Daniel Krappmann

Constitutive nuclear factor-kappaB-RelA activation is required for proliferation and survival of Hodgkin's disease tumor cells.

The pathogenesis and etiology of Hodgkins disease, a common human malignant lymphoma, is still unresolved. As a unique characteristic, we have identified constitutive activation of the transcription factor nuclear factor (NF)-kappaB p50-RelA in Hodgkin/Reed-Sternberg (H/RS) cells, which discriminates these neoplastic cells from most cell types studied to date. In contrast to other lymphoid and nonlymphoid cell lines tested, proliferation of H/RS cells depended on activated NF-kappaB. Furthermore, constitutive NF-kappaB p50-RelA prevented Hodgkins lymphoma cells from undergoing apoptosis under stress conditions. Consistent with this dual function, Hodgkins lymphoma cells depleted of constitutive nuclear NF-kappaB revealed strongly impaired tumor growth in severe combined immunodeficient mice. Our findings identify NF-kappaB as an important component for understanding the pathogenesis of Hodgkins disease and for developing new therapeutic strategies against it.

NF-κB Function in Growth Control: Regulation of Cyclin D1 Expression and G0/G1-to-S-Phase Transition

ABSTRACT Nuclear factor kappa B (NF-κB) has been implicated in the regulation of cell proliferation, transformation, and tumor development. We provide evidence for a direct link between NF-κB activity and cell cycle regulation. NF-κB was found to stimulate transcription of cyclin D1, a key regulator of G1checkpoint control. Two NF-κB binding sites in the human cyclin D1 promoter conferred activation by NF-κB as well as by growth factors. Both levels and kinetics of cyclin D1 expression during G1phase were controlled by NF-κB. Moreover, inhibition of NF-κB caused a pronounced reduction of serum-induced cyclin D1-associated kinase activity and resulted in delayed phosphorylation of the retinoblastoma protein. Furthermore, NF-κB promotes G1-to-S-phase transition in mouse embryonal fibroblasts and in T47D mammary carcinoma cells. Impaired cell cycle progression of T47D cells expressing an NF-κB superrepressor (IκBαΔN) could be rescued by ectopic expression of cyclin D1. Thus, NF-κB contributes to cell cycle progression, and one of its targets might be cyclin D1.

NF-kappaB and the innate immune response.

In the innate immune reaction, microbial pathogens activate phylogenetically conserved cellular signal transduction pathways that regulate the ubiquitous nuclear factor-kappaB (NFkappaB). NF-kappaB has pleiotropic functions in immunity; however, it is also critical for development and cellular survival. Many aspects of how the different pathways utilize a common kinase complex that ultimately activates NF-kappaB have been clarified by gene inactivation and biochemical analysis.

Aberrantly expressed c‐Jun and JunB are a hallmark of Hodgkin lymphoma cells, stimulate proliferation and synergize with NF‐κB

AP‐1 family transcription factors have been implicated in the control of proliferation, apoptosis and malignant transformation. However, their role in oncogenesis is unclear and no recurrent alterations of AP‐1 activities have been described in human cancers. Here, we show that constitutively activated AP‐1 with robust c‐Jun and JunB overexpression is found in all tumor cells of patients with classical Hodgkins disease. A similar AP‐1 activation is present in anaplastic large cell lymphoma (ALCL), but is absent in other lymphoma types. Whereas c‐Jun is up‐regulated by an autoregulatory process, JunB is under control of NF‐κB. Activated AP‐1 supports proliferation of Hodgkin cells, while it suppresses apoptosis of ALCL cells. Furthermore, AP‐1 cooperates with NF‐κB and stimulates expression of the cell‐cycle regulator cyclin D2, proto‐oncogene c‐met and the lymphocyte homing receptor CCR7, which are all strongly expressed in primary HRS cells. Together, these data suggest an important role of AP‐1 in lymphoma pathogenesis.

Molecular mechanisms of constitutive NF-kappaB/Rel activation in Hodgkin/Reed-Sternberg cells

A common characteristic of malignant cells derived from patients with Hodgkins disease (HD) is a high level of constitutive nuclear NF-κB/Rel activity, which stimulates proliferation and confers resistance to apoptosis. We have analysed the mechanisms that account for NF-κB activation in a panel of Hodgkin/Reed-Sternberg (H-RS) cell lines. Whereas two cell lines (L428 and KMH-2) expressed inactive IκBα, no significant changes in NF-κB or IκB expression were seen in other H-RS cells (L591, L1236 and HDLM-2). Constitutive NF-κB was susceptible to inhibition by recombinant IκBα, suggesting that neither mutations in the NF-κB genes nor posttranslational modifications of NF-κB were involved. Endogenous IκBα was bound to p65 and displayed a very short half-life. IκBα degradation could be blocked by inhibitors of the NF-κB activating pathway. Proteasomal inhibition caused an accumulation of phosphorylated IκBα and a reduction of NF-κB activity in HDLM-2 and L1236 cells. By in vitro kinase assays we demonstrate constitutive IκB kinase (IKK) activity in H-RS cells, indicating ongoing signal transduction. Furthermore, H-RS cells secrete one or more factor(s) that were able to trigger NF-κB activation. We conclude that aberrant activation of IKKs, and in some cases defective IκBs, lead to constitutive nuclear NF-κB activity, which in turn results in a growth advantage of Hodgkins disease tumor cells.

Nuclear Factor κB–dependent Gene Expression Profiling of Hodgkin's Disease Tumor Cells, Pathogenetic Significance, and Link to Constitutive Signal Transducer and Activator of Transcription 5a Activity

Constitutive nuclear nuclear factor (NF)-κB activity is observed in a variety of hematopoietic and solid tumors. Given the distinctive role of constitutive NF-κB for Hodgkin and Reed-Sternberg (HRS) cell viability, we performed molecular profiling in two Hodgkins disease (HD) cell lines to identify NF-κB target genes. We recognized 45 genes whose expression in both cell lines was regulated by NF-κB. The NF-κB–dependent gene profile comprises chemokines, cytokines, receptors, apoptotic regulators, intracellular signaling molecules, and transcription factors, the majority of which maintain a marker-like expression in HRS cells. Remarkably, we found 17 novel NF-κB target genes. Using chromatin immunoprecipitation we demonstrate that NF-κB is recruited directly to the promoters of several target genes, including signal transducer and activator of transcription (STAT)5a, interleukin-13, and CC chemokine receptor 7. Intriguingly, NF-κB positively regulates STAT5a expression and signaling pathways in HRS cells, and promotes its persistent activation. In fact, STAT5a overexpression was found in most tumor cells of tested patients with classical HD, indicating a critical role for HD. The gene profile underscores a central role of NF-κB in the pathogenesis of HD and potentially of other tumors with constitutive NF-κB activation.

Transcription factor NF-κB is constitutively activated in acute lymphoblastic leukemia cells

The pleiotropic transcription factor NF-κB controls cellular apoptotic and growth processes and increasing evidence suggests a role in tumorigenesis. We describe here that constitutively activated NF-κB complexes are found in the vast majority (39 out of 42 samples) of childhood acute lymphoblastic leukemia (ALL) without any subtype restriction. Electrophoretic shift analysis further demonstrates that these complexes are composed of p50-p50 and p65-p50 dimers. Proteasome inhibition in primary ALL cultures results in a hyperphosphorylated form of IκBα, indicating that activation of upstream kinases, which trigger IκBα degradation, has led to nuclear translocation of NF-κB. Careful inhibition of cellular proteolytic activities is of importance when analyzing extracts from primary ALL cells. Degradation of p65 and other proteins in ALL samples could be specifically suppressed by α-1 antitrypsin. Constitutive NF-κB activation is thus a common characteristic of childhood ALL and strongly suggests a critical role of this factor for leukemia cell survival.

NF-κB p105 is a target of IκB kinases and controls signal induction of Bcl-3–p50 complexes

The NF‐κB precursor p105 has dual functions: cytoplasmic retention of attached NF‐κB proteins and generation of p50 by processing. It is poorly understood whether these activities of p105 are responsive to signalling processes that are known to activate NF‐κB p50–p65. We propose a model that p105 is inducibly degraded, and that its degradation liberates sequestered NF‐κB subunits, including its processing product p50. p50 homodimers are specifically bound by the transcription activator Bcl‐3. We show that TNFα, IL‐1β or phorbolester (PMA) trigger rapid formation of Bcl‐3–p50 complexes with the same kinetics as activation of p50–p65 complexes. TNF‐α‐induced Bcl‐3–p50 formation requires proteasome activity, but is independent of p50–p65 released from IκBα, indicating a pathway that involves p105 proteolysis. The IκB kinases IKKα and IKKβ physically interact with p105 and inducibly phosphorylate three C‐terminal serines. p105 is degraded upon TNF‐α stimulation, but only when the IKK phospho‐acceptor sites are intact. Furthermore, a p105 mutant, lacking the IKK phosphorylation sites, acts as a super‐repressor of IKK‐induced NF‐κB transcriptional activity. Thus, the known NF‐κB stimuli not only cause nuclear accumulation of p50–p65 heterodimers but also of Bcl‐3–p50 and perhaps further transcription activator complexes which are formed upon IKK‐mediated p105 degradation.

OTULIN Antagonizes LUBAC Signaling by Specifically Hydrolyzing Met1-Linked Polyubiquitin

Summary The linear ubiquitin (Ub) chain assembly complex (LUBAC) is an E3 ligase that specifically assembles Met1-linked (also known as linear) Ub chains that regulate nuclear factor κB (NF-κB) signaling. Deubiquitinases (DUBs) are key regulators of Ub signaling, but a dedicated DUB for Met1 linkages has not been identified. Here, we reveal a previously unannotated human DUB, OTULIN (also known as FAM105B), which is exquisitely specific for Met1 linkages. Crystal structures of the OTULIN catalytic domain in complex with diubiquitin reveal Met1-specific Ub-binding sites and a mechanism of substrate-assisted catalysis in which the proximal Ub activates the catalytic triad of the protease. Mutation of Ub Glu16 inhibits OTULIN activity by reducing kcat 240-fold. OTULIN overexpression or knockdown affects NF-κB responses to LUBAC, TNFα, and poly(I:C) and sensitizes cells to TNFα-induced cell death. We show that OTULIN binds LUBAC and that overexpression of OTULIN prevents TNFα-induced NEMO association with ubiquitinated RIPK1. Our data suggest that OTULIN regulates Met1-polyUb signaling.

Requirement of Hsp90 activity for IκB kinase (IKK) biosynthesis and for constitutive and inducible IKK and NF-κB activation

The molecular chaperone Hsp90 affects the function and fate of a number of signaling molecules. We have investigated the Hsp90 requirement for constitutive and inducible activity of the IκB kinase (IKK) complex and of NF-κB. Inhibition by the Hsp90 ATPase inhibitors, geldanamycin (GA) and radicicol (RC), revealed that Hsp90 controls IKKs at two levels, inducibility of enzymatic activity and biogenesis, which can be discriminated by short- and long-time GA incubation, respectively. Short-time inhibition of Hsp90 resulted in impaired IKK kinase activation by TNFα, IL-1β or phorbolester PMA. Furthermore, GA inhibited constitutive activation of IKK and NF-κB in Hodgkins lymphoma cells. Hsp90 function was also required for trans- and autophosphorylation of transfected IKKβ. GA exposure for several hours resulted in a downmodulation of IKK complex α, β and γ subunits to various extent. Proteasome inhibition interfered with GA mediated IKK depletion and Hsp90 inhibition induced polyubiquitination of IKKα and β during protein synthesis. In fact, GA blocked biogenesis of IKKα and IKKβ but did not interfere with post-translational turnover. Together, these results define a dual requirement for Hsp90 as a regulator of NF-κB signaling by its general involvement in IKK activation and by its role in IKK homeostasis.