cataract.

Download Full-text

Seasonal expressions of SPAG11A and androgen receptor in the epididymis of the wild ground squirrels (Citellus dauricus Brandt)

European Journal of Histochemistry ◽

10.4081/ejh.2020.3111 ◽

2020 ◽

Vol 64 (2) ◽

Cited By ~ 1

Author(s):

Wenyang Yu ◽

Ziwen Zhang ◽

Pei Liu ◽

Xiaoying Yang ◽

Haolin Zhang ◽

...

Keyword(s):

Smooth Muscle ◽

Androgen Receptor ◽

Luteinizing Hormone ◽

Epithelial Cells ◽

Breeding Season ◽

Seasonal Changes ◽

Follicle Stimulating Hormone ◽

Ground Squirrels ◽

Mrna Levels ◽

Real Time Quantitative Pcr

Sperm-associated antigen 11A (SPAG11A) is expressed exclusively in the epididymis, which can specifically regulate sperm maturation. The aim of this study was to investigate the seasonal expressions of beta-defensin (SPAG11A) and androgen receptor (AR) in the epididymis of the wild ground squirrels (Citellus dauricus Brandt). Morphologically, the results showed that epididymis length and weight in the breeding season were significantly higher than those of the non-breeding season. Histologically, the results revealed that enlarged lumen diameters, thickened epithelial and abundant sperm in the breeding season while reduced lumen diameters and epithelial with no sperm in the non-breeding season. SPAG11A was intensely expressed in cytoplasm and nucleus of epithelial cells in the breeding season, and weaker staining in the non-breeding season. The immunostaining of AR was only presented in nucleus of smooth muscle cells and epithelial cells in the whole epididymis with stronger staining in the breeding season. The results of real-time quantitative PCR also showed that the mRNA levels of SPAG11A and AR in the epididymis during the breeding season were significantly higher than those of the non-breeding season. In addition, the levels of testosterone (T), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the serum were higher during the breeding season. Taken together, these results suggested that SPAG11A might play an important role to regulate seasonal changes in epididymal function of the wild ground squirrels.

Download Full-text

Allelic variant at −79 (C>T) in CDKN1B (p27Kip1) confers an increased risk of thyroid cancer and alters mRNA levels

Endocrine Related Cancer ◽

10.1677/erc-09-0016 ◽

2010 ◽

Vol 17 (2) ◽

pp. 317-328 ◽

Cited By ~ 26

Author(s):

Iñigo Landa ◽

Cristina Montero-Conde ◽

Donatella Malanga ◽

Silvia De Gisi ◽

Guillermo Pita ◽

...

Keyword(s):

Thyroid Cancer ◽

Cancer Susceptibility ◽

Recessive Model ◽

Luciferase Reporter ◽

Mrna Levels ◽

Luciferase Reporter Gene ◽

Reverse Transcription Pcr ◽

First Case ◽

Common Genetic Variants ◽

Increased Risk

The aim of this study is to assess if common genetic variants located in the CDKN1B locus, coding for the cell cycle inhibitor p27Kip1, are involved in thyroid cancer susceptibility. Based on the literature and functional predictions, we selected three polymorphisms within the CDKN1B gene (rs2066827 (T326G, V109G), rs34330 (−79C>T) and rs36228499 (−838C>A)) to perform the first case–control study in thyroid cancer involving this locus. We had 649 Spanish patients with sporadic thyroid cancer and 385 healthy representative controls available. Luciferase reporter gene assays, real-time quantitative reverse transcription-PCR and immunoblot experiments were carried out to demonstrate the putative effect of the associated variant. The polymorphism rs34330 (−79C>T) was identified as a risk factor for developing the follicular variant of papillary thyroid carcinoma (FVPTC), fitting a recessive model (odds ratio=2.12; 95% confidence interval=1.09–4.15; P value=0.023). The risk allele (T) of this single nucleotide polymorphism led to a lower transcription rate in cells transfected with a luciferase reporter driven by the polymorphic p27Kip1 promoter (P value <0.001). This effect was observed in −79TT genotype control carriers, who showed a tendency towards lower CDKN1B mRNA levels in lymphocytes, as well as at the protein level. This is the first study that identifies CDKN1B as a low-penetrance gene in thyroid cancer, and specifically in FVPTC subtype. We propose a reduced CDKN1B gene transcription depending on the genotype of the −79C>T (rs34330) variant as a novel mechanism underlying p27Kip1 downregulation.

Download Full-text