Oxidative Stress Induces Protein Phosphatase 2A-dependent Dephosphorylation of the Pocket Proteins pRb, p107, and p130

Oxidative stress induces cell death and growth arrest. In this study, the regulation and the functional role of the retinoblastoma family proteins pRb, p107, and p130 in the cellular response to oxidative stress were investigated. Treatment of endothelial cells with H2O2 induced rapid hypophosphorylation of the retinoblastoma family proteins. This event did not require p53 or p21Waf1/Cip1/Sdi1 and was not associated with cyclin/cyclin-dependent kinase down-modulation. Four lines of evidence indicate that H2O2-induced hypophosphorylation of pRb, p107, and p130 was because of the activity of protein phosphatase 2A (PP2A). First, cell treatment with two phosphatase inhibitors, okadaic acid and calyculin A, prevented the hypophosphorylation of the retinoblastoma family proteins, at concentrations that specifically inhibit PP2A. Second, SV40 small t, which binds and inhibits PP2A, when overexpressed prevented H2O2-induced dephosphorylation of the retinoblastoma family proteins, whereas a SV40 small t mutant unable to bind PP2A was totally inert. Third, PP2A core enzyme physically interacted with pRb and p107, both in H2O2-treated and untreated cells. Fourth, a PP2A phosphatase activity was co-immunoprecipitated with pRb, and the activity of pRb-associated PP2A was positively modulated by cell treatment with H2O2. Because DNA damaging agents inhibit DNA synthesis in a pRb-dependent manner, it was determined whether the PP2A-mediated dephosphorylation of the retinoblastoma family proteins played a role in this S-phase response. Indeed, it was found that inhibition of PP2A by SV40 small t over-expression prevented DNA synthesis inhibition induced by H2O2

The laminA/NF-Y protein complex reveals an unknown transcriptional mechanism on cell proliferation

Lamin A is a component of the nuclear matrix that also controls proliferation by largely unknown mechanisms. NF-Y is a ubiquitous protein involved in cell proliferation composed of three subunits (-YA -YB -YC) all required for the DNA binding and transactivation activity. To get clues on new NF-Y partner(s) we performed a mass spectrometry screening of proteins that co-precipitate with the regulatory subunit of the complex, NF-YA. By this screening we identified lamin A as a novel putative NF-Y interactor. Co-immunoprecipitation experiments and confocal analysis confirmed the interaction between the two endogenous proteins. Interestingly, this association occurs on euchromatin regions, too. ChIP experiments demonstrate lamin A enrichment in several promoter regions of cell cycle related genes in a NF-Y dependent manner. Gain and loss of function experiments reveal that lamin A counteracts NF-Y transcriptional activity. Taking advantage of a recently generated transgenic reporter mouse, called MITO-Luc, in which an NF-Y–dependent promoter controls luciferase expression, we demonstrate that lamin A counteracts NF-Y transcriptional activity not only in culture cells but also in living animals. Altogether, our data demonstrate the occurrence of lamin A/NF-Y interaction and suggest a possible role of this protein complex in regulation of NF-Y function in cell proliferatio

CCAAT binding transcription factor binds and regulates human COL1A1 promoter activity in human dermal fibroblasts: demonstration of increased binding in systemic sclerosis fibroblasts

OBJECTIVE: To determine the binding factors that interact with the proximal promoter region of the human type I collagen gene, COL1A1, and to examine their involvement in its transcriptional regulation in normal and systemic sclerosis (SSc) dermal fibroblasts. METHODS: Nuclear extracts from dermal fibroblasts from 4 patients with SSc and 4 age- and sex-matched control individuals were examined by electrophoresis mobility shift assays with a COL1A1 promoter fragment encompassing nucleotides -174 to -50 bp. Supershift assays with antibodies specific to various transcription factors, and competition experiments using consensus, wild-type, or mutated oligonucleotides corresponding to their specific binding sites, were performed. The effects of specific oligonucleotides as intracellular competitors were examined by transient transfection experiments in SSc fibroblasts using a COL1A1 construct containing -174 bp of the promoter. RESULTS: The findings demonstrate that the CCAAT binding transcription factor (CBF) binds the proximal CCAAT box located at -100 to -96 bp, but not the distal CCAAT box at -125 to -121 bp, of the human COL1A1 promoter in both SSc and normal fibroblasts. CBF binding activity was 3-5-fold higher in the SSc fibroblasts. Moreover, the promoter activity of the -174-bp COL1A1 construct was decreased by up to 50% when specific oligonucleotides were used as intracellular competitors. In addition, Sp1 and Sp3 were other transcription factors found to be involved in the formation of the DNA-protein complexes within this region of the COL1A1 promoter. CONCLUSION: These results indicate that the transcription factor CBF binds the human COL1A1 proximal promoter region in human dermal fibroblasts, and its binding activity is higher in SSc fibroblasts

B-Myb acts as a repressor of human COL1A1 collagen gene expression by interacting with Sp1 and CBF factors in scleroderma fibroblasts.

We investigated the role of B-Myb, a cell-cycle-regulated transcription factor, in the expression of the alpha1 (I) pro-collagen gene (COL1A1) in scleroderma fibroblasts. Scleroderma or SSc (systemic sclerosis) is a fibrotic disease characterized by excessive production of extracellular matrix components, especially type I collagen. Northern-blot analysis showed an inverse relationship between COL1A1 mRNA expression and that of B-Myb during exponential cell growth and during quiescence in human SSc fibroblasts. Overexpression of B-Myb in SSc fibroblasts was correlated with decreased COL1A1 mRNA expression. Transient transfections localized the down-regulatory effect of B-Myb to a region containing the proximal 174 bp of the COL1A1 promoter that does not contain B-Myb consensus binding sites. Gel-shift analysis, using nuclear extracts from normal and SSc fibroblasts transfected with B-Myb, showed no differences in DNA-protein complex formation when compared with the nuclear extracts from mock-transfected cells. However, we found that B-Myb decreases Sp1 (specificity protein 1) and CBF (CCAAT-binding factor) binding for their specific sites localized in the 174 bp COL1A1 proximal promoter. These results were also confirmed using B-Myb-immunodepleted nuclear extracts. Furthermore, immunoprecipitation assays using SSc nuclear extracts demonstrated a physical interaction of B-Myb with Sp1 and CBF transcription factors, and also an interaction between Sp1 and CBF. In addition, by employing full-length or deleted B-Myb cDNA construct, we found that B-Myb down-regulates the COL1A1 proximal promoter through its C-terminal domain. Thus these results suggest that B-Myb may be an important factor in the pathway(s) regulating collagen production in SSc fibroblasts

Abstract C231: A great mime produced by nature: The case of paclitaxel

Paclitaxel is widely used in the medical treatment of solid tumors. Despite the clinical success still now the exact mechanism(s) through which the drug kills cancer cells are unknown. In this work we provide evidence, through Surface Plasmon Resonance technology, that paclitaxel beside to microtubules is able also to target mitochondria by directly interacting with the disordered loop of Bcl‐2, thereby stimulating the opening of the permeability transition pore channel and ultimately reverting Bcl‐2 function from antiapoptotic to proapoptotic. Studying the paclitaxel binding site in Bcl‐2, we discovered through molecular modeling that it is positioned in the disordered loop domain and is extraordinarily similar to that experimentally defined in beta‐tubulin, thereby prompting us to speculate that paclitaxel is a peptidomimetic factor. Since to our knowledge, the only factor able to revert the Bcl‐2 function from protector to cell killer is Nur77, we tested the hypothesis that paclitaxel mimics the activity of Nur77. To address this issue, coimmunoprecipitation experiments demonstrated that like paclitaxel Nur77 interacts with Bcl‐2 in the disordered loop domain and with beta‐tubulin. Moreover, stably transformed cells with Nur77 show the classical slow band isoform of Bcl‐2 detectable in cells treated with paclitaxel. Finally, interaction between Nur‐77 and Bcl‐2 was inhibited by paclitaxel, thus demonstrating that the drug and Nur77 compete for the binding to Bcl‐2. The discovery of the molecular component mimicked by paclitaxel could promote the development of novel agonists of the paclitaxel receptor with a nontaxane structure