tretinoin and cobaltous-chloride

Hypoxia has recently been proposed as one of the most important factors in progressive renal injury. Hypoxia-induced vascular endothelial growth factor (VEGF) expression may play a critical role in maintaining peritubular capillary endothelium in renal disease. This study was designed to investigate the effect and underlying mechanism of all-trans retinoic acid (ATRA) on hypoxia-induced injury in NRK52E cells.. For mimicking hypoxia, cells were treated with 100 µM of cobalt chloride (CoCl2). The cell viability, expression of VEGF, p65, transforming growth factor-β2 (TGF-β2) and serine carboxypeptidase 1 (Scpep1), and nuclear factor of kappaB (NF-x03BA;B) activities after ATRA treatment were determined by MTT, western blot and electrophoretic mobility shift assay. Co-immunoprecipitation analysis was performed to demonstrate whether Scpep1 interacted with TGF-β2.. It was found that CoCl2 triggered hypoxia injury and significantly reduced cell viability. ATRA pretreatment increased the cell survival rate. Under hypoxic conditions, the expression of VEGF, p65 and TGF-β2 increased. Addition of ATRA significantly attenuated the expression of VEGF, p65 and TGF-β2. There was a corresponding variation of NF-x03BA;B/DNA binding activities. In addition, ATRA stimulated Scpep1 expression under normoxic and hypoxia condition. Furthermore, TGF-β2 interacted with Scpep1.. This study indicated that ATRA may attenuate hypoxia-induced injury in NRK52E cells via inhibiting NF-x03BA;B/VEGF and TGF-β2/VEGF pathway.

Topics: Animals; Antineoplastic Agents; Cathepsin A; Cell Hypoxia; Cell Line; Cell Survival; Cobalt; Electrophoretic Mobility Shift Assay; Gene Expression Regulation; Immunoprecipitation; NF-kappa B; Protein Binding; Rats; Signal Transduction; Transcription Factor RelA; Transforming Growth Factor beta2; Tretinoin; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor B

It has been reported that the all-trans retinoic acid (atRA)-mediated protective effects in various cells are related to the inhibition of nuclear factor (NF)-κB activities. There exists some evidence that an increase in vascular endothelial growth factor (VEGF), which is expressed by proximal tubular epithelial cells and regulated by NFκB, may play a critical role in maintaining peritubular capillary endothelium in renal disease. By stimulating the production of VEGF, hypoxia is involved in tubulointerstitial fibrosis processes in various renal diseases.. NRK52E cells survival rate was proportional to absorbance in dimethyl-thiazol-diphenyltetrazoliumbromide tests. Quantitative real-time polymerase chain reaction and Western blot were performed to assay the expression of VEGF, p65, and Scpep1. The activation of NFκB was determined by electrophoretic mobility shift assay. Co-immunoprecipitation analysis demonstrates that whether the Scpep1 and NFκB protein interacted.. We demonstrated that the hypoxia-mimicking agent CoCl2 triggered hypoxia injury of rat proximal tubular epithelial cells and significantly reduced cell viability. Addition of atRA increased the cell survival rate. Under CoCl(2)-mimicking hypoxic conditions, the expression of VEGF and p65 increased. The addition of atRA significantly attenuated the expression of VEGF and p65. There was a similar variation of NFκB/DNA binding activities. atRA not only activated distinct pathways to stimulate the expression of Scpep1, a retinoid-inducible gene, under normoxic conditions, but also acted as a CoCl(2)-mimicking hypoxia.. The protective effects of atRA against hypoxia-induced injury might be involved in suppression of VEGF expression via stimulating Scpep1 distinct pathways and inhibiting the NFκB pathway.

Topics: Animals; Blotting, Western; Carboxypeptidases; Cell Hypoxia; Cell Line; Cell Survival; Cobalt; Cytoprotection; Dose-Response Relationship, Drug; Electrophoretic Mobility Shift Assay; Epithelial Cells; Fibrosis; Gene Expression Regulation; Immunoprecipitation; Kidney Tubules, Proximal; Rats; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transcription Factor RelA; Tretinoin; Vascular Endothelial Growth Factor A

Macrophage inhibitory cytokine-1/growth differentiation factor 15 (MIC-1/GDF15), a divergent member of the TGF-beta superfamily is induced by a range of proinflammatory cytokines and oxidized low-density lipoprotein (oxLDL) and is highly expressed in macrophages in atherosclerotic and tumor lesions. MIC-1/GDF15, a major p53 target gene, is largely described to have anti-tumorigenic activity and more recently high MIC-1/GDF15 serum levels in late stage cancer were shown to be the major cause of cancer-associated weight loss. MIC-1/GDF15 serum levels independently predict both atherosclerotic events and severity of rheumatoid arthritis (RA), suggesting serum levels are important in modifying disease expression. Controlling serum levels is the ratio of latent unprocessed MIC-1/GDF15 stromal stores to soluble mature MIC-1/GDF15 generated by the cell. Here, we investigate MIC-1/GDF15 secretion from U937 monocytoid cells and identify novel mechanisms designed to ensure secretion of unprocessed cytokine and creation of latent stromal stores. We find that endogenous MIC-1/GDF15 is secreted as both processed and unprocessed forms. Pulse chase analysis of MIC-1/GDF15 secretion reveals that unprocessed MIC-1/GDF15 precursor is rapidly secreted, while mature MIC-1/GDF15 generated within the cell by intracellular processing is secreted much slower, possibly via an alternate secretory route. The COOH-T 47 amino acids of the propeptide are responsible for rapid secretion of MIC-1/GDF15 precursor and this effect occurs in the trans-Golgi network (TGN)/post TGN compartment. Thus, variations in MIC-1/GDF15 intracellular processing, regulating the presence or absence of propeptide, are a powerful mechanism modulating rate of MIC-1/GDF15 secretion and proMIC-1/GDF15 stromal storage, with major impact on circulating levels of mature MIC-1/GDF15.

Topics: Arthritis, Rheumatoid; Atherosclerosis; Cell Differentiation; Cell Hypoxia; Cloning, Molecular; Cobalt; Growth Differentiation Factor 15; Humans; Immunization; Lipopolysaccharides; Macrophages; Neoplasms; Protein Processing, Post-Translational; Secretory Pathway; Transforming Growth Factor beta; Transgenes; Tretinoin; U937 Cells

Previous studies have shown that the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) and its specific hydrolase dimethylarginine dimethylaminohydrolase (DDAH) are involved in the regulation of apoptosis in different cell types. In the present study, we investigated the role of the DDAH/ADMA pathway in cobalt chloride (CoCl(2))-induced apoptosis and the antiapoptotic effect of all-trans retinoic acid (atRA) in undifferentiated pheochromocytoma (PC12) cells. Treatment of CoCl(2) (125 microM) for 48 hr significantly induced the apoptosis of PC12 cells, concomitantly with increased intracellular reactive oxygen species (ROS) production and caspase-3 activity. CoCl(2) treatment also decreased the activity of DDAH and the expression of DDAH2 (mRNA and protein), resulting in an increased level of ADMA. All these alterations induced by CoCl(2) were attenuated by atRA (0.1, 1, or 10 microM). Interestingly, the antiapoptotic effects of atRA were inhibited by DDAH2 small RNA interference. In contrast, DDAH2 overexpression inhibited the proapoptotic effects of CoCl(2). We also found that treatment of exogenous ADMA (3, 10, or 30 microM) induced the apoptosis of PC12 cells in a concentration- and time-dependent manner, which was inhibited by the antioxidant or the caspase-3 inhibitor. These findings suggest that the modulation of the DDAH/ADMA/ROS pathway plays an important role in CoCl(2)-induced apoptosis and the antiapoptotic effects of atRA in undifferentiated PC12 cells.

Topics: Amidohydrolases; Animals; Apoptosis; Arginine; Caspase 3; Cobalt; Dose-Response Relationship, Drug; Neurons; Neuroprotective Agents; Neurotoxins; Oxidative Stress; PC12 Cells; Rats; Reactive Oxygen Species; Signal Transduction; Tretinoin

The effects of CoCl(2) on retinoic acid (RA)-treated embryoid bodies (EBs) were investigated. Four-day EBs were treated with 5x10(-6) M of RA for 4 d, then subjected to attached culturing for 7 d in the presence of CoCl(2) at 0, 20, and 100 microM. Differentiation into MAP2- and GFAP-immunopositive cells was inhibited by CoCl(2) in a dose-dependent manner. Next, RA-treated EBs were dissociated into single cells and cultured for 7 d at an initial cell density of 1x10(3)/cm(2). The number of cells increased in a CoCl(2)-dose dependent fashion. In cultures with 100 microM of CoCl(2), more than 90% of the cells were immunopositive for nestin and nestin-immunopositive cells formed clusters, while there were few cells immunopositive for MAP2 or GFAP. These results suggest that CoCl(2) inhibits neural differentiation of RA-treated EB cells and promotes the proliferation of nestin-immunopositive cells, i.e., embryonic stem (ES)-derived neural stem-like cells.

Topics: Animals; Base Sequence; Cell Differentiation; Cell Proliferation; Cobalt; DNA Primers; Dose-Response Relationship, Drug; Embryonic Stem Cells; Immunohistochemistry; Neurons; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin

Topics: Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Cell Differentiation; Cell Division; Cell Hypoxia; Cobalt; Deferoxamine; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Leukemia, Promyelocytic, Acute; Neoplasm Proteins; Oncogene Proteins, Fusion; Oxides; Tretinoin; U937 Cells

We recently reported that hypoxia-mimetic agents cobalt chloride (CoCl2 CoCl2 ) and desferrioxamine (DFO) could induce differentiation of acute myeloid leukemic (AML) cells. Here, we investigate whether these two agents influence the in vitro differentiation-inducing effect of arsenic trioxide (As2O3) on AML cells, an effective drug for the treatment of acute promyelocytic leukemia (APL) that is a unique subtype of AML with a specific fusion protein, PML-RARalpha.. The APL cell line NB4 and non-APL promonocytic leukemic cell line U937 were treated with As2O3 (0.5 microM) combined with CoCl2 (50 microM) or DFO (10 microM). The U937/PR9 subclone, whose expression of PML-RARalpha protein can be induced by Zn2+, was also investigated. Cellular differentiation was evaluated by morphological criteria and myeloid differentiation-related antigens and marker gene expression. The hypoxia-inducible factor-1alpha (HIF-1alpha) mRNA and protein were detected, respectively, by semi-quantitative/real-time quantitative reverse transcription polymerase chain reaction and immunoblots. PML-RARalpha protein was also analyzed.. CoCl2 and DFO potentiated the growth-inhibiting and differentiation-inducing effects of low-dose As2O3, the latter enhancing CoCl2 and DFO-induced accumulation of HIF-1alpha protein in NB4 cells but not in U937 cells. These two hypoxia-mimetic agents also accelerated As2O3-induced modulation and degradation of PML-RARalpha protein in NB4 cells. Furthermore, inducible expression of the fusion gene restored the co-operative effects of As2O3 and CoCl2/DFO on U937/PR9 cells in terms of growth arrest, differentiation induction and HIF-1alpha protein accumulation.. Mimicked hypoxia enhanced As2O3-induced differentiation, in which HIF-1alpha and PML/RARalpha proteins played an important role. These data provide new insights into the understanding of the mechanisms of the action of As2O3 in the treatment of patients with APL.

Topics: Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Bone Marrow; Cell Differentiation; Cell Division; Cell Hypoxia; Cell Line, Tumor; Cobalt; Deferoxamine; Drug Synergism; Gene Expression Regulation, Leukemic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Leukemia, Promyelocytic, Acute; Neoplasm Proteins; Oncogene Proteins, Fusion; Oxides; Oxygen; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin; U937 Cells

Ascorbic acid has been shown to stimulate collagen synthesis in monolayer cultures of human dermal fibroblasts. In the present studies, we examined whether the presence of a collagen matrix influences this response of dermal fibroblasts to ascorbic acid. Fibroblasts and collagen were mixed and allowed to gel and contract for 6 days to form a matrix prior to determining the concentration and time dependence for ascorbic acid to affect collagen synthesis by fibroblasts within the matrix. Collagen synthesis was stimulated at levels at or above 10 microM ascorbic acid and was maximal after 2 days of treatment. This concentration and time dependence is similar to that of cells grown in monolayer cultures. The effects of transforming growth factor-beta (TGF-beta) and fibroblast growth factor (FGF) were also examined in this model. TGF-beta increased and FGF inhibited collagen synthesis in the gels, as has been shown for cells in monolayer cultures. The effects of potential inhibitors of lipid peroxidation induced by ascorbic acid were also examined in these matrices and compared to previous results obtained in monolayer cultures. Propyl gallate, cobalt chloride, alpha,alpha-dipyridyl, and alpha-tocopherol inhibited the ascorbic acid-mediated stimulation of collagen synthesis while mannitol had no effect. Natural retinoids inhibited total protein synthesis without the specific effect on collagen synthesis that was seen in monolayer cultures. These results indicate that ascorbic acid stimulates collagen synthesis in fibroblasts grown in a collagen matrix in a manner similar to that found in monolayer cultures. In contracting collagen gels, however, the magnitude of the effect is less and retinoids do not specifically inhibit collagen synthesis.

Topics: 2,2'-Dipyridyl; Ascorbic Acid; Cells, Cultured; Cobalt; Collagen; Dose-Response Relationship, Drug; Ethanol; Fibroblast Growth Factors; Fibroblasts; Gels; Growth Substances; Humans; Infant, Newborn; Kinetics; Lipid Peroxidation; Mannitol; Propyl Gallate; Skin; Time Factors; Transforming Growth Factor beta; Tretinoin; Vitamin A

This study examined the morphology and the development of inward currents in the course of differentiation of a stem cell toward a neuronal phenotype. Using the P19 embryonal cell line, whole-cell current profiles of P19 cells before, during and after retinoic acid-induced differentiation were matched with their morphology as well as with the expression of neuron-specific enolase-like immunoreactivity. Prior to and during the initial 48 hr of retinoic acid treatment, P19 cells either lacked detectable currents or expressed a voltage-dependent outward potassium current, did not display neuron-like morphology and did not express neuron-specific enolase-like immunoreactivity. Upon completion of retinoic acid treatment, the current profile of fully differentiated P19 cells was hallmarked by a large voltage-dependent inward current which consisted of a sodium current and a smaller cobalt-sensitive calcium component, in addition to the potassium current observed earlier. Such cells invariably emitted neurites and displayed neuron-specific enolase-like immunoreactivity. Interestingly, coupling was prevalent among P19 cells in the undifferentiated state but was absent in the fully differentiated cultures. In studying cells undergoing neuronal differentiation, these results underscore the importance of taking into account both electrical properties and morphological considerations in determining the degree of differentiation.

Topics: Animals; Cell Aggregation; Cell Differentiation; Cell Division; Cell Line; Cobalt; Electrophysiology; Evoked Potentials; Immunoenzyme Techniques; Membrane Potentials; Neurons; Phenotype; Phosphopyruvate Hydratase; Stem Cells; Tetraethylammonium; Tetraethylammonium Compounds; Tetrodotoxin; Tretinoin