Calpain restrains the stem cells compartment in breast cancer.

CAPNS1 is essential for the stability and function of ubiquitous CAPN1 and CAPN2. Calpain modulates by proteolytic cleavage many cellular substrates and its activity is often deregulated in cancer cells, therefore calpain inhibition has been proposed as a therapeutical strategy for a number of malignancies. Here we show that CAPNS1 depletion is coupled to impairment of MCF7 and MCF10AT cell lines growth on plate and defective architecture of mammary acini derived from MCF10A cells. In soft agar CAPNS1 depletion leads to cell growth increase in MCF7, and decrease in MCF10AT cells. In both MCF7 and MCF10AT, CAPNS1 depletion leads to the enlargement of the stem cell compartment, as demonstrated by mammosphere formation assays and evaluation of stem cell markers by means of FACS and western blot analysis. Accordingly, activation of calpain by thapsigargin treatment leads to a decrease in the stem cell reservoir. The expansion of the cancer stem cell population in CAPNS1 depleted cells is coupled to a defective shift from symmetric to asymmetric division during mammosphere growth coupled to a decrease in NUMB protein level.

Calpain small-1 modulates Akt/FoxO3A signaling and apoptosis through PP2A.

Here, we show that FoxO3A transcription factor is upregulated upon calpain small-1 (CAPNS1) depletion both in mouse embryonic fibroblasts (MEFs) and in the human mammary carcinoma cell line MCF-7. On starvation, CAPNS1 depletion is associated with a higher rate of FoxO3A dephosphorylation and translocation to the nucleus and to a sharper increase in the levels of p27Kip1 and Bim, the products of two FoxO target genes. Notably, FoxO3A depletion in CAPNS1-/- MEFs reduces both the induction of Bim and apoptosis. Both okadaic acid treatment and silencing of the protein phosphatase 2A (PP2A) catalytic subunit can partially reduce starvation-induced FoxO3A activation and apoptosis in CAPNS1-/- fibroblasts. PP2A associates more tightly with Akt in CAPNS1 knockout cells, indicating that PP2A is involved in calpain-mediated FoxO regulation. Finally, we show that PP2A regulatory subunits B56 alpha and gamma are in vitro substrates of calpain, and calpain regulates B56 alpha stability in vivo, suggesting a direct role of calpain in the regulation of PP2A function. In conclusion, for the first time we report that CAPNS1 interferes with PP2A-Akt interaction consequently affecting FoxO3A-dependent cell death. Calpain inhibition might therefore be exploited as a tool to induce apoptosis in tumors sensitive to FoxO activation.

Ceramide triggers an NF-kappaB-dependent survival pathway through calpain.

We have shown that C2 ceramide, a cell-permeable analog of this lipid second messenger, triggers an NF-kappaB dependent survival pathway that counteracts cell death. Activation of NF-kappaB and subsequent induction of prosurvival genes relies on calpain activity and is prevented on silencing of the calpain small subunit (Capn4) that is required for the function of ubiquitous calpains. We have demonstrated that p105 (NF-kappaB1) and its proteolytic product p50 can be targets of micro- and milli-calpain in vitro and that a p50 deletion mutant, lacking both the N- and the C-terminal ends, is resistant to calpain-mediated degradation. Capn4 silencing results in stabilization of endogenous p105 and p50 in diverse human cell lines. Furthermore, p105 processing and activation of NF-kappaB survival genes in response to C2 ceramide is impaired in Capn4-/- mouse embryonic fibroblasts defective in calpain activity. Altogether, these data argue for the existence of a ceramide-calpain-NF-kappaB axis with prosurvival functions.

Gas6 anti-apoptotic signaling requires NF-kappa B activation.

The growth arrest-specific 6 gene product Gas6 is a growth and survival factor related to protein S. Gas6 is the ligand of Axl receptor tyrosine kinase; upon binding to its receptor Gas6 activates the phosphatidylinositol 3-OH kinase (PI3K) and its downstream targets S6K and Akt. Gas6 anti-apoptotic signaling was previously shown to require functional PI3K and Akt and to involve Bad phosphorylation in serum-starved NIH 3T3 cells. Here we demonstrate that Gas6 induces a rapid and transient increase in nuclear NF-kappa B binding activity coupled to transcription activation from NF-kappa B-responsive promoters and increase in Bcl-x(L) protein level. Gas6 survival function is impaired in cells lacking p65/RelA and in NIH 3T3 cells transfected with a dominant negative I kappa B, indicating that NF-kappa B activation plays a central role in promoting survival in this system. Moreover, NF-kappa B activation can be blocked by a dominant negative Akt and by wortmannin, an inhibitor of PI3K, thus suggesting that NF-kappa B activation is a downstream event with respect to PI3K and Akt, as already described for other growth factors. In addition, we show that glycogen synthase kinase 3, which is phosphorylated in response to Gas6, can physically associate with NFKB1/p105 in living cells and can phosphorylate it in vitro. Furthermore, Gas6 treatment is coupled to a decrease in p105 protein level. Altogether these data suggest the involvement of NF-kappa B and glycogen synthase kinase 3 in Gas6 anti-apoptotic signaling and unveil a possible link between these survival pathways.

Human immunodeficiency virus type 1 tat protein activates transcription factor NF-kappaB through the cellular interferon-inducible, double-stranded RNA-dependent protein kinase, PKR.

The transactivator protein of human immunodeficiency virus type 1 (HIV-1) (Tat) is a powerful activator of nuclear factor-kappaB (NF-kappaB), acting through degradation of the inhibitor IkappaB-alpha (F. Demarchi, F. d'Adda di Fagagna, A. Falaschi, and M. Giacca, J. Virol. 70:4427-4437, 1996). Here, we show that this activity of Tat requires the function of the cellular interferon-inducible protein kinase PKR. Tat-mediated NF-kappaB activation and transcriptional induction of the HIV-1 long terminal repeat were impaired in murine cells in which the PKR gene was knocked out. Both functions were restored by cotransfection of Tat with the cDNA for PKR. Expression of a dominant-negative mutant of PKR specifically reduced the levels of Tat transactivation in different human cell types. Activation of NF-kappaB by Tat required integrity of the basic domain of Tat; previous studies have indicated that this domain is necessary for specific Tat-PKR interaction.

Transcriptional interference perturbs the binding of Sp1 to the HIV-1 promoter.

Transcriptional interference between adjacent genes has been demonstrated in a variety of biological systems. To study this process in RNA polymerase II (pol II) transcribed genes we have analysed the effect of transcription on tandem HIV-1 promoters integrated into the genome of HeLa cells. We show that transcriptional activation at the upstream promoter reduces transcription from the downstream promoter, as compared with basal transcription conditions (in the absence of an activator). Furthermore, insertion of a strong transcriptional termination element between the two promoters alleviates this transcriptional interference, resulting in elevated levels of transcription from the downstream promoter. Actual protein interactions with the downstream (occluded) promoter were analysed by in vivo footprinting. Binding of Sp1 transcription factors to the occluded promoter was reduced, when compared with the footprint pattern of the promoter protected by the terminator. This suggests that promoter occlusion is due to disruption of certain transcription factors and that it can be blocked by an intervening transcriptional terminator. Chromatin mapping with DNase I indicates that a factor binds to the termination element under both basal and induced conditions.

Activation of transcription factor NF-kappaB by the Tat protein of human immunodeficiency virus type 1.

A recombinant Tat protein was used to investigate the molecular mechanisms of transcriptional activation of the human immunodeficiency virus type 1 long terminal repeat (LTR). Liposome-mediated delivery of this protein to responsive cells results in dose-dependent LTR activation. As evaluated by mRNA quantitation with competitive PCR, the activation response is rapid and transient, peaking at 5 h after the beginning of Tat treatment. In vivo footprinting experiments at the LTR showed that transcriptional activation is concomitant with a modification of the protein-DNA interaction pattern at the downstream kappaB site of the enhancer and at the adjacent Sp1 boxes. The effects of Tat on the enhancer are mediated by Tat-induced nuclear translocation of NF-kappaB, which parallels the kinetics of transcriptional activation. This induction results from degradation of the inhibitor IkappaB-alpha, is blocked under antioxidant conditions and by a protease inhibitor, and occurs as a rapid response in different cell types. The functional response to Tat is impaired upon cell treatment with a kappaB site decoy or with sodium salicylate, an inhibitor of NF-kappaB activation. These results show that NF-kappaB activation by Tat is important for LTR transcriptional activation. Furthermore, they suggest that some of the pleiotropic effects of Tat on cellular functions can be mediated by induction of NF-kappaB.

In vivo protein-DNA interactions at human DNA replication origin.

Protein-DNA interactions were studied in vivo at the region containing a human DNA replication origin, located at the 3' end of the lamin B2 gene and partially overlapping the promoter of another gene, located downstream. DNase I treatment of nuclei isolated from both exponentially growing and nonproliferating HL-60 cells showed that this region has an altered, highly accessible, chromatin structure. High-resolution analysis of protein-DNA interactions in a 600-bp area encompassing the origin was carried out by the in vivo footprinting technique based on the ligation-mediated polymerase chain reaction. In growing HL-60 cells, footprints at sequences homologous to binding sites for known transcription factors (members of the basic-helix-loop-helix family, nuclear respiratory factor 1, transcription factor Sp1, and upstream binding factor) were detected in the region corresponding to the promoter of the downstream gene. Upon conversion of cells to a nonproliferative state, a reduction in the intensity of these footprints was observed that paralleled the diminished transcriptional activity of the genomic area. In addition to these protections, in close correspondence to the replication initiation site, a prominent footprint was detected that extended over 70 nucleotides on one strand only. This footprint was absent from nonproliferating HL-60 cells, indicating that this specific protein-DNA interaction might be involved in the process of origin activation.

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.

In vivo footprinting analysis of constitutive and inducible protein-DNA interactions at the long terminal repeat of human immunodeficiency virus type 1.

The regulation of the rate of transcription of human immunodeficiency virus type 1 is mainly exerted through the long terminal repeat (LTR) at the 5' end of the provirus. A large number of cis-acting regulatory elements have been identified in the LTR by in vitro binding studies; the biological role of these sites within living infected cells, however, is still not clear. We have studied the interactions of nuclear proteins with the LTR in the U1 monocytic cell line by in vivo dimethylsulfate footprinting, using the ligation-mediated polymerase chain reaction technique. In this cell line, transcription of the virus, which is very low under basal conditions, is highly inducible by treatment with phorbol esters; therefore, this system is likely to represent a suitable cellular model to study viral latency. Independently of the level of viral transcription, major in vivo footprints appear at the two Sp1 sites adjacent to the enhancer, the downstream-positioned enhancer repeat, the NFAT binding site, and one of the purine-rich sites of the negative regulatory element. Upon transcriptional activation by phorbol myristate acetate, the only perturbation in the footprinting pattern is a dramatic increase in dimethylsulfate sensitivity of guanine at position -92 in the downstream enhancer repeat. This modification is correlated with the transient induction of two enhancer-binding activities, as determined by gel retardation assays. While the transcriptional rate is still increasing and the in vivo footprinting pattern is unchanged at up to 24 h postactivation, these enhancer-binding factors are considerably reduced at this time. Therefore, further levels of regulation have to be considered to explain the maintenance of the induced state.

Probing protein-DNA interactions at the long terminal repeat of human immunodeficiency virus type 1 by in vivo footprinting.

We have analyzed protein-DNA interactions at the long terminal repeat of human immunodeficiency virus type 1 in a productively infected T-cell line by in vivo dimethyl sulfate footprinting. Major footprints are evident at the basal promoter and enhancer elements. In particular, proteins appear to occupy the TATA box, the Sp1 sites, and the two repeats of the enhancer region. In the negative regulatory element, protections are detected over the USF/MLTF and NFAT-1 sites. Furthermore, two previously unrecognized sites, from nucleotides -260 to -275 and from nucleotides -205 to -216, respectively, appear to be involved in protein-DNA interactions. These two sites are purine rich and share a common sequence motif.

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.