Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis.

Autophagy-related gene (Atg) 5 is a gene product required for the formation of autophagosomes. Here, we report that Atg5, in addition to the promotion of autophagy, enhances susceptibility towards apoptotic stimuli. Enforced expression of Atg5-sensitized tumour cells to anticancer drug treatment both in vitro and in vivo. In contrast, silencing the Atg5 gene with short interfering RNA (siRNA) resulted in partial resistance to chemotherapy. Apoptosis was associated with calpain-mediated Atg5 cleavage, resulting in an amino-terminal cleavage product with a relative molecular mass of 24,000 (Mr 24K). Atg5 cleavage was observed independent of the cell type and the apoptotic stimulus, suggesting that calpain activation and Atg5 cleavage are general phenomena in apoptotic cells. Truncated Atg5 translocated from the cytosol to mitochondria, associated with the anti-apoptotic molecule Bcl-xL and triggered cytochrome c release and caspase activation. Taken together, calpain-mediated Atg5 cleavage provokes apoptotic cell death, therefore, represents a molecular link between autophagy and apoptosis--a finding with potential importance for clinical anticancer therapies.

RhoH/TTF negatively regulates leukotriene production in neutrophils.

Leukotriene B(4) (LTB(4)) is an important proinflammatory lipid mediator generated by neutrophils upon activation. GM-CSF stimulation is known to enhance agonist-mediated LTB(4) production of neutrophils within minutes, a process called "priming". In this study, we demonstrate that GM-CSF also limits the production of LTB(4) by neutrophils via a transcriptional mechanism at later time points. We identified hemopoietic-specific Ras homologous (RhoH)/translocation three four (TTF), which was induced following GM-CSF stimulation in neutrophils, as a key regulator in this process. Neutrophils derived from RhoH/TTF-deficient (Rhoh(-/-)) mice demonstrated increased LTB(4) production upon activation compared with normal mouse neutrophils. Moreover, neutrophils from cystic fibrosis patients expressed enhanced levels of RhoH/TTF and generated less LTB(4) upon activation compared with normal human neutrophils. Taken together, these data suggest that RhoH/TTF represents an inducible feedback inhibitor in neutrophils that is involved in the limitation of innate immune responses.

Deregulated ephrin-B2 expression in the mammary gland interferes with the development of both the glandular epithelium and vasculature and promotes metastasis formation.

Eph receptor tyrosine kinases and their membrane-bound ephrin ligands play key roles during morphogenesis and adult tissue homeostasis. Receptor-ligand interactions result in forward and reverse signalling from the receptor and ligand respectively. To delineate the role(s) of forward and reverse signalling in mammary gland biology we have established transgenic mice exhibiting mammary epithelial-specific overexpression of either the native ephrin-B2 or a dominant negative ephrin-B2 mutant incapable of reverse signalling. During pregnancy and lactation overexpression of the native ephrin-B2 resulted in precocious differentiation, whereas overexpression of mutated ephrin-B2 caused delayed epithelial differentiation and in disturbed tissue architecture. Both transgenes affected also mammary vascularisation. Whereas ephrin-B2 induced superfluous but organised capillaries, mutant ephrin-B2 overexpression resulted in an irregular vasculature with blind-ending capillaries. Mammary tumours were not observed in either transgenic line, however, the crossing with NeuT transgenic animals revealed that mutated ephrin-B2 expression significantly accelerated tumour growth and imposed a metastatic phenotype.

Mammary epithelial-specific knockout of the ephrin-B2 gene leads to precocious epithelial cell death at lactation.

The family of Eph receptor tyrosine kinases and their membrane bound ligands, the ephrins, are involved in a wide variety of morphogenic processes during embryonic development and adult tissue homeostasis. Receptor-ligand interaction requires direct cell-cell contact and results in forward and reverse signaling originating from the receptor and ligand, respectively. We have previously shown that EphB4 and ephrinB2 are differentially expressed during the development of the adult mammary parenchyma. Overexpression of EphB4 in the mammary epithelium of transgenic mice leads to perturbations in mammary epithelial morphology, motility and growth. To investigate the role of ephrinB2 signaling in mammary gland biology, we have established transgenic mice exhibiting conditional ephrinB2 knockout in the mammary epithelium. In homozygote double transgenic CreLox mice, specific knockout of ephrinB2 occurred in the mammary epithelium during the first pregnancy-lactating period. Abolishing ephrinB2 function led to severe interference with the architecture and functioning of the mammary gland at lactation. The morphology of the transgenic lactating glands resembled that of involuting controls, with decreased epithelial cell number and collapsed lobulo-alveolar structures. Accordingly, massive epithelial cell death and expression of involution-specific genes were observed. Interestingly, in parallel to cell death, significant cell proliferation was apparent, suggestive of tissue regeneration.

The death-associated protein kinase 2 is up-regulated during normal myeloid differentiation and enhances neutrophil maturation in myeloid leukemic cells.

The death-associated protein kinase 2 (DAPK2) belongs to a family of Ca(2+)/calmodulin-regulated serine/threonine kinases involved in apoptosis. During investigation of candidate genes operative in granulopoiesis, we identified DAPK2 as highly expressed. Subsequent investigations demonstrated particularly high DAPK2 expression in normal granulocytes compared with monocytes/macrophages and CD34(+) progenitor cells. Moreover, significantly increased DAPK2 mRNA levels were seen when cord blood CD34(+) cells were induced to differentiate toward neutrophils in tissue culture. In addition, all-trans retinoic acid (ATRA)-induced neutrophil differentiation of two leukemic cell lines, NB4 and U937, revealed significantly higher DAPK2 mRNA expression paralleled by protein induction. In contrast, during differentiation of CD34(+) and U937 cells toward monocytes/macrophages, DAPK2 mRNA levels remained low. In primary leukemia, low expression of DAPK2 was seen in acute myeloid leukemia samples, whereas chronic myeloid leukemia samples in chronic phase showed intermediate expression levels. Lentiviral vector-mediated expression of DAPK2 in NB4 cells enhanced, whereas small interfering RNA-mediated DAPK2 knockdown reduced ATRA-induced granulocytic differentiation, as evidenced by morphology and neutrophil stage-specific maturation genes, such as CD11b, G-CSF receptor, C/EBPepsilon, and lactoferrin. In summary, our findings implicate a role for DAPK2 in granulocyte maturation.

GPI-specific phospholipase D (GPI-PLD) is expressed during mouse development and is localized to the extracellular matrix of the developing mouse skeleton.

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is abundant in serum and has a well-characterized biochemistry; however, its physiological role is completely unknown. Previous investigations into GPI-PLD have focused on the adult animal or on in vitro systems and a putative role in development has been neither proposed nor investigated. We describe the first evidence of GPI-PLD expression during mouse embryonic ossification. GPI-PLD expression was detected predominantly at sites of skeletal development, increasing during the course of gestation. GPI-PLD was observed during both intramembraneous and endochondral ossification and localized predominantly to the extracellular matrix of chondrocytes and to primary trabeculae of the skeleton. In addition, the mouse chondrocyte cell line ATDC5 expressed GPI-PLD after experimental induction of differentiation. These results implicate GPI-PLD in the process of bone formation during mouse embryogenesis.

EphB4 receptor tyrosine kinase transgenic mice develop glomerulopathies reminiscent of aglomerular vascular shunts.

We have established transgenic mice over-expressing the EphB4 receptor tyrosine kinase in the kidney. The EphB4 protein was localised to the developing tubular system of both control and transgenic newborn mice. In transgenic adults, transgene expression persisted in the proximal tubules and the Bowman's capsules, structures, which were not stained in control kidneys. The glomeruli of control animals consisted of regular, round vascular baskets with clearly discernable afferent and efferent arterioles. In contrast, approximately 40% of the transgenic glomeruli had an irregular shrivelled appearance and many exhibited fused, horse shoe-like afferent and efferent arterioles bypassing the glomerulus. These abnormal glomerular structures are very reminiscent of aglomerular vascular shunts, a human degenerative glomerulopathy of unknown aetiology.

Regulation and function of the cytokine-inducible SH-2 domain proteins, CIS and SOCS3, in mammary epithelial cells.

The cytokine-inducible src homology 2 (SH-2) proteins, CIS (cytokine inducible SH-2 domain protein) and SOCS3 (suppressor of cytokine signaling 3), are implicated in the negative regulation of prolactin (PRL) receptor-mediated activation of signal transducer and activator of transcription 5 (STAT5). We have studied the expression and function of CIS and SOCS3 proteins in the mouse mammary gland and in HC11 mammary epithelial cells. CIS and SOCS3 were differentially regulated: high expression levels of CIS mRNA were measured during the second half of pregnancy, whereas SOCS3 expression was high during the first 12 d post conceptum. SOCS3 levels increased, whereas CIS levels decreased, in the initial phase of involution. At the beginning of the lactation period both CIS and SOCS3 were high. PRL and epidermal growth factor (EGF) were able to induce CIS and SOCS3, whereas glucocorticoids inhibited their expression in mammary epithelial cells. The effect of EGF was much stronger on SOCS3 than on CIS. Ectopic expression of both SOCS3 and CIS inhibited STAT5 activation. Our data indicate that in the mammary gland CIS and SOCS3 are involved in regulating STAT5 signaling at three different instances: 1) SOCS3 serves as a mediator of the inhibitory EGF effect on PRL-induced STAT5 activation; 2) CIS and SOCS3 play a role as negative feedback inhibitors of PRL action; 3) Inhibition of CIS and SOCS3 expression by glucocorticoids contributes to the positive effect of glucocorticoids on PRL-induced STAT5 activation.

Tumor cell specific expression of MMP-2 correlates with tumor vascularisation in breast cancer.

The metastatic potential of tumors is dependent on the ability of tumor cells to degrade extracellular matrix components by the expression of matrix metalloproteinases (MMPs) and to induce vascularisation of the tumor tissue. Thus, expression of MMPs and the number of blood vessel in tumor tissue may serve as prognostic markers of aggressive and metastasizing tumor growth. We have determined the vascularisation and the expression of MMP-2 by immuno-histochemical staining of 19 benign and 75 malignant breast tissue specimens with CD31- and MMP-2 specific antisera. The degree of vascularisation was expressed by intratumoral microvascular density (IMD), which takes into account all vessels present in a hot spot irrespective of their size. In addition, we have introduced a novel parameter, vascular grading (VG), which describes the percentage of small microvessels of <20 microm in diameter in the total number of blood vessels. IMD tended to indicate an elevated risk for metastasis formation and disease recurrence, while VG did not correlate with metastasis formation. Similarly, MMP-2 expression neither correlated with the clinical outcome of the disease nor with the classical histo-pathological parameters such as stage, grade, lymph node involvement and estrogen receptor status. Tumor cell-specific MMP-2 expression, however, showed a highly significant correlation with VG but not with IMD. These results indicate that MMP-2 expression is rather involved in the formation of small capillaries than in vessel maturation and tumor cell invasion. Thus, MMP-2 expression by tumor cells may serve as indicator of strong angiogenic induction potential of breast tumor cells.

Engineered fibrin matrices for functional display of cell membrane-bound growth factor-like activities: study of angiogenic signaling by ephrin-B2.

With the rapid increase in approaches to pro- or anti-angiogenic therapy, new and effective methodologies for administration of cell-bound growth factors will be required. We sought to develop the natural hydrogel matrix fibrin as platform for extensive interactions and continuous signaling by the vascular morphogen ephrin-B2 that normally resides in the plasma membrane and requires multivalent presentation for ligation and activation of Eph receptors on apposing endothelial cell surfaces. Using fibrin and protein engineering technology to induce multivalent ligand presentation, a recombinant mutant ephrin-B2 receptor binding domain was covalently coupled to fibrin networks at variably high densities. The ability of fibrin-bound ephrin-B2 to act as ligand for endothelial cells was preserved, as demonstrated by a concomitant, dose-dependent increase of endothelial cell binding to engineered ephrin-B2-fibrin substrates in vitro. The therapeutic relevance of ephrin-B2-fibrin implant matrices was demonstrated by a local angiogenic response in the chick embryo chorioallontoic membrane evoked by the local and prolonged presentation of matrix-bound ephrin-B2 to tissue adjacing the implant. This new knowledge on biomimetic fibrin vehicles for precise local delivery of membrane-bound growth factor signals may help to elucidate specific biological growth factor function, and serve as starting point for development of new treatment strategies.

Loss of EphB4 receptor tyrosine kinase protein expression during carcinogenesis of the human breast.

Members of the Eph family of receptor tyrosine kinase have been implicated in cell-cell communication and tissue integrity during embryogenesis. We have previously demonstrated cell type specific and hormone dependent EphB4 expression in the mouse mammary parenchyma suggesting involvement in the homeostasis of this organ. Since disruption of tissue organization is crucial for metastatic dissemination, we have investigated the expression of EphB4 during carcinogenesis of the human breast. Immunohistochemical analysis of 24 normal human breast samples and 124 consecutive breast carcinomas was correlated with tumor characteristics (stage, histology, grade, lymph node involvement) and the expression of ER, PR, Ki-67, p53 and HER2. In normal breast tissue, the EphB4 protein was expressed exclusively in parenchymal cells. Strikingly, a drastic reduction in the number of EphB4 protein expressing cells was observed in almost all invasive carcinomas analyzed, irrespective of the tumor type (p<0.0001). Furthermore, we found a highly significant correlation between EphB4 positivity and low histological grading of the tumor cells (p=0.002) suggesting that in breast cancer, EphB4 expression is not compatible with tumor progression. This raises the possibility that EphB4 could represent a potent tool for therapeutic intervention.

Hint2, a mitochondrial apoptotic sensitizer down-regulated in hepatocellular carcinoma.

BACKGROUND & AIMS: Hints, histidine triad nucleotide-binding proteins, are adenosine monophosphate-lysine hydrolases of uncertain biological function. Here we report the characterization of human Hint2. METHODS: Tissue distribution was determined by real-time quantitative polymerase chain reaction and immunoblotting, cellular localization by immunocytochemistry, and transfection with green fluorescent protein constructs. Enzymatic activities for protein kinase C and adenosine phosphoramidase in the presence of Hint2 were measured. HepG2 cell lines with Hint2 overexpressed or knocked down were established. Apoptosis was assessed by immunoblotting for caspases and by flow cytometry. Tumor growth was measured in SCID mice. Expression in human tumors was investigated by microarrays. RESULTS: Hint2 was predominantly expressed in liver and pancreas. Hint2 was localized in mitochondria. Hint2 hydrolyzed adenosine monophosphate linked to an amino group (AMP-pNA; k(cat):0.0223 s(-1); Km:128 micromol/L). Exposed to apoptotic stress, fewer HepG2 cells overexpressing Hint2 remained viable (32.2 +/- 0.6% vs 57.7 +/- 4.6%), and more cells displayed changes of the mitochondrial membrane potential (87.8 +/- 2.35 vs 49.7 +/- 1.6%) with more cleaved caspases than control cells. The opposite was observed in HepG2 cells with knockdown expression of Hint2. Subcutaneous injection of HepG2 cells overexpressing Hint2 in SCID mice resulted in smaller tumors (0.32 +/- 0.13 g vs 0.85 +/- 0.35 g). Microarray analyses revealed that HINT2 messenger RNA is downregulated in hepatocellular carcinomas (-0.42 +/- 0.58 log2 vs -0.11 +/- 0.28 log2). Low abundance of HINT2 messenger RNA was associated with poor survival. CONCLUSION: Hint2 defines a novel class of mitochondrial apoptotic sensitizers down-regulated in hepatocellular carcinoma.

PDZ interaction site in ephrinB2 is required for the remodeling of lymphatic vasculature.

The transmembrane ligand ephrinB2 and its cognate Eph receptor tyrosine kinases are important regulators of embryonic blood vascular morphogenesis. However, the molecular mechanisms required for ephrinB2 transduced cellular signaling in vivo have not been characterized. To address this question, we generated two sets of knock-in mice: ephrinB2DeltaV mice expressed ephrinB2 lacking the C-terminal PDZ interaction site, and ephrinB2(5F) mice expressed ephrinB2 in which the five conserved tyrosine residues were replaced by phenylalanine to disrupt phosphotyrosine-dependent signaling events. Our analysis revealed that the homozygous mutant mice survived the requirement of ephrinB2 in embryonic blood vascular remodeling. However, ephrinB2DeltaV/DeltaV mice exhibited major lymphatic defects, including a failure to remodel their primary lymphatic capillary plexus into a hierarchical vessel network, hyperplasia, and lack of luminal valve formation. Unexpectedly, ephrinB2(5F/5F) mice displayed only a mild lymphatic phenotype. Our studies define ephrinB2 as an essential regulator of lymphatic development and indicate that interactions with PDZ domain effectors are required to mediate its functions.

Eph and ephrin signaling in mammary gland morphogenesis and cancer.

The Eph family of receptor tyrosine kinases and their membrane-bound ligands, the ephrins, play a central role in pattern formation during embryonic development and there is growing evidence that they are also instrumental in the control of tissue dynamics in the adult. The mammary gland is a paradigm for morphogenic processes occurring in the adult, since the gland develops predominantly postnatally and is subjected to continuous cyclic remodeling according to functional demands. Thus, pattern formation and the establishment of a functional organ structure are permanent themes in the mammary gland life cycle. In this paper we summarize the experimental evidence and discuss possible mechanisms by which Ephs and ephrins are modulating mammary epithelial cell adhesion, communication, and migration. Furthermore, we speculate on the different aspects of their influence on normal mammary gland development, function, and carcinogenesis.

Fibre-type specific concentration of focal adhesion kinase at the sarcolemma: influence of fibre innervation and regeneration.

In skeletal muscles, focal adhesion complexes (FACs) form part of the costamere, a sarcolemmal protein complex that enables lateral transfer of forces and ensures the stability of the sarcolemma. The present investigation tested whether localisation of a major assembly factor of FACs, focal adhesion kinase (FAK), to the sarcolemma parallels the known modulation of FACs by fibre type (innervation pattern) and fibre regeneration. Immunohistochemical experiments indicated that FAK is preferentially associated with the sarcolemma in a high proportion (>74 %) of the (slow-twitch) type I and (fast-twitch) type IIA fibres in normal rat soleus (N-SOL) muscle and of the type IIA fibres in extensor digitorum longus (N-EDL) muscle. In contrast, a low proportion (<15 %) of fast-twitch type IIB and type I fibres in N-EDL showed sarcolemmal FAK immunoreactivity. Cross-reinnervation of slow-twitch rat SOL muscle with the fast EDL nerve induced slow-to-fast fibre transformation and led to a significant reduction in sarcolemmal FAK immunoreactivity in type I and type IIA fibres. Transplantation of the fast EDL into the slow SOL bed with regeneration and reinnervation of the muscle by the SOL nerve (T-EDL) caused a significant increase in sarcolemmal FAK immunoreactivity in new type I and hybrid I/II fibres and a corresponding reduction in sarcolemmal FAK immunoreactivity in 'normal' IIA and IIB fibres. Conversely, sarcolemmal FAK immunoreactivity in small IIB fibres of T-EDL muscle was increased. Correspondingly, the transplanted and regenerated SOL (reinnervated by the fast EDL nerve) maintained the percentage of FAK-positive sarcolemma in the (regenerated) type I and IIA fibres. Thus, the expression and association of FAK with the sarcolemma are regulated (i) by factors that determine the fibre type and (ii) during fibre regeneration. Our data suggest that the integrity of sarcolemmal FACs is dependent on the fibre type and that FAC turnover is increased during regeneration of muscle fibres.

Anti-mouse GPI-PLD antisera highlight structural differences between murine and bovine GPI-PLDs.

Despite its well characterised biochemistry, the physiological role of glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is unknown. Most of the previous studies investigating the distribution of GPI-PLD have focused on the human and bovine forms of the enzyme. Studies on mouse GPI-PLD are rare, partly due to the lack of a specific anti-mouse GPI-PLD antibody, but also due to the apparent low reactivity of existing antibodies to rodent GPI-PLDs. Here we describe the isolation of a mouse liver cDNA, the construction and expression of a recombinant enzyme and the generation of an affinity-purified rabbit anti-mouse GPI-PLD antiserum. The antibody shows good reactivity to partially purified murine and purified bovine GPI-PLD. In contrast, a rat anti-bovine GPI-PLD antibody shows no reactivity with the mouse enzyme and the two antibodies recognise different proteolytic fragments of the bovine enzyme. Comparison between the rodent, bovine and human enzymes indicates that small changes in the amino acid sequence of a short peptide in the mouse and bovine GPI-PLDs may contribute to the different reactivities of the two antisera. We discuss the implications of these results and stress the importance of antibody selection while investigating GPI-PLD in the mouse.

Altered mammary epithelial development, pattern formation and involution in transgenic mice expressing the EphB4 receptor tyrosine kinase.

We have previously documented the cell-type-specific and hormone-dependent expression of the EphB4 receptor in the mouse mammary gland. To investigate its role in the biology of the mammary gland, we have established transgenic mice bearing the EphB4 receptor under the control of the MMTV-LTR promoter, which represents the first transgenic mouse model to investigate the effect(s) of unscheduled expression of EphB4 in adult organisms. Transgene expression in the mammary epithelium was induced at puberty, increased during pregnancy, culminated at early lactation and persisted until day three of post-lactational involution. In contrast, expression of the endogenous EphB4 gene is downregulated during pregnancy, is essentially absent during lactation and is re-induced after day three of post-lactational involution. The unscheduled expression of EphB4 led to a delayed development of the mammary epithelium at puberty and during pregnancy. During pregnancy, less lobules were formed, these however exhibited more numerous but smaller alveolar units. Transgenic mammary glands were characterized by a fragile, irregular morphology at lactation; however, sufficient functionality was maintained to nourish the young. Transgenic mammary glands exhibited untimely epithelial apoptotic cell death during pregnancy and abnormal epithelial DNA synthesis at early post-lactational involution, indicating a disturbed response to proliferative/apoptotic signals. Mammary tumours were not observed in the EphB4 transgenic animals; however, in double transgenic animals expressing both EphB4 and the neuT genes, tumour appearance was significantly accelerated and, in contrast to neuT-only animals, metastases were observed in the lung. These results implicate EphB4 in the regulation of tissue architecture, cellular growth response and establishment of the invasive phenotype in the adult mammary gland.