TGF-β1 Regulation of Estrogen Production in Mature Rat Leydig Cells

Background

Besides androgens, estrogens produced in Leydig cells are also crucial for mammalian germ cell differentiation. Transforming growth factor-β1 (TGF-β1) is now known to have multiple effects on regulation of Leydig cell function. The objective of the present study is to determine whether TGF-β1 regulates estradiol (E₂) synthesis in adult rat Leydig cells and then to assess the impact of TGF-β1 on Cx43-based gap junctional intercellular communication (GJIC) between Leydig cells.

Methodology/Principal Findings

Primary cultured Leydig cells were incubated in the presence of recombinant TGF-β1 and the production of E₂ as well as testosterone (T) were measured by RIA. The activity of P450arom was addressed by the tritiated water release assay and the expression of Cyp19 gene was evaluated by Western blotting and real time RT-PCR. The expression of Cx43 and GJIC were investigated with immunofluorescence and fluorescence recovery after photo-bleaching (FRAP), respectively. Results from this study show that TGF-β1 down-regulates the level of E₂ secretion and the activity of P450arom in a dose-dependent manner in adult Leydig cells. In addition, the expression of Cx43 and GJIC was closely related to the regulation of E₂ and TGF-β1, and E₂ treatment in turn restored the inhibition of TGF-β1 on GJIC.

Conclusions

Our results indicate, for the first time in adult rat Leydig cells, that TGF-β1 suppresses P450arom activity, as well as the expression of the Cyp19 gene, and that depression of E₂ secretion leads to down-regulation of Cx43-based GJIC between Leydig cells.     

Vision-Related Quality of Life in Patients with Inactive HLA-B27–Associated-Spectrum Anterior Uveitis

We investigated the vision-related quality of life (VR-QOL) in patients with HLA-B27 associated anterior uveitis (AU). The study was conducted in 2012 at the ophthalmology department of the University Medical Center of Groningen. We included AU patients who were HLA-B27 positive and/or were diagnosed by a rheumatologist with an HLA-B27 associated systemic disease. Sixty-one of 123 (50%) adult patients participated. All patients filled-out the National Eye Institute Visual Functioning Questionnaire-25 (NEI VFQ-25), Beck Depression Inventory (BDI-II), social support lists and an additional questionnaire for gathering general information. Medical records were reviewed for clinical characteristics. Analyses were conducted on various patient and ocular characteristics. We compared our NEI VFQ-25 scores with those previously found in the literature. Our main outcome measures were VR-QOL scores and their associations with various general patient and ocular characteristics. We found that the NEI VFQ-25 mean overall composite score was 88.9±8.8, which is relatively high, but lower than that found in a normal working population. The mean general health score was 47.4±20.8, which is lower than in patients with other ocular diseases. Patients with a systemic disease scored significantly lower on general health and VR-QOL, compared to patients without a systemic disease. Patients with a depression (6/59 (10%)) frequently had ankylosing spondylitis (5/6 patients) and they scored significantly worse on VR-QOL. We concluded that patients with HLA-B27 associated AU have a relatively high VR-QOL. However, the presence of a systemic disease is associated with lower VR-QOL and general health scores. In addition, depression is associated with a lower VR-QOL.     

Programmed Cell Death in 2′,3′-dideoxycytidine-resistant Human Monoblastoid U937 Cells

2′,3′-Dideoxycytidine is a powerful in vitro inhibitor of human immunodeficiency virus and is currently used in the treatment of acquired immunodeficiency syndrome. A long-term exposure of U937 monoblastoid cells to dideoxycytidine induces the selection of drug-resistant cells (U937-R). In previous studies, we investigated some important biochemical properties and functional activities, such as basal respiration, protein kinase C activity, superoxide anion release, and the level of reduced glutathione, which were found to be higher in the drug-resistant cell line, compared to the parental one. In the present study, we evaluated the response of the two cell lines to the induction of apoptosis by treatment with staurosporine and okadaic acid, which interfere with the protein kinase and phosphatase pathways, respectively. Moreover, knowing that GSH plays a crucial role in the regulation of nitric oxide-dependent apoptosis, U937-R and parental lines have been treated with SIN-1, which is known to generate significant amounts of O₂ and nitric oxide. Resistant and parental cells have been analysed by light and electron microscopy and agarose gel electrophoresis of isolated DNA has been performed. The obtained results demonstrate a different susceptibility of U937-R cell line to apoptosis induced with the three triggers. U937-R cells show more advanced apoptotic features if compared with parental cells, after staurosporine treatment. Differently, the okadaic acid does not induce a different behaviour in the two models. On the contrary, the agent SIN-1 determines an increased number of apoptotic cells in the U937 line. The results suggest that a higher level of protein kinase C and glutathione could prevent programmed cell death in U937-R.     

Regulation of Weibel-Palade Body Exocytosis by α-Synuclein in Endothelial Cells

α-Synuclein is a small presynaptic protein implicated in the pathogenesis of Parkinson disease. Nevertheless, its physiological roles and mechanisms remain incompletely understood. α-Synuclein is not only expressed in neurons but also in the vascular endothelium, which contains intracellular granules called Weibel-Palade bodies (WPBs) that contain a number of chemokines, adhesive molecules, and inflammatory cytokines. This study explored whether the exocytosis of WPB is regulated by α-synuclein. Phorbol 12-myristate 13-acetate-, thrombin-, or forskolin-induced von Willebrand factor release or translocation of P-selectin from endothelial cells were inhibited by α- and β-synuclein but not γ-synuclein. Three point mutants (A30P, A53T, and E46K) found in familial Parkinson disease also inhibited WPB exocytosis similar to that of wild-type α-synuclein. Furthermore, the negative regulation of WPB exocytosis required the N terminus or the nonamyloid β-component of Alzheimer disease amyloid region of α-synuclein, but not the C-terminal acidic tail, and α-synuclein affected WPB exocytosis through interference with RalA activation by enhancing the interaction of RalGDS-β-arrestin complexes. Immuno-EM analysis revealed that α-synuclein was localized close to WPBs. These findings imply that α-synuclein plays as a negative regulator in WPB exocytosis in endothelial cells.     

Regulation of Glucokinase by Intracellular Calcium Levels in Pancreatic β Cells

Glucokinase (GCK) controls the rate of glucose metabolism in pancreatic β cells, and its activity is rate-limiting for insulin secretion. Posttranslational GCK activation can be stimulated through either G protein-coupled receptors or receptor tyrosine kinase signaling pathways, suggesting a common mechanism. Here we show that inhibiting Ca²⁺ release from the endoplasmic reticulum (ER) decouples GCK activation from receptor stimulation. Furthermore, pharmacological release of ER Ca²⁺ stimulates activation of a GCK optical biosensor and potentiates glucose metabolism, implicating rises in cytoplasmic Ca²⁺ as a critical regulatory mechanism. To explore the potential for glucose-stimulated GCK activation, the GCK biosensor was optimized using circularly permuted mCerulean3 proteins. This new sensor sensitively reports activation in response to insulin, glucagon-like peptide 1, and agents that raise cAMP levels. Transient, glucose-stimulated GCK activation was observed in βTC3 and MIN6 cells. An ER-localized channelrhodopsin was used to manipulate the cytoplasmic Ca²⁺ concentration in cells expressing the optimized FRET-GCK sensor. This permitted quantification of the relationship between cytoplasmic Ca²⁺ concentrations and GCK activation. Half-maximal activation of the FRET-GCK sensor was estimated to occur at ∼400 nm Ca²⁺. When expressed in islets, fluctuations in GCK activation were observed in response to glucose, and we estimated that posttranslational activation of GCK enhances glucose metabolism by ∼35%. These results suggest a mechanism for integrative control over GCK activation and, therefore, glucose metabolism and insulin secretion through regulation of cytoplasmic Ca²⁺ levels.     

Therapeutic Effect of Human iPS-Cell–Derived Myeloid Cells Expressing IFN-β against Peritoneally Disseminated Cancer in Xenograft Models

We recently developed a method to generate myeloid cells with proliferation capacity from human iPS cells. iPS-ML (iPS-cell–derived myeloid/macrophage line), generated by introducing proliferation and anti-senescence factors into iPS-cell–derived myeloid cells, grew continuously in an M-CSF–dependent manner. A large number of cells exhibiting macrophage-like properties can be readily obtained by using this technology. In the current study, we evaluated the possible application of iPS-ML in anti-cancer therapy. We established a model of peritoneally disseminated gastric cancer by intraperitoneally injecting NUGC-4 human gastric cancer cells into SCID mice. When iPS-ML were injected intraperitoneally into the mice with pre-established peritoneal NUGC-4 tumors, iPS-ML massively accumulated and infiltrated into the tumor tissues. iPS-ML expressing IFN-β (iPS-ML/IFN-β) significantly inhibited the intra-peritoneal growth of NUGC-4 cancer. Furthermore, iPS-ML/IFN-β also inhibited the growth of human pancreatic cancer MIAPaCa-2 in a similar model. iPS-ML are therefore a promising treatment agent for peritoneally disseminated cancers, for which no standard treatment is currently available.     

BRCA1 and Estrogen Receptor α Expression Regulation in Breast Cancer Cells

Molecular Biology - BRCA1 (breast cancer 1) protein is involved in the genome stability maintenance participating in homologous recombination-dependent DNA repair. Disruption of BRCA1 functioning...     

The Regulation of Autophagy in Eukaryotic Cells: Do All Roads Pass through Atg1?

The induction of autophagy appears to be tightly controlled in all eukaryotic cells. This highly conserved, degradative process is induced by a variety of signals, including nutrient deprivation, and is generally thought to be refractory to cell growth. Recent work in the budding yeast, Saccharomyces cerevisiae, has suggested that the Atg1 protein kinase is at the center of this control. Atg1, and its associated proteins, appear to be directly targeted by multiple signaling pathways important for the control of both autophagy and cell growth. These pathways involve the small GTP-binding Ras proteins, the Tor protein kinases and the AMP-activated protein kinase, Snf1, respectively. A key question that remains is whether this regulatory paradigm has been evolutionarily conserved. In other words, is Atg1 the primary target of those signalingpathways responsible for coordinating growth with environmental influences in other eukaryotes? Here, we suggest that Atg1 is very likely to fulfill this role but that a truly definitive answer will require that we develop a better understanding of this protein kinase and its targets in all eukaryotes.     

Regulation and Identification of Na,K-ATPase α1 Subunit Phosphorylation in Rat Parotid Acinar Cells

The stimulation of fluid and electrolyte secretion in salivary cells results in ionic changes that promote rapid increases in the activity of the Na,K-ATPase. In many cell systems, there are conflicting findings concerning the regulation of the phosphorylation of the Na,K-ATPase α subunit, which is the catalytic moiety. Initially, we investigated the phosphorylation sites on the α1 subunit in native rat parotid acinar cells using tandem mass spectrometry and identified two new phosphorylation sites (Ser²²², Ser⁴⁰⁷), three sites (Ser²¹⁷, Tyr²⁶⁰, Ser⁴⁷) previously found from large scale proteomic screens, and two sites (Ser²³, Ser¹⁶) known to be phosphorylated by PKC. Subsequently, we used phospho-specific antibodies to examine the regulation of phosphorylation on Ser²³ and Ser¹⁶ and measured changes in ERK phosphorylation in parallel. The G-protein-coupled muscarinic receptor mimetic carbachol, the phorbol ester phorbol 12-myristate 13-acetate, the Ca²⁺ ionophore ionomycin, and the serine/threonine phosphatase inhibitor calyculin A increased Ser²³ α1 phosphorylation. Inhibition of classical PKC proteins blocked carbachol-stimulated Ser²³ α1 subunit phosphorylation but not ERK phosphorylation, which was blocked by an inhibitor of novel PKC proteins. The carbachol-initiated phosphorylation of Ser²³ α1 subunit was not modified by ERK or PKA activity. The Na,K-ATPase inhibitor ouabain reduced and enhanced the carbachol-promoted phosphorylation of Ser²³ and Ser¹⁶, respectively, the latter because ouabain itself increased Ser¹⁶ phosphorylation; thus, both sites display conformational-dependent phosphorylation changes. Ouabain-initiated phosphorylation of Ser¹⁶ α1 was not blocked by PKC inhibitors, unlike carbachol- or phorbol 12-myristate 13-acetate-initiated phosphorylations, suggesting that this site was also a substrate for a kinase other than PKC.     

Regulation of anthocyanin biosynthesis by nitrogen in TTG1–GL3/TT8–PAP1-programmed red cells of Arabidopsis thaliana

Nitrogen nutrients can regulate anthocyanin biosynthesis in Arabidopsis thaliana. In this investigation, we report the nitrogen regulation of anthocyanin biosynthesis activated by TTG1-GL3/TT8-PAP1 in red pap1-D cells. To understand the mechanisms of nitrogen regulation, we employed red pap1-D cells and wild-type cells (as a control) to examine the effects of different nitrogen treatments on anthocyanin biosynthesis. In general, the higher concentrations of ammonium and high total nitrogen tested (e.g., 58.8 and 29.8?mM total nitrogen consisting of NH(4)NO(3) and KNO(3)) reduced the levels and molecular diversity of anthocyanins; in contrast, the lower concentrations of ammonium and total nitrogen conditions (e.g., 9.4?mM KNO(3) and the depletion of nitrogen) increased the levels and molecular diversity of anthocyanins. An expression analysis of the main regulatory and pathway genes showed that at conditions of higher concentrations of ammonium and total nitrogen, the expression levels of PAP1 and TT8 decreased, but the expression levels of LBD37, 38 and 39, three negative regulators of anthocyanin biosynthesis, increased. In addition, the expression levels of the main pathway genes decreased. In contrast, at conditions of lower concentrations of ammonium and total nitrogen, the expression levels of PAP1, TT8 and the main pathway genes increased, whereas those of LBD37, 38 and 39 decreased. These results show that nitrogen regulation of anthocyanin biosynthesis in red cells undergoes a mechanism by which nitrogen controls the expression of genes encoding both main components of the TTG1-GL3/TT8-PAP1 complex and negative regulators. Based on these observations, we propose that the regulatory mechanism of nitrogen may occur via two pathways to control the expression of genes encoding positive and negative regulators in red pap1-D cells.     

Regulation of CCAAT/Enhancer-binding Protein Homologous Protein (CHOP) Expression by Interleukin-1β in Pancreatic β Cells

Apoptosis contributes to immune-mediated pancreatic β cell destruction in type I diabetes. Exposure of β cells to interleukin-1β (IL-1β) causes endoplasmic reticulum stress and activates proapoptotic networks. Here, we show that nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways regulate the expression of CCAAT/enhancer-binding protein homologous protein (CHOP), which mediates endoplasmic reticulum stress-induced apoptosis. Both CHOP mRNA and protein increase in β cells treated with IL-1β. In addition, prolonged exposure to high glucose further increases IL-1β-triggered CHOP expression. IL-1β also causes increased expression of C/EBP-β and a reduction of MafA, NFATc2, and Pdx-1 expression in β cells. Inhibition of the NF-κB and MAPK signaling pathways differentially attenuates CHOP expression. Knocking down CHOP by RNA interference protects β cells from IL-1β-induced apoptosis. These studies provide direct mechanistic links between cytokine-induced signaling pathways and CHOP-mediated apoptosis of β cells.     

The Role of Altered Cell–Cell Communication in Melanoma Progression

Under normal homeostasis, melanocyte growth and behaviour is tightly controlled by the surrounding keratinocytes. Keratinocytes regulate melanocyte behaviour through a complex system of paracrine growth factors and cell-cell adhesion molecules. Pathological changes, leading to development of malignant melanoma, upset this delicate homeostatic balance and can lead to altered expression of cell-cell adhesion and cell-cell communication molecules. In particular, there is a switch from the E-cadherin-mediated keratinocyte-melanocyte partnership to the N-cadherin-mediated melanoma-melanoma and melanoma-fibroblast interaction. Other changes include the alteration in the gap junctions formed between the melanocyte and keratinocyte. Changes in the connexin expression, in particular the loss of connexin 43, may result in a reduction or a loss of gap junctional activity, which is thought to contribute towards tumour progression. In the current review we describe the alterations in cell-cell adhesion and communication associated with melanoma development and progression, and discuss how a greater understanding of these processes may aid the future therapy of this disease.     

Differential Regulation of Extracellular Matrix and Soluble Fibulin-1 Levels by TGF-β1 in Airway Smooth Muscle Cells

Fibulin-1 (FBLN-1) is a secreted glycoprotein that is associated with extracellular matrix (ECM) formation and rebuilding. Abnormal and exaggerated deposition of ECM proteins is a hallmark of many fibrotic diseases, such as chronic obstructive pulmonary disease (COPD) where small airway fibrosis occurs. The aim of this study was to investigate the regulation of FBLN-1 by transforming growth factor beta 1 (TGF-β₁) (a pro-fibrotic stimulus) in primary human airway smooth muscle (ASM) cells from volunteers with and without COPD. Human ASM cells were seeded at a density of 1×10⁴ cells/cm², and stimulated with or without TGF-β₁ (10 ng/ml) for 72 hours before FBLN-1 deposition and soluble FBLN-1 were measured. Fold change in FBLN-1 mRNA was measured at 4, 8, 24, 48, 72 hours. In some experiments, cycloheximide (0.5 µg/ml) was used to assess the regulation of FBLN-1 production. TGF-β₁ decreased the amount of soluble FBLN-1 both from COPD and non-COPD ASM cells. In contrast, the deposition of FBLN-1 into the ECM was increased in ASM cells obtained from both groups. TGF-β₁ did not increase FBLN-1 gene expression at any of the time points. There were no differences in the TGF-β₁ induced FBLN-1 levels between cells from people with or without COPD. Cycloheximide treatment, which inhibits protein synthesis, decreased both the constitutive release of soluble FBLN-1, and TGF-β₁ induced ECM FBLN-1 deposition. Furthermore, in cycloheximide treated cells addition of soluble FBLN-1 resulted in incorporation of FBLN-1 into the ECM. Therefore the increased deposition of FBLN-1 by ASM cells into the ECM following treatment with TGF-β₁ is likely due to incorporation of soluble FBLN-1 rather than de-novo synthesis.