Hsp70 regulates erythropoiesis by preventing caspase-3-mediated cleavage of GATA-1.

Caspase-3 is activated during both terminal differentiation and erythropoietin-starvation-induced apoptosis of human erythroid precursors. The transcription factor GATA-1, which performs an essential function in erythroid differentiation by positively regulating promoters of erythroid and anti-apoptotic genes, is cleaved by caspases in erythroid precursors undergoing cell death upon erythropoietin starvation or engagement of the death receptor Fas. In contrast, by an unknown mechanism, GATA-1 remains uncleaved when these cells undergo terminal differentiation upon stimulation with Epo. Here we show that during differentiation, but not during apoptosis, the chaperone protein Hsp70 protects GATA-1 from caspase-mediated proteolysis. At the onset of caspase activation, Hsp70 co-localizes and interacts with GATA-1 in the nucleus of erythroid precursors undergoing terminal differentiation. In contrast, erythropoietin starvation induces the nuclear export of Hsp70 and the cleavage of GATA-1. In an in vitro assay, Hsp70 protects GATA-1 from caspase-3-mediated proteolysis through its peptide-binding domain. The use of RNA-mediated interference to decrease the Hsp70 content of erythroid precursors cultured in the presence of erythropoietin leads to GATA-1 cleavage, a decrease in haemoglobin content, downregulation of the expression of the anti-apoptotic protein Bcl-X(L), and cell death by apoptosis. These effects are abrogated by the transduction of a caspase-resistant GATA-1 mutant. Thus, in erythroid precursors undergoing terminal differentiation, Hsp70 prevents active caspase-3 from cleaving GATA-1 and inducing apoptosis.

Effect of tyrosine kinase inhibitor STI571 on the kinase activity of wild-type and various mutated c-kit receptors found in mast cell neoplasms.

Systemic mastocytosis (SM) is a rare disease caused by an abnormal mast cell accumulation in various tissues. Two classes of constitutive activating c-kit mutations are found in SM. The most frequent class occurs in the catalytic pocket coding region with substitutions at codon 816 and the other in the intracellular juxtamembrane coding region. Therefore, kinase inhibitors that block mutated c-kit activity might be used as therapeutic agents in SM. Here, we show that STI571 inhibits both wild-type and juxtamembrane mutant c-kit kinase activity, but has no effect on the activity of the D816 V mutant. Accordingly, STI571 selectively decreases the survival of normal mast cell and of mast cell lines either with juxtamembrane c-kit mutations, but not that of tumoral mast cell from patient with SM or of mast cell lines with the D816 V mutation. Therefore, STI571 is not a good candidate to treat SM and specific kinase inhibitors should be designed to inhibit constitutive activating mutations at codon 816.

Human T-cell lymphotropic virus oncoprotein Tax represses TGF-beta 1 signaling in human T cells via c-Jun activation: a potential mechanism of HTLV-I leukemogenesis.

Human T-cell leukemia virus I is the etiologic agent of adult T-cell leukemia (ATL), an aggressive T-cell malignancy. The viral oncoprotein Tax, through the activation of nuclear factorkappaB (NF-kappaB), CCAAT-enhancer binding protein (CREB), and activated protein-1 (AP-1) pathways, is a transcriptional regulator of critical genes for T-cell homeostasis. In ATL cells, activated AP-1 complexes induce the production of transforming growth factor beta1 (TGF-beta1). TGF-beta1 is an inhibitor of T-cell proliferation and cytotoxicity. Here we show that, in contrast to normal peripheral T cells, ATL cells are resistant to TGF-beta1-induced growth inhibition. The retroviral transduction of the Tax protein in peripheral T cells resulted in the loss of TGF-beta1 sensitivity. Transient transfection of Tax in HepG2 cells specifically inhibited Smad/TGF-beta1 signaling in a dose-dependent manner. In the presence of Tax transfection, increasing amounts of Smad3 restored TGF-beta1 signaling. Tax mutants unable to activate NF-kappaB or CREB pathways were also able to repress Smad3 transcriptional activity. Next we have demonstrated that Tax inhibits TGF-beta1 signaling by reducing the Smad3 DNA binding activity. However, Tax did not decrease the expression and the nuclear translocation of Smad3 nor did it interact physically with Smad3. Rather, Tax induced c-Jun N-terminal kinase (JNK) activity and c-Jun phosphorylation, leading to the formation of Smad3/c-Jun complexes. Whereas c-Jun alone abrogates Smad3 DNA binding, cotransfection of Tax and of a dominant-negative form of JNK or a c-Jun antisense-restored Smad3 DNA binding activity and TGF-beta1 responsiveness. In ATL and in normal T cells transduced by Tax, c-Jun was constitutively phosphorylated. Thus, we describe a new function of Tax, as a repressor of TGF-beta1 signaling through JNK/c-Jun constitutive activation, which may play a critical role in ATL leukemogenesis.