Malini Sen

Blockade of Wnt-5A/Frizzled 5 signaling inhibits rheumatoid synoviocyte activation

OBJECTIVE It is not understood why cultured fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) often display a persistently activated phenotype, despite removal from an inflammatory environment. Previously, we found that these FLS expressed high levels of both Wnt-5A and Frizzled 5 (Fz5), a receptor-ligand pair implicated in both limb bud and bone marrow stem cell development. The objective of the present experiments was to determine whether Wnt-5A/FzS signaling contributes to FLS activation. METHODS Wnt-5A expression in FLS was inhibited by transfection with both antisense and dominant negative (dn) vectors. Fz5 signaling was blocked with an antibody to the extracellular domain of the receptor. The effects of these treatments on the expression of the proinflammatory cytokines interleukin-6 (IL-6) and IL-15 and on the expression of receptor activator of nuclear factor kappaB ligand (RANKL) were assessed by reverse transcriptase-polymerase chain reaction and immunoblotting. RESULTS Both antisense Wnt-5A and dnWnt-5A vectors, but not empty vector, diminished IL-6 and IL-15 expression in RA FLS. Anti-Fz5 antibody exerted similar effects and also reduced RANKL expression. CONCLUSION Wnt-5A/Fz5 signaling may contribute to the activated state of FLS in RA. Receptor antagonists of Fz5 should be considered for the treatment of refractory synovitis.

Transcriptional Outcome of Wnt-Frizzled Signal Transduction in Inflammation: Evolving Concepts

Wnt-Frizzled signaling was first identified as a key event in Drosophila development. Over the years, ample evidence has accumulated regarding the multiple roles of Wnt-Frizzled signaling in mammalian cell differentiation and tissue/organ morphogenesis. It is thus not surprising that variations in the regulatory network of the Wnt signaling scheme would lead to alterations in cellular organization and cell activation and to the development of pathogenic conditions. Several reports have accordingly implied the involvement of Wnt-Frizzled signaling in the activation of proinflammatory mediators in inflammatory disorders. We will discuss how Wnt-Frizzled signaling may initiate/augment inflammation, focusing on its transcriptional outcome.

A methylthioadenosine phosphorylase (MTAP) fusion transcript identifies a new gene on chromosome 9p21 that is frequently deleted in cancer

Homozygous deletions of human chromosome 9p21 occur frequently in malignant cell lines, and are also common in primary gliomas, lung cancers, and leukemias. Moving from the centromere to the telomere, this complex region encodes the tumor suppressor genes p15INK4B (CDKN2B), p14ARF, p16INK4A (CDKN2A), and the housekeeping gene methylthioadenosine phosphorylase (MTAP). However, not all chromosome 9p21 deletions in tumors include these tumor suppressor genes. Here we describe the partial sequence and the exact localization of a new gene on chromosome 9p21 centromeric of p15INK4B, that formed an in frame fusion transcript with MTAP in a glioma xenograft, and that is homozygously deleted in various malignant cell lines. Northern blot revealed corresponding 1.5 kb transcript in non-deleted cell lines as well as in normal lymphocytes. Using a RNA master blot membrane including 50 different tissues, we could show that this new transcript is expressed in all tissues of the adult but not or only at very low levels in most of the fetal tissues tested. The expression pattern is similar to that of p16INK4A. The localization as well as the deletion pattern makes this transcript a candidate for a new tumor suppressor gene.

The Wingless homolog Wnt5a stimulates phagocytosis but not bacterial killing

Phagocytosis is a primary defense program orchestrated by monocytes/macrophages. Unregulated phagocytosis can lead to pathological conditions. In the current study we have demonstrated that Wnt5a stimulates phagocytosis through PI3 kinase–Rac1 and lipid-raft-dependent processes. Wnt5a-mediated augmentation in phagocytosis is suppressed by blocking expression of the putative Wnt5a receptor Frizzled 5. Enhanced phagocytosis of bacteria by Wnt5a–Fz5 signaling increases the secretion of proinflammatory cytokines, but not the bacterial killing rate. Furthermore, a small molecule inhibitor of Wnt production, IWP-2, which reduces secretion of functionally active Wnt5a, not only suppresses both phagocytosis and the secretion of proinflammatory cytokines but also accelerates the bacterial killing rate.

Wnt5a–Rac1–NF-κB Homeostatic Circuitry Sustains Innate Immune Functions in Macrophages

Macrophages play a critical role in innate immunity. Differentiation Ags present on macrophages such as CD14 orchestrate the first line of defense against infection. The basal/homeostatic signaling scheme that keeps macrophages thus groomed for innate immune functions remains unresolved. Wnt5a–Fz5 signaling being a primordial event during cell differentiation, we examined the involvement of Wnt5a–Fz5 signaling in the maintenance of innate immune functions. In this study, we demonstrate that innate immune functions of macrophages ensue at least partly through a homeostatic Wnt5a–Fz5–NF-κB (p65) circuit, which is Rac1 dependent. The autocrine/paracrine Wnt5a–Fz5–Rac1–p65 signaling cascade not only maintains basal levels of the immune defense modulating IFNs and CD14; it also supports macrophage survival. Wnt5a–Fz5–Rac1 signaling mediated p65 homeostasis in turn sustains Wnt5a expression in a feed-forward mode. The natural immune response of macrophages to Escherichia coli/LPS and virus is accordingly sustained. The depiction of sustenance of innate immune functions as an outcome of a homeostatic Wnt5a–p65 axis unfolds previously unidentified details of immune regulation and provides new insight into homeostatic cell signaling.

WISP3–IGF1 interaction regulates chondrocyte hypertrophy

Summary WISP3 (Wnt induced secreted protein 3) is a multi-domain protein of mesenchymal origin. Mutations in several domains of WISP3 cause PPRD (progressive pseudo rheumatoid dysplasia), which is associated with cartilage loss and restricted skeletal development. Despite several studies focusing on the functional characterization of WISP3, the molecular details underlying the course of PPRD remain unresolved. We are interested in analyzing the function of WISP3 in the context of cartilage integrity. The current study demonstrates that WISP3 binds to insulin-like growth factor 1 (IGF1) and inhibits IGF1 secretion. Additionally, WISP3 curbs IGF1-mediated collagen X expression, accumulation of reactive oxygen species (ROS) and alkaline phosphatase activity, all of which are associated with the induction of chondrocyte hypertrophy. Interestingly, both IGF1 and ROS in turn trigger an increase in WISP3 expression. Together, our results are indicative of an operational WISP3–IGF1 regulatory loop whereby WISP3 preserves cartilage integrity by restricting IGF1-mediated hypertrophic changes in chondrocytes, at least partly, upon interaction with IGF1.

Wnt signaling in rheumatoid synoviocyte activation

Abstract Rheumatoid arthritis (RA) is a joint-specific disease with complex pathogenesis. It is characterized by synovial inflammation, cartilage loss, and joint destruction. The reasons why joint damage recurs when therapy is discontinued are not clearly understood. Several lines of evidence suggest that cartilage damage is promoted by the transformed and invasive fibroblast-like synoviocytes (FLS) of the rheumatoid joint. It has been demonstrated in several systems that aberrant wnt-mediated signaling causes blockade of cartilage differentiation and malformation of joints. In this review, we have discussed the importance of wnt–frizzled-mediated signaling in the autonomous activation of FLS in patients with RA. Anti-wnt/anti-frizzled antibodies, frizzled receptor antagonists, or small molecule inhibitors of wnt–frizzled signaling might be useful for therapeutic interventions in RA.

CCN6 regulates mitochondrial function

ABSTRACT Despite established links of CCN6, or Wnt induced signaling protein-3 (WISP3), with progressive pseudo rheumatoid dysplasia, functional characterization of CCN6 remains incomplete. In light of the documented negative correlation between accumulation of reactive oxygen species (ROS) and CCN6 expression, we investigated whether CCN6 regulates ROS accumulation through its influence on mitochondrial function. We found that CCN6 localizes to mitochondria, and depletion of CCN6 in the chondrocyte cell line C-28/I2 by using siRNA results in altered mitochondrial electron transport and respiration. Enhanced electron transport chain (ETC) activity of CCN6-depleted cells was reflected by increased mitochondrial ROS levels in association with augmented mitochondrial ATP synthesis, mitochondrial membrane potential and Ca2+. Additionally, CCN6-depleted cells display ROS-dependent PGC1α (also known as PPARGC1A) induction, which correlates with increased mitochondrial mass and volume density, together with altered mitochondrial morphology. Interestingly, transcription factor Nrf2 (also known as NFE2L2) repressed CCN6 expression. Taken together, our results suggest that CCN6 acts as a molecular brake, which is appropriately balanced by Nrf2, in regulating mitochondrial function. Summary: CCN6, or Wnt-induced signaling protein 3 (WISP3), regulates mitochondrial electron transport and ATP synthesis. The influence of CCN6 on mitochondrial function might impact cell growth and differentiation.

Wnt5a Signaling Promotes Host Defense against Leishmania donovani Infection

Leishmania donovani infects macrophages, disrupting immune homeostasis. The underlying mechanism that sustains infection remains unresolved. In view of the potential of Wnt5a signaling to support immune homeostasis, we evaluated the interrelationship of Wnt5a signaling and Leishmania donovani infection. Upon infecting macrophages separately with antimony drug–sensitive and –resistant L. donovani, we noted disruption in the steady-state level of Wnt5a. Moreover, inhibition of Wnt5a signaling by small interfering RNA transfection in vitro or by use of inhibitor of Wnt production in vivo led to an increase in cellular parasite load. In contrast, treatment of macrophages with recombinant Wnt5a caused a decrease in the load of antimony-sensitive and -resistant parasites, thus confirming that Wnt5a signaling antagonizes L. donovani infection. Using inhibitors of the Wnt5a signaling intermediates Rac1 and Rho kinase, we demonstrated that Wnt5a-mediated inhibition of parasite infection in macrophages is Rac1/Rho dependent. Furthermore, phalloidin staining and reactive oxygen species estimation of Wnt5a-treated macrophages suggested that a Wnt5a-Rac/Rho–mediated decrease in parasite load is associated with an increase in F- actin assembly and NADPH oxidase activity. Moreover, live microscopy of L. donovani–infected macrophages treated with Wnt5a demonstrated increased endosomal/lysosomal fusions with parasite-containing vacuoles (parasitophorous vacuoles [PV]). An increase in PV–endosomal/lysosomal fusion accompanied by augmented PV degradation in Wnt5a-treated macrophages was also apparent from transmission electron microscopy of infected cells. Our results suggest that, although L. donovani evades host immune response, at least in part through inhibition of Wnt5a signaling, revamping Wnt5a signaling can inhibit L. donovani infection, irrespective of drug sensitivity or resistance.

Wnt5A Signaling Promotes Defense Against Bacterial Pathogens by Activating a Host Autophagy Circuit

Bacterial pathogens are associated with severe infections (e.g., sepsis) and exacerbation of debilitating conditions such as chronic obstructive pulmonary disease (COPD). The interactions of bacterial pathogens with macrophages, a key component of innate immunity and host defense, are not clearly understood and continue to be intensively studied. Having previously demonstrated a role of Wnt5A signaling in phagocytosis, we proceeded to decipher the connection of Wnt5A signaling with infection by pathogenic bacteria, namely Pseudomonas aeruginosa (PA) and Streptococcus pneumoniae (SP), which are related with the progression of COPD and sepsis. We found that during the initial hours of infection with PA and SP, there is decrease in the steady state levels of the Wnt5A protein in macrophages. Suppression of Wnt5A signaling, moreover, impairs macrophage clearance of the bacterial infection both in vitro and in vivo. Activation of Wnt5A signaling, on the other hand, enhances clearance of the infection. Macrophage-mediated containment of bacterial infection in our study is dependant on Wnt5A-induced Rac1/Disheveled activation and cytochalasin D inhibitable actin assembly, which is associated with ULK1 kinase activity and LC3BII accumulation. Our experimental findings are consistent with Wnt5A signaling-dependent induction of autophagic killing (xenophagy) of PA and SP, as further substantiated by transmission electron microscopy. Overall, our study unveils the prevalence of a Wnt5A—Rac1—Disheveled-mediated actin-associated autophagy circuit as an important component of innate immunity in host macrophages that may turn out crucial for restricting infection by leading bacterial pathogens.