NEMO and RIP1 control cell fate in response to extensive DNA damage via TNF-α feedforward signaling.

Upon DNA damage, ataxia telangiectasia mutated (ATM) kinase triggers multiple events to promote cell survival and facilitate repair. If damage is excessive, ATM stimulates cytokine secretion to alert neighboring cells and apoptosis to eliminate the afflicted cell. ATM augments cell survival by activating nuclear factor (NF)-κB; however, how ATM induces cytokine production and apoptosis remains elusive. Here we uncover a p53-independent mechanism that transmits ATM-driven cytokine and caspase signals upon strong genotoxic damage. Extensive DNA lesions stimulated two sequential NF-κB activation phases, requiring ATM and NEMO/IKK-γ: The first phase induced TNF-α-TNFR1 feedforward signaling, promoting the second phase and driving RIP1 phosphorylation. In turn, RIP1 kinase triggered JNK3/MAPK10-dependent interleukin-8 secretion and FADD-mediated proapoptotic caspase-8 activation. Thus, in the context of excessive DNA damage, ATM employs NEMO and RIP1 kinase through autocrine TNF-α signaling to switch on cytokine production and caspase activation. These results shed light on cell-fate regulation by ATM.

The neurological phenotype of ataxia-telangiectasia: solving a persistent puzzle.

Human genomic instability syndromes affect the nervous system to different degrees of severity, attesting to the vulnerability of the CNS to perturbations of genomic integrity and the DNA damage response (DDR). Ataxia-telangiectasia (A-T) is a typical genomic instability syndrome whose major characteristic is progressive neuronal degeneration but is also associated with immunodeficiency, cancer predisposition and acute sensitivity to ionizing radiation and radiomimetic chemicals. A-T is caused by loss or inactivation of the ATM protein kinase, which mobilizes the complex, multi-branched cellular response to double strand breaks in the DNA by phosphorylating numerous DDR players. The link between ATM's function in the DDR and the neuronal demise in A-T has been questioned in the past. However, recent studies of the ATM-mediated DDR in neurons suggest that the neurological phenotype in A-T is indeed caused by deficiency in this function, similar to other features of the disease. Still, major issues concerning this phenotype remain open, including the presumed differences between the DDR in post-mitotic neurons and proliferating cells, the nature of the damage that accumulates in the DNA of ATM-deficient neurons under normal life conditions, the mode of death of ATM-deficient neurons, and the lack of a major neuronal phenotype in the mouse model of A-T. A-T remains a prototype disease for the study of the DDR's role in CNS development and maintenance.

The role of the DNA damage response in neuronal development, organization and maintenance.

The DNA damage response is a key factor in the maintenance of genome stability. As such, it is a central axis in sustaining cellular homeostasis in a variety of contexts: development, growth, differentiation, and maintenance of the normal life cycle of the cell. It is now clear that diverse mechanisms encompassing cell cycle regulation, repair pathways, many aspects of cellular metabolism, and cell death are inter-linked and act in consort in response to DNA damage. Defects in the DNA damage response in proliferating cells can lead to cancer while defects in neurons result in neurodegenerative pathologies. Neurons are highly differentiated, post-mitotic cells that cannot be replenished after disease or trauma. Their high metabolic activity that generates large amounts of reactive oxygen species with DNA damaging capacity and their intense transcriptional activity increase the potential for damage of their genomic DNA. Neurons ensure their longevity and functionality in the face of these threats by elaborate mechanisms that defend the integrity of their genome. This review focuses on the DNA damage response in neuronal cells and points to the importance of this elaborate network to the integrity of the nervous system from its early development and throughout the lifetime of the organism.

Differential roles of ATM- and Chk2-mediated phosphorylations of Hdmx in response to DNA damage.

The p53 tumor suppressor plays a major role in maintaining genomic stability. Its activation and stabilization in response to double strand breaks (DSBs) in DNA are regulated primarily by the ATM protein kinase. ATM mediates several posttranslational modifications on p53 itself, as well as phosphorylation of p53's essential inhibitors, Hdm2 and Hdmx. Recently we showed that ATM- and Hdm2-dependent ubiquitination and subsequent degradation of Hdmx following DSB induction are mediated by phosphorylation of Hdmx on S403, S367, and S342, with S403 being targeted directly by ATM. Here we show that S367 phosphorylation is mediated by the Chk2 protein kinase, a downstream kinase of ATM. This phosphorylation, which is important for subsequent Hdmx ubiquitination and degradation, creates a binding site for 14-3-3 proteins which controls nuclear accumulation of Hdmx following DSBs. Phosphorylation of S342 also contributed to optimal 14-3-3 interaction and nuclear accumulation of Hdmx, but phosphorylation of S403 did not. Our data indicate that binding of a 14-3-3 dimer and subsequent nuclear accumulation are essential steps toward degradation of p53's inhibitor, Hdmx, in response to DNA damage. These results demonstrate a sophisticated control by ATM of a target protein, Hdmx, which itself is one of several ATM targets in the ATM-p53 axis of the DNA damage response.

ATM-mediated response to DNA double strand breaks in human neurons derived from stem cells.

Ataxia-telangiectasia (A-T) is a multi-system genomic instability syndrome that is caused by loss or inactivation of the ATM protein kinase. ATM is largely nuclear in proliferating cells, and activates an extensive network of pathways in response to double strand breaks (DSBs) in the DNA by phosphorylating key proteins in these pathways. The prominent symptom of A-T is neuronal degeneration, making the elucidation of ATM's functions in neurons essential to understanding the disease. It has been suggested that ATM is cytoplasmic in neurons and functions in processes that are not associated with the DNA damage response. Recently we showed that in human neuron-like cells obtained by in vitro differentiation of neuroblastomas, ATM was largely nuclear and mediated the DSB response as in proliferating cells. We have now extended these studies to two additional model systems: neurons derived from human embryonic stem cells, and cortical neurons derived from neural stem cells. The results substantiate the notion that ATM is nuclear in human neurons and mediates the DSB response, the same as it does in proliferating cells. We present here unique and powerful model systems to further study the ATM-mediated network in neurons.

Analysis of the ataxia telangiectasia mutated-mediated DNA damage response in murine cerebellar neurons.

The DNA damage response is a network of signaling pathways that affects many aspects of cellular metabolism after the induction of DNA damage. The primary transducer of the cellular response to the double-strand break, a highly cytotoxic DNA lesion, is the nuclear protein kinase ataxia telangiectasia (A-T) mutated (ATM), which phosphorylates numerous effectors that play key roles in the damage response pathways. Loss or inactivation of ATM leads to A-T, an autosomal recessive disorder characterized by neuronal degeneration, particularly the loss of cerebellar granule and Purkinje cells, immunodeficiency, genomic instability, radiosensitivity, and cancer predisposition. The reason for the cerebellar degeneration in A-T is not clear. It has been ascribed by several investigators to cytoplasmic functions of ATM that may not be relevant to the DNA damage response. We set out to examine the subcellular localization of ATM and characterize the ATM-mediated damage response in mouse cerebellar neurons. We found that ATM is essentially nuclear in these cells and that various readouts of the ATM-mediated damage response are similar to those seen in commonly used cell lines. These include the autophosphorylation of ATM, which marks its activation, and phosphorylation of several of its downstream substrates. Importantly, all of these responses are detected in the nuclei of granule and Purkinje cells, suggesting that nuclear ATM functions in these cells similar to other cell types. These results support the notion that the cerebellar degeneration in A-T patients results from defective DNA damage response.

Widespread occurrence of antisense transcription in the human genome.

An increasing number of eukaryotic genes are being found to have naturally occurring antisense transcripts. Here we study the extent of antisense transcription in the human genome by analyzing the public databases of expressed sequences using a set of computational tools designed to identify sense-antisense transcriptional units on opposite DNA strands of the same genomic locus. The resulting data set of 2,667 sense-antisense pairs was evaluated by microarrays containing strand-specific oligonucleotide probes derived from the region of overlap. Verification of specific cases by northern blot analysis with strand-specific riboprobes proved transcription from both DNA strands. We conclude that > or =60% of this data set, or approximately 1,600 predicted sense-antisense transcriptional units, are transcribed from both DNA strands. This indicates that the occurrence of antisense transcription, usually regarded as infrequent, is a very common phenomenon in the human genome. Therefore, antisense modulation of gene expression in human cells may be a common regulatory mechanism.

hCHL2, a novel chordin-related gene, displays differential expression and complex alternative splicing in human tissues and during myoblast and osteoblast maturation.

Chordin-like cysteine-rich repeats (CRs) are conserved domains present in an expanding family of secreted proteins that associate with members of the TGF beta superfamily. In this study, we report the molecular cloning and characterization of CHL2 (chordin-like 2), a novel protein closely related to CHL (chordin-like). Both are members of the chordin family of proteins, and contain a signal peptide and three CR domains. We found that recombinant human CHL2 (hCHL2) protein is secreted and binds activin A, but not BMP-2, -4, or -6. Expression of hCHL2 mRNA and protein was detected in a variety of human tissues and is particularly abundant in the uterus. Extensive and complex alternative splicing of hCHL2 was observed in different tissues, resulting in several distinct protein isoforms that vary substantially in the presence of a signal peptide and their content of CR domains. Differential expression of CHL2 variants was observed during myoblast and osteoblast differentiation, implying a role for this gene in these physiological processes.

A senescence-inflammatory switch from cancer-inhibitory to cancer-promoting mechanism.

Senescence, perceived as a cancer barrier, is paradoxically associated with inflammation, which promotes tumorigenesis. Here, we characterize a distinct low-grade inflammatory process in stressed epithelium that is related to para-inflammation; this process either represses or promotes tumorigenesis, depending on p53 activity. Csnk1a1 (CKIα) downregulation induces a senescence-associated inflammatory response (SIR) with growth arrest in colorectal tumors, which loses its growth control capacity in the absence of p53 and instead, accelerates growth and invasiveness. Corresponding processes occur in CKIα-deleted intestinal organoids, assuming tumorigenic transformation properties ex vivo, upon p53 loss. Treatment of organoids and mice with anti-inflammatory agents suppresses the SIR and prevents p53-deficient organoid transformation and mouse carcinogenesis. SIR/para-inflammation suppression may therefore constitute a key mechanism in the anticarcinogenic effects of nonsteroidal anti-inflammatory drugs.

Nuclear ataxia-telangiectasia mutated (ATM) mediates the cellular response to DNA double strand breaks in human neuron-like cells.

The protein kinase ATM (ataxia-telangiectasia mutated) activates the cellular response to double strand breaks (DSBs), a highly cytotoxic DNA lesion. ATM is activated by DSBs and in turn phosphorylates key players in numerous damage response pathways. ATM is missing or inactivated in the autosomal recessive disorder ataxia-telangiectasia (A-T), which is characterized by neuronal degeneration, immunodeficiency, genomic instability, radiation sensitivity, and cancer predisposition. The predominant symptom of A-T is a progressive loss of movement coordination due to ongoing degeneration of the cerebellar cortex and peripheral neuropathy. A major deficiency in understanding A-T is the lack of information on the role of ATM in neurons. It is unclear whether the ATM-mediated DSB response operates in these cells similarly to proliferating cells. Furthermore, ATM was reported to be cytoplasmic in neurons and suggested to function in these cells in capacities other than the DNA damage response. Recently we obtained genetic molecular evidence that the neuronal degeneration in A-T does result from defective DNA damage response. We therefore undertook to investigate this response in a model system of human neuron-like cells (NLCs) obtained by neuronal differentiation in culture. ATM was largely nuclear in NLCs, and their ATM-mediated responses to DSBs were similar to those of proliferating cells. Knocking down ATM did not interfere with neuronal differentiation but abolished ATM-mediated damage responses in NLCs. We concluded that nuclear ATM mediates the DSB response in NLCs similarly to in proliferating cells. Attempts to understand the neurodegeneration in A-T should be directed to investigating the DSB response in the nervous system.

Phosphorylation of Hdmx mediates its Hdm2- and ATM-dependent degradation in response to DNA damage.

Maintenance of genomic stability depends on the DNA damage response, an extensive signaling network that is activated by DNA lesions such as double-strand breaks (DSBs). The primary activator of the mammalian DSB response is the nuclear protein kinase ataxia-telangiectasia, mutated (ATM), which phosphorylates key players in various arms of this network. The activation and stabilization of the p53 protein play a major role in the DNA damage response and are mediated by ATM-dependent posttranslational modifications of p53 and Mdm2, a ubiquitin ligase of p53. p53's response to DNA damage also depends on Mdm2-dependent proteolysis of Mdmx, a homologue of Mdm2 that represses p53's transactivation function. Here we show that efficient damage-induced degradation of human Hdmx depends on functional ATM and at least three sites on the Hdmx that are phosphorylated in response to DSBs. One of these sites, S403, is a direct ATM target. Accordingly, each of these sites is important for Hdm2-mediated ubiquitination of Hdmx after DSB induction. These results demonstrate a sophisticated mechanism whereby ATM fine-tunes the optimal activation of p53 by simultaneously modifying each player in the process.

In situ activation pattern of Met docking site following renal injury and hypertrophy.

BACKGROUND: Hepatocyte growth factor/scatter factor (HGF/SF) binds to its tyrosine kinase receptor, Met, thereby stimulating diverse cellular responses. The multifunctional docking site in the C-terminal domain mediates the signal of phosphorylated Met receptors to multiple transducers. The tyrosine at position 1356 of the Met docking site is crucial for cell motility and morphogenesis. METHODS: We examined the in situ distribution patterns of the Tyr1356-phosphorylated form of Met with a novel monoclonal antibody following renal injury and renal hypertrophy in rats. Sections of the kidney following either sham operation, transient ischaemia of one kidney or unilateral nephrectomy were analysed using indirect immunofluorescence staining and confocal laser scanning microscopy analysis of total Met protein levels and Tyr1356-phosphorylated Met (Met and pMet, respectively). RESULTS: At 6 h post-treatment, pMet increases in ischaemic kidneys compared with sham-operated kidneys, and these changes become substantial after 48 h in both medulla and cortex of ischaemic kidneys (P < 0.001). We also show significant up-regulation of Met predominantly in the medulla of ischaemic kidneys, 48 h following injury (P < 0.009). Inter-estingly, the stimulus for hypertrophy in the remnant kidney after uninephrectomy and the contra-lateral kidney during ischaemia is not accom-panied by significant up-regulation of Met or pMet staining compared with sham operation at both time points. CONCLUSIONS: We demonstrate in this work, for the first time, in situ detection of tyrosine kinase growth factor receptor docking site activation during pathological processes in the kidney. Using this methodology, we show a significant increase in Met docking site activity in both renal medulla and cortex solely following stimulation by ischaemia and repair.

Unusual alternative splicing within the human kallikrein genes KLK2 and KLK3 gives rise to novel prostate-specific proteins.

Prostate-specific antigen (PSA) and human kallikrein 2 are closely related products of the human kallikrein genes KLK3 and KLK2, respectively. Both PSA and human kallikrein 2 are produced and secreted in the prostate and have important applications in the diagnosis of prostate cancer. We report here the identification of unusual mRNA splice variants of the KLK2 and KLK3 genes that result from inclusion of intronic sequences adjacent to the first exon. The novel proteins encoded by these transcripts, named PSA-linked molecule (PSA-LM) and hK2-linked molecule (K-LM), share only the signal peptide with the original protein product of the respective gene. The mature proteins are entirely different and bear no similarity to the kallikrein family or to other proteins in the databases. As is the case with PSA, PSA-LM is expressed in the secretory epithelial cells of the prostate and is up-regulated in response to androgenic stimulation. A similar pattern of expression is suggested for K-LM.