losartan-potassium and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

Tumor growth factor-beta1 (TGF-beta1) plays a pivotal role in processes like kidney epithelial-mesenchymal transition (EMT) and interstitial fibrosis, which correlate well with progression of renal disease. Little is known about underlying mechanisms that regulate EMT. Based on the anatomical relationship between erythropoietin (EPO)-producing interstitial fibroblasts and adjacent tubular cells, we investigated the role of EPO in TGF-beta1-mediated EMT and fibrosis in kidney injury.. We examined apoptosis and EMT in TGF-beta1-treated LLC-PK1 cells in the presence or absence of EPO. We examined the effect of EPO on TGF-beta1-mediated Smad signaling. Apoptosis and cell proliferation were assessed with flow cytometry and hemocytometry. We used Western blotting and indirect immunofluorescence to evaluate expression levels of TGF-beta1 signal pathway proteins and EMT markers.. We demonstrated that ZVAD-FMK (a caspase inhibitor) inhibited TGF-beta1-induced apoptosis but did not inhibit EMT. In contrast, EPO reversed TGF-beta1-mediated apoptosis and also partially inhibited TGF-beta1-mediated EMT. We showed that EPO treatment suppressed TGF-beta1-mediated signaling by inhibiting the phosphorylation and nuclear translocation of Smad 3. Inhibition of mitogen-activated protein kinase kinase 1 (MEK 1) either directly with PD98059 or with MEK 1 siRNA resulted in inhibition of EPO-mediated suppression of EMT and Smad signal transduction in TGF-beta1-treated cells.. EPO inhibited apoptosis and EMT in TGF-beta1-treated LLC-PK1 cells. This effect of EPO was partially mediated by a mitogen-activated protein kinase-dependent inhibition of Smad signal transduction.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase Inhibitors; Caspases; Cell Line; Cell Proliferation; Cysteine Proteinase Inhibitors; Down-Regulation; Epithelium; Erythropoietin; Kidney; Kidney Tubules, Proximal; Mesoderm; Mitogen-Activated Protein Kinase Kinases; Signal Transduction; Smad Proteins; Swine; Transforming Growth Factor beta1

SCL/Tal-1 is a helix-loop-helix (HLH) transcription factor required for blood cell development, whose abnormal expression is responsible for induction of T-cell acute lymphoblastic leukemia. We show here that SCL/Tal-1 is a key target of caspases in developing erythroblasts. SCL/Tal-1 degradation occurred rapidly after caspase activation and preceded the cleavage of the major erythroid transcription factor GATA-1. Expression of a caspase-resistant SCL/Tal-1 in erythroid progenitors was able to prevent amplification of caspase activation, GATA-1 degradation and impaired erythropoiesis induced by growth factor deprivation or death receptor triggering. The potent proerythropoietic activity of uncleavable SCL/Tal-1 was clearly evident in the absence of erythropoietin, a condition that did not allow survival of normal erythroid cells or expansion of erythroblasts expressing caspase-resistant GATA-1. In the absence of erythropoietin, cells expressing caspase-resistant SCL/Tal-1 maintain high levels of Bcl-X(L), which inhibits amplification of the caspase cascade and mediates protection from apoptosis. Thus, SCL/TAL-1 is a survival factor for erythroid cells, whereas caspase-mediated cleavage of SCL/Tal-1 results in amplification of caspase activation, GATA-1 degradation and impaired erythropoiesis.

Topics: Amino Acid Chloromethyl Ketones; Antibodies; Apoptosis; B-Lymphocytes; Basic Helix-Loop-Helix Transcription Factors; bcl-X Protein; Blotting, Western; Caspase 3; Caspase 7; Caspase 8; Caspases; Cell Division; Cloning, Molecular; DNA-Binding Proteins; Down-Regulation; Enzyme Precursors; Erythroblasts; Erythroid-Specific DNA-Binding Factors; Erythropoiesis; Erythropoietin; fas Receptor; GATA1 Transcription Factor; GATA2 Transcription Factor; Gene Expression Regulation; Green Fluorescent Proteins; Helix-Loop-Helix Motifs; Humans; Luminescent Proteins; Microscopy, Fluorescence; Mutagenesis, Site-Directed; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Recombinant Fusion Proteins; T-Cell Acute Lymphocytic Leukemia Protein 1; Transcription Factors

The prevention of apoptosis is a key function of growth factors in the regulation of erythropoiesis. This study examined the role of the constitutively active serine/threonine kinase glycogen synthase kinase-3 (GSK3), a target of the phosphoinositide-3-kinase (PI3K)/Akt pathway, in the regulation of apoptosis in primary human erythroid progenitors. GSK3 phosphorylation at its key regulatory residues S21 (alpha isoform) and S9 (beta isoform) was high in steady-state culture, disappeared on growth factor withdrawal, and returned in response to treatment of cells with either erythropoietin or stem cell factor. Phosphorylation correlated with a PI3K-dependent reduction of 25% to 30% in measured GSK3 activity. LY294002, a specific inhibitor of PI3K, induced apoptosis in growth factor-replete erythroid cells to a degree similar to growth factor deprivation, whereas the Mek1 inhibitor U0126 had no effect, implicating PI3K and not mitogen-activated protein kinase in survival signaling. Growth factor-deprived erythroblasts, which undergo apoptosis rapidly, were protected from apoptosis by both lithium chloride, a GSK3 selective inhibitor, and inhibition of caspase activity. However, the clonogenic potential of single cells, which more accurately reflects cell survival, was maintained by lithium chloride, but not by caspase inhibition. Furthermore, lithium chloride, but not caspase inhibition, prevented the appearance of the conformational form of Bax associated with apoptosis induction. In summary, GSK3 activity is suppressed by erythropoietin and stem cell factor in human erythroid progenitor cells, and increased GSK3 activity, brought about by growth factor withdrawal, may regulate commitment to cell death through a caspase-independent pathway that results in a conformational change in Bax.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; bcl-2-Associated X Protein; Calcium-Calmodulin-Dependent Protein Kinases; Caspase 3; Caspase Inhibitors; Cells, Cultured; Chromones; Colony-Forming Units Assay; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Erythroid Precursor Cells; Erythropoietin; Flavonoids; Glycogen Synthase Kinase 3; Glycogen Synthase Kinases; Humans; Isoenzymes; Lithium Chloride; MAP Kinase Kinase 1; MAP Kinase Signaling System; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase Kinases; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Conformation; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Stem Cell Factor

P39/Tsugane is a myelomonocytoid cell line derived from a patient with myelodysplastic syndrome (MDS). The cells readily undergo apoptosis in response to various agents, and the cell line has been suggested as a useful model to study apoptosis in MDS. The aims of the present study were to assess differentiation and apoptosis induced with all-trans retinoic acid (ATRA) and etoposide, to characterize the mode of apoptosis in these two model systems, and to assess the influence of granulocyte colony-stimulating factor (G-CSF), which in combination with erythropoietin has been shown to inhibit apoptosis in MDS. ATRA induced differentiation and apoptosis in a concentration- and time-dependent manner. Differentiated cells were partially rescued (by 50%) from apoptosis with G-CSF. Etoposide induced apoptosis in a concentration- and time-dependent manner, but no signs of preceding maturation or G-CSF rescue were detected. ATRA- and etoposide-induced apoptosis were both mediated through the caspase pathway and were partially blocked with the general caspase inhibitor zVAD-fmk. Simultaneous treatment with G-CSF and zVAD-fmk additively blocked ATRA-induced apoptosis. However, the two pathways differed in terms of substrate cleavage during apoptosis. ATRA-induced apoptosis caused actin cleavage, which was not affected by G-CSF, and Bcl-2 downregulation. Etoposide induced a caspase-dependent cleavage of Bcl-2, while actin remained intact. The Fas system did not seem to play a major role in any of these apoptotic pathways. Our results may provide new tools to study the mechanisms of apoptosis in MDS.

Topics: Actins; Acute Disease; Amino Acid Chloromethyl Ketones; Antibodies, Monoclonal; Apoptosis; Blast Crisis; Caspase Inhibitors; Caspases; Cell Differentiation; Cysteine Proteinase Inhibitors; Cytoskeleton; Erythropoietin; Etoposide; fas Receptor; Granulocyte Colony-Stimulating Factor; Humans; Leukemia, Myeloid; Myelodysplastic Syndromes; Neoplasm Proteins; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tretinoin; Tumor Cells, Cultured