Claudio Brancolini

The protein encoded by a growth arrest-specific gene (gas6) is a new member of the vitamin K-dependent proteins related to protein S, a negative coregulator in the blood coagulation cascade.

A set of growth arrest-specific genes (gas) whose expression is negatively regulated after serum induction has previously been described (C. Schneider, R. M. King, and L. Philipson, Cell 54:787-793, 1988). The detailed analysis of one of them, gas6, is reported here, gas6 mRNA (2.6 kb) is abundantly expressed in serum-starved (48 h in 0.5% fetal calf serum) NIH 3T3 cells but decreases dramatically after fetal calf serum or basic fibroblast growth factor stimulation. The human homolog of gas6 was also cloned and sequenced, revealing a high degree of homology and a similar pattern of expression in IMR90 human fibroblasts. Computer analysis of the protein encoded by murine and human gas6 cDNAs showed significant homology (43 and 44% amino acid identity, respectively) to human protein S, a negative coregulator in the blood coagulation pathway. By using an anti-human Gas6 monospecific affinity-purified antibody, we show that the biosynthetic level of human Gas6 fully reflects mRNA expression in IMR90 human fibroblasts. This finding thus defines a new member of vitamin K-dependent proteins that is expressed in many human and mouse tissues and may be involved in the regulation of a protease cascade relevant in growth regulation.

Caspase-2 can trigger cytochrome C release and apoptosis from the nucleus.

The cysteine proteases specific for aspartic residues, known as caspases, are localized in different subcellular compartments and play specific roles during the regulative and the executive phase of the cell death process. Here we investigated the subcellular localization of caspase-2 in healthy cells and during the execution of the apoptotic program. We have found that caspase-2 is a nuclear resident protein and that its import into the nucleus is regulated by two different nuclear localization signals. We have shown that in an early phase of apoptosis caspase-2 can trigger mitochondrial dysfunction from the nucleus without relocalizing into the cytoplasm. Release of cytochrome c occurs in the absence of overt alteration of the nuclear pores and changes of the nuclear/cytoplasmic barrier. Addition of leptomycin B, an inhibitor of nuclear export, did not interfere with the ability of caspase-2 to trigger cytochromec release. Only during the late phase of the apoptotic process can caspase-2 relocalize in the cytoplasm, as consequence of an increase in the diffusion limits of the nuclear pores. Taken together these data indicate the existence of a nuclear/mitochondrial apoptotic pathway elicited by caspase-2.

Role of caspases, Bid, and p53 in the apoptotic response triggered by histone deacetylase inhibitors trichostatin-A (TSA) and suberoylanilide hydroxamic acid (SAHA)

Histone deacetylase activity is potently inhibited by hydroaximc acid derivatives such as suberoylanilide hydroxamic acid (SAHA) and trichostatin-A (TSA). These inhibitors specifically induce differentiation/apoptosis of transformed cellsin vitro and suppress tumor growth in vivo. Because of its low toxicity, SAHA is currently evaluated in clinical trials for the treatment of cancer. SAHA and TSA induce apoptosis, which is characterized by mitochondrial stress, but so far, the critical elements of this apoptotic program remain poorly defined. To characterize in more detail this apoptotic program, we used human cell lines containing alterations in important elements of apoptotic response such as: p53, Bcl-2, caspase-9, and caspase-3. We demonstrate that caspase-9 is critical for apoptosis induced by SAHA and TSA and that efficient proteolytic activation of caspase-2, caspase-8, and caspase-7 strictly depends on caspase-9. Bcl-2 efficiently antagonizes cytochrome c release and apoptosis in response to both histone deacetylase inhibitors. We provide evidences that translocation into the mitochondria of the Bcl-2 family member Bid depends on caspase-9 and that this translocation is a late event during TSA-induced apoptosis. We also demonstrate that the susceptibility to TSA- and SAHA-induced cell death is regulated by p53.

Calpain is required for macroautophagy in mammalian cells

Ubiquitously expressed micro- and millicalpain, which both require the calpain small 1 (CAPNS1) regulatory subunit for function, play important roles in numerous biological and pathological phenomena. We have previously shown that the product of GAS2, a gene specifically induced at growth arrest, is an inhibitor of millicalpain and that its overexpression sensitizes cells to apoptosis in a p53-dependent manner (Benetti, R., G. Del Sal, M. Monte, G. Paroni, C. Brancolini, and C. Schneider. 2001. EMBO J. 20:2702–2714). More recently, we have shown that calpain is also involved in nuclear factor κB activation and its relative prosurvival function in response to ceramide, in which calpain deficiency strengthens the proapoptotic effect of ceramide (Demarchi, F., C. Bertoli, P.A. Greer, and C. Schneider. 2005. Cell Death Differ. 12:512–522). Here, we further explore the involvement of calpain in the apoptotic switch and find that in calpain-deficient cells, autophagy is impaired with a resulting dramatic increase in apoptotic cell death. Immunostaining of the endogenous autophagosome marker LC3 and electron microscopy experiments demonstrate that autophagy is impaired in CAPNS1-deficient cells. Accordingly, the enhancement of lysosomal activity and long-lived protein degradation, which normally occur upon starvation, is also reduced. In CAPNS1-depleted cells, ectopic LC3 accumulates in early endosome-like vesicles that may represent a salvage pathway for protein degradation when autophagy is defective.

The death substrate Gas2 binds m‐calpain and increases susceptibility to p53‐dependent apoptosis

Gas2 is a caspase‐3 substrate that plays a role in regulating microfilament and cell shape changes during apoptosis. Here we provide evidence that overexpression of Gas2 efficiently increases cell susceptibility to apoptosis following UV irradiation, etoposide and methyl methanesulfonate treatments, and that these effects are dependent on increased p53 stability and transcription activity. To investigate possible pathways linking Gas2 to p53, a yeast two‐hybrid screen swas performed, indicating m‐calpain as a strong Gas2‐ interacting protein. Moreover, we demonstrate that Gas2 physically interacts with m‐calpain in vivo and that recombinant Gas2 inhibits calpain‐dependent processing of p53. Importantly, the Gas2 dominant‐negative form (Gas2Δ171–314) that binds calpain but is unable to inhibit its activity abrogates Gas2s ability to stabilize p53, to enhance p53 transcriptional activity and to induce p53‐dependent apoptosis. Finally, we show that Gas2 is able to regulate the levels of p53 independently of Mdm2 status, suggesting that, like calpastatin, it may enhance p53 stability by inhibiting calpain activity.

Proteolytic processing of the adherens junctions components beta-catenin and gamma-catenin/plakoglobin during apoptosis.

Apoptotic cells undergo specific morphological changes that include loss of cell-cell interactions. Cellular adhesiveness is dependent on members of the cadherin family of adhesion receptors and on the cytoplasmic adaptor proteins α-catenin, β-catenin and γ-catenin/plakoglobin. The caspase family of cystein proteases play a key role during the execution phase of the apoptotic program. These proteolytic enzymes, once activated, cleave cellular proteins which are important for the maintenance of cell integrity. Here we report that γ-catenin is cleaved at different sites during apoptosis in various cell lines. The major apoptotic product of γ-catenin still retains the ability to bind α-catenin but loses the carboxy-terminal region. We also show that γ-catenin is cleaved by caspase-3 in vitro although with lower affinity when compared to PARP or β-catenin. These findings indicate that multiple proteolytic events regulate the dismantling of the cell-cell junctional complexes during apoptosis.

Identification of USP18 as an Important Regulator of the Susceptibility to IFN-α and Drug-Induced Apoptosis

Gene products that modify the apoptotic susceptibility of cancer cells may offer novel drug response markers or therapeutic targets. In this study, we probed the contribution of 53 different isopeptidases to apoptosis triggered by bortezomib and etoposide. USP18, a type I IFN-induced protein that deconjugates the ubiquitin-like modifier ISG15 from target proteins, was found to limit apoptotic susceptibility to IFN-alpha or bortezomib. Ablating USP18 in cells treated with IFN-alpha increased tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) production; upregulated expression of transcription factors IFN-regulatory factor (IRF)-1, IRF-7, and IRF-9; and promoted the extrinsic pathway of apoptosis. The proapoptotic effects of ablating USP18 were abrogated by FLIP overexpression or TRAIL silencing. However, in bortezomib-treated cells, weak spontaneous signaling from type I IFNs was implicated in the proapoptotic effect of USP18 ablation. Ectopic USP18 repressed apoptotic signaling by IFN-alpha, TRAIL, or bortezomib. Similar effects were produced by a catalytically inactive USP18 mutant, indicating that the antiapoptotic function of USP18 is independent of its catalytic activity. These findings suggest that USP18 may significantly limit operation of the extrinsic apoptotic pathway triggered by type I IFN and drugs.

Class IIa HDACs: from important roles in differentiation to possible implications in tumourigenesis.

•  Introduction •  General concepts on class IIa HDACs ‐  Regulation of subcellular localization ‐  Binding partners ‐  Catalytic activity ‐  Class IIa HDAC orthologues in model organisms •  HDAC4 ‐  HDAC4 and differentiation ‐  HDAC4 and cancer •  HDAC5 ‐  HDAC5 and differentiation ‐  HDAC5 and cancer •  HDAC7 ‐  HDAC7 and vascular endothelium ‐  Is HDAC7 a negative regulator of cell proliferation? ‐  HDAC7 and cancer •  HDAC9 ‐  HDAC9 and differentiation ‐  HDAC9 and cancer •  Conclusions

Caspase activation and apoptosis in response to proteasome inhibitors.

Several studies have indicated that proteasome inhibitors (PIs) are promising anticancer agents. We have discovered that PIs have the unique ability to activate effector caspases through a mitochondrial Bcl-2 inhibitable but caspase-9 independent pathway. Stabilization of released Smac induced by blockade of the proteasome could explain the apoptosome-independent cell death induced by PIs. Infact, Smac/DIABLO critically supports this PIs-dependent caspase activation. By using a new assay, we confirm that at a single cell level both Smac and PIs can activate caspases in the absence of the apoptosome. Moreover, we have observed two PIs-induced kinetics of caspase activation, with caspase-9 being still required for the rapid caspase activation in response to mitochondrial depolarization, but dispensable for the slow DEVDase activation. In summary, our data indicate that PIs can activate downstream caspases at least in part through Smac/DIABLO stabilization.

Twist is substrate for caspase cleavage and proteasome-mediated degradation

Twist is a member of the basic helix–loop–helix family of transcription factors. An aberrant Twist expression has been found in diverse types of cancer, including sarcomas, carcinomas and lymphomas, supporting a role for Twist in tumor progression. Twist is known to be essential for mesodermal development. However, since a prolonged Twist expression results in a block of muscle, cartilage and bone differentiation, Twist has to be excluded from somites during late embryogenesis for terminal differentiation to occur. This implies that Twist expression must be target of a tight control. Here we provide evidence that Twist undergoes post-transcriptional regulation. Twist is substrate for cleavage by caspases during apoptosis and its cleavage results in ubiquitin-mediated proteasome degradation. Our findings suggest that Twist post-transcriptional regulation may play an important role in tissue determination and raise the possibility that alterations in the protein turnover may account for Twist overexpression observed in tumors.