TGF-beta-activating kinase-1 inhibits cell cycle and expression of cyclin D1 and A in LLC-PK1 cells.
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BACKGROUND: Transforming growth factor-beta (TGF-beta) is known to play an important role in the pathophysiology of renal tubular disease. Researchers have recently identified a novel mitogen-activated protein kinase kinase kinase (MAPKKK), TAK (TGF-beta activated kinase)1, which stimulates the MKK3/6-p38K pathway. The purpose of our study was to investigate the functional role of the TAK1-MKK3/6-p38K pathway and classical MAPK cascades in the progression of the cell cycle in renal tubular cells. METHODS: The constitutive active form and negative form of TAK1 (TAK1dN and TAK1K63W, respectively), and active and negative forms of the p42/44 MAPK-activator, MKK1 (S222E and S222A, respectively) were transfected to LLC-PK1 cells. Western blot analyses and promoter-luciferase assay of cyclins D1, D2, D3, E, and A were performed, and cell cycle progression was analyzed by FACS scan. RESULTS: TAK1dN stimulated MKK6 and p38K activity and inhibited the percentage of the S and G2/M phases. TAK1K63 W inhibited TGF-beta-stimulated MKK6 and p38K activity. Cyclin D1 and cyclin A protein levels and promoter activities were negatively regulated by TAK1dN. In contrast, overexpression of the active form of p42/44 MAPK-activator, MKK1, increased cyclin D1 and A promoter activity and protein levels. CONCLUSION: The growth-inhibitory effects of TGF-beta are at least partially mediated by the TAK1-MKK6-p38K pathway. Cyclin D1 and A promoter activity and cell cycle progression in renal tubular cells are negatively regulated by the TAK1-MKK6-p38K pathway and positively regulated by the MKK1-p42/44MAPK pathway. (+info)
TGF-beta2 in aqueous humor suppresses S-phase entry in cultured corneal endothelial cells.
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PURPOSE: Corneal endothelium in vivo is arrested in G1, the phase of the cell cycle that prepares cells for DNA synthesis. In many cell types, transforming factor (TGF)-beta inhibits proliferation by inducing G1-phase arrest. Evidence indicates that corneal endothelial cells synthesize mRNA for TGF-beta1 and are also bathed in aqueous humor that contains TGF-beta2 (mainly in a latent form). As such, this cytokine may maintain the corneal endothelium in a G1-phase-arrested state in vivo. The purpose of these studies was to determine the effect of exogenous TGF-beta2 and TGF-beta2 in aqueous humor on DNA synthesis in cultured corneal endothelial cells. METHODS: Rat corneal endothelial cells were grown in explant culture and identified by morphology and reverse transcription-polymerase chain reaction using primers for specific corneal cell markers. Subconfluent cells were synchronized in the G0 phase (quiescence) by serum starvation for 24 hours. Serum was then added to the cells in the presence or absence of exogenous TGF-beta2 or activated rat aqueous humor. [3H]Thymidine was added, and radioactivity was measured at various time points to detect DNA synthesis. Preincubation of exogenous TGF-beta2 or activated rat aqueous humor with neutralizing antibody was used to test for cytokine specificity. RESULTS: A linear increase in [3H]thymidine incorporation began approximately 16 hours after serum addition, and peak incorporation occurred at approximately 24 hours. Exposure of cells to serum plus TGF-beta2 suppressed [3H]thymidine incorporation in a dose-dependent manner at concentrations ranging from 5 pg/ml to 5 ng/ml. [3H]Thymidine incorporation was also suppressed in cells exposed to serum plus rat aqueous humor diluted 1:10. Neutralizing antibody reversed the effects of both exogenous TGF-beta2 and aqueous humor. CONCLUSIONS: Exogenous TGF-beta2 and TGF-beta2 in aqueous humor suppress S-phase entry of rat corneal endothelial cells. These results suggest that this cytokine in aqueous humor could help maintain the corneal endothelium in a G1-phase-arrested state in vivo. (+info)
PLA(2) stimulation of Na(+)/H(+) antiport and proliferation in rat aortic smooth muscle cells.
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The proliferative properties and the ability to stimulate the Na(+)/H(+) antiport activity of a secretory phospholipase A(2) were studied in rat aortic smooth muscle cells in culture. The requirement of the enzymatic activity of phospholipase A(2) to elicit mitogenesis was assessed by the use of ammodytin L, a Ser(49) phospholipase A(2) from the venom of Vipera ammodytes, devoid of hydrolytic activity. We propose that the proliferative effect is mediated by the same transduction pathway for both proteins. In particular, 1) both secretory phospholipase A(2) and ammodytin L stimulated thymidine incorporation in a dose-dependent manner; 2) both proteins affected the cell cycle, as assessed by cell growth and fluorescence-activated cell sorting experiments; 3) both phospholipase A(2) and ammodytin L increased intracellular pH, a permissive factor for cell proliferation, through activation of the Na(+)/H(+) antiport; 4) ammodytin L was able to displace the (125)I-labeled phospholipase A(2) from specific binding sites in a concentration range consistent with that capable of eliciting a cellular response; and 5) the inhibition by heparin was similar for both proteins, taking into account the ratio of heparin to protein. In conclusion, the enzymatic activity of phospholipase A(2) is not required for the stimulation of mitogenesis. The inhibitory effect of heparin combined with its therapeutic potential could help to clarify the role of phospholipase A(2) in the pathogenesis of several preinflammatory situations. (+info)
Impaired wound healing and angiogenesis in eNOS-deficient mice.
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A role for nitric oxide (NO) in wound healing has been proposed; however, the absolute requirement of NO for wound healing in vivo and the contribution of endothelial NO synthase (eNOS) have not been determined. Experiments were carried out using eNOS gene knockout (KO) mice to determine the requirement for eNOS on wound closure and wound strength. Excisional wound closure was significantly delayed in the eNOS KO mice (29.4 +/- 2.2 days) compared with wild-type (WT) controls (20.2 +/- 0.4 days). At 10 days, incisional wound tensile strength demonstrated a 38% reduction in the eNOS KO mice. Because effective wound repair requires growth factor-stimulated angiogenesis, in vitro and in vivo angiogenesis assays were performed in the mice to assess the effects of eNOS deficiency on angiogenesis. Endothelial cell sprouting assays confirmed in vitro that eNOS is required for proper endothelial cell migration, proliferation, and differentiation. Aortic segments harvested from eNOS KO mice cultured with Matrigel demonstrated a significant reduction in endothelial cell sprouting and [(3)H]thymidine incorporation compared with WT mice at 5 days. Capillary ingrowth into subcutaneously implanted Matrigel plugs was significantly reduced in eNOS KO mice (2.67 +/- 0.33 vessels/plug) compared with WT mice (10.17 +/- 0.79 vessels/plug). These results clearly show that eNOS plays a significant role in facilitating wound repair and growth factor-stimulated angiogenesis. (+info)
Radiolabeled thymidine: a sensitive tracer for early tumor response and recurrence after irradiation.
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This study evaluated the sensitivity of a radiolabeled thymidine tracer for assessment of early tumor response and recurrence after irradiation. METHODS: SW707 human colon carcinoma implanted into nude mice was irradiated with 6 or 20 Gy. Tumor volume was determined for an interval of 14 d. At 4, 8 and 24 h and at 2, 3, 7, 10 and 14 d after irradiation, [14C]thymidine uptake into the tumor was determined with a liquid scintillation counter and the intratumoral distribution of [14C]thymidine was visualized and evaluated semiquantitatively by autoradiography using a phosphor imager. RESULTS: In both groups, tumor volume decreased until day 7 after irradiation; afterward, regrowth occurred in only the group that had received 6 Gy. A decrease in thymidine uptake was found as early as 8 h after irradiation. On day 3 after irradiation, thymidine uptake increased again in the 6-Gy group, before the increase in tumor volume, but remained unchanged in the 20-Gy group. Also on day 3, multiple foci of thymidine uptake suggesting proliferation preceding tumor recurrence were seen on autoradiographs from the 6-Gy group but not from the 20-Gy group. Histological findings correlated with the results of autoradiography. CONCLUSION: The results show that radiolabeled thymidine is a sensitive tracer for assessment of early tumor response and recurrence after irradiation. The rapid decrease in uptake, however, does not allow any prediction about tumor recurrence. (+info)
Simulated ischemia in flow-adapted endothelial cells leads to generation of reactive oxygen species and cell signaling.
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We have previously shown that increased reactive oxygen species (ROS) generation occurs with ischemia in the oxygenated lung and have hypothesized that mechanotransduction is the initiating event. In the present study, we developed an in vitro model of oxygenated ischemia by interrupting medium flow to flow-adapted bovine pulmonary artery endothelial cells in an artificial capillary system. Cellular oxygenation during the "ischemic" period was maintained by perfusing medium over the abluminal surface of porous capillaries. Cells were assessed for ROS generation, nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) binding activities, and DNA synthesis using dichlorofluorescein fluorescence by flow cytometry and spectrofluorometry, electrophoretic mobility shift assay of nuclear extracts with NF-kappaB-specific or AP-1-specific (32)P-labeled oligonucleotides, and (3)H-thymidine incorporation into DNA. Cells that were flow adapted for 2 to 7 days with 1 to 2 dyne/cm(2) shear stress exhibited a 1.6- to 1.9-fold increase in ROS generation during 1 hour of simulated ischemia compared with continuously perfused cells. This effect was abolished by diphenyleneiodonium chloride (DPI), indicating a role for a flavoprotein such as NADPH oxidase. The increase in ROS generation with ischemia was similar for cells from low and high passages. With ischemia, flow-adapted cells exhibited increases of 1.7-fold in nuclear NF-kappaB and 1.5-fold in nuclear AP-1; these changes were abolished by pretreatment with N-acetylcysteine or DPI. Ischemia for 24 hours resulted in a 1.8-fold increase of (3)H-thymidine incorporation into DNA and a significant increase of cells entering the cell cycle, as indicated by flow cytometry with propidium iodide. We conclude that flow-adapted endothelial cells generate ROS with ischemia that results in activation of NF-kappaB and AP-1 and an increase of DNA synthesis. This effect is not mediated by hypoxia, implicating a role for mechanotransduction in ischemia-mediated cell signaling. (+info)
Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) present in fish oils have been ascribed as having significant antithrombotic and antiatherosclerotic effects. Vascular smooth muscle cell (SMC) proliferation plays an important role in the pathogenesis of atherosclerosis and restenosis. Recent studies have indicated that serotonin at concentrations present at sites of vascular injury stimulates SMC proliferation and may contribute to the restenotic process. In the present study we demonstrate that among the fatty acids tested, only EPA and DHA could block the mitogenic effect of serotonin on vascular SMC. Further, when added together these fatty acids act synergistically in blocking the mitogenic effect of serotonin. EPA and DHA blocked the 5HT-induced increase in the 5-HT(2) receptor mRNA. This antimitogenic effect of EPA and DHA may partially explain some of the beneficial effects of fish oils. (+info)
Biological effects of 1alpha-hydroxy- and 1beta-(hydroxymethyl)-vitamin D compounds relevant for potential colorectal cancer therapy.
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1alpha,25-dihydroxyvitamin D(3) and two synthetic analogs, 1alpha, 25-dihydroxy-16-ene-23-yne-vitamin D(3) (Ro 23-7553) and 1alpha, 25-dihydroxy-16-ene-24-oxo-vitamin D(3) (JK-1624-3), were tested for their ability to specifically inhibit growth and promote differentiation of human colon cancer cells in comparison with a series of 1beta-(hydroxymethyl) congeners of the natural hormone, such as 1beta-(hydroxymethyl)-3alpha,25(OH)(2)-16-ene,24-oxo-vitamin D(3) (JK-1624-2), 1beta-(hydroxymethyl)-3alpha, 25-dihydroxy-16-ene-26,27-dihomo vitamin D(3) (JK-1626-2), and 1beta-(hydroxymethyl)-3alpha,25-dihydroxy-22,24-diene-26,27- dihomo vitamin D(3) (MCW-EE). Western blot analysis revealed that reduction of cyclin D1 levels is a key mechanism by which the vitamin D compounds under investigation inhibit Caco-2 tumor cell growth. Both the 1alpha-hydroxy- as well as the 1beta-hydroxymethyl-type vitamin D compounds, which exhibit only low affinity for the vitamin D receptor, significantly reduced [(3)H]thymidine DNA labeling in confluent Caco-2 cell cultures. This suggests that high-affinity binding to the vitamin D receptor is not an absolute prerequisite for genomic action on tumor cell growth. Hybrid analogs JK-1624-2 and MCW-EE, although antimitotically active, were rather ineffective in promoting phenotypic differentiation of human colon cancer cells. However, because both compounds also do not promote osteoclast differentiation from hematopoetic bone marrow cells, they still could be used as antimitotic agents in cancer therapy, even at dose levels that, with other analogs, could cause hypercalcemia. (+info)