A Role for Cadherin-5 in Regulation of Vascular Endothelial Growth Factor Receptor 2 Activity in Endothelial Cells

FLK-1/vascular endothelial growth factor receptor 2 (VEGFR-2) is one of the receptors for VEGF. In this study we examined the effect of cell density on activation of VEGFR-2. VEGF induces only very slight tyrosine phosphorylation of VEGFR-2 in confluent (95–100% confluent) pig aortic endothelial (PAE) cells. In contrast, robust VEGF-dependent tyrosine phosphorylation of VEGFR-2 was observed in cells plated in sparse culture conditions (60–65% confluent). A similar cell density-dependent phenomenon was observed in different endothelial cells but not in NIH-3T3 fibroblast cells expressing VEGFR-2. Stimulating cells with high concentrations of VEGF or replacing the extracellular domain of VEGFR-2 with that of the colony-stimulating factor 1 receptor did not alleviate the sensitivity of VEGFR-2 to cell density, indicating that the confluent cells were probably not secreting an antagonist to VEGF. Furthermore, in PAE cells, ectopically introduced platelet-derived growth factor α receptor could be activated at both high and low cell density conditions, indicating that the density effect was not universal for all receptor tyrosine kinases expressed in endothelial cells. In addition to lowering the density of cells, removing divalent cations from the medium of confluent cells potentiated VEGFR-2 phosphorylation in response to VEGF. These findings suggested that cell–cell contact may be playing a role in regulating the activation of VEGFR-2. To this end, pretreatment of confluent PAE cells with a neutralizing anti-cadherin-5 antibody potentiated the response of VEGFR-2 to VEGF. Our data demonstrate that endothelial cell density plays a critical role in regulating VEGFR-2 activity, and that the underlying mechanism appears to involve cadherin-5.

Enhancing leptin response by preventing SH2-containing phosphatase 2 interaction with Ob receptor

Leptin is an adipocyte-derived cytokine that regulates food intake and body weight via interaction with its Ob receptor (ObR). Serum leptin levels are chronically elevated in obese humans, suggesting that obesity may be associated with leptin resistance and the inability to generate an adequate ObR response. Evidence suggests that transcriptional activation of target genes by STAT3 (signal transducer and activator of transcription) in the hypothalamus is a critical pathway that mediates leptin’s action. Herein we report that activation of ObR induces the tyrosine phosphorylation of the tyrosine phosphatase SH2-containing phosphatase 2 (SHP-2) and demonstrate that Tyr986 within the ObR cytoplasmic domain is essential to mediate phosphorylation of SHP-2 and binding of SHP-2 to ObR. Surprisingly, mutation of Tyr986 to Phe, which abrogates SHP-2 phosphorylation and binding to the receptor, dramatically increases gene induction mediated by STAT3. Our findings indicate that SHP-2 is a negative regulator of STAT3-mediated gene induction after activation of ObR and raise the possibility that blocking the interaction of SHP-2 with ObR could overcome leptin resistance by boosting leptin’s weight-reducing effects in obese individuals.

Bax ablation prevents dopaminergic neurodegeneration in the 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) damages dopaminergic neurons in the substantia nigra pars compacta (SNpc) as seen in Parkinson's disease. Here, we show that the pro-apoptotic protein Bax is highly expressed in the SNpc and that its ablation attenuates SNpc developmental neuronal apoptosis. In adult mice, there is an up-regulation of Bax in the SNpc after MPTP administration and a decrease in Bcl-2. These changes parallel MPTP-induced dopaminergic neurodegeneration. We also show that mutant mice lacking Bax are significantly more resistant to MPTP than their wild-type littermates. This study demonstrates that Bax plays a critical role in the MPTP neurotoxic process and suggests that targeting Bax may provide protective benefit in the treatment of Parkinson's disease.

Bc1-2 protects mice against fatal alphavirus encephalitis.

Virus-induced apoptosis has been well characterized in vitro, but the role of apoptosis in viral pathogenesis is not well understood. The suicide of a cell in response to viral infection is postulated to be an important host defense for the organism, leading to a reduction in its total viral burden. However, virus-induced death of nonregenerating cells in the central nervous system may be detrimental to the host. Therefore, to investigate the role of apoptosis in the pathogenesis of fatal encephalitis, we constructed a recombinant alphavirus chimera that expresses the antiapoptotic gene, bcl-2, in virally infected neural cells. Infection of neonatal mice with the alphavirus chimera expressing human bcl-2 [Sindbis virus (SIN)/bcl-2] resulted in a significantly lower mortality rate (7.5%) as compared with infection with control chimeric viruses containing a chloramphenicol acetyltransferase (CAT) reporter gene (SIN/CAT) (78.1%) or bcl-2 containing a premature stop codon (SIN/bcl-2stop) (72.1%) (P < 0.001). Viral titers were reduced 5-fold 1 day after infection and 10-fold 6 days after infection in the brains of SIN/bcl-2-infected mice as compared to SIN/CAT or SIN/bcl-2stop-infected mice. In situ end labeling to detect apoptotic nuclei demonstrated a reduction in the number of foci of apoptotic cells in the brains of mice infected with SIN/bcl-2 as compared with SIN/bcl-2stop. The reduction in apoptosis was associated with a reduction in the number of foci of cells expressing alphavirus RNA. Thus, the antiapoptotic gene, bcl-2, suppresses viral replication and protects against a lethal viral disease, suggesting an interaction between cellular genetic control of viral replication and cell death.

A recombinant bcl-x s adenovirus selectively induces apoptosis in cancer cells but not in normal bone marrow cells.

Many cancers overexpress a member of the bcl-2 family of inhibitors of apoptosis. To determine the role of these proteins in maintaining cancer cell viability, an adenovirus vector that expresses bcl-xs, a functional inhibitor of these proteins, was constructed. Even in the absence of an exogenous apoptotic signal such as x-irradiation, this virus specifically and efficiently kills carcinoma cells arising from multiple organs including breast, colon, stomach, and neuroblasts. In contrast, normal hematopoietic progenitor cells and primitive cells capable of repopulating severe combined immunodeficient mice were refractory to killing by the bcl-xs adenovirus. These results suggest that Bcl-2 family members are required for survival of cancer cells derived from solid tissues. The bcl-xs adenovirus vector may prove useful in killing cancer cells contaminating the bone marrow of patients undergoing autologous bone marrow transplantation.

Membrane-associated CD19-LYN complex is an endogenous p53-independent and Bc1-2-independent regulator of apoptosis in human B-lineage lymphoma cells.

CD19 receptor is expressed at high levels on human B-lineage lymphoid cells and is physically associated with the Src protooncogene family protein-tyrosine kinase Lyn. Recent studies indicate that the membrane-associated CD19-Lyn receptor-enzyme complex plays a pivotal role for survival and clonogenicity of immature B-cell precursors from acute lymphoblastic leukemia patients, but its significance for mature B-lineage lymphoid cells (e.g., B-lineage lymphoma cells) is unknown. CD19-associated Lyn kinase can be selectively targeted and inhibited with B43-Gen, a CD19 receptor-specific immunoconjugate containing the naturally occurring protein-tyrosine kinase inhibitor genistein (Gen). We now present experimental evidence that targeting the membrane-associated CD19-Lyn complex in vitro with B43-Gen triggers rapid apoptotic cell death in highly radiation-resistant p53-Bax- Ramos-BT B-lineage lymphoma cells expressing high levels of Bcl-2 protein without affecting the Bcl-2 expression level. The therapeutic potential of this membrane-directed apoptosis induction strategy was examined in a scid mouse xenograft model of radiation-resistant high-grade human B-lineage lymphoma. Remarkably, in vivo treatment of scid mice challenged with an invariably fatal number of Ramos-BT cells with B43-Gen at a dose level < 1/10 the maximum tolerated dose resulted in 70% long-term event-free survival. Taken together, these results provide unprecedented evidence that the membrane-associated anti-apoptotic CD19-Lyn complex may be at least as important as Bcl-2/Bax ratio for survival of lymphoma cells.

bcl-x is expressed in embryonic and postnatal neural tissues and functions to prevent neuronal cell death.

Previous studies have implicated the bcl-2 protooncogene as a potential regulator of neuronal survival. However, mice lacking functional bcl-2 exhibited normal development and maintenance of the central nervous system (CNS). Since bcl-2 appears dispensable for neuronal survival, we have examined the expression and function of bcl-x, another member of the bcl-2 family of death regulatory genes. Bcl-2 is expressed in neuronal tissues during embryonic development but is down-regulated in the adult CNS. In contrast, Bcl-xL expression is retained in neurons of the adult CNS. Two different forms of bcl-x mRNA and their corresponding products, Bcl-xL and Bcl-x beta, were expressed in embryonic and adult neurons of the CNS. Microinjection of bcl-xL and bcl-x beta cDNAs into primary sympathetic neurons inhibited their death induced by nerve growth factor withdrawal. Thus, Bcl-x proteins appear to play an important role in the regulation of neuronal survival in the adult CNS.

Activities and response to DNA damage of latent and active sequence-specific DNA binding forms of mouse p53

The mouse p53 protein generated by alternative splicing (p53as) has amino acid substitutions at its C terminus that result in constitutively active sequence-specific DNA binding (active form), whereas p53 protein itself binds inefficiently (latent form) unless activated by C-terminal modification. Exogenous p53as expression activated transcription of reporter plasmids containing p53 binding sequences and inhibited growth of mouse and human cells lacking functional endogenous p53. Inducible p53as in stably transfected p53 null fibroblasts increased p21WAF1/Cip-1/Sdi and decreased bcl-2 protein steady-state levels. Endogenous p53as and p53 proteins differed in response to cellular DNA damage. p53 protein was induced transiently in normal keratinocytes and fibroblasts whereas p53as protein accumulation was sustained in parallel with induction of p21WAF1/Cip-1/Sdi protein and mRNA, in support of p53as transcriptional activity. Endogenous p53 and p53as proteins in epidermal tumor cells responded to DNA damage with different kinetics of nuclear accumulation and efficiencies of binding to a p53 consensus DNA sequence. A model is proposed in which C-terminally distinct p53 protein forms specialize in functions, with latent p53 forms primarily for rapid non-sequence-specific binding to sites of DNA damage and active p53 forms for sustained regulation of transcription and growth.

MRIT, a novel death-effector domain-containing protein, interacts with caspases and BclXL and initiates cell death

Activation of the cascade of proteolytic caspases has been identified as the final common pathway of apoptosis in diverse biological systems. We have isolated a gene, termed MRIT, that possesses overall sequence homology to FLICE (MACH), a large prodomain caspase that links the aggregated complex of the death domain receptors of the tumor necrosis factor receptor family to downstream caspases. However, unlike FLICE, the C-terminal domain of MRIT lacks the caspase catalytic consensus sequence QAC(R/Q)G. Nonetheless MRIT activates caspase-dependent death. Using yeast two-hybrid assays, we demonstrate that MRIT associates with caspases possessing large and small prodomains (FLICE, and CPP32/YAMA), as well as with the adaptor molecule FADD. In addition, MRIT simultaneously and independently interacts with BclXL and FLICE in mammalian cells. Thus, MRIT is a mammalian protein that interacts simultaneously with both caspases and a Bcl-2 family member.

Rapid elimination of mature autoreactive B cells demonstrated by Cre-induced change in B cell antigen receptor specificity in vivo

Developing autoreactive B cells edit their B cell antigen receptor (BCR) in the bone marrow and are clonally deleted when they fail to reexpress an innocent BCR. Here, inducible Cre-loxP-mediated gene inversion is used to change the specificity of the BCR on mature IgM+ IgD+ B cells in vivo to address the fate of lymphocytes encountering self-antigens at this developmental stage. Expression of an autoreactive BCR on mature B cells leads to their rapid elimination from the periphery, a process that is inhibited by constitutive bcl-2 transgene expression in an antigen dose-dependent manner. Thus, selection of mature B cells into the long-lived peripheral pool does not prevent their deletion upon encounter of self-antigens.

Cytokine-mediated Bax deficiency and consequent delayed neutrophil apoptosis: A general mechanism to accumulate effector cells in inflammation

Neutrophils are important effector cells in immunity to microorganisms, particularly bacteria. Here, we show that the process of neutrophil apoptosis is delayed in several inflammatory diseases, suggesting that this phenomenon may represent a general feature contributing to the development of neutrophilia, and, therefore, in many cases to host defense against infection. The delay of neutrophil apoptosis was associated with markedly reduced levels of Bax, a pro-apoptotic member of the Bcl-2 family. Such Bax-deficient cells were also observed upon stimulation of normal neutrophils with cytokines present at sites of neutrophilic inflammation, such as granulocyte and granulocyte–macrophage colony-stimulating factors, in vitro. Moreover, Bax-deficient neutrophils generated by using Bax antisense oligodeoxynucleotides demonstrated delayed apoptosis, providing direct evidence for a role of Bax as a pro-apoptotic molecule in these cells. Interestingly, the Bax gene was reexpressed in Bax-deficient neutrophils under conditions of cytokine withdrawal. Thus, both granulocyte expansion and the resolution of inflammation appear to be regulated by the expression of the Bax gene in neutrophils.

Activation of human monocytes induces differential resistance to apoptosis with rapid down regulation of caspase-8/FLICE

Cells of the monocyte/macrophage lineage play a central role in both innate and acquired immunity of the host. However, the acquisition of functional competence and the ability to respond to a variety of activating or modulating signals require maturation and differentiation of circulating monocytes and entail alterations in both biochemical and phenotypic profiles of the cells. The process of activation also confers survival signals essential for the functional integrity of monocytes enabling the cells to remain viable in microenvironments of immune or inflammatory lesions that are rich in cytotoxic inflammatory mediators and reactive free-radical species. However, the molecular mechanisms of activation-induced survival signals in monocytes remain obscure. To define the mechanistic basis of activation-induced resistance to apoptosis in human monocytes at the molecular level, we evaluated the modulation of expression profiles of genes associated with the cellular apoptotic pathways upon activation and demonstrate the following: (i) activation results in selective resistance to apoptosis particularly to that induced by signaling via death receptors and DNA damage; (ii) concurrent with activation, the most apical protease in the death receptor pathway, caspase-8/FLICE is rapidly down-regulated at the mRNA level representing a novel regulatory mechanism; and (iii) activation of monocytes also leads to dramatic induction of the Bfl-1 gene, an anti apoptotic member of the Bcl-2 family. Our findings thus provide a potential mechanistic basis for the activation-induced resistance to apoptosis in human monocytes.

The cancer growth suppressor gene mda-7 selectively induces apoptosis in human breast cancer cells and inhibits tumor growth in nude mice

A differentiation induction subtraction hybridization strategy is being used to identify and clone genes involved in growth control and terminal differentiation in human cancer cells. This scheme identified melanoma differentiation associated gene-7 (mda-7), whose expression is up-regulated as a consequence of terminal differentiation in human melanoma cells. Forced expression of mda-7 is growth inhibitory toward diverse human tumor cells. The present studies elucidate the mechanism by which mda-7 selectively suppresses the growth of human breast cancer cells and the consequence of ectopic expression of mda-7 on human breast tumor formation in vivo in nude mice. Infection of wild-type, mutant, and null p53 human breast cancer cells with a recombinant type 5 adenovirus expressing mda-7, Ad.mda-7 S, inhibited growth and induced programmed cell death (apoptosis). Induction of apoptosis correlated with an increase in BAX protein, an established inducer of programmed cell death, and an increase in the ratio of BAX to BCL-2, an established inhibitor of apoptosis. Infection of breast carcinoma cells with Ad.mda-7 S before injection into nude mice inhibited tumor development. In contrast, ectopic expression of mda-7 did not significantly alter cell cycle kinetics, growth rate, or survival in normal human mammary epithelial cells. These data suggest that mda-7 induces its selective anticancer properties in human breast carcinoma cells by promoting apoptosis that occurs independent of p53 status. On the basis of its selective anticancer inhibitory activity and its direct antitumor effects, mda-7 may represent a new class of cancer suppressor genes that could prove useful for the targeted therapy of human cancer.

Withdrawal of IL-7 induces Bax translocation from cytosol to mitochondria through a rise in intracellular pH

IL-7 functions as a trophic factor during T lymphocyte development by a mechanism that is partly based on the induction of Bcl-2, which protects cells from apoptosis. Here we report a mechanism by which cytokine withdrawal activates the prodeath protein Bax. On loss of IL-7 in a dependent cell line, Bax protein translocated from the cytosol to the mitochondria, where it integrated into the mitochondrial membrane. This translocation was attributable to a conformational change in the Bax protein itself. We show that a rise in intracellular pH preceded mitochondrial translocation and triggered the change in Bax conformation. Intracellular pH in the IL-7-dependent cells rose steadily to peak over pH 7.8 by 6 hr after cytokine withdrawal, paralleling the time point of Bax translocation (a similar alkalinization and Bax translocation was also observed after IL-3 withdrawal from a dependent cell line). The conformation of Bax was directly altered by pH of 7.8 or higher and was demonstrated by increased protease sensitivity, exposure of N terminus epitopes, and exposure of a hydrophobic domain in the C terminus. Eliminating charged amino acids at the C or N termini of Bax induced a conformational change similar to that induced by raising pH, implicating these residues in the pH effect. Therefore, we have shown that by either cytokine withdrawal, experimental manipulation of pH, or site-directed mutagenesis, Bax protein changes conformation, exposing membrane-seeking domains, thereby inducing mitochondrial translocation and initiating the cascade of events leading to apoptotic death.

Prevention of lymphocyte cell death in sepsis improves survival in mice

Sepsis induces extensive lymphocyte apoptosis, a process which may be beneficial to host survival by down-regulating the inflammatory response or, alternatively, harmful by impairing host defenses. To determine the beneficial vs. adverse effects of lymphocyte apoptosis in sepsis, we blocked lymphocyte apoptosis either by N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl) fluoromethyl ketone (z-VAD), a broad-spectrum caspase inhibitor, or by use of Bcl-2 Ig transgenic mice that selectively overexpress the antiapoptotic protein Bcl-2 in a lymphoid pattern. Both z-VAD and Bcl-2 prevented lymphocyte apoptosis and resulted in a marked improvement in survival. z-VAD did not decrease lymphocyte tumor necrosis factor-α production. Considered together, these two studies employing different methods of blocking lymphocyte apoptosis provide compelling evidence that immunodepression resulting from the loss of lymphocytes is a central pathogenic event in sepsis, and they challenge the current paradigm that regards sepsis as a disorder resulting from an uncontrolled inflammatory response. Caspase inhibitors may represent a treatment strategy in this highly lethal disorder.