Roles of the ERK1/2 and PI3K/PKB signaling pathways in regulating the expression of extracellular matrix genes in rat pulmonary artery smooth muscle cells

Abstract Purpose: To investigate the mechanisms by which PD98059 and LY294002 interfere with the abnormal deposition of extracellular matrix regulated by connective tissue growth factor (CTGF) of rat pulmonary artery smooth muscle cells (PASMCs). Methods: Rat PASMCs were cultured and separated into a control group. Real-time fluorescence quantitative PCR was performed to detect the expression of collagen III and fibronectin mRNA. Immunohistochemistry and western blot analyses were performed to detect the expression of collagen III protein. Results: The expression of collagen III and fibronectin mRNA was greater in PASMCs stimulated with CTGF for 48 h, than in the control group. After 72h of stimulation, the expression of collagen III protein in the PASMCs was greater than in the control. The equivalent gene and protein expression of the CPL group were much more significant. Conclusions: CTGF can stimulate the gene expression of collagen III and fibronectin in PASMCs, which may be one of the factors that promote pulmonary vascular remodeling (PVR) under the conditions of pulmonary arterial hypertension (PAH). PD98059 and LY294002 can inhibit the ERK1/2 and PI3K/PKB signaling pathways, respectively, thus interfering with the biological effects of CTGF. This may be a new way to reduce PAH-PVR.

Avances en el tratamiento de cáncer de próstata resistente a la castración: énfasis en nuevas terapias hormonales / Advances in the treatment of castration-resistant prostate cancer: emphasis in new hormonal therapies

Prostate cancer represents the second cancer-related cause of death in North American and Chilean men. The main treatment for incurable stages of disease is surgical or pharmacological castration. However, with time and despite the addition of anti-androgens, the disease progresses to a clinical state that has been commonly referred to as “hormone refractory”. In recent years, the concept of hormone refractoriness has been challenged and replaced by “castration resistance”, acknowledging that further and optimal hormonal manipulation can be attained, beyond achieving testosterone levels at castration range. The purpose of this review is to summarize the recent therapeutic breakthroughs in the management of metastatic castrate resistant prostate cancer (mCRPC), with greater emphasis in the newer hormonal therapy agents such as Abiraterone and Enzalutamide. Future combination and sequential treatment strategies are contextualized in the current era of personalized cancer medicine and genomic characterization of prostate cancer.

Gamma Linolenic Acid Exerts Anti-Inflammatory and Anti-Fibrotic Effects in Diabetic Nephropathy

PURPOSE: This study was undertaken to investigate the effects of gamma linolenic acid (GLA) on inflammation and extracellular matrix (ECM) synthesis in mesangial and tubular epithelial cells under diabetic conditions. MATERIALS AND METHODS: Sprague-Dawley rats were intraperitoneally injected with either a diluent [n=16, control (C)] or streptozotocin [n=16, diabetes (DM)], and eight rats each from the control and diabetic groups were treated with evening primrose oil by gavage for three months. Rat mesangial cells and NRK-52E cells were exposed to medium containing 5.6 mM glucose and 30 mM glucose (HG), with or without GLA (10 or 100 microM). Intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1), and fibronectin (FN) mRNA and protein expression levels were evaluated. RESULTS: Twenty-four-hour urinary albumin excretion was significantly increased in DM compared to C rats, and GLA treatment significantly reduced albuminuria in DM rats. ICAM-1, MCP-1, FN mRNA and protein expression levels were significantly higher in DM than in C kidneys, and these increases were significantly abrogated by GLA treatment. In vitro, GLA significantly inhibited increases in MCP-1 mRNA expression and protein levels under high glucose conditions in HG-stimulated mesangial and tubular epithelial cells (p<0.05, respectively). ICAM-1 and FN expression showed a similar pattern to the expression of MCP-1. CONCLUSION: GLA attenuates not only inflammation by inhibiting enhanced MCP-1 and ICAM-1 expression, but also ECM accumulation in diabetic nephropathy.

Mechanism of the transforming growth factor-â induction of fibronectin expression in hepatic stem-like cells

Transforming growth factor-â1 (TGF-â1) plays an important role in the fibrogenic process in the liver. The aim of the present study was to explore the action of TGF-â1 on fibronectin expression in rat hepatic stem-like cells and the underlying mechanisms. The level of fibronectin expression was determined in hepatic stem-like cells (WB cells) before and after TGF-â1 stimulation by RT-PCR and Western blot methods. Using immunogold transmission electron microscopy and the Western blot method, we observed the result of the expression and the distribution of cAMP, phosphorylated Smad3 and Smad7 before and after TGF-â1 treatment. The levels of fibronectin expression in both mRNA and protein increased 4- to 5-fold after TGF-â1 stimulation, reaching an optimum level after 8 h and then gradually falling back. Similarly, TGF-â1 stimulation resulted in an increase of cAMP in WB cells, peaking at 8 h. After treatment with TGF-â1 for 24 h, the expression of cAMP gradually decreased. In addition, we found that TGF-â1 treatment also contributed to the increased expression and to changes in cellular distribution of phosphorylated Smad3 (translocation from the cytoplasm to the nucleus) and Smad7 (translocation from the nucleus to the cytoplasm) in WB cells. The present study demonstrates that TGF-â is involved in the fibrogenic process in hepatic stem cells through up-regulation of fibronectin expression, and the mechanisms underlying this process may be associated with the activation of cAMP and Smad pathways.

IGF-1 Counteracts TGF-beta-Mediated Enhancement of Fibronectin for in Vitro Human Lens Epithelial Cells

PURPOSE: To determine whether insulin-like growth factor (IGF-1) affects transforming growth factor (TGF-beta)- mediated fibronectin accumulation in human lens epithelial cell line (HLE B-3) cells. MATERIALS AND METHODS: HLE B-3 cells were incubated for 24 hours with TGF-beta (10ng/ mL), IGF-1 (10ng/mL), or both. Expression of the fibronectin gene was determined using a real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Fibronectin levels were examined using western blot analysis and immunofluorescence staining. RESULTS: Expression of the fibronectin gene was not different between the TGF-beta/IGF-1 treated group and the TGF-beta treated group (p= 0.116). However, western blot analysis demonstrated decreased fibronectin levels in human lens epithelial cells treated with TGF-beta and IGF-1 compared to those treated with TGF-beta only (p < 0.01). Immunofluorescence staining disclosed inhibition of TGF-beta-induced fibronectin in the presence of IGF-1. CONCLUSION: This study suggests that IGF-1 counteracts TGF-beta-mediated fibronectin accumulation in human lens epithelial cells.

The relationship between expression of FN in human skin injury and early injury time / 法医学杂志

OBJECTIVE@#In order to supply an effective reference of early injury time estimation and explore the time limit of detection of EDA\EDB mRNA in human skin samples, the expression of alternative splicing segment of fibronectin--EDA\EDB in incised wound of human skin were studied.@*METHODS@#Using in situ hybridization with DIG-labeled anti-sense RNA probe, the expression of FN EDA\EDB domain was detected in human skin incised wound at the early stage of injury (from 30 min to 3 h).@*RESULTS@#The positive expression rates of FN-EDA\EDB immediately after injury and area far away from wound were same as the control group. The expression of FN-EDA\EDB in human skin incised wound showed a gradually increased tendency in early injury time (within 3 h). The positive expression cells were mainly distributed in basement cells of epidermis and the expression of EDA is much higher than EDB. It's difficult to detect EDA\EDB mRNA when the samples were deposited in air for 4 hour.@*CONCLUSION@#FN-EDA\EDB may be used as a sensitive mark for the estimation of early injury time. The in-situ hybridization technique is not applicable in the application.

Expression of EIIIA-fibronectin in injured rat skin used in estimation of wound interval / 法医学杂志

OBJECTIVE@#To observe the means of fibronectin(FN) alternative splicing and the expression of EIIIA-FN variant in rat skin after bruise, for the sake of providing some help for forensic estimation of wound interval.@*METHODS@#Total RNA was isolated from wounded skin, and reverse transcription polymerase chain reaction was conducted to amplify target segments.@*RESULTS@#Detectable EIIIA+(526 bp) segments, lacked in normal organize, was amplified at 1 h after experimental wound, and the levels were increased within 24 h.@*CONCLUSION@#The alternative splicing EIIIA-fibronectin variant would be a satisfied criterion for research of skin injury.

The in situ hybridization effort estimation between antisense RNA probe and random primed DNA probe / 法医学杂志

OBJECTIVE@#To estimate the effort between antisense RNA probe and random primed DNA probe.@*METHODS@#The in situ hybridization was conducted on parafin section from wounding model of rat skin.@*RESULTS@#Although both probes appeared positive staining, RNA probes was superior to DNA probes in terms of depth of staining and background.@*CONCLUSION@#RNA probe showed more satisfactorily on ISH.

The expression of FN-IIIcs in the wound healing and its relationship with injury time / 法医学杂志

OBJECTIVE@#To research the expression of alternative splicing segment of fibronectin-IIIcs in incised wound of skin, to offer the experimental data for early injury time judgement.@*METHODS@#Using in situ hybridization with DIG-labeled anti-sense RNA probe, the expression of FN-IIIcs domain was detected in both human and rat skin incised wound.@*RESULTS@#(1) The expression of FN-IIIcs improved from the time of 30 min after injury, reached peak at 6 h and then reduced gradually. (2) The positive expression cells were mainly distributed in hair follicles, sebaceous glands and endothelium, however, in human beings they were basement cells of epidermis.@*CONCLUSION@#The expression of FN-IIIcs domain would be a supposed useful criteria for early injury time judgement.

Induction of fibronectin gene expression by inhibitors of protein phosphatase type 2B in normal and transformed fibroblasts

Two intracellular signal pathways mediated by cAMP and protein kinase C (PKC) were involved in the regulation of FN gene expression (Lee et al., Exp. Mol. Med. 30: 240, 1998). In this study, a possible involvement of protein phosphatase-dependent pathways in the regulation of FN gene expression was investigated by using protein phosphatase type 2B (PP2B) inhibitors, cyclosporin A and ascomycin. Both cyclosporin A and ascomycin increased the levels of FN mRNA in WI-38 human lung fibroblasts and the SV40-transformed WI-38 cells but not in MC3T3-E1 osteoblasts. The expression of FN appears to increase from six hours up to 48 hours after treatment suggesting that it is not an immediate effect. In addition, this effect required a new protein synthesis. Neither cyclosporin A nor ascomycin affects the phorbol myristate acetate (PMA)-induced stimulation of FN gene expression and the same result occurred in vice versa suggesting the mechanism of PMA and cyclosporin A/ascomycin in the regulation of FN gene expression may share a common downstream pathway. Taken together, this study suggests that PP2B is involved in the regulation of FN gene expression in normal and transformed fibroblasts but not in osteoblasts.

Regulation of fibronectin gene expression by cyclic AMP and phorbol myristate acetate in HT-1080 human fibrosarcoma cells

We studied the regulation of fibronectin (FN) gene expression by cAMP and phorbol-12-myristate-13-acetate (PMA) in HT-1080 human fibrosarcoma cells. Dibutyryl cAMP increased FN synthesis and mRNA levels, while PMA inhibited the cAMP-induced FN synthesis. In transient transfection assays, cAMP increased FN promoter activity, while PMA paradoxically enhanced the cAMP-induced promoter activity. Stable transfection experiments, however, showed that neither cAMP or PMA alone nor together affected FN promoter activity. These results suggest that PMA antagonizes the cAMP-induced FN gene expression and that both the action of cAMP and the inhibition of its action by PMA may occur at the posttranscriptional level in HT-1080 cells.