Although circumstantial evidence supports enhanced Toll-like receptor 7 (TLR7) signalling as a mechanism of human systemic autoimmune disease
, evidence of lupus-causing TLR7 gene variants is ...lacking. Here we describe human systemic lupus erythematosus caused by a TLR7 gain-of-function variant. TLR7 is a sensor of viral RNA
,
and binds to guanosine
-
. We identified a de novo, previously undescribed missense TLR7
variant in a child with severe lupus and additional variants in other patients with lupus. The TLR7
variant selectively increased sensing of guanosine and 2',3'-cGMP
, and was sufficient to cause lupus when introduced into mice. We show that enhanced TLR7 signalling drives aberrant survival of B cell receptor (BCR)-activated B cells, and in a cell-intrinsic manner, accumulation of CD11c
age-associated B cells and germinal centre B cells. Follicular and extrafollicular helper T cells were also increased but these phenotypes were cell-extrinsic. Deficiency of MyD88 (an adaptor protein downstream of TLR7) rescued autoimmunity, aberrant B cell survival, and all cellular and serological phenotypes. Despite prominent spontaneous germinal-centre formation in Tlr7
mice, autoimmunity was not ameliorated by germinal-centre deficiency, suggesting an extrafollicular origin of pathogenic B cells. We establish the importance of TLR7 and guanosine-containing self-ligands for human lupus pathogenesis, which paves the way for therapeutic TLR7 or MyD88 inhibition.
Activation of JAK-STAT signaling is one of the hallmarks of myelofibrosis, a myeloproliferative neoplasm that leads to inflammation, progressive bone marrow failure, and a risk of leukemic ...transformation. Around 90% of patients with myelofibrosis have a mutation in JAK2, MPL, or CALR: so-called 'driver' mutations that lead to activation of JAK2. Ruxolitinib, and other JAK2 inhibitors in clinical use, provide clinical benefit but do not have a major impact on the abnormal hematopoietic clone. This phenomenon is termed 'persistence', in contrast to usual patterns of resistance. Multiple groups have shown that type 1 inhibitors of JAK2, which bind the active conformation of the enzyme, lead to JAK2 becoming resistant to degradation with consequent accumulation of phospho-JAK2. In turn, this can lead to exacerbation of inflammatory manifestations when the JAK inhibitor is discontinued, and it may also contribute to disease persistence. The ways in which JAK2 V617F and CALR mutations lead to activation of JAK-STAT signaling are incompletely understood. We summarize what is known about pathological JAK-STAT activation in myelofibrosis and how this might lead to future novel therapies for myelofibrosis with greater disease-modifying potential.
Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key drivers of blood and lymph vessel formation in development, but also in several pathological processes. VEGF-C ...signaling through VEGFR-3 promotes lymphangiogenesis, which is a clinically relevant target for treating lymphatic insufficiency and for blocking tumor angiogenesis and metastasis. The extracellular domain of VEGFRs consists of seven Ig homology domains; domains 1–3 (D1-3) are responsible for ligand binding, and the membrane-proximal domains 4–7 (D4-7) are involved in structural rearrangements essential for receptor dimerization and activation. Here we analyzed the crystal structures of VEGF-C in complex with VEGFR-3 domains D1-2 and of the VEGFR-3 D4-5 homodimer. The structures revealed a conserved ligand-binding interface in D2 and a unique mechanism for VEGFR dimerization and activation, with homotypic interactions in D5. Mutation of the conserved residues mediating the D5 interaction (Thr446 and Lys516) and the D7 interaction (Arg737) compromised VEGF-C induced VEGFR-3 activation. A thermodynamic analysis of VEGFR-3 deletion mutants showed that D3, D4-5, and D6-7 all contribute to ligand binding. A structural model of the VEGF-C/VEGFR-3 D1-7 complex derived from small-angle X-ray scattering data is consistent with the homotypic interactions in D5 and D7. Taken together, our data show that ligand-dependent homotypic interactions in D5 and D7 are essential for VEGFR activation, opening promising possibilities for the design of VEGFR-specific drugs.
RATIONALE:Lymphatic vessels in the respiratory tract normally mature into a functional network during the neonatal period, but under some pathological conditions they can grow as enlarged, dilated ...sacs that result in the potentially lethal condition of pulmonary lymphangiectasia.
OBJECTIVE:We sought to determine whether overexpression of the lymphangiogenic growth factor (vascular endothelial growth factor-C VEGF-C) can promote lymphatic growth and maturation in the respiratory tract. Unexpectedly, perinatal overexpression of VEGF-C in the respiratory epithelium led to a condition resembling human pulmonary lymphangiectasia, a life-threatening disorder of the newborn characterized by respiratory distress and the presence of widely dilated lymphatics.
METHODS AND RESULTS:Administration of doxycycline to Clara cell secretory protein-reverse tetracycline-controlled transactivator/tetracycline operator-VEGF-C double-transgenic mice during a critical period from embryonic day 15.5 to postnatal day 14 was accompanied by respiratory distress, chylothorax, pulmonary lymphangiectasia, and high mortality. Enlarged sac-like lymphatics were abundant near major airways, pulmonary vessels, and visceral pleura. Side-by-side comparison revealed morphological features similar to pulmonary lymphangiectasia in humans. The condition was milder in mice given doxycycline after age postnatal day 14 and did not develop after postnatal day 35. Mechanistic studies revealed that VEGF recptor (VEGFR)-3 alone drove lymphatic growth in adult mice, but both VEGFR-2 and VEGFR-3 were required for the development of lymphangiectasia in neonates. VEGFR-2/VEGFR-3 heterodimers were more abundant in the dilated lymphatics, consistent with the involvement of both receptors. Despite the dependence of lymphangiectasia on VEGFR-2 and VEGFR-3, the condition was not reversed by blocking both receptors together or by withdrawing VEGF-C.
CONCLUSIONS:The findings indicate that VEGF-C overexpression can induce pulmonary lymphangiectasia during a critical period in perinatal development.
Antibodies that block vascular endothelial growth factor (VEGF) have become an integral part of antiangiogenic tumor therapy, and antibodies targeting other VEGFs and receptors (VEGFRs) are in ...clinical trials. Typically receptor-blocking antibodies are targeted to the VEGFR ligand-binding site. Here we describe a monoclonal antibody that inhibits VEGFR-3 homodimer and VEGFR-3/VEGFR-2 heterodimer formation, signal transduction, as well as ligand-induced migration and sprouting of microvascular endothelial cells. Importantly, we show that combined use of antibodies blocking ligand binding and receptor dimerization improves VEGFR inhibition and results in stronger inhibition of endothelial sprouting and vascular network formation in vivo. These results suggest that receptor dimerization inhibitors could be used to enhance antiangiogenic activity of antibodies blocking ligand binding in tumor therapy.
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► We report a monoclonal antibody that blocks VEGF receptor dimerization ► This antibody shows enhanced VEGF receptor inhibition at high ligand concentrations ► Blocking both ligand binding and receptor dimerization improves VEGFR inhibition ► This antibody combination should outperform current inhibitors of tumor angiogenesis
Vascular endothelial growth factors (VEGFs) regulate blood and lymphatic vessel development through VEGF receptors (VEGFRs). The VEGFR immunoglobulin homology domain 2 (D2) is critical for ligand ...binding, and D3 provides additional interaction sites. VEGF-B and placenta growth factor (PlGF) bind to VEGFR-1 with high affinity, but only PlGF is angiogenic in most tissues. We show that VEGF-B, unlike other VEGFs, did not require D3 interactions for high-affinity binding. VEGF-B with a PlGF-derived L1 loop (B-L1P) stimulated VEGFR-1 activity, whereas PlGF with a VEGF-B-derived L1 loop (P-L1B) did not. Unlike P-L1B and VEGF-B, B-L1P and PlGF were also angiogenic in mouse skeletal muscle. Furthermore, B-L1P also bound to VEGFR-2 and activated downstream signaling. These results establish a role for L1-mediated D3 interactions in VEGFR activation in endothelial cells and indicate that VEGF-B is a high-affinity VEGFR-1 ligand that, unlike PlGF, cannot efficiently induce signaling downstream of VEGFR-1.
Cardiac hypertrophy accompanies many forms of heart disease, including ischemic disease, hypertension, heart failure, and valvular disease, and it is a strong predictor of increased cardiovascular ...morbidity and mortality. Deletion of bone marrow kinase in chromosome X (Bmx), an arterial nonreceptor tyrosine kinase, has been shown to inhibit cardiac hypertrophy in mice. This finding raised the possibility of therapeutic use of Bmx tyrosine kinase inhibitors, which we have addressed here by analyzing cardiac hypertrophy in gene-targeted mice deficient in Bmx tyrosine kinase activity. We found that angiotensin II (Ang II)-induced cardiac hypertrophy is significantly reduced in mice deficient in Bmx and in mice with inactivated Bmx tyrosine kinase compared with WT mice. Genome-wide transcriptomic profiling showed that Bmx inactivation suppresses myocardial expression of genes related to Ang II-induced inflammatory and extracellular matrix responses whereas expression of RNAs encoding mitochondrial proteins after Ang II administration was maintained in Bmx-inactivated hearts. Very little or no Bmx mRNA was expressed in human cardiomyocytes whereas human cardiac endothelial cells expressed abundant amounts. Ang II stimulation of endothelial cells increased Bmx phosphorylation, and Bmx gene silencing inhibited downstream STAT3 signaling, which has been implicated in cardiac hypertrophy. Furthermore, activation of the mechanistic target of rapamycin complex 1 pathway by Ang II treatment was decreased in the Bmx-deficient hearts. Our results demonstrate that inhibition of the cross-talk between endothelial cells and cardiomyocytes by Bmx inactivation suppresses Ang II-induced signals for cardiac hypertrophy. These results suggest that the endothelial Bmx tyrosine kinase could provide a target to attenuate the development of cardiac hypertrophy.
The significance of ErbB4 in tumor biology is poorly understood. The ERBB4 gene is alternatively spliced producing juxtamembrane (JM-a and JM-b) and cytoplasmic (CYT-1 and CYT-2) isoforms. Here, ...signaling via the two alternative ErbB4 JM isoforms (JM-a CYT-2 and JM-b CYT-2) was compared. Fibroblasts expressing ErbB4 JM-a demonstrated enhanced ErbB4 autophosphorylation, growth, and survival. In contrast, cells overexpressing ErbB4 JM-b underwent starvation-induced death. Both pro- and antisurvival responses to the two ErbB4 isoforms were sensitive to an ErbB kinase inhibitor. Platelet-derived growth factor receptor-alpha (PDGFRA) was identified as an ErbB4 target gene that was differentially regulated by the two ErbB4 isoforms. The soluble intracellular domain of ErbB4, released from the JM-a but not from the JM-b isoform, associated with the transcription factor AP-2 and promoted its potential to enhance PDGFRA transcription. Survival of cells expressing JM-a was suppressed by targeting either PDGFR-α or AP-2, whereas cells expressing JM-b were rescued from cell death by the PDGFR-α agonist, PDGF-BB. These findings indicate that two alternative ErbB4 isoforms may promote antagonistic cellular responses and suggest that pharmacological inhibition of ErbB4 kinase activity may lead to either suppression or promotion of cellular growth.
Treatment of patients with myelofibrosis with the type I JAK (Janus kinase) inhibitor ruxolitinib paradoxically induces JAK2 activation loop phosphorylation and is associated with a life-threatening ...cytokine-rebound syndrome if rapidly withdrawn. We developed a time-dependent assay to mimic ruxolitinib withdrawal in primary JAK2
and CALR mutant myelofibrosis patient samples and observed notable activation of spontaneous STAT signaling in JAK2
samples after drug washout. Accumulation of ruxolitinib-induced JAK2 phosphorylation was dose dependent and correlated with rebound signaling and the presence of a JAK2
mutation. Ruxolitinib prevented dephosphorylation of a cryptic site involving Tyr
in JAK2 blocking ubiquitination and degradation. In contrast, a type II JAK inhibitor, CHZ868, did not induce JAK2 phosphorylation, was not associated with withdrawal signaling, and was superior in the eradication of flow-purified JAK2
mutant CD34
progenitors after drug washout. Type I inhibitor-induced loop phosphorylation may act as a pathogenic signaling node released upon drug withdrawal, especially in JAK2
patients.
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