Jennifer S. Michaelson

Critical role for Daxx in regulating Mdm2

The tumour suppressor p53 induces apoptosis or cell-cycle arrest in response to genotoxic and other stresses. In unstressed cells, the anti-proliferative effects of p53 are restrained by mouse double minute 2 (Mdm2), a ubiquitin ligase (E3) that promotes p53 ubiquitination and degradation. Mdm2 also mediates its own degradation through auto-ubiquitination. It is unclear how the cis- and trans-E3 activities of Mdm2, which have opposing effects on cell fate, are differentially regulated. Here, we show that death domain-associated protein (Daxx) is required for Mdm2 stability. Downregulation of Daxx decreases Mdm2 levels, whereas overexpression of Daxx strongly stabilizes Mdm2. Daxx simultaneously binds to Mdm2 and the deubiquitinase Hausp, and it mediates the stabilizing effect of Hausp on Mdm2. In addition, Daxx enhances the intrinsic E3 activity of Mdm2 towards p53. On DNA damage, Daxx dissociates from Mdm2, which correlates with Mdm2 self-degradation. These findings reveal that Daxx modulates the function of Mdm2 at multiple levels and suggest that the disruption of the Mdm2–Daxx interaction may be important for p53 activation in response to DNA damage.

TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration

Inflammation participates in tissue repair through multiple mechanisms including directly regulating the cell fate of resident progenitor cells critical for successful regeneration. Upon surveying target cell types of the TNF ligand TWEAK, we observed that TWEAK binds to all progenitor cells of the mesenchymal lineage and induces NF‐κB activation and the expression of pro‐survival, pro‐proliferative and homing receptor genes in the mesenchymal stem cells, suggesting that this pro‐inflammatory cytokine may play an important role in controlling progenitor cell biology. We explored this potential using both the established C2C12 cell line and primary mouse muscle myoblasts, and demonstrated that TWEAK promoted their proliferation and inhibited their terminal differentiation. By generating mice deficient in the TWEAK receptor Fn14, we further showed that Fn14‐deficient primary myoblasts displayed significantly reduced proliferative capacity and altered myotube formation. Following cardiotoxin injection, a known trigger for satellite cell‐driven skeletal muscle regeneration, Fn14‐deficient mice exhibited reduced inflammatory response and delayed muscle fiber regeneration compared with wild‐type mice. These results indicate that the TWEAK/Fn14 pathway is a novel regulator of skeletal muscle precursor cells and illustrate an important mechanism by which inflammatory cytokines influence tissue regeneration and repair. Coupled with our recent demonstration that TWEAK potentiates liver progenitor cell proliferation, the expression of Fn14 on all mesenchymal lineage progenitor cells supports a broad involvement of this pathway in other tissue injury and disease settings.

β-catenin is a downstream effector of Wnt-mediated tumorigenesis in the mammary gland

The Wnt signal transduction pathway has been implicated in mammary tumorigenesis in the mouse. β-catenin, a key downstream effector of this pathway interacts with and thus activates the Tcf/Lef family of transcription factors. Elevated levels of β-catenin have been found in many human tumors, notably colon carcinomas. Recently, elevated levels of β-catenin have been associated with poor prognosis in human adenocarcinoma of the breast. In order to assess the possible role of β-catenin in mammary carcinoma, we have created transgenic mice bearing the MMTV–LTR driving an activated form of β-catenin. These mice develop mammary gland hyperplasia and mammary adenocarcinoma, a phenotype very similar to that of transgenic mice expressing an MMTV-driven Wnt gene. Indeed, the histopathology of the mammary tumors in Wnt-mediated adenocarcinoma is identical to that observed in our β-catenin-mediated disease model. Furthermore, putative β-catenin transcriptional targets, cyclin D1 and c-myc, are elevated in β-catenin-mediated mammary tumors and cell lines. These observations support the notion that the oncogenic Wnt pathway operates via β-catenin and its targets in the context of mammary hyperplasia and carcinoma.

Proinflammatory Effects of Tweak/Fn14 Interactions in Glomerular Mesangial Cells

TNF-like weak inducer of apoptosis, or TWEAK, is a relatively new member of the TNF-ligand superfamily. Ligation of the TWEAK receptor Fn14 by TWEAK has proinflammatory effects on fibroblasts, synoviocytes, and endothelial cells. Several of the TWEAK-inducible cytokines are important in the pathogenesis of kidney diseases; however, whether TWEAK can induce a proinflammatory effect on kidney cells is not known. We found that murine mesangial cells express cell surface TWEAK receptor. TWEAK stimulation of mesangial cells led to a dose-dependent increase in CCL2/MCP-1, CCL5/RANTES, CXCL10/IFN-γ-induced protein 10 kDa, and CXCL1/KC. The induced levels of chemokines were comparable to those found following mesangial cell exposure to potent proinflammatory stimuli such as TNF-α + IL-1β. CXCL11/interferon-inducible T cell α chemoattractant, CXCR5, mucosal addressin cell adhesion molecule-1, and VCAM-1 were up-regulated by TWEAK as well. TWEAK stimulation of mesangial cells resulted in an increase in phosphorylated Iκ-B, while pretreatment with an Iκ-B phosphorylation inhibitor significantly blocked chemokine induction, implicating activation of the NF-κB signaling pathway in TWEAK-induced chemokine secretion. Importantly, the Fn14-mediated proinflammatory effects of TWEAK on kidney cells were confirmed using mesangial cells derived from Fn14-deficient mice and by injection in vivo of TWEAK into wild-type vs Fn14-deficient mice. Finally, TWEAK-induced chemokine secretion was prevented by treatment with novel murine anti-TWEAK Abs. We conclude that TWEAK induces mesangial cells to secrete proinflammatory chemokines, suggesting a prominent role for TWEAK in the pathogenesis of renal injury. Our results support Ab inhibition of TWEAK as a potential new approach for the treatment of chemokine-dependent inflammatory kidney diseases.

Increased Fibroblast Growth Factor-Inducible 14 Expression Levels Promote Glioma Cell Invasion via Rac1 and Nuclear Factor-κB and Correlate with Poor Patient Outcome

Glial tumors progress to malignant grades by heightened proliferation and relentless dispersion throughout the central nervous system. Understanding genetic and biochemical processes that foster these behaviors is likely to reveal specific and effective targets for therapeutic intervention. Our current report shows that the fibroblast growth factor-inducible 14 (Fn14), a member of the tumor necrosis factor (TNF) receptor superfamily, is expressed at high levels in migrating glioma cells in vitro and invading glioma cells in vivo. Forced Fn14 overexpression stimulates glioma cell migration and invasion, and depletion of Rac1 by small interfering RNA inhibits this cellular response. Activation of Fn14 signaling by the ligand TNF-like weak inducer of apoptosis (TWEAK) stimulates migration and up-regulates expression of Fn14; this TWEAK effect requires Rac1 and nuclear factor-kappaB (NF-kappaB) activity. The Fn14 promoter region contains NF-kappaB binding sites, which mediate positive feedback causing sustained overexpression of Fn14 and enduring glioma cell invasion. Furthermore, Fn14 gene expression levels increase with glioma grade and inversely correlate with patient survival. These results show that the Fn14 cascade operates as a positive feedback mechanism for elevated and sustained Fn14 expression. Such a feedback loop argues for aggressive targeting of the Fn14 axis as a unique and specific driver of glioma malignant behavior.

RNAi reveals anti-apoptotic and transcriptionally repressive activities of DAXX

The function of DAXX, a highly conserved mammalian gene, has remained controversial; this is due, in part, to its identification in a variety of yeast two-hybrid screens. Targeted deletion in the mouse revealed that DAXX is essential for embryonic development. Furthermore, the increased levels of apoptosis observed in Daxx-knockout embryos and embryonic stem cell lines suggested that DAXX functions in an anti-apoptotic capacity. In contrast, overexpression studies showed that DAXX may promote apoptosis. Additional studies showed that, when overexpressed, DAXX could function as a transcriptional repressor. To clarify these matters, we have used RNAi to deplete endogenous DAXX and thereby assess DAXX function in cell lines previously tested in overexpression studies. Increased apoptosis was observed in DAXX-depleted cells, showing DAXX to be anti-apoptotic. The apoptosis induced by the absence of DAXX was rescued by Bcl-2 overexpression. In addition, transcriptional derepression was observed in RNAi-treated cells, indicating the ability of endogenous DAXX to repress gene expression and allowing for the identification of novel targets of DAXX repression, including nuclear factor κB (NF-κB)- and E2F1- regulated targets. Thus, depletion of DAXX by RNAi has verified the crucial role of endogenous DAXX as an anti-apoptotic regulator, and has allowed the identification of probable physiological targets of DAXX transcriptional repression.

Urinary TWEAK as a biomarker of lupus nephritis: a multicenter cohort study

IntroductionTNF-like weak inducer of apoptosis (TWEAK) has been implicated as a mediator of chronic inflammatory processes via prolonged activation of the NF-κB pathway in several tissues, including the kidney. Evidence for the importance of TWEAK in the pathogenesis of lupus nephritis (LN) has been recently introduced. Thus, TWEAK levels may serve as an indication of LN presence and activity.MethodsMulticenter cohorts of systemic lupus erythematosus (SLE) patients and controls were recruited for cross-sectional and longitudinal analysis of urinary TWEAK (uTWEAK) and/or serum TWEAK (sTWEAK) levels as potential biomarkers of LN. The performance of TWEAK as a biomarker for nephritis was compared with routinely used laboratory tests in lupus patients, including anti-double stranded DNA antibodies and levels of C3 and C4.ResultsuTWEAK levels were significantly higher in LN patients than in non-LN SLE patients and other disease control groups (P = 0.039). Furthermore, uTWEAK was better at distinguishing between LN and non-LN SLE patients than anti-DNA antibodies and complement levels, while high uTWEAK levels predicted LN in SLE patients with an odds ratio of 7.36 (95% confidence interval = 2.25 to 24.07; P = 0.001). uTWEAK levels peaked during LN flares, and were significantly higher during the flare than at 4 and 6 months prior to or following the flare event. A linear mixed-effects model showed a significant association between uTWEAK levels in SLE patients and their disease activity over time (P = 0.008). sTWEAK levels, however, were not found to correlate with the presence of LN or the degree of nephritis activity.ConclusionsHigh uTWEAK levels are indicative of LN, as opposed to non-LN SLE and other healthy and disease control populations, and reflect renal disease activity in longitudinal follow-up. Thus, our study further supports a role for TWEAK in the pathogenesis of LN, and provides strong evidence for uTWEAK as a candidate clinical biomarker for LN.

TWEAK/Fn14 Interactions Are Instrumental in the Pathogenesis of Nephritis in the Chronic Graft-versus-Host Model of Systemic Lupus erythematosus

TNF-like weak inducer of apoptosis (TWEAK), a member of the TNF superfamily, is a prominent inducer of proinflammatory cytokines in vitro and in vivo. We previously found that kidney cells display the TWEAK receptor Fn14, and that TWEAK stimulation of mesangial cells and podocytes induces a potent proinflammatory response. Several of the cytokines up-regulated in the kidney in response to TWEAK are instrumental in Lupus nephritis; we therefore hypothesized that TWEAK/Fn14 interactions may be important in the cascade(s) leading to renal damage in systemic Lupus erythematosus. In this study, we analyzed the effects of Fn14 deficiency in the chronic graft-vs-host model of SLE, and the benefits of treatment with an anti-TWEAK mAb in this mouse model. We found that anti-nuclear Ab titers were no different between C57BL/6 Fn14 wild-type and deficient mice injected with alloreactive bm12 splenocytes. However, kidney disease was significantly less severe in Fn14 knockout mice. Furthermore, kidney IgG deposition, IL-6, MCP-1, RANTES, and IP-10, as well as macrophage infiltration, were significantly decreased in Fn14-deficient mice with induced lupus. Similarly, mice with induced Lupus treated with an anti-TWEAK neutralizing mAb had significantly diminished kidney expression of IL-6, MCP-1, IL-10, as well as proteinuria, but similar autoantibody titers, as compared with control-treated mice. We conclude that TWEAK is an important mediator of kidney damage that acts by promoting local inflammatory events, but without impacting adaptive immunity in this experimental LN model. Thus, TWEAK blockade may be a novel therapeutic approach to reduce renal damage in SLE.

TWEAK/Fn14 pathway: an immunological switch for shaping tissue responses

Summary:  Our immune system performs the vital function of recognizing and eliminating invading pathogens and malignancies. There is an increasing appreciation that the immune system also actively mediates tissue responses under both physiological and pathological conditions, significantly impacting the inflammatory, fibrogenic, and regenerative components. Likewise, there is a growing understanding of how epithelial, endothelial, and other non‐hematopoietic tissue cell types actively contribute to the interplay that shapes tissue responses. While much of the molecular basis underlying the immune regulation of tissue responses remains to be delineated, the tumor necrosis factor (TNF) superfamily ligand/receptor pair of TNF‐like weak inducer of apoptosis (TWEAK) and fibroblast growth factor‐inducible molecule 14 (Fn14) has now emerged as a key piece of this puzzle. In this review, we first discuss how the usually ‘dormant’ TWEAK/Fn14 pathway becomes activated specifically in injury and disease contexts. We then summarize how TWEAK‐mediated Fn14 signaling triggers a wide range of activities in tissue parenchymal and stromal cells as well as progenitor cells. Finally, we review recent experimental evidence that further supports the functional dichotomy of TWEAK/Fn14 activation in physiological versus pathological tissue responses and its potential therapeutic implications. Whereas transient TWEAK/Fn14 activation promotes productive tissue responses after injury, excessive or persistent TWEAK/Fn14 activation drives pathological tissue responses, leading to progressive damage and degeneration.

Anti-tumor activity of stability-engineered IgG-like bispecific antibodies targeting TRAIL-R2 and LTβR

Bispecific antibodies (BsAbs) represent an emerging class of biologics that achieve dual targeting with a single agent. Recombinant DNA technologies have facilitated a variety of creative bispecific designs with many promising therapeutic applications; however, practical methods for producing high quality BsAbs that have good product stability, long serum half-life, straightforward purification, and scalable production have largely been limiting. Here we describe a protein-engineering approach for producing stable, scalable tetravalent IgG-like BsAbs. The stability-engineered IgG-like BsAb was envisioned to target and crosslink two TNF family member receptors, TRAIL-R2 (TNF-Related Apoptosis Inducing Ligand Receptor-2) and LTβR (Lymphotoxin-beta Receptor), expressed on the surface of epithelial tumor cells with the goal of triggering an enhanced anti-tumor effect. Our IgG-like BsAbs consists of a stability-engineered anti- LTβR single chain Fv (scFv) genetically fused to either the N- or C-terminus of the heavy chain of a full-length anti-TRAIL-R2 IgG1 monoclonal antibody. Both N- or C-terminal BsAbs were active in inhibiting tumor cell growth in vitro, and with some cell lines demonstrated enhanced activity relative to the combination of parental Abs. Pharmacokinetic studies in mice revealed long serum half-lives for the BsAbs. In murine tumor xenograft models, therapeutic treatment with the BsAbs resulted in reduction in tumor volume either comparable to or greater than the combination of parental antibodies, indicating that simultaneously targeting and cross-linking receptor pairs is an effective strategy for treating tumor cells. These studies support that stability-engineering is an enabling step for producing scalable IgG-like BsAbs with properties desirable for biopharmaceutical development.