A novel member of the Rhomboid family, RHBDD1, regulates BIK-mediated apoptosis

Rhomboid family members are widely conserved and found in all three kingdoms of life. They are serine proteases and serve important regulatory functions. In the present study, a novel gene highly expressed in the testis, RHBDD1, is shown to be a new member of the Rhomboid family, participating in the cleavage of BIK, a proapoptotic member of the Bcl-2 family. The RHBDD1-involved proteolytic modification is upstream of the BIK protein degradation pathway. Mutagenesis studies show that the amino acid residues glycine142 and serine144 of RHBDD1 are crucial for its activity in cleaving BIK at a site located in the transmembrane region. Overexpression or knock-down of RHBDD1 in HEK 293T cells can reduce or enhance BIK-mediated apoptosis, respectively. The present findings suggest that, by acting as a serine protease, RHBDD1 modulates BIK-mediated apoptotic activity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 31 July 2008; received after revision 16 September 2008; accepted 19 September 2008     

Indole-3-carbinol enhances ultraviolet B-induced apoptosis by sensitizing human melanoma cells

Indole-3-carbinol (I3C) has been found to act against several types of cancer, while ultraviolet B (UVB) is known to induce the apoptosis of human melanoma cells. Here, we investigated whether I3C can sensitize G361 human melanoma cells to UVB-induced apoptosis. We examined the effects of combined I3C and UVB (I3C/UVB) at various dosages. I3C (200 μM)/UVB (50 mJ/cm²) synergistically reduced melanoma cell viability, whereas I3C (200 μM) or UVB (50 mJ/cm²), separately, had little effect on cell viability. DNA fragmentation assays indicated that I3C/UVB induced apoptosis. Further results show that I3C/UVB activates caspase-8, −3, and Bid and causes the cleavage of poly(ADP-ribose) polymerase. Moreover, I3C decreased the expression of the anti-apoptotic protein, Bcl-2, whereas UVB increased the translocation of Bax to mitochondria. Thus, an increased Bax/Bcl-2 ratio by I3C/UVB may result in melanoma apoptosis. In conclusion, our study demonstrated that I3C sensitizes human melanoma cells by down-regulating Bcl-2. Received 5 July 2006; received after revision 25 August 2006; accepted 11 September 2006     

A combined artificial chromosome-stem cell therapy method in a model experiment aimed at the treatment of Krabbe’s disease in the Twitcher mouse

Mammalian artificial chromosomes (MACs) are safe, stable, non-integrating genetic vectors with almost unlimited therapeutic transgene-carrying capacity. The combination of MAC and stem cell technologies offers a new strategy for stem cell-based therapy, the efficacy of which was confirmed and validated by using a mouse model of a devastating monogenic disease, galactocerebrosidase deficiency (Krabbe’s disease). Therapeutic MACs were generated by sequence-specific loading of galactocerebrosidase transgenes into a platform MAC, and stable, pluripotent mouse embryonic stem cell lines were established with these chromosomes. The transgenic stem cells were thoroughly characterized and used to produce chimeric mice on the mutant genetic background. The lifespan of these chimeras was increased twofold, verifying the feasibility of the development of MAC-stem cell systems for the delivery of therapeutic genes in stem cells to treat genetic diseases and cancers, and to produce cell types for cell replacement therapies. Received 29 July 2008; received after revision 22 September 2008; accepted 24 September 2008     

Progesterone inhibits human endothelial cell proliferation through a p53-dependent pathway

Previous studies have shown that progesterone inhibits endothelial cell proliferation through a nuclear receptor-mediated mechanism. Here, we further demonstrate that progesterone at physiologic levels (5 – 500 nM) dose- and time-dependently inhibited DNA synthesis of cultured human umbilical vein endothelial cells (HUVEC). The mRNA and protein levels of p21, p27, and p53 in HUVEC were increased by progesterone. The formation of CDK2-p21 and CDK2-p27 were increased and the CDK2 activity was decreased in the progesterone-treated HUVEC. The progesterone-inhibited [3H]thymidine incorporation was completely blocked when the expressions of p21 and p27 were knocked-down together. Transfection of HUVEC with dominant negative p53 cDNA prevented the progesterone-induced increases in p21 and p27 promoter activity and protein level, decreases in thymidine incorporation, and capillary-like tube formation. Matrigel angiogenesis assay in mice demonstrated the antiangiogenic effect of progesterone in vivo. These findings demonstrate for the first time that progesterone inhibited endothelial cell proliferation through a p53-dependent pathway. Received 28 July 2008; received after revision 25 September 2008; accepted 26 September 2008     

Proteinases in cutaneous wound healing

Cutaneous wound healing is a complex and highly coordinated process where a number of different cell types participate to renew the damaged tissue under the strict regulation of soluble and insoluble factors. One of the most versatile processes involved in wound repair is proteolysis. During cell migration, proteins of extracellular matrix are cleaved, often creating biologically active cleavage products, and proteolysis of cellular contacts leads to increased cell motility and division. Moreover, proteases activate various growth factors and other proteases in wound and regulate growth factor signaling by shedding growth factor receptors on cell surface. Normally, proteolysis is strictly controlled, and changes in protease activity are associated with alterations in wound closure and scar formation. Here, we present the current view on the role of metalloproteinases and the plasmin-plasminogen system in normal and aberrant cutaneous wound repair and discuss their role as potential therapeutic targets for chronic ulcers or fibrotic scars. Received 07 July 2008; received after revision 11 August 2008; accepted 13 August 2008     

Overexpression of HAb18G/CD147 promotes invasion and metastasis via α3β1 integrin mediated FAK-paxillin and FAK-PI3K-Ca2+ pathways

Mechanism of HAb18G/CD147 underlying the metastasis process of human hepatoma cells has not been determined. In the present study, we found that integrin α3β1 colocalizes with HAb18G/CD147 in human 7721 hepatoma cells. The enhancing effect of HAb18G/CD147 on adhesion, invasion capacities and matrix metalloproteinases (MMPs) secretion was decreased by integrin α3β1 antibodies (p<0.01). The expressions of integrin downstream molecules including focal adhesion kinase (FAK), phospho-FAK (p-FAK), paxillin, and phospho-paxillin (p-paxillin) were increased in human hepatoma cells overexpressing HAb18G/CD147. Deletion of HAb18G/CD147 reduces the quantity of focal adhesions and rearranges cytoskeleton. Wortmannin and LY294002, specific phosphatidylinositol kinase (PI3K) inhibitors, reversed the effect of HAb18G/CD147 on the regulation of intracellular Ca²⁺ mobilization, significantly reducing cell adhesion, invasion and MMPs secretion potential (p<0.01). Together, these results suggest that HAb18G/CD147 enhances the invasion and metastatic potentials of human hepatoma cells via integrin α3β1-mediated FAK-paxillin and FAKPI3K-Ca²⁺ signal pathways. Received 5 June 2008; received after revision 16 July 2008; accepted 23 July 2008     

Mcl-1 is a potential therapeutic target in multiple types of cancer

Resistance to apoptosis is a common challenge in human malignancies contributing to both progress of cancer and resistance to conventional therapeutics. Abnormalities in a variety of cell intrinsic and extrinsic molecular mechanisms cooperatively promote tumor formation. Therapeutic approaches that specifically target components of these molecular mechanisms are getting widespread attention. Mcl-1 is a highly expressed pro-survival protein in human malignancies and its cellular expression is tightly regulated via multiple mechanisms. Mcl-1 differs from other members of the Bcl-2 family in having a very short half-life. So inhibition of its expression and/or neutralization of its anti-apoptotic function will rapidly make Mcl-1-dependent cells more susceptible to apoptosis and provide an opportunity to combat several types of cancers. This review summarizes the current knowledge on the regulation of Mcl-1 expression and discusses the alternative approaches targeting Mcl-1 in human cancer cells whose survivals mainly depend on Mcl-1. Received 6 October 2008; received after revision 21 October 2008; accepted 10 November 2008     

Cadmium-induced autophagy and apoptosis are mediated by a calcium signaling pathway

The cytotoxicity of cadmium (Cd) induced autophagy and apoptosis in MES-13 cells was determined by flow cytometry. Autophagy was also assessed by formation of autophagosomes and processing of LC3. Pharmacological inhibition of autophagy resulted in increased of cell viability, suggesting autophagy plays a role in cell death in Cd-treated mesangial cells. Cd also induced a rapid elevation in cytosolic calcium ([Ca²⁺]_i ), and modulation of [Ca²⁺]_i via treatment with IP ₃R inhibitor or knockdown of calcineurin resulted in a change in the proportion of cell death, suggesting that the release of calcium from the ER plays a crucial role in Cd-induced cell death. Inhibition of Cd-induced ERK activation by PD 98059 suppressed Cd-induced autophagy, and BAPTA-AM eliminated activation of ERK. BAPTA-AM also inhibited Cd-induced mitochondrial depolarization and activation of caspases. These findings demonstrated that Cd induces both autophagy and apoptosis through elevation of [Ca²⁺]_i, followed by Ca²⁺-ERK and Ca²⁺-mitochondria-caspase signaling pathways. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 05 July 2008; received after revision 25 August 2008; accepted 17 September 2008     

Transient cytoskeletal alterations after SOD1 depletion in neuroblastoma cells

We have studied the effects of superoxide production after Cu,Zn superoxide dismutase (SOD1) down-regulation by RNA interference. We demonstrated that SOD1 depletion induced, only in neuroblastoma cells, a decrease in actin and β-tubulin content and accumulation of neurofilament light chain and Tau proteins. Alterations of cell morphology and the microfilament network were also observed, together with the up-regulation of the Cdk5/p35 pathway, which is involved in the regulation of actin polymerization. The decrease of filamentous actin was transient and was recovered through the activation of p38/Hsp27 MAPK pathway, as well as after treatment with N-acetyl-L-cysteine. The importance of p38 in the recovery of cytoskeleton was confirmed by experiments carried out in the presence of its inhibitor SB203580, which induced cell death. Our data demonstrate that SOD1 is essential for the preservation of cytoskeleton integrity, by maintaining physiological concentration of reactive oxygen species and inhibiting the activation of the neuronal specific Cdk5/p35 pathway. P. Vigilanza, K. Aquilano: Both authors equally contributed to this work. Received 15 November 2007; received after revision 19 January 2008; accepted 22 January 2008     

Inhibition of protein deacetylation by trichostatin A impairs microtubule-kinetochore attachment

Inhibition of protein deacetylation arrests cells in mitosis, but the mechanism is unknown. To understand why inhibiting protein deacetylation causes cell cycle arrest, we treated HeLa cells beyond G1/S transition with trichostatin A (TSA), a potent protein deacetylase inhibitor, and found that the cells arrested at prometaphase with ectopic spindles and unaligned chromosomes. The hyper-acetylated cells encountered a serious microtubule (MT)-kinetochore attachment problem, although the kinetochores are intact at ultrastructural level. By immunofluorescence staining of kinetochore proteins, we found that the pericentromeric H3K9Me3-HP1 pathway was disrupted and that the CENP-A-dependent outer plate protein dynamics of kinetochores was greatly diminished by the drug treatment. The treatment also caused the loss of chromosome passenger complex (CPC), the proposed error checking system, from centromere and impaired the microtubule dynamics of the cells. Overall, we propose that deacetylation inhibition impairs MT-kinetochore attachment through disrupting the centromere function and altering the kinetochore composition and MT dynamics. Received 30 April 2008; received after revision 28 July 2008; accepted 14 August 2008     

Cell migration to the chemokine CXCL8: Paxillin is activated and regulates adhesion and cell motility

The chemokine CXCL8 is a powerful inducer of directional cell motility, primarily during inflammation. In this study, we found that CXCL8 stimulation led to paxillin phosphorylation in normal neutrophils, and that both CXCL8 receptors (CXCR1 and CXCR2) mediated CXCL8-induced paxillin phosphorylation. In CXCR2-transfected cells, the process depended on G_αi and G_αs coupling to CXCR2. Dominant negative (DN) paxillin increased CXCL8-induced adhesion and migration, indicating that endogenous paxillin keeps migration at submaximal levels. Furthermore, using activating antibodies to β1 integrins, analyses with focal adhesion kinase (FAK) DN variant (FRNK) and co-immunoprecipitations of FAK and paxillin, we found that β1 integrin ligation cooperates with CXCL8-induced stimulation, leading to FAK activation and thereafter to FAK-mediated paxillin phosphorylation. Our findings indicate that paxillin keeps directional motility at a restrained magnitude, and suggest that perturbations in its activation may lead to chemotactic imbalance and to pathological conditions associated with excessive or reduced leukocyte migration. R. Mintz, T. Meshel: These authors contributed equally to this work. Received 31 July 2008; received after revision 14 December 2008; accepted 16 December 2008     

Rheological behavior of mammalian cells

Rheological properties of living cells determine how cells interact with their mechanical microenvironment and influence their physiological functions. Numerous experimental studies have show that mechanical contractile stress borne by the cytoskeleton and weak power-law viscoelasticity are governing principles of cell rheology, and that the controlling physics is at the level of integrative cytoskeletal lattice properties. Based on these observations, two concepts have emerged as leading models of cytoskeletal mechanics. One is the tensegrity model, which explains the role of the contractile stress in cytoskeletal mechanics, and the other is the soft glass rheology model, which explains the weak power-law viscoelasticity of cells. While these two models are conceptually disparate, the phenomena that they describe are often closely associated in living cells for reasons that are largely unknown. In this review, we discuss current understanding of cell rheology by emphasizing the underlying biophysical mechanism and critically evaluating the existing rheological models. Received 25 May 2008; received after revision 19 June 2008; accepted 1 July 2008     

Different carbon sources affect lifespan and protein redox state during Saccharomyces cerevisiae chronological ageing

In this study, a proteomic approach that combines selective labelling of proteins containing reduced cysteine residues with two-dimensional electrophoresis/mass spectrometry was used to evaluate the redox state of protein cysteines during chronological ageing in Saccharomyces cerevisiae. The procedure was developed on the grounds that biotinconjugated iodoacetamide (BIAM) specifically reacts with reduced cysteine residues. BIAM-labelled proteins can then be selectively isolated by streptavidin affinity capture. We compared cells grown on 2% glucose in the exponential phase and during chronological ageing and we found that many proteins undergo cysteine oxidation. The target proteins include enzymes involved in glucose metabolism. Both caloric restriction and growth on glycerol resulted in a decrease in the oxidative modification. Furthermore, in these conditions a reduced production of ROS and a more negative glutathione half cell redox potential were observed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 15 September 2008; received after revision 17 December 2008; accepted 06 January 2009     

Role of full-length osteoprotegerin in tumor cell biology

Osteoprotegerin (OPG) is a soluble tumor necrosis factor receptor family member, which potently inhibits RANKL-mediated osteoclastogenesis. Numerous constructs have been created for therapeutic purposes in which the heparin-binding and death homology domains of OPG were removed and the remaining peptide (amino acids 22–194) was fused to the Fc domain of human IgG1 (OPG-Fc). The administration of OPG-Fc efficiently counteracted bone loss in a variety of preclinical models of cancers. However, several in vitro studies have shown that native or recombinant full-length OPG not only neuralizes RANKL, but also the death-inducing ligand TRAIL, suggesting that OPG might potentially counteract the anti-tumor activity of TRAIL. Additional evidence suggests that full-length OPG possesses RANKL- and TRAIL-independent biological properties, mainly related to the promotion of endothelial cell survival and angiogenesis. Finally, breast tumor cells overexpressing OPG have shown increased bone metastatic potential in vivo. The relevance of these apparently conflicting findings in tumor cell biology is highlighted. Received 2 September 2008; received after revision 29 September 2008; accepted 13 October 2008     

Unscheduled CDK1 activity in G1 phase of the cell cycle triggers apoptosis in X-irradiated lymphocytic leukemia cells

Cyclin-dependent kinase 1 (CDK1) is a major component of the cell cycle progression engine. Recently, several investigations provided evidence demonstrating that unscheduled CDK1 activation may also be involved in apoptosis in cancerous cells. In this article, we demonstrate that X-ray irradiation induced G1 arrest in MOLT-4 lymphocytic leukemia cells, the arrest being accompanied by reduction in the activity of CDK2, but increased CDK1 activity and cell apoptosis in the G1 phase. Interestingly, this increase in CDK1 and apoptosis by ionizing radiation was prevented by pretreatment with the CDK1 inhibitor, roscovitine, suggesting that CDK1 kinase activity is required for radiation-induced apoptotic cell death in this model system. Furthermore, cyclin B1 and CDK1 were detected co-localizing and associating in G1 phase MOLT-4 cells, with the cellular lysates from these cells revealing a genotoxic stress-induced increase in CDK1 phosphorylation (Thr-161) and dephosphorylation (Tyr-15), as analyzed by postsorting immunoprecipitation and immunoblotting. Finally, X-irradiation was found to increase Bcl-2 phosphorylation in G1 phase cells. Taken together, these novel findings suggest that CDK1 is activated by unscheduled accumulation of cyclin B1 in G1 phase cells exposed to X-ray, and that CDK1 activation, at the wrong time and in the wrong phase, may directly or indirectly trigger a Bcl-2-dependent signaling pathway leading to apoptotic cell death in MOLT-4 cells. Received 30 March 2006; received after revision 23 June 2006; accepted 24 August 2006 J. Wu and Y. Feng contributed equally to this work.     

Lack of Na+,K+-ATPase expression in intercalated cells may be compensated by Na+-ATPase: A study on MDCK – C11 cells

The lack of Na⁺,K⁺-ATPase expression in intercalated cells (IC) is an intriguing condition due to its fundamental role in cellular homeostasis. In order to better understand this question we compared the activities of Na⁺,K⁺-ATPase and Na⁺-ATPase in two MDCK cell clones: the C11, with IC characteristics, and the C7, with principal cells (PC) characteristics. The Na⁺,K⁺-ATPase activity found in C11 cells is far lower than in C7 cells and the expression of its β-subunit is similar in both cells. On the other hand, a subset of C11 without α-subunit expression has been found. In C11 cells the Na⁺-ATPase activity is higher than that of the Na⁺,K⁺-ATPase, and it is increased by medium alkalinization, suggesting that it could account for the cellular Na⁺-homeostasis. Although further studies are necessary for a better understanding of these findings, the presence of Na⁺-ATPase may explain the adequate survival of cells that lack Na⁺,K⁺-ATPase. Received 09 July 2008; received after revision 03 August 2008; accepted 12 August 2008     

Syncoilin, an intermediate filament-like protein linked to the dystrophin associated protein complex in skeletal muscle

Syncoilin is a member of the intermediate filament protein family, highly expressed in skeletal and cardiac muscle. Syncoilin binds α-dystrobrevin, a component of the dystrophin associated protein complex (DAPC) located at the muscle cell membrane, and desmin, a muscle-specific intermediate filament protein, thus providing a link between the DAPC and the muscle intermediate filament network. This link may be important for muscle integrity and force transduction during contraction, a theory that is supported by the reduced force-generating capacity of muscles from syncoilin-null mice. Additionally, syncoilin is found at increased levels in the regenerating muscle fibres of patients with muscular dystrophies and mouse models of muscle disease. Therefore, syncoilin may be important for muscle regeneration in response to injury. The aims of this article are to review current knowledge about syncoilin and to discuss its possible functions in skeletal muscle. Received 21 May 2008; received after revision 10 July 2008; accepted 18 July 2008     

Differential regulation of collagen types I and III expression in cardiac fibroblasts by AGEs through TRB3/MAPK signaling pathway

Advanced glycation end products (AGEs) play an important role in collagen deposition in diabetic cardiomyopathy. TRB3, a mammalian homolog of Drosophila tribbles, functions to increase glucose intolerance and regulates cell proliferation. We demonstrated that AGEs induce collagen type I expression but inhibit collagen type III expression, accompanied by increased TRB3 expression. Furthermore, the collagen type I induced byAGEs was down-regulated after inhibition of ERK and p38-MAPK, the collagen type III reduced by AGEs was up-regulated after inhibition of ERK. The expression of collagen types I and III regulated by AGEs through MAPK was partly reversed after treatment with TRB3 siRNA. It suggests that the TRB3/MAPK signaling pathway participates in the regulation of collagen types I and III by AGEs and may provide new therapeutic strategies for diabetic cardiomyopathy. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 08 May 2008; received after revision 25 June 2008; accepted 22 July 2008 M. Tang, M. Zhong: These two authors contributed equally to this work.     

Proliferating cell nuclear antigen: a proteomics view

Proliferating cell nuclear antigen (PCNA), a cell cycle marker protein, is well known as a DNA sliding clamp for DNA polymerase delta and as an essential component for eukaryotic chromosomal DNA replication and repair. Due to its mobility inside nuclei, PCNA is dynamically presented in a soluble or chromatin-associated form. The heterogeneity and specific modifications of PCNA may reflect its multiple functions and the presence of many binding partners in the cell. The recent proteomics approaches applied to characterizing PCNA interactions revealed multiple PCNA partners with a wide spectrum of activity and unveiled the possible existence of new PCNA functions. Since more than 100 PCNA-interacting proteins and several PCNA modifications have already been reported, a proteomics point of view seems exactly suitable to better understand the role of PCNA in cellular functions. Received 29 May 2008; received after revision 7 July 2008; accepted 16 July 2008