Biliary atresia: insights into mechanisms using a toxic model of the disease including Wnt and Hippo signaling pathways and microtubules.

BACKGROUND: Mechanisms underlying bile duct injury in biliary atresia (BA) remain unclear and mechanisms of bile duct repair are unknown. This study aimed to explore the roles of microtubule instability and Wnt and Hippo signaling pathways in a biliatresone-induced BA model. METHODS: Using primary murine neonatal cholangiocytes in both 2D and 3D cultures, and ex-vivo extra hepatic bile ducts (EHBD) which also has peri-cholangiocyte area, we analyzed injury and recovery processes. Injury was induced by the toxin biliatresone and recovery was induced by toxin wash-out. RESULTS: Microtubule stabilizer paclitaxel prevented biliatresone-induced injury, both to cholangiocytes as well as reduced periductal αSMA stain, this process is mediated by decreased glutathione levels. RhoU and Wnt11 (Wnt signaling) and Pard6g and Amotl1 (Hippo signaling) are involved in both injury and recovery processes, with the latter acting upstream to Wnt signaling. CONCLUSIONS: Early stages of biliatresone-induced EHBD injury in cholangiocytes and periductal structures are reversible. Wnt and Hippo signaling pathways play crucial roles in injury and recovery, providing insights into BA injury mechanisms and potential recovery avenues. IMPACT: Microtubule stabilization prevents cholangiocyte injury and lumen obstruction in a toxic model of biliary atresia (biliatresone induced). Early stages of biliatresone-induced injury, affecting both cholangiocytes and periductal structures, are reversible. Both Wnt and Hippo signaling pathways play a crucial role in bile duct injury and recovery, with a noted interplay between the two. Understanding mechanisms of cholangiocyte recovery is imperative to unveil potential therapeutic avenues.

Tissue markers may predict treatment response to antitumor necrosis factor-α agents in children with Crohn's disease.

OBJECTIVES: Patients with moderate-severe Crohn's disease (CD) who are treated with antitumor necrosis factor alpha (TNF-α) agents may be subjected to primary nonresponse or partial response. We aimed to identify tissue markers that may predict response to these agents. METHODS: Pediatric patients (6-18 years) with either ileal or ileo-colonic CD who were treated with anti-TNF-α were stratified into three different groups based on their overall response to therapy at the end of induction including clinical and laboratory parameters (group 1-full responders [FR], group 2-partial responders [PR], group 3-nonresponders [NR]). Seven tissue markers (fibronectin, interleukin [IL]-23R, IL-23, TNF-α, collagen-III, IL-13R, and hypoxia-inducible factors [HIF]-1α) were evaluated. Immunofluorescence (IF) analyses were performed on biopsies from the terminal ileum, which were retrieved up to 6 months before treatment initiation. RESULTS: Twenty-six CD patients (16 [61.5%] males; age 13.9 ± 2.9 years), including 8 (30.8%) with ileal disease and 18 (69.2%) with ileo-colonic disease, were enrolled. Terminal ileum biopsies from nine patients from group 1, nine from group 2, and eight from group 3 were evaluated. Three antibodies were found to be significantly different between NR and FR groups; Collagen III and fibronectin stains were significantly more prominent in NR patients, while TNF-α stain was significantly more pronounced in FR, p < 0.05 for each. PR could not have been predicted with neither of markers. CONCLUSIONS: Decreased tissue IF intensity of fibronectin and collagen III and increased intensity of TNF-α may predict response to anti-TNF-α treatment.

Periductal bile acid exposure causes cholangiocyte injury and fibrosis.

INTRODUCTION: Bile duct integrity is essential for the maintenance of the structure and function of the biliary tree. We previously showed that cholangiocyte injury in a toxic model of biliary atresia leads to increased monolayer permeability. Increased epithelial permeability was also shown in other cholangiopathies. We hypothesized that after initial cholangiocyte injury, leakage of bile acids into the duct submucosa propagates cholangiocyte damage and fibrosis. We thus aimed to determine the impact of bile acid exposure on cholangiocytes and the potential therapeutic effect of a non-toxic bile acid. MATERIALS AND METHODS: Extrahepatic bile duct explants were isolated from adult and neonatal BALB/c mice. Explants were cultured with or without glycochenodeoxycholic acid and ursodeoxycholic acid. They were then fixed and stained. RESULTS: Explants treated with glycochenodeoxycholic acid demonstrated cholangiocyte injury with monolayer disruption and partial lumen obstruction compared to control ducts. Masson's trichrome stains revealed increased collagen fibers. Myofibroblast marker α-SMA stains were significantly elevated in the periductal region. The addition of ursodeoxycholic acid resulted in decreased cholangiocyte injury and reduced fibrosis. CONCLUSIONS: Bile acid leakage into the submucosa after initial cholangiocyte injury may serve as a possible mechanism of disease propagation and progressive fibrosis in cholangiopathies.

Targeting the actin nucleation promoting factor WASp provides a therapeutic approach for hematopoietic malignancies.

Cancer cells depend on actin cytoskeleton rearrangement to carry out hallmark malignant functions including activation, proliferation, migration and invasiveness. Wiskott-Aldrich Syndrome protein (WASp) is an actin nucleation-promoting factor and is a key regulator of actin polymerization in hematopoietic cells. The involvement of WASp in malignancies is incompletely understood. Since WASp is exclusively expressed in hematopoietic cells, we performed in silico screening to identify small molecule compounds (SMCs) that bind WASp and promote its degradation. We describe here one such identified molecule; this WASp-targeting SMC inhibits key WASp-dependent actin processes in several types of hematopoietic malignancies in vitro and in vivo without affecting naïve healthy cells. This small molecule demonstrates limited toxicity and immunogenic effects, and thus, might serve as an effective strategy to treat specific hematopoietic malignancies in a safe and precisely targeted manner.

Salivary C-reactive protein-a possible predictor of serum levels in pediatric acute respiratory illness.

Identifying the etiology of an acute respiratory infection in children is a well-known challenge. In this study, we evaluated the correlation between salivary C-reactive protein (CRP) and its serum counterpart, which is known to be higher in bacterial infections but necessitates a venipuncture. Salivary and serum CRPs were measured in children with an acute respiratory illness, aged 2 months to 18 years. Pearson's correlation coefficients were used to measure correlation. Discrimination of the salivary CRP levels for predicting serum levels above 100 mg/L was calculated and compared to serum CRP levels. Sensitivity and specificity were similarly calculated. Salivary CRP was measured in 104 samples. Levels correlated significantly and positively with serum CRP levels (r = 0.670, p<0.001). Area under the curve for predicting serum CRP levels of 100 mg/L was 0.848. For a salivary CRP concentration above 32,610 ng/L, the sensitivity and specificity were 69% and 93%, respectively, for accurately predicting a serum CRP level ≥100 mg/L.Conclusions: Salivary CRP can be used in the pediatric acute setting due to its high specificity for predicting elevated serum levels without the need for venipuncture. Further studies are required to achieve higher sensitivity rates. What is known: • Salivary C-reactive protein has shown correlation to its serum counterpart, mainly in healthy children, adults, and ill neonates. What is new: • In a large population of children with acute respiratory illness, aged 2 months to 18 years, salivary C-reactive protein showed high specificity for predicting elevated serum levels, thus indicating its potential as a diagnostic tool.

Extrahepatic cholangiocyte obstruction is mediated by decreased glutathione, Wnt and Notch signaling pathways in a toxic model of biliary atresia.

Biliary atresia is a neonatal liver disease with extrahepatic bile duct obstruction and progressive liver fibrosis. The etiology and pathogenesis of the disease are unknown. We previously identified a plant toxin, biliatresone, responsible for biliary atresia in naturally-occurring animal models, that causes cholangiocyte destruction in in-vitro models. Decreases in reduced glutathione (GSH) mimic the effects of biliatresone, and agents that replenish cellular GSH ameliorate the effects of the toxin. The goals of this study were to define signaling pathways downstream of biliatresone that lead to cholangiocyte destruction and to determine their relationship to GSH. Using cholangiocyte culture and 3D cholangiocyte spheroid cultures, we found that biliatresone and decreases in GSH upregulated RhoU/Wrch1, a Wnt signaling family member, which then mediated an increase in Hey2 in the NOTCH signaling pathway, causing downregulation of the transcription factor Sox17. When these genes were up- or down-regulated, the biliatresone effect on spheroids was phenocopied, resulting in lumen obstruction. Biopsies of patients with biliary atresia demonstrated increased RhoU/Wrch1 and Hey2 expression in cholangiocytes. We present a novel pathway of cholangiocyte injury in a model of biliary atresia, which is relevant to human BA and may suggest potential future therapeutics.

Actin retrograde flow controls natural killer cell response by regulating the conformation state of SHP-1.

Natural killer (NK) cells are a powerful weapon against viral infections and tumor growth. Although the actin-myosin (actomyosin) cytoskeleton is crucial for a variety of cellular processes, the role of mechanotransduction, the conversion of actomyosin mechanical forces into signaling cascades, was never explored in NK cells. Here, we demonstrate that actomyosin retrograde flow (ARF) controls the immune response of primary human NK cells through a novel interaction between ß-actin and the SH2-domain-containing protein tyrosine phosphatase-1 (SHP-1), converting its conformation state, and thereby regulating NK cell cytotoxicity. Our results identify ARF as a master regulator of the NK cell immune response. Since actin dynamics occur in multiple cellular processes, this mechanism might also regulate the activity of SHP-1 in additional cellular systems.

New Structural Insights into Formation of the Key Actin Regulating WIP-WASp Complex Determined by NMR and Molecular Imaging.

Wiskott-Aldrich syndrome protein (WASp) is exclusively expressed in hematopoietic cells and responsible for actin-dependent processes, including cellular activation, migration, and invasiveness. The C-terminal domain of WASp-Interacting Protein (WIP) binds to WASp and regulates its activity by shielding it from degradation in a phosphorylation dependent manner as we previously demonstrated. Mutations in the WAS-encoding gene lead to the primary immunodeficiencies Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT). Here, we shed a first structural light upon this function of WIP using nuclear magnetic resonance (NMR) and in vivo molecular imaging. Coexpression of fragments WASp(20-158) and WIP(442-492) allowed the purification and structural characterization of a natively folded complex, determined to form a characteristic pleckstrin homology domain with a mixed α/ß-fold and central two-winged ß-sheet. The WIP-derived peptide, unstructured in its free form, wraps around and interacts with WASp through short structural elements. Förster resonance energy transfer (FRET) and biochemical experiments demonstrated that, of these elements, WIP residues 454-456 are the major contributor to WASp affinity, and the previously overlooked residues 449-451 were found to have the largest effect upon WASp ubiquitylation and, presumably, degradation. Results obtained from this complementary combination of technologies link WIP-WASp affinity to protection from degradation. Our findings about the nature of WIP·WASp complex formation are relevant for ongoing efforts to understand hematopoietic cell behavior, paving the way for new therapeutic approaches to WAS and XLT.

A conformational change within the WAVE2 complex regulates its degradation following cellular activation.

WASp family Verprolin-homologous protein-2 (WAVE2), a member of the Wiskott-Aldrich syndrome protein (WASp) family of actin nucleation promoting factors, is a central regulator of actin cytoskeleton polymerization and dynamics. Multiple signaling pathways operate via WAVE2 to promote the actin-nucleating activity of the actin-related protein 2/3 (Arp2/3) complex. WAVE2 exists as a part of a pentameric protein complex known as the WAVE regulatory complex (WRC), which is unstable in the absence of its individual proteins. While the involvement of WAVE2 in actin polymerization has been well documented, its negative regulation mechanism is poorly characterized to date. Here, we demonstrate that WAVE2 undergoes ubiquitylation in a T-cell activation dependent manner, followed by proteasomal degradation. The WAVE2 ubiquitylation site was mapped to lysine 45, located at the N-terminus where WAVE2 binds to the WRC. Using Förster resonance energy transfer (FRET), we reveal that the autoinhibitory conformation of the WRC maintains the stability of WAVE2 in resting cells; the release of autoinhibition following T-cell activation facilitates the exposure of WAVE2 to ubiquitylation, leading to its degradation. The dynamic conformational structures of WAVE2 during cellular activation dictate its degradation.

Dephosphorylation of the adaptor LAT and phospholipase C-γ by SHP-1 inhibits natural killer cell cytotoxicity.

Natural killer (NK) cells discriminate between healthy cells and virally infected or transformed self-cells by tuning activating and inhibitory signals received through cell surface receptors. Inhibitory receptors inhibit NK cell function by recruiting and activating the tyrosine phosphatase Src homology 2 (SH2) domain-containing protein tyrosine phosphatase-1 (SHP-1) to the plasma membrane. However, to date, the guanine nucleotide exchange factor VAV1 is the only direct SHP-1 substrate identified in NK cells. We reveal that the adaptor protein linker for activation of T cells (LAT) as well as phospholipase C-γ1 (PLC-γ1) and PLC-γ2 are SHP-1 substrates. Dephosphorylation of Tyr(132) in LAT by SHP-1 in NK cells abrogated the recruitment of PLC-γ1 and PLC-γ2 to the immunological synapse between the NK cell and a cancer cell target, which reduced NK cell degranulation and target cell killing. Furthermore, the ubiquitylation of LAT by the E3 ubiquitin ligases c-Cbl and Cbl-b, which was induced by LAT phosphorylation, led to the degradation of LAT in response to the engagement of inhibitory receptors on NK cells, which abrogated NK cell cytotoxicity. Knockdown of the Cbl proteins blocked LAT ubiquitylation, which promoted NK cell function. Expression of a ubiquitylation-resistant mutant LAT blocked inhibitory receptor signaling, enabling cells to become activated. Together, these data identify previously uncharacterized SHP-1 substrates and inhibitory mechanisms that determine the response of NK cells.

WIP: more than a WASp-interacting protein.

WIP plays an important role in the remodeling of the actin cytoskeleton, which controls cellular activation, proliferation, and function. WIP regulates actin polymerization by linking the actin machinery to signaling cascades. WIP binding to WASp and to its homolog, N-WASp, which are central activators of the actin-nucleating complex Arp2/3, regulates their cellular distribution, function, and stability. By binding to WASp, WIP protects it from degradation and thus, is crucial for WASp retention. Indeed, most mutations that result in WAS, an X-linked immunodeficiency caused by defective/absent WASp activity, are located in the WIP-binding region of WASp. In addition, by binding directly to actin, WIP promotes the formation and stabilization of actin filaments. WASp-independent activities of WIP constitute a new research frontier and are discussed extensively in this article. Here, we review the current information on WIP in human and mouse systems, focusing on its associated proteins, its molecular-regulatory mechanisms, and its role as a key regulator of actin-based processes in the immune system.

Triple-color FRET analysis reveals conformational changes in the WIP-WASp actin-regulating complex.

Wiskott-Aldrich syndrome protein (WASp) is a key regulator of the actin cytoskeletal machinery. Binding of WASp-interacting protein (WIP) to WASp modulates WASp activity and protects it from degradation. Formation of the WIP-WASp complex is crucial for the adaptive immune response. We found that WIP and WASp interacted in cells through two distinct molecular interfaces. One interaction occurred between the WASp-homology-1 (WH1) domain of WASp and the carboxyl-terminal domain of WIP that depended on the phosphorylation status of WIP, which is phosphorylated by protein kinase C θ (PKCθ) in response to T cell receptor activation. The other interaction occurred between the verprolin homology, central hydrophobic region, and acidic region (VCA) domain of WASp and the amino-terminal domain of WIP. This latter interaction required actin, because it was inhibited by latrunculin A, which sequesters actin monomers. With triple-color fluorescence resonance energy transfer (3FRET) technology, we demonstrated that the WASp activation mechanism involved dissociation of the first interaction, while leaving the second interaction intact. This conformation exposed the ubiquitylation site on WASp, leading to degradation of WASp. Together, these data suggest that the activation and degradation of WASp are delicately balanced and depend on the phosphorylation state of WIP. Our molecular analysis of the WIP-WASp interaction provides insight into the regulation of actin-dependent processes.

WIP remodeling actin behind the scenes: how WIP reshapes immune and other functions.

Actin polymerization is a fundamental cellular process regulating immune cell functions and the immune response. The Wiskott-Aldrich syndrome protein (WASp) is an actin nucleation promoting factor, which is exclusively expressed in hematopoietic cells, where it plays a key regulatory role in cytoskeletal dynamics. WASp interacting protein (WIP) was first discovered as the binding partner of WASp, through the use of the yeast two hybrid system. WIP was later identified as a chaperone of WASp, necessary for its stability. Mutations occurring at the WASp homology 1 domain (WH1), which serves as the WIP binding site, were found to cause the Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT). WAS manifests as an immune deficiency characterized by eczema, thrombocytopenia, recurrent infections, and hematopoietic malignancies, demonstrating the importance of WIP for WASp complex formation and for a proper immune response. WIP deficiency was found to lead to different abnormalities in the activity of various lymphocytes, suggesting differential cell-dependent roles for WIP. Additionally, WIP deficiency causes cellular abnormalities not found in WASp-deficient cells, indicating that WIP fulfills roles beyond stabilizing WASp. Indeed, WIP was shown to interact with various binding partners, including the signaling proteins Nck, CrkL and cortactin. Recent studies have demonstrated that WIP also takes part in non immune cellular processes such as cancer invasion and metastasis, in addition to cell subversion by intracellular pathogens. Understanding of numerous functions of WIP can enhance our current understanding of activation and function of immune and other cell types.