Francesca Demarchi

p65/RelA modulates BECN1 transcription and autophagy.

ABSTRACT Recently, autophagy has emerged as a critical process in the control of T-cell homeostasis. Given the pivotal role of NF-κB in the signaling events of T cells, we have analyzed and unveiled a conserved NF-κB binding site in the promoter of the murine and human BECN1 autophagic gene (Atg6). Accordingly, we demonstrate that the NF-κB family member p65/RelA upregulates BECN1 mRNA and protein levels in different cellular systems. Moreover, p65-mediated upregulation of BECN1 is coupled to increased autophagy. The newly identified κB site in the BECN1 promoter specifically interacts with p65 both in vitro and in living Jurkat cells upon phorbol myristate acetate (PMA)-ionomycin stimulation, where p65 induction is coupled to BECN1 upregulation and autophagy induction. Finally, anti-CD3- and PMA-ionomycin-mediated activation of T-cell receptor signaling in peripheral T cells from lymph nodes of healthy mice results in an upregulation of BECN1 expression that can be blocked by the NF-κB inhibitor BAY 11-7082. Altogether, these data suggest that autophagy could represent a novel route modulated by p65 to regulate cell survival and control T-cell homeostasis.

Glycogen Synthase Kinase-3β Regulates NF-κB1/p105 Stability

A number of different kinases have been implicated in NF-κB regulation and survival function. Here we investigated the molecular cross-talk between glycogen synthase kinase-3β (GSK-3β) and the p105 precursor of the NF-κB p50 subunit. GSK-3β forms an in vivo complex with and specifically phosphorylates NF-κB1/p105 at Ser-903 and Ser-907 in vitro. In addition, the p105 phosphorylation level is reduced in fibroblasts lacking GSK-3β as compared with wild-type cells. GSK-3β has a dual effect on p105: it stabilizes p105 under resting conditions and primes p105 for degradation upon tumor necrosis factor (TNF)-α treatment. Indeed, constitutive processing of p105 to p50 occurs at a higher rate in cells lacking GSK-3β with respect to wild-type cells and can be reduced upon reintroduction of GSK-3β by transfection. Moreover, p105 degradation in response to TNF-α is prevented in GSK-3β–/– fibroblasts and by a Ser to Ala point mutation on p105 at positions 903 or 907. Interestingly, the increased sensitiveness to TNF-α-induced death occurring in GSK-3β–/– fibroblasts, which is coupled to a perturbation of p50/105 ratio, can be reproduced by p105 silencing in wild-type fibroblasts.

GSK-3β regulates NF-κB1/p105 stability

A number of different kinases have been implicated in NF-κB regulation and survival function. Here we investigated the molecular cross-talk between glycogen synthase kinase-3β (GSK-3β) and the p105 precursor of the NF-κB p50 subunit. GSK-3β forms an in vivo complex with and specifically phosphorylates NF-κB1/p105 at Ser-903 and Ser-907 in vitro. In addition, the p105 phosphorylation level is reduced in fibroblasts lacking GSK-3β as compared with wild-type cells. GSK-3β has a dual effect on p105: it stabilizes p105 under resting conditions and primes p105 for degradation upon tumor necrosis factor (TNF)-α treatment. Indeed, constitutive processing of p105 to p50 occurs at a higher rate in cells lacking GSK-3β with respect to wild-type cells and can be reduced upon reintroduction of GSK-3β by transfection. Moreover, p105 degradation in response to TNF-α is prevented in GSK-3β–/– fibroblasts and by a Ser to Ala point mutation on p105 at positions 903 or 907. Interestingly, the increased sensitiveness to TNF-α-induced death occurring in GSK-3β–/– fibroblasts, which is coupled to a perturbation of p50/105 ratio, can be reproduced by p105 silencing in wild-type fibroblasts.

The calpain system as a modulator of Stress/Damage response

Ubiquitously expressed _- and m-calpain proteases consist of 80-kDa catalytic subunits encoded by the Capn1 and Capn2 genes, respectively, and a common 28-kDa regulatory subunit encoded by the calpain small 1 (Capns1) gene.The _- and m-calpain proteases have been implicated in both pro- or anti-apoptotic functions. We have found that Capns1 depletion is coupled to increased sensitivity to apoptosis triggered by a number of autophagy-inducing stimuli in mammalian cells. Therefore we investigated the involvement of calpains in autophagy using MEFs derived from Capns1 knockout mice and Capns1 depleted human cells as model systems.We found that autophagy is impaired in Capns1 deficient cells by immunostaining of the endogenous autophagosome marker LC3 and electron microscopy experiments. Accordingly, the enhancement of lysosomal activity and long-lived proteins degradation, normally occurring upon starvation, are also reduced.In Capns1 depleted cells ectopic LC3 accumulates in early endosome-like vesicles that might represent a salvage pathway for protein degradation when autophagy is defective.Calpain represents a promising target for cancer therapy since its activity is tightly linked to tumor progression. Indeed it is elevated during transformation, it is required for autophagy and survival of cancer cells and plays a key role in metastatic cell migration and angiogenesis.

p65/RelA binds and activates the beclin 1 promoter.

We unveiled novel p65/RelA consensus sites in the promoter of the beclin 1 gene and demonstrate that p65/RelA positively modulates canonical autophagy in various human cell lines both under basal conditions and upon induction by ceramide. Interestingly, we find that T cell receptor-dependent activation of Jurkat cells triggers an increase in the binding of p65/RelA to the beclin 1 promoter accompanied by enhanced autophagy, suggesting that p65/RelA could regulate T-cell activation and homeostasis through autophagy.

The Isopeptidase Inhibitor G5 Triggers a Caspase-independent Necrotic Death in Cells Resistant to Apoptosis A COMPARATIVE STUDY WITH THE PROTEASOME INHIBITOR BORTEZOMIB

Inhibitors of the ubiquitin-proteasome system (UPSIs) promote apoptosis of cancer cells and show encouraging anti-tumor activities in vivo. In this study, we evaluated the death activities of two different UPSIs: bortezomib and the isopeptidase inhibitor G5. To unveil whether these compounds elicit different types of death, we compared their effect both on apoptosis-proficient wild type mouse embryo fibroblasts and on cells defective for apoptosis (double-deficient Bax/Bak mouse embryo fibroblasts) (double knockout; DKO). We have discovered that (i) both inhibitors induce apoptosis in a Bax and Bak-dependent manner, (ii) both inhibitors elicit autophagy in WT and DKO cells, and (iii) only G5 can kill apoptosis-resistant DKO cells by activating a necrotic response. The induction of necrosis was confirmed by different experimental approaches, including time lapse analysis, HMGB1 release, and electron microscopy studies. Neither treatment with antinecrotic agents, such as antioxidants, poly(ADP-ribose) polymerase and JNK inhibitors, necrostatin, and the intracellular Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester, nor overexpression of Bcl-2 and Bcl-xL prevented necrosis induced by G5. This necrotic death is characterized by the absence of protein oxidation and by the rapid cyclosporin A-independent dissipation of the mitochondrial membrane potential. Notably, a peculiar feature of the G5-induced necrosis is an early and dramatic reorganization of the actin cytoskeleton, coupled to an alteration of cell adhesion. The importance of cell adhesion impairment in the G5-induced necrotic death of DKO cells was confirmed by the antagonist effect of the extracellular matrix-adhesive components, collagen and fibronectin.

DNA damage response links calpain to cellular senescence.

Senescence represents an important barrier against cellular transformation. Here we show that CAPNS1 depletion impairs senescence induction both in BJ-ET H-Rasv12 inducible human fibroblasts upon Ras induction and in HT1080 targeted to senescence by treatment with low doses of doxorubicin. We further show that CAPNS1 depletion is coupled to reduced levels of H2AX phosphorylation, not only in Rasv12 induced BJ-ET fibroblasts, but also in a number of cellular systems upon genotoxic stress. In particular CAPNS1 depletion affects γ-H2AX appearance or persistence in U2OS osteosarcoma cells 24 hours after MMC addition or UV light exposure; HT1080 upon camptothecin treatment for 4 hours and 48 hours after addition of MMC; MDA-MB-231, 24 hours after UV light exposure and 2 hours after bleomycin addition. Overall this study unveils a novel link between calpain, cellular senescence and DNA damage response.

A protein target site in an early replicated human DNA sequence: a highly conserved binding motif.

We have previously reported that a human nuclear factor, probably corresponding to the USF/MLTF protein [1,2], is able to bind specifically to a DNA sequence present in DNA replicated at the onset of S-phase [3]. Here we demonstrate that the same factor binds also to several other similar sequences, present in eukaryotic and viral genomes. Mutations or methylation in a CpG dinucleotide, central in the palindromic binding site, completely abolish binding. Furthermore, we present evidence for the existence of at least two other nuclear proteins in human cells with the same DNA binding specificity. The data presented suggest a strong evolutionary conservation, among distantly related organisms, of the binding motif, which is probably the target of a number of nuclear factors that share the same DNA binding specificity albeit in the context of different functions.

Human Synaptobrevin-like 1 Gene Basal Transcription Is Regulated through the Interaction of Selenocysteine tRNA Gene Transcription Activating Factor-Zinc Finger 143 Factors with Evolutionary Conserved Cis-elements

The synaptobrevin-like 1 (SYBL1) gene is ubiquitously expressed and codes for an unusual member of the v-SNAREs molecules implicated in cellular exocytosis. This X-linked gene has the peculiarity of also being present on the Y chromosome in a transcriptional inactive status. Moreover, although ubiquitous, the function of SYBL1 is prominent in specific tissues, such as brain. As a first insight into the molecular mechanisms controlling SYBL1 expression, in this report we describe the extent and role of SYBL1 upstream regions and characterize the binding of trans-acting factors. In vivo foot-printing experiments identify three protected regions. Band shift and transient reporter gene assays indicate a strong role of two of these evolutionary conserved regions in regulating SYBL1 transcription. Because one site is the classical CAAT box, we characterized the binding to the other site of the mammalian homologues of the selenocysteine tRNA gene transcription activating factor (Staf) family, zinc-finger transcription factors, and their role in regulating SYBL1 expression. The results reported here clarify that a Staf-zinc finger family factor, together with the CAAT factor, is the major nuclear protein bound to the SYBL1 promoter region and is responsible for its regulation in HeLa cells, thus identifying the basic control of SYBL1 transcription. In vivo binding of Staf proteins to the SYBL1 promoter is confirmed by chromatin immunoprecipitation assays. Our results identify a fourth mRNA promoter stimulated by a member of the Staf-zinc finger family, the function of which on mRNA polymerase II promoters is still very poorly understood.

Transcriptional Activation of Human Immunodeficiency Virus Type 1 by Herpesvirus Infection: An in vivo Footprinting Study

The process of transcriptional activation directed by the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) was investigated by in vivo footprinting using ligation-mediated polymerase chain reaction in a human epithelial cell line infected with human herpes simplex virus type 1 (HSV-1) or human herpes virus 6 (HHV-6). Infection with both viruses induces a remarkable enhancement in LTR-mediated gene expression that correlates with a change in the pattern of protein binding to the downstream kappa B site of the enhancer region. In HHV-6 infected cells, this change in the genomic footprinting pattern is concomitant with the induction of specific enhancer-binding proteins in the nucleus. The similarity of these events to those detected in other previously investigated experimental systems suggests that the LTR enhancer region is the ultimate target for the induction of the HIV-1 transcriptional response upon stimuli acting through different upstream pathways.