Conditional and specific NF-kappaB blockade protects pancreatic beta cells from diabetogenic agents.

Type 1 diabetes is characterized by the infiltration of inflammatory cells into pancreatic islets of Langerhans, followed by the selective and progressive destruction of insulin-secreting beta cells. Islet-infiltrating leukocytes secrete cytokines such as IL-1beta and IFN-gamma, which contribute to beta cell death. In vitro evidence suggests that cytokine-induced activation of the transcription factor NF-kappaB is an important component of the signal triggering beta cell apoptosis. To study the in vivo role of NF-kappaB in beta cell death, we generated a transgenic mouse line expressing a degradation-resistant NF-kappaB protein inhibitor (DeltaNIkappaBalpha), acting specifically in beta cells, in an inducible and reversible manner, by using the tet-on regulation system. In vitro, islets expressing the DeltaNIkappaBalpha protein were resistant to the deleterious effects of IL-1beta and IFN-gamma, as assessed by reduced NO production and beta-cell apoptosis. This effect was even more striking in vivo, where nearly complete protection against multiple low-dose streptozocin-induced diabetes was observed, with reduced intraislet lymphocytic infiltration. Our results show in vivo that beta cell-specific activation of NF-kappaB is a key event in the progressive loss of beta cells in diabetes. Inhibition of this process could be a potential effective strategy for beta-cell protection.

c-Abl regulates p53 levels under normal and stress conditions by preventing its nuclear export and ubiquitination.

The p53 protein is subject to Mdm2-mediated degradation by the ubiquitin-proteasome pathway. This degradation requires interaction between p53 and Mdm2 and the subsequent ubiquitination and nuclear export of p53. Exposure of cells to DNA damage results in the stabilization of the p53 protein in the nucleus. However, the underlying mechanism of this effect is poorly defined. Here we demonstrate a key role for c-Abl in the nuclear accumulation of endogenous p53 in cells exposed to DNA damage. This effect of c-Abl is achieved by preventing the ubiquitination and nuclear export of p53 by Mdm2, or by human papillomavirus E6. c-Abl null cells fail to accumulate p53 efficiently following DNA damage. Reconstitution of these cells with physiological levels of c-Abl is sufficient to promote the normal response of p53 to DNA damage via nuclear retention. Our results help to explain how p53 is accumulated in the nucleus in response to DNA damage.

ABL1 methylation in Ph-positive ALL is exclusively associated with the P210 form of BCR-ABL.

In human Ph-positive leukemia there is a clear association of different forms of the BCR-ABL oncogene with distinct types of leukemia. The P190 form of BCR-ABL is rarely observed in chronic myeloid leukemia (CML) but is present in 50% of Ph-positive acute lymphoblastic leukemia (ALL). In contrast, the P210 form is observed both in CML and 50% of Ph-positive ALL. Methylation of the proximal promoter of the ABL1 gene has been shown to be a nearly universal event associated with clinical progression of CML. This raises the question of whether methylation of the ABL1 promoter is an epigenetic modification also associated with Ph-positive ALL. To study this issue, we used methylation-specific PCR and bisulfite sequencing to determine the methylation status of the ABL1 promoter in 18 Ph-positive ALL samples. We report here that gene-specific ABL1 promoter methylation is associated mainly with the P210 form of BCR-ABL and not the P190 form. While six out of the seven P210-positive ALL samples had ABL1 promoter methylation, none of the 11 P190-positive ALL samples demonstrated ABL1 promoter methylation. In addition, we estimated the extent and relative abundance of ABL1 promoter methylation in several Ph-positive ALL samples and compared it to the methylation pattern in chronic, accelerated and blastic crisis phases of CML. We put forth a model that correlates the different types of leukemias with the different levels of ABL1 promoter methylation.

Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity.

NF-kappaB (nuclear factor-kappaB) is a collective name for inducible dimeric transcription factors composed of members of the Rel family of DNA-binding proteins that recognize a common sequence motif. NF-kappaB is found in essentially all cell types and is involved in activation of an exceptionally large number of genes in response to infections, inflammation, and other stressful situations requiring rapid reprogramming of gene expression. NF-kappaB is normally sequestered in the cytoplasm of nonstimulated cells and consequently must be translocated into the nucleus to function. The subcellular location of NF-kappaB is controlled by a family of inhibitory proteins, IkappaBs, which bind NF-kappaB and mask its nuclear localization signal, thereby preventing nuclear uptake. Exposure of cells to a variety of extracellular stimuli leads to the rapid phosphorylation, ubiquitination, and ultimately proteolytic degradation of IkappaB, which frees NF-kappaB to translocate to the nucleus where it regulates gene transcription. NF-kappaB activation represents a paradigm for controlling the function of a regulatory protein via ubiquitination-dependent proteolysis, as an integral part of a phosphorylationbased signaling cascade. Recently, considerable progress has been made in understanding the details of the signaling pathways that regulate NF-kappaB activity, particularly those responding to the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1. The multisubunit IkappaB kinase (IKK) responsible for inducible IkappaB phosphorylation is the point of convergence for most NF-kappaB-activating stimuli. IKK contains two catalytic subunits, IKKalpha and IKKbeta, both of which are able to correctly phosphorylate IkappaB. Gene knockout studies have shed light on the very different physiological functions of IKKalpha and IKKbeta. After phosphorylation, the IKK phosphoacceptor sites on IkappaB serve as an essential part of a specific recognition site for E3RS(IkappaB/beta-TrCP), an SCF-type E3 ubiquitin ligase, thereby explaining how IKK controls IkappaB ubiquitination and degradation. A variety of other signaling events, including phosphorylation of NF-kappaB, hyperphosphorylation of IKK, induction of IkappaB synthesis, and the processing of NF-kappaB precursors, provide additional mechanisms that modulate the level and duration of NF-kappaB activity.

High susceptibility to bacterial infection, but no liver dysfunction, in mice compromised for hepatocyte NF-kappaB activation.

Based on the essential involvement of NF-kappaB in immune and inflammatory responses and its apoptosis-rescue function in normal and malignant cells, inhibitors of this transcription factor are potential therapeutics for the treatment of a wide range of diseases, from bronchial asthma to cancer. Yet, given the essential function of NF-kappaB in the embryonic liver, it is important to determine its necessity in the liver beyond embryogenesis. NF-kappaB is normally retained in the cytoplasm by its inhibitor IkappaB, which is eliminated upon cell stimulation through phosphorylation-dependent ubiquitin degradation. Here, we directed a degradation-resistant IkappaBalpha transgene to mouse hepatocytes in an inducible manner and showed substantial tissue specificity using various means, including a new method for live-animal imaging. Transgene expression resulted in obstruction of NF-kappaB activation, yet produced no signs of liver dysfunction, even when implemented over 15 months. However, the transgene-expressing mice were very vulnerable both to a severe immune challenge and to a systemic bacterial infection. Despite having intact immunocytes and inflammatory cells, these mice were unable to clear Listeria monocytogenes from the liver and succumbed to sepsis. These findings indicate the essential function of the hepatocyte through NF-kappaB activation in certain systemic infections, possibly by coordinating innate immunity in the liver.

ABL1 methylation is a distinct molecular event associated with clonal evolution of chronic myeloid leukemia.

Methylation of the proximal promoter of the ABL1 oncogene is a common epigenetic alteration associated with clinical progression of chronic myeloid leukemia (CML). In this study we queried whether both the Ph'-associated and normal ABL1 alleles undergo methylation; what may be the proportion of hematopoietic progenitors bearing methylated ABL1 promoters in chronic versus acute phase disease; whether methylation affects the promoter uniformly or in patches with discrete clinical relevance; and, finally, whether methylation of ABL1 reflects a generalized process or is gene-specific. To address these issues, we adapted the techniques of methylation-specific PCR and bisulfite-sequencing to study the regulatory regions of ABL1 and other genes with a role in DNA repair or genotoxic stress response. In cell lines established from CML blast crisis, which only carry a single ABL1 allele nested within the BCR-ABL fusion gene, ABL1 promoters were universally methylated. By contrast, in clinical samples from patients at advanced stages of disease, both methylated and unmethylated promoter alleles were detectable. To distinguish between allele-specific methylation and a mixed cell population pattern, we studied the methylation status of ABL1 in colonies derived from single hematopoietic progenitors. Our results showed that both methylated and unmethylated promoter alleles coexisted in the same colony. Furthermore, ABL1 methylation was noted in the vast majority of colonies from blast crisis, but not chronic-phase CML. Both cell lines and clinical samples from acute-phase CML showed nearly uniform hypermethylation along the promoter region. Finally, we showed that ABL1 methylation does not reflect a generalized process and may be unique among DNA repair/genotoxic stress response genes. Our data suggest that specific methylation of the Ph'-associated ABL1 allele accompanies clonal evolution in CML.

c-Abl neutralizes the inhibitory effect of Mdm2 on p53.

Upon exposure to stress signals, the p53 tumor suppressor protein is stabilized and induces growth suppression. p53 activities are efficiently inhibited by the Mdm2 oncoprotein through an autoregulatory feedback loop. In addition, Mdm2 promotes p53 degradation, thereby terminating its growth inhibitory signal. Hence, p53 exerts its effects during the interval between p53 activation and the subsequent inhibition by Mdm2. Modulation of this interval by regulatory proteins may determine the extent and duration of p53 activity. Recent studies have shown that the c-Abl protein-tyrosine kinase binds p53 and enhances its transcriptional activity. Here we provide an explanation for the cooperation between these proteins. We demonstrate that c-Abl increases the expression level of the p53 protein. The enhanced expression is achieved by inhibiting Mdm2-mediated degradation of p53. This provides a likely mechanistic explanation for the findings that c-Abl overcomes the inhibitory effects of Mdm2 on p53-mediated transcriptional activation and apoptosis. These results suggest that c-Abl modulates the time window within which p53 remains active. The ability of c-Abl to neutralize the inhibitory effects of Mdm2 on p53 may be important for its growth inhibitory function.

Identification of the receptor component of the IkappaBalpha-ubiquitin ligase.

NF-kappaB, a ubiquitous, inducible transcription factor involved in immune, inflammatory, stress and developmental processes, is retained in a latent form in the cytoplasm of non-stimulated cells by inhibitory molecules, IkappaBs. Its activation is a paradigm for a signal-transduction cascade that integrates an inducible kinase and the ubiquitin-proteasome system to eliminate inhibitory regulators. Here we isolate the pIkappaBalpha-ubiquitin ligase (pIkappaBalpha-E3) that attaches ubiquitin, a small protein which marks other proteins for degradation by the proteasome system, to the phosphorylated NF-kappaB inhibitor pIkappaBalpha. Taking advantage of its high affinity to pIkappaBalpha, we isolate this ligase from HeLa cells by single-step immunoaffinity purification. Using nanoelectrospray mass spectrometry, we identify the specific component of the ligase that recognizes the pIkappaBalpha degradation motif as an F-box/WD-domain protein belonging to a recently distinguished family of beta-TrCP/Slimb proteins. This component, which we denote E3RSIkappaB (pIkappaBalpha-E3 receptor subunit), binds specifically to pIkappaBalpha and promotes its in vitro ubiquitination in the presence of two other ubiquitin-system enzymes, E1 and UBC5C, one of many known E2 enzymes. An F-box-deletion mutant of E3RS(IkappaB), which tightly binds pIkappaBalpha but does not support its ubiquitination, acts in vivo as a dominant-negative molecule, inhibiting the degradation of pIkappaBalpha and consequently NF-kappaB activation. E3RS(IkappaB) represents a family of receptor proteins that are core components of a class of ubiquitin ligases. When these receptor components recognize their specific ligand, which is a conserved, phosphorylation-based sequence motif, they target regulatory proteins containing this motif for proteasomal degradation.

Co-stimulation-dependent activation of a JNK-kinase in T lymphocytes.

Previously we implicated c-Jun N-terminal kinase (JNK) as an element that is involved in signal integration during co-stimulation of T lymphocytes. This pathway has now been traced to an upper level, comprising MAPKK SEK1/MKK4/JNKK1 which, similarly to JNK, must receive input both from the TCR and CD28. A large portion of this input is probably integrated at the level of the Rho-family protein CDC42 which, here, activates SEK1 and JNK to the level reached by TCR and CD28 stimulation. We have identified another putative SEK/ JNK pathway regulator, PKCtheta, which in contrast to CDC42, activates SEK and JNK maximally only in conjunction with a calcium signal delivered through calcineurin. Signals originating at the TCR and CD28 may travel down the JNK pathway via PKCtheta, calcineurin, CDC42, MEKK1 and SEK1.

Primary B cells essentially lack constitutive NF-kappa B activity.

Based primarily on the analysis of B cell lines, mature B cells are considered distinct from non-B cells and immature B cells by having constitutive nuclear NF-kappaB activity. By their comparison to splenic non-B cells or activated B cells we show here that primary resting B cells lack cell-autonomous NF-kappaB activity. This finding indicates that the role of the transcription factor in B cells is similar to that in other cells, namely a common mediator of activation and stress signals. Whereas the absence of constitutive NF-kappaB activity in mature B cells does not argue against a role of NF-kappaB in B cell development, it does not support the notion of the essential function of the factor in maintaining the unique transcriptional activities in B cells.

Inhibition of NF-kappa-B cellular function via specific targeting of the I-kappa-B-ubiquitin ligase.

Activation of the transcription factor NF-kappa B is a paradigm for signal transduction through the ubiquitin-proteasome pathway: ubiquitin-dependent degradation of the transcriptional inhibitor I kappa B in response to cell stimulation. A major issue in this context is the nature of the recognition signal and the targeting enzyme involved in the proteolytic process. Here we show that following a stimulus-dependent phosphorylation, and while associated with NF-kappa B, I kappa B is targeted by a specific ubiquitin-ligase via direct recognition of the signal-dependent phosphorylation site; phosphopeptides corresponding to this site specifically inhibit ubiquitin conjugation of I kappa B and its subsequent degradation. The ligase recognition signal is functionally conserved between I kappa B alpha and I kappa B beta, and does not involve the nearby ubiquitination site. Microinjection of the inhibitory peptides into stimulated cells abolished NF-kappa B activation in response to TNF alpha and the consequent expression of E-selectin, an NF-kappa B-dependent cell-adhesion molecule. Inhibition of NF-kappa B function by specific blocking of ubiquitin ligase activity provides a novel approach for intervening in cellular processes via regulation of unique proteolytic events.

Molecular follow-up of disease progression and interferon therapy in chronic myelocytic leukemia.

We previously reported that the abl promoter (Pa) undergoes de novo DNA methylation in the course of chronic myelocytic leukemia (CML). The clinical implications of this finding are the subject of the present study in which samples of CML patients, including a group treated with interferon alpha (IFNalpha) were surveyed. The methylation status of the abl promoter was monitored by polymerase chain reaction (PCR) amplification of the Pa region after digestion with several site-methylation sensitive restriction enzymes. Some 74% of the DNA samples from blood and marrow drawn in the chronic phase were nonmethylated, similar to control samples from non-CML patients. The remaining 26% were partially methylated in the abl Pa region. The latter samples were derived from patients who were indistinguishable from the others on the basis of clinical presentation. Methylated samples were mostly derived from patients known to have a disease of longer duration (26 months v 7.5 months, P = .01). Samples of 30 IFNalpha-treated patients were sequentially analyzed in the course of treatment. Fifteen patients with no evidence of Pa methylation before treatment remained methylation-free. The remainder, who displayed Pa methylation before treatment, reverted to the methylation-free status. The outcome is attributed to IFNalpha therapy, as the Pa methylation status was not reversed in any of the patients treated with hydroxyurea. Methylation of the abl promoter indicates a disease of long-standing, most likely associated with a higher probability of imminent blastic transformation. It appears to predict the outcome of IFNalpha therapy far better than the cytogenetic response.

Stimulation-dependent I kappa B alpha phosphorylation marks the NF-kappa B inhibitor for degradation via the ubiquitin-proteasome pathway.

The nuclear translocation of NF-kappa B follows the degradation of its inhibitor, I kappa B alpha, an event coupled with stimulation-dependent inhibitor phosphorylation. Prevention of the stimulation-dependent phosphorylation of I kappa B alpha, either by treating cells with various reagents or by mutagenesis of certain putative I kappa B alpha phosphorylation sites, abolishes the inducible degradation of I kappa B alpha. Yet, the mechanism coupling the stimulation-induced phosphorylation with the degradation has not been resolved. Recent reports suggest a role for the proteasome in I kappa B alpha degradation, but the mode of substrate recognition and the involvement of ubiquitin conjugation as a targeting signal have not been addressed. We show that of the two forms of I kappa B alpha recovered from stimulated cells in a complex with RelA and p50, only the newly phosphorylated form, pI kappa B alpha, is a substrate for an in vitro reconstituted ubiquitin-proteasome system. Proteolysis requires ATP, ubiquitin, a specific ubiquitin-conjugating enzyme, and other ubiquitin-proteasome components. In vivo, inducible I kappa B alpha degradation requires a functional ubiquitin-activating enzyme and is associated with the appearance of high molecular weight adducts of I kappa B alpha. Ubiquitin-mediated protein degradation may, therefore, constitute an integral step of a signal transduction process.

Costimulation requirement for AP-1 and NF-kappa B transcription factor activation in T cells.

The transcriptional activity of the IL-2 promoter requires T-cell costimulation delivered by the TCR and the auxiliary receptor CD28. Several transcription factors participate in IL-2 promoter activation, among which are AP-1-like factors and NF-kappa B. Protein phosphorylation has an important role in the regulation of these two factors: (1) it induces the transactivating capacity of the AP-1 protein c-Jun; and (2) it is involved in the release of the cytoplasmic inhibitor, I kappa B, from NF-kappa B, allowing translocation of the latter into the nucleus. We have recently shown that both phosphorylation processes require T-cell costimulation. Furthermore, in activated T cells, the kinetics of the two phosphorylation events are essentially similar. According to our results, however, the kinases responsible for the two processes are distinct entities. Whereas TPCK inhibits phosphorylation of I kappa B and, consequently, activation of NF-kappa B, it markedly enhances the activity of JNK, the MAP kinase-related kinase that phosphorylates the transactivation domain of c-Jun. We, therefore, propose the activation scheme presented in FIGURE 3 for T-cell costimulation. Costimulation results in the activation of a signaling pathway that leads to the simultaneous induction of the two transcription factors, AP-1 and NF-kappa B. Integration of the signals generated by TCR and CD28 engagement occurs along this pathway, which then bifurcates to induce I kappa B phosphorylation and NF-kappa B activation on the one hand, and JNK activation and c-Jun phosphorylation on the other. We are currently engaged in defining where the two signals integrate along the AP-1/NF-kappa B pathway.

In vivo stimulation of I kappa B phosphorylation is not sufficient to activate NF-kappa B.

NF-kappa B is a major inducible transcription factor in many immune and inflammatory reactions. Its activation involves the dissociation of the inhibitory subunit I kappa B from cytoplasmic NF-kappa B/Rel complexes, following which the Rel proteins are translocated to the nucleus, where they bind to DNA and activate transcription. Phosphorylation of I kappa B in cell-free experiments results in its inactivation and release from the Rel complex, but in vivo NF-kappa B activation is associated with I kappa B degradation. In vivo phosphorylation of I kappa B alpha was demonstrated in several recent studies, but its role is unknown. Our study shows that the T-cell activation results in rapid phosphorylation of I kappa B alpha and that this event is a physiological one, dependent on appropriate lymphocyte costimulation. Inducible I kappa B alpha phosphorylation was abolished by several distinct NF-kappa B blocking reagents, suggesting that it plays an essential role in the activation process. However, the in vivo induction of I kappa B alpha phosphorylation did not cause the inhibitory subunit to dissociate from the Rel complex. We identified several protease inhibitors which allow phosphorylation of I kappa B alpha but prevent its degradation upon cell stimulation, presumably through inhibition of the cytoplasmic proteasome. In the presence of these inhibitors, phosphorylated I kappa B alpha remained bound to the Rel complex in the cytoplasm for an extended period of time, whereas NF-kappa B activation was abolished. It appears that activation of NF-kappa B requires degradation of I kappa B alpha while it is a part of the Rel cytoplasmic complex, with inducible phosphorylation of the inhibitory subunit influencing the rate of degradation.

Progressive de novo DNA methylation at the bcr-abl locus in the course of chronic myelogenous leukemia.

De novo methylation of CpG islands is a rare event in mammalian cells. It has been observed in the course of developmental processes, such as X chromosome inactivation and genomic imprinting. The methylation of DNA, an important factor in the epigenetic control of gene expression, may also be involved in tumorigenesis. After the t(9;22) chromosomal translocation and generation of the Philadelphia chromosome, the initiating event in chronic myelogenous leukemia (CML), most of the abl coding sequence is fused to the 5' region of the bcr gene. Expression of the hybrid bcr-abl gene is, therefore, regulated by the bcr promoter. In most cases of CML, one of the two abl promoters (Pa) is nested within the bcr-abl transcriptional unit and should be able to transcribe the type Ia 6-kb normal abl mRNA from the Philadelphia chromosome. However, we have found that the 6-kb transcript is present only in CML cell lines containing a normal abl allele and that the apparent inactivation of the nested Pa promoter is associated with allele-specific methylation. Furthermore, we have noticed that the Pa promoter is contained within a CpG island and undergoes progressive de novo methylation in the course of the disease. This is attested to by the fact that DNA samples from CML patients that are methylation-free at the time of diagnosis invariably become methylated in advanced CML. Since tumor progression in CML cannot always be inferred from the clinical presentation, assessment of de novo CpG methylation may prove to be of critical value in management of the disease. It could herald blastic transformation at a stage when bone marrow transplantation, the only potentially curative therapeutic procedure in CML, is still effective.