Activation of NF-κB is required for hypertrophic growth of primary rat neonatal ventricular cardiomyocytes

The transcription factor NF-κB regulates expression of genes that are involved in inflammation, immune response, viral infection, cell survival, and division. However, the role of NF-κB in hypertrophic growth of terminally differentiated cardiomyocytes is unknown. Here we report that NF-κB activation is required for hypertrophic growth of cardiomyocytes. In cultured rat primary neonatal ventricular cardiomyocytes, the nuclear translocation of NF-κB and its transcriptional activity were stimulated by several hypertrophic agonists, including phenylephrine, endothelin-1, and angiotensin II. The activation of NF-κB was inhibited by expression of a “supersuppressor” IκBα mutant that is resistant to stimulation-induced degradation and a dominant negative IκB kinase (IKKβ) mutant that can no longer be activated by phosphorylation. Furthermore, treatment with phenylephrine induced IκBα degradation in an IKK-dependent manner, suggesting that NF-κB is a downstream target of the hypertrophic agonists. Importantly, expression of the supersuppressor IκBα mutant or the dominant negative IKKβ mutant blocked the hypertrophic agonist-induced expression of the embryonic gene atrial natriuretic factor and enlargement of cardiomyocytes. Conversely, overexpression of NF-κB itself induced atrial natriuretic factor expression and cardiomyocyte enlargement. These findings suggest that NF-κB plays a critical role in the hypertrophic growth of cardiomyocytes and may serve as a potential target for the intervention of heart disease.

CD95/Fas induces cleavage of the GrpL/Gads adaptor and desensitization of antigen receptor signaling

The balance between cell survival and cell death is critical for normal lymphoid development. This balance is maintained by signals through lymphocyte antigen receptors and death receptors such as CD95/Fas. In some cells, ligating the B cell antigen receptor can protect the cell from apoptosis induced by CD95. Here we report that ligation of CD95 inhibits antigen receptor-mediated signaling. Pretreating CD40-stimulated tonsillar B cells with anti-CD95 abolished B cell antigen receptor-mediated calcium mobilization. Furthermore, CD95 ligation led to the caspase-dependent inhibition of antigen receptor-induced calcium mobilization and to the activation of mitogen-activated protein kinase pathways in B and T cell lines. A target of CD95-mediated caspase 3-like activity early in the apoptotic process is the adaptor protein GrpL/Gads. GrpL constitutively interacts with SLP-76 via its C-terminal SH3 domain to regulate transcription factors such as NF-AT. Cleavage of GrpL removes the C-terminal SH3 domain so that it is no longer capable of recruiting SLP-76 to the membrane. Transfection of a truncated form of GrpL into Jurkat T cells blocked T cell antigen receptor-induced activation of NF-AT. These results suggest that CD95 signaling can desensitize antigen receptors, in part via cleavage of the GrpL adaptor.

Akt suppresses androgen-induced apoptosis by phosphorylating and inhibiting androgen receptor

Whereas several apoptosis-related proteins have been linked to the antiapoptotic effects of Akt serine–threonine kinase, the search continues to explain the Akt signaling role in promoting cell survival via antiapoptotic effects. Here, we demonstrate that Akt phosphorylates the androgen receptor (AR) at Ser-210 and Ser-790. A mutation at AR Ser-210 results in the reversal of Akt-mediated suppression of AR transactivation. Activation of the phosphatidylinositol-3-OH kinase/Akt pathway results in the suppression of AR target genes, such as p21, and the decrease of androgen/AR-mediated apoptosis, which may involve the inhibition of interaction between AR and AR coregulators. Together, these findings provide a molecular basis for cross-talk between two signaling pathways at the level of Akt and AR–AR coregulators that may help us to better understand the roles of Akt in the androgen/AR-mediated apoptosis.

Involvement of p38 in Apoptosis-associated Membrane Blebbing and Nuclear Condensation

The stress-activated protein kinase p38 is often induced by cytotoxic agents, but its contribution to cell death is ill defined. In Rat-1 cells, we found a strong correlation between activation of p38 and induction of c-Myc–dependent apoptosis. In cells with deregulated c-Myc expression but not in control cells, cis-diamminedichloroplatinum induced p38 activity and typical features of apoptosis, including internucleosomal DNA degradation, induction of caspase activities, and both nuclear (nuclear condensation and fragmentation) and extranuclear (cell blebbing) morphological alterations. The pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone did not block p38 activation and the p38 inhibitor SB203580 had no detectable effect on the activation of caspases or the in vivo cleavage of several caspase substrates, suggesting that p38 and caspase activation can contribute distinct features of apoptosis. Accordingly, we found that cell blebbing was independent of caspase activity and, rather, depended on p38-sensitive changes in microfilament dynamics likely mediated by heat shock protein 27 phosphorylation. Furthermore, p38 activity contributed to both caspase-dependent and caspase-independent nuclear condensation and fragmentation, suggesting a role in an early event triggering both mechanisms of apoptosis or sensitizing the cells to the action of both types of apoptosis executioners. Inhibiting p38 also resulted in a significant enhancement in cell survival estimated by colony formation. This capacity to modulate the sensitivity to apoptosis in cells with deregulated c-Myc expression suggests an important role for p38 in tumor cell killing by chemotherapeutic agents.

Fas (CD95) expression and death-mediating function are induced by CD4 cross-linking on CD4+ T cells.

The CD4 receptor contributes to T-cell activation by coligating major histocompatibility complex class II on antigen presenting cells with the T-cell receptor (TCR)/CD3 complex, and triggering a cascade of signaling events including tyrosine phosphorylation of intracellular proteins. Paradoxically, CD3 cross-linking prior to TCR stimulation results in apoptotic cell death, as does injection of anti-CD4 antibodies in vivo of CD4 ligation by HIV glycoprotein (gp) 120. In this report we investigate the mechanism by which CD4 cross-linking induces cell death. We have found that CD4 cross-linking results in a small but rapid increase in levels of cell surface Fas, a member of the tumor necrosis factor receptor family implicated in apoptotic death and maintenance of immune homeostasis. Importantly, CD4 cross-linking triggered the ability of Fas to function as a death molecule. Subsequent to CD4 cross-linking, CD4+ splenocytes cultured overnight became sensitive to Fas-mediated death. Death was Fas-dependent, as demonstrated by cell survival in the absence of plate-bound anti-Fas antibody, and by the lack of CD4-induced death in cells from Fas-defective lymphoproliferative (lpr) mice. We demonstrate here that CD4 regulates the ability of Fas to induce cell death in Cd4+ T cells.

Wild-type and mutant p53 differentially regulate transcription of the insulin-like growth factor I receptor gene.

The insulin-like growth factor I receptor (IGF-I-R) plays a critical role in transformation events. It is highly overexpressed in most malignant tissues where it functions as an anti-apoptotic agent by enhancing cell survival. Tumor suppressor p53 is a nuclear transcription factor that blocks cell cycle progression and induces apoptosis. p53 is the most frequently mutated gene in human cancer. Cotransfection of Saos-2 (os-teosarcoma-derived cells) and RD (rhabdomyosarcoma-derived cells) cells with IGF-I-R promoter constructs driving luciferase reporter genes and with wild-type p53 expression vectors suppressed promoter activity in a dose-dependent manner. This effect of p53 is mediated at the level of transcription and it involves interaction with TBP, the TATA box-binding component of TFIID. On the other hand, three tumor-derived mutant forms of p53 (mut 143, mut 248, and mut 273) stimulated the activity of the IGF-I-R promoter and increased the levels of IGF-I-R/luciferase fusion mRNA. These results suggest that wild-type p53 has the potential to suppress the IGF-I-R promoter in the postmitotic, fully differentiated cell, thus resulting in low levels of receptor gene expression in adult tissues. Mutant versions of p53 protein, usually associated with malignant states, can derepress the IGF-I-R promoter, with ensuing mitogenic activation by locally produced or circulating IGFs.

BCL2 regulates neural differentiation.

A main function attributed to the BCL2 protein is its ability to confer resistance against apoptosis. In addition to the constitutively high expression of BCL2, caused by gene rearrangement in follicular lymphomas, elevated expression of the BCL2 gene has been found in differentiating hematopoietic, neural, and epithelial tissues. To address the question of whether the expression of BCL2 is a cause or consequence of cell differentiation, we used a human neural-crest-derived tumor cell line, Paju, that undergoes spontaneous neural differentiation in vitro. The Paju cell line displays moderate expression of BCL2, the level of which increases in parallel with further neural differentiation induced by treatment with phorbol 12-myristate 13-acetate. Transfection of normal human BCL2 cDNA in sense and antisense orientations had a dramatic impact on the differentiation of the Paju cells. Overexpression of BCL2 cDNA induced extensive neurite outgrowth, even in low serum concentrations, together with an increased expression of neuron-specific enolase. Paju cells expressing the anti-sense BCL2 cDNA construct, which reduced the endogenous levels of BCL2, did not undergo spontaneous neural differentiation. These cells acquired an epithelioid morphology and up-regulated the intermediate filament protein nestin, typically present in primitive neuroectodermal cells. The manipulated levels of BCL2 did not have appreciable impact on cell survival in normal culture. Our findings demonstrate that the BCL2 gene product participates in the regulation of neural differentiation.

Inhibition of apoptosis by overexpressing Bcl-2 enhances gene amplification by a mechanism independent of aphidicolin pretreatment.

To study the effect of apoptosis on gene amplification, we have constructed HeLa S3 cell lines in which the expression of bcl-2 (BCL2) can be controlled by tetracycline in the growth medium. Induction of Bcl-2 expression caused a temporary delay of apoptosis and resulted in roughly a 3-fold increase in the frequency of resistant colonies when cells were selected with trimetrexate. This resistance was due to amplification of the dihydrofolate reductase gene. Cells grown out of the pooled resistant colonies retained the same level of resistance to trimetrexate whether Bcl-2 was induced or repressed, consistent with the theory that Bcl-2 functions by facilitating gene amplification, rather than being the resistance mechanism per se. Pretreating cells with aphidicolin is another method to increase gene amplification frequency. When Bcl-2-expressing cells were pretreated with aphidicolin, the resulting increase in gene amplification frequency was approximately the product of the increases caused by aphidicolin pretreatment or Bcl-2 expression alone, indicating that Bcl-2 increases gene amplification through a mechanism independent of that of aphidicolin pretreatment. These results are consistent with the concept that gene amplification occurs at a higher frequency during drug-induced cell cycle perturbation. Bcl-2 evidently increases the number of selected amplified colonies by prolonging cell survival during the perturbation.

Apoptosis of hemopoietic cells by the human granulocyte-macrophage colony-stimulating factor mutant E21R.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) binds to a high-affinity heterodimeric receptor composed of a specific alpha chain and a common beta chain (beta(c)), which is shared with the receptors for interleukins 3 and 5. Hemopoietic cell survival requires GM-CSF binding this high-affinity receptor. We have recently developed the GM-CSF mutant E21R, which selectively binds to the alpha chain and behaves as a competitive GM-CSF antagonist. We have now examined the role of E21R on the survival of hemopoietic cells and found that E21R causes apoptosis (programmed cell death) of normal and malignant cells directly in the absence of GM-CSF. The direct apoptotic effect of E21R occurred in a dose- and time-dependent manner. Apoptosis by E21R was dependent on cells expressing the high-affinity GM-CSF receptor and could be blocked by GM-CSF. Significantly, apoptosis of the cells occurred even in the presence of the survival factors granulocyte CSF and stem cell factor but was prevented by engagement of beta(c) with interleukin 3. The initiation of apoptosis required phosphorylation, transcriptional activity, and protein synthesis. These findings support a model whereby binding of E21R to the alpha chain leads to apoptosis, while beta(c) plays an important role in cell survival. This model may be applicable to other multimeric cytokine receptors and offers a novel approach for the treatment of human leukemia.

An X chromosome-linked gene encoding a protein with characteristics of a rhoGAP predominantly expressed in hematopoietic cells.

An increasingly large number of proteins involved in signal transduction have been identified in recent years and shown to control different steps of cell survival, proliferation, and differentiation. Among the genes recently identified at the tip of the long arm of the human X chromosome, a novel gene, C1, encodes a protein that appears to represent a newly discovered member of the group of signaling proteins involved in regulation of the small GTP binding proteins of the ras superfamily. The protein encoded by C1, p115, is synthesized predominantly in cells of hematopoietic origin. It is characterized by two regions of similarity to motifs present in known proteins: GAP and SH3 homologous regions. Its localization in a narrow cytoplasmic region just below the plasma membrane and its inhibitory effect on stress fiber organization indicate that p115 may down regulate rho-like GTPases in hematopoietic cells.

The tRNA processing enzyme RNase T is essential for maturation of 5S RNA.

The maturation of 5S RNA in Escherichia coli is poorly understood. Although it is known that large precursors of 5S RNA accumulate in mutant cells lacking the endoribonuclease-RNase E, almost nothing is known about how the mature 5' and 3' termini of these molecules are generated. We have examined 5S RNA maturation in wild-type and single- or multiple-exoribonuclease-deficient cells by Northern blot and primer-extension analysis. Our results indicate that no mature 5S RNA is made in RNase T-deficient strains. Rather, 5S RNA precursors containing predominantly 2 extra nucleotides at the 3' end accumulate. Apparently, these 5S RNAs are functional inasmuch as mutant cells are viable, growing only slightly slower than wild type. Purified RNase T can remove the extra 3' residues, showing that it is directly involved in the trimming reaction. In contrast, mutations affecting other 3' exoribonucleases have no effect on 5S RNA maturation. Approximately 90% of the 5S RNAs in both wild-type and RNase T- cells contain mature 5' termini, indicating that 5' processing is independent of RNase T action. These data identify the enzyme responsible for generating the mature 3' terminus of 5S RNA molecules and also demonstrate that a completely processed 5S RNA molecule is not essential for cell survival.

Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos

Proximal spinal muscular atrophy is an autosomal recessive human disease of spinal motor neurons leading to muscular weakness with onset predominantly in infancy and childhood. With an estimated heterozygote frequency of 1/40 it is the most common monogenic disorder lethal to infants; milder forms represent the second most common pediatric neuromuscular disorder. Two candidate genes—survival motor neuron (SMN) and neuronal apoptosis inhibitory protein have been identified on chromosome 5q13 by positional cloning. However, the functional impact of these genes and the mechanism leading to a degeneration of motor neurons remain to be defined. To analyze the role of the SMN gene product in vivo we generated SMN-deficient mice. In contrast to the human genome, which contains two copies, the mouse genome contains only one SMN gene. Mice with homozygous SMN disruption display massive cell death during early embryonic development, indicating that the SMN gene product is necessary for cellular survival and function.

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.

Hypersensitivity of Ku80-deficient cell lines and mice to DNA damage: The effects of ionizing radiation on growth, survival, and development

We recently have shown that mice deficient for the 86-kDa component (Ku80) of the DNA-dependent protein kinase exhibit growth retardation and a profound deficiency in V(D)J (variable, diversity, and joining) recombination. These defects may be related to abnormalities in DNA metabolism that arise from the inability of Ku80 mutant cells to process DNA double-strand breaks. To further characterize the role of Ku80 in DNA double-strand break repair, we have generated embryonic stem cells and pre-B cells and examined their response to ionizing radiation. Ku80−/− embryonic stem cells are more sensitive than controls to γ-irradiation, and pre-B cells derived from Ku80 mutant mice display enhanced spontaneous and γ-ray-induced apoptosis. We then determined the effects of ionizing radiation on the survival, growth, and lymphocyte development in Ku80-deficient mice. Ku80−/− mice display a hypersensitivity to γ-irradiation, characterized by loss of hair pigmentation, severe injury to the gastrointestinal tract, and enhanced mortality. Exposure of newborn Ku80−/− mice to sublethal doses of ionizing radiation enhances their growth retardation and results in the induction of T cell-specific differentiation. However, unlike severe combined immunodeficient mice, radiation-induced T cell development in Ku80−/− mice is not accompanied by extensive thymocyte proliferation. The response of Ku80-deficient cell lines and mice to DNA-damaging agents provides important insights into the role of Ku80 in growth regulation, lymphocyte development, and DNA repair.