Machine learning-based prediction of relapse in rheumatoid arthritis patients using data on ultrasound examination and blood test.

Recent effective therapies enable most rheumatoid arthritis (RA) patients to achieve remission; however, some patients experience relapse. We aimed to predict relapse in RA patients through machine learning (ML) using data on ultrasound (US) examination and blood test. Overall, 210 patients with RA in remission at baseline were dichotomized into remission (n = 150) and relapse (n = 60) based on the disease activity at 2-year follow-up. Three ML classifiers [Logistic Regression, Random Forest, and extreme gradient boosting (XGBoost)] and data on 73 features (14 US examination data, 54 blood test data, and five data on patient information) at baseline were used for predicting relapse. The best performance was obtained using the XGBoost classifier (area under the receiver operator characteristic curve (AUC) = 0.747), compared with Random Forest and Logistic Regression (AUC = 0.719 and 0.701, respectively). In the XGBoost classifier prediction, ten important features, including wrist/metatarsophalangeal superb microvascular imaging scores, were selected using the recursive feature elimination method. The performance was superior to that predicted by researcher-selected features, which are conventional prognostic markers. These results suggest that ML can provide an accurate prediction of relapse in RA patients, and the use of predictive algorithms may facilitate personalized treatment options.

Region specificity of rheumatoid foot symptoms associated with ultrasound-detected synovitis and joint destruction.

OBJECTIVES: We aimed to clarify the clinical implication of ultrasound (US)-detected foot joint inflammation in tightly controlled patients with rheumatoid arthritis (RA). METHODS: We evaluated bilateral foot joints (second to fifth metatarsophalangeal joints of forefoot; tarsometatarsal, cuneonavicular and midtarsal joints of midfoot) of 430 RA patients for synovitis using Power Doppler (PD) imaging by US. We made a cross-sectional and a 3-year longitudinal analysis about the associations of US-detected synovitis with clinical, laboratory and radiographic data as well as foot-specific outcomes using a self-administered foot evaluation questionnaire (SAFE-Q). RESULTS: The US-detected foot synovitis was seen in 28% of patients. The US-detected synovitis was closely related to 28 joint-disease activity score (DAS28) more in the forefoot than in the midfoot, while related to joint destruction in both. Multiple regression analyses showed significant associations between midfoot PD positivity and SAFE-Q in the remission group. SAFE-Q was worsened after the 3-year interval, but PD positivity at baseline did not contribute to the changes. On the other hand, destruction of the joints with US-detected synovitis significantly progressed in 3 years than with not. CONCLUSIONS: US-detected synovitis on foot joints were related to systemic inflammation, clinical symptoms, and future joint destruction with region specificity.

Positive rate and prognostic significance of the superb microvascular imaging signal in joints of rheumatoid arthritis patients in remission with normal C-reactive protein levels and erythrocyte sedimentation rates.

PURPOSE: This study aimed to evaluate the positive rate and prognostic significance of superb microvascular imaging (SMI) in rheumatoid arthritis (RA) patients in remission with normal C-reactive protein (CRP) levels and erythrocyte sedimentation rates (ESR). METHODS: The study enrolled 112 RA patients, and ultrasound (US) assessment was performed on 28 joints of each patient. RESULTS: The SMI signal-positive rates for each joint were: metacarpophalangeal (MCP) joints: 20.5%, wrist joints: 43.8%, metatarsophalangeal (MTP) joints: 17.0%, and other foot joints: 25.0%. Investigation of the prognostic significance of the SMI signal in each joint revealed that only in the MTP joints was the total score of the SMI signal in the patients with relapse significantly higher than that in the patients with remission (P = 0.01). Comparison of the receiver operating characteristics curves for predicting relapse showed that the area under the curve (AUC) of the MTP joints was the highest (AUC = 0.66) of the investigated joints. The optimal threshold for the total MTP SMI score was 1 (accuracy = 83.3%). Positive/negative data of the SMI signal in the MTP joints were not significantly associated with the values of conventional disease activity markers. CONCLUSION: In RA patients in remission with normal CRP and ESR levels, the percentage of positive SMI signal was highest in the wrist joints. However, the accuracy of the SMI signal for predicting relapse was greatest for the MTP joints, suggesting that US assessment of the MTP joints by SMI is useful for predicting relapse in these patients.

Severe joint deformity and patient global assessment of disease are associated with discrepancies between sonographic and clinical remission: A cross-sectional study of rheumatoid arthritis patients.

OBJECTIVE: Although recent clinical trials showed that ultrasound (US) remission is not required to achieve good outcomes at the group level, it currently remains unclear whether the prognosis of individual patients in clinical remission, but not US remission, i.e. those with subclinical sonographic synovitis (SSS), is favorable. However, it is no longer acceptable to perform US on all patients in order to identify those with SSS. Therefore, the present study was initiated to elucidate the conditions under which SSS is frequently detected. METHODS: In total, 563 consecutive RA patients were recruited. Bilateral 2-5 MCP, wrist, ankle, and 2-5 MTP joints were scanned by US, and Gray scale and Power Doppler (PD) images were scored semi-quantitatively. Clinical data were obtained by physicians who were blind to US results. Changes in the modified Total Sharp Score (mTSS) of tocilizumab (TCZ) users were calculated. RESULTS: A total of 402 patients were included. SSS was more frequently detected in patients with more severe joint deformity, even if they were in remission. In contrast, a high Patient Global Assessment of Disease (PtGA) did not reflect SSS. Furthermore, the relationship between PtGA and PD scores was weak. Although the frequency of SSS was high in TCZ user, the presence of SSS in TCZ users not always results in the progression of mTSS. CONCLUSIONS: While remission is overestimated in patients with severe joint deformity, underestimations may occur in those who do not fulfill remission criteria because of a high PtGA.

Prediction of recurrence and remission using superb microvascular imaging in rheumatoid arthritis.

PURPOSE: Ultrasound is commonly used to assess the degree of synovitis in patients with rheumatoid arthritis (RA); however, it is unclear which joints are optimal for evaluating and predicting recurrence and remission. PATIENTS AND METHODS: In 293 RA patients enrolled in the KURAMA cohort, 28 joints were assessed by ultrasound. RESULTS: Results from patients in remission in both 2015 and 2017 (Group 1, n = 152) were compared with those from patients in remission in 2015 and non-remission in 2017 (Group 2, n = 60). The SMI scores for total (3.1 vs. 6.3, P = 0.004), MCP2-5 (1.1 vs. 2.4, P = 0.03), wrist (0.9 vs. 2.1, P = 0.0003), MTP2-5 (0.4 vs. 1.0, P = 0.03), and Lisfranc joints (0.07 vs. 0.25, P = 0.04) were significantly higher for Group 2. When those in non-remission in 2015 and remission in 2017 (Group 3, n = 27) were compared with those in remission in 2015 and non-remission in both 2015 and 2017 (Group 4, n = 54), the GS-SMI combined score (3.0 vs. 5.0, P = 0.04) and SMI score (1.5 vs. 2.9, P = 0.04) for MCP2-5 joints were significantly higher for Group 4. Multivariate logistic regression analysis identified "wrist SMI score ≧ 1" as an independent prognostic factor for recurrence (odds ratio 3.08, P = 0.001) and "MCP2-5 GS-SMI combined score ≦ 4" as an independent prognostic factor for remission (odds ratio 3.25, P = 0.048). CONCLUSION: We identified the optimal joint cut-off scores for predicting recurrence and remission in RA patients. Risk-stratification therapy based on the ultrasound scores may improve outcome and quality of life for patients with RA.

Coordinating Tissue Regeneration Through Transforming Growth Factor-ß Activated Kinase 1 Inactivation and Reactivation.

Aberrant wound healing presents as inappropriate or insufficient tissue formation. Using a model of musculoskeletal injury, we demonstrate that loss of transforming growth factor-ß activated kinase 1 (TAK1) signaling reduces inappropriate tissue formation (heterotopic ossification) through reduced cellular differentiation. Upon identifying increased proliferation with loss of TAK1 signaling, we considered a regenerative approach to address insufficient tissue production through coordinated inactivation of TAK1 to promote cellular proliferation, followed by reactivation to elicit differentiation and extracellular matrix production. Although the current regenerative medicine paradigm is centered on the effects of drug treatment ("drug on"), the impact of drug withdrawal ("drug off") implicit in these regimens is unknown. Because current TAK1 inhibitors are unable to phenocopy genetic Tak1 loss, we introduce the dual-inducible COmbinational Sequential Inversion ENgineering (COSIEN) mouse model. The COSIEN mouse model, which allows us to study the response to targeted drug treatment ("drug on") and subsequent withdrawal ("drug off") through genetic modification, was used here to inactivate and reactivate Tak1 with the purpose of augmenting tissue regeneration in a calvarial defect model. Our study reveals the importance of both the "drug on" (Cre-mediated inactivation) and "drug off" (Flp-mediated reactivation) states during regenerative therapy using a mouse model with broad utility to study targeted therapies for disease. Stem Cells 2019;37:766-778.

Compound mutations in Bmpr1a and Tak1 synergize facial deformities via increased cell death.

BMP signaling plays a critical role in craniofacial development. Augmentation of BMPR1A signaling through neural crest-specific expression of constitutively active Bmpr1a (caBmpr1a) results in craniofacial deformities in mice. To investigate whether deletion of Tak1 may rescue the craniofacial deformities caused by enhanced Smad-dependent signaling through caBMPR1A, we generated embryos to activate transcription of caBmpr1a transgene and ablate Tak1 in neural crest derivatives at the same time. We found that deformities of the double mutant mice showed more severe than those with each single mutation, including median facial cleft and cleft palate. We found higher levels of cell death in the medial nasal and the lateral nasal processes at E10.5 in association with higher levels of p53 in the double mutant embryos. We also found higher levels of pSmad1/5/9 in the lateral nasal processes at E10.5 in the double mutant embryos. Western analyses revealed that double mutant embryos showed similar degrees of upregulation of pSmad1/5/9 with caBmpr1a or Tak1-cKO embryos while the double mutant embryos showed higher levels of phospho-p38 than caBmpr1a or Tak1-cKO embryos at E17.5, but not at E10.5. It suggested that deletion of Tak1 aggravates the craniofacial deformities of the caBmpr1a mutants by increasing p53 and phospho-p38 at different stage of embryogenesis.

Tak1, Smad4 and Trim33 redundantly mediate TGF-ß3 signaling during palate development.

Transforming growth factor-beta3 (TGF-ß3) plays a critical role in palatal epithelial cells by inducing palatal epithelial fusion, failure of which results in cleft palate, one of the most common birth defects in humans. Recent studies have shown that Smad-dependent and Smad-independent pathways work redundantly to transduce TGF-ß3 signaling in palatal epithelial cells. However, detailed mechanisms by which this signaling is mediated still remain to be elucidated. Here we show that TGF-ß activated kinase-1 (Tak1) and Smad4 interact genetically in palatal epithelial fusion. While simultaneous abrogation of both Tak1 and Smad4 in palatal epithelial cells resulted in characteristic defects in the anterior and posterior secondary palate, these phenotypes were less severe than those seen in the corresponding Tgfb3 mutants. Moreover, our results demonstrate that Trim33, a novel chromatin reader and regulator of TGF-ß signaling, cooperates with Smad4 during palatogenesis. Unlike the epithelium-specific Smad4 mutants, epithelium-specific Tak1:Smad4- and Trim33:Smad4-double mutants display reduced expression of Mmp13 in palatal medial edge epithelial cells, suggesting that both of these redundant mechanisms are required for appropriate TGF-ß signal transduction. Moreover, we show that inactivation of Tak1 in Trim33:Smad4 double conditional knockouts leads to the palatal phenotypes which are identical to those seen in epithelium-specific Tgfb3 mutants. To conclude, our data reveal added complexity in TGF-ß signaling during palatogenesis and demonstrate that functionally redundant pathways involving Smad4, Tak1 and Trim33 regulate palatal epithelial fusion.

TGF-ß-activated kinase 1 (Tak1) mediates agonist-induced Smad activation and linker region phosphorylation in embryonic craniofacial neural crest-derived cells.

BACKGROUND: The role of Smad-independent TGF-ß signaling in craniofacial development is poorly elucidated. RESULTS: In craniofacial mesenchymal cells, Tak1 regulates both R-Smad C-terminal and linker region phosphorylation in TGF-ß signaling. CONCLUSION: Tak1 plays an irreplaceable role in craniofacial ecto-mesenchyme during embryogenesis. SIGNIFICANCE: Understanding the mechanisms of TGF-ß signaling contributes to knowledge of pathogenetic mechanisms underlying common craniofacial birth defects. Although the importance of TGF-ß superfamily signaling in craniofacial growth and patterning is well established, the precise details of its signaling mechanisms are still poorly understood. This is in part because of the concentration of studies on the role of the Smad-dependent (so-called "canonical") signaling pathways relative to the Smad-independent ones in many biological processes. Here, we have addressed the role of TGF-ß-activated kinase 1 (Tak1, Map3k7), one of the key mediators of Smad-independent (noncanonical) TGF-ß superfamily signaling in craniofacial development, by deleting Tak1 specifically in the neural crest lineage. Tak1-deficient mutants display a round skull, hypoplastic maxilla and mandible, and cleft palate resulting from a failure of palatal shelves to appropriately elevate and fuse. Our studies show that in neural crest-derived craniofacial ecto-mesenchymal cells, Tak1 is not only required for TGF-ß- and bone morphogenetic protein-induced p38 Mapk activation but also plays a role in agonist-induced C-terminal and linker region phosphorylation of the receptor-mediated R-Smads. Specifically, we demonstrate that the agonist-induced linker region phosphorylation of Smad2 at Thr-220, which has been shown to be critical for full transcriptional activity of Smad2, is dependent on Tak1 activity and that in palatal mesenchymal cells TGFßRI and Tak1 kinases mediate both overlapping and distinct TGF-ß2-induced transcriptional responses. To summarize, our results suggest that in neural crest-derived ecto-mesenchymal cells, Tak1 provides a critical point of intersection in a complex dialogue between the canonical and noncanonical arms of TGF-ß superfamily signaling required for normal craniofacial development.

TAK1 (MAP3K7) signaling regulates hematopoietic stem cells through TNF-dependent and -independent mechanisms.

A cytokine/stress signaling kinase Tak1 (Map3k7) deficiency is known to impair hematopoietic progenitor cells. However, the role of TAK1 signaling in the stem cell function of the hematopoietic system is not yet well defined. Here we characterized hematopoietic stem cells (HSCs) harboring deletion of Tak1 and its activators, Tak1 binding proteins 1 and 2 (Tab1 and Tab2) using a competitive transplantation assay in a mouse model. Tak1 single or Tab1/Tab2 double deletions completely eliminated the reconstitution activity of HSCs, whereas Tab1 or Tab2 single deletion did not cause any abnormality. Tak1 single or Tab1/Tab2 double deficient lineage-negative, Sca-1(+), c-Kit(+) (LSK) cells did not proliferate and underwent cell death. We found that Tnfr1 deficiency restored the reconstitution activity of Tak1 deficient bone marrow cells for 6-18 weeks. However, the reconstitution activity of Tak1- and Tnfr1-double deficient bone marrow cells declined over the long term, and the number of phenotypically identified long-term hematopoietic stem cells were diminished. Our results indicate that TAB1- or TAB2-dependent activation of TAK1 is required for maintenance of the hematopoietic system through two mechanisms: one is prevention of TNF-dependent cell death and the other is TNF-independent maintenance of long-term HSC.

TAK1 kinase signaling regulates embryonic angiogenesis by modulating endothelial cell survival and migration.

TGF-ß activated kinase 1 (TAK1) is a mediator of various cytokine signaling pathways. Germline deficiency of Tak1 causes multiple abnormalities, including dilated blood vessels at midgestation. However, the mechanisms by which TAK1 regulates vessel formation have not been elucidated. TAK1 binding proteins 1 and 2 (TAB1 and TAB2) are activators of TAK1, but their roles in embryonic TAK1 signaling have not been determined. In the present study, we characterized mouse embryos harboring endothelial-specific deletions of Tak1, Tab1, or Tab2 and found that endothelial TAK1 and TAB2, but not TAB1, were critically involved in vascular formation. TAK1 deficiency in endothelial cells caused increased cell death and vessel regression at embryonic day 10.5 (E10.5). Deletion of TNF signaling largely rescued endothelial cell death in TAK1-deficient embryos at E10.5. However, embryos deficient in both TAK1 and TNF signaling still exhibited dilated capillary networks at E12.5. TAB2 deficiency caused reduced TAK1 activity, resulting in abnormal capillary blood vessels, similar to the compound deficiency of TAK1 and TNF signaling. Ablation of either TAK1 or TAB2 impaired cell migration and tube formation. Our results show that endothelial TAK1 signaling is important for 2 biologic processes in angiogenesis: inhibiting TNF-dependent endothelial cell death and promoting TNF-independent angiogenic cell migration.

Epithelial transforming growth factor ß-activated kinase 1 (TAK1) is activated through two independent mechanisms and regulates reactive oxygen species.

Dysregulation in cellular redox systems results in accumulation of reactive oxygen species (ROS), which are causally associated with a number of disease conditions. Transforming growth factor ß-activated kinase 1 (TAK1) is a signaling intermediate of innate immune signaling pathways and is critically involved in the redox regulation in vivo. Ablation of TAK1 causes accumulation of ROS, resulting in epithelial cell death and inflammation. Here we determine the mechanism by which TAK1 kinase is activated in epithelial tissues. TAB1 and TAB2 are structurally unrelated TAK1 binding protein partners. TAB2 is known to mediate polyubiquitin chain-dependent TAK1 activation in innate immune signaling pathways, whereas the role of TAB1 is not defined. We found that epithelial-specific TAB1 and TAB2 double- but not TAB1 or TAB2 single-knockout mice phenocopied epithelial-specific TAK1 knockout mice. We demonstrate that phosphorylation-dependent basal activity of TAK1 is dependent on TAB1. Ablation of both TAB1 and TAB2 diminished the activity of TAK1 in vivo and causes accumulation of ROS in the epithelial tissues. These results demonstrate that epithelial TAK1 activity is regulated through two unique, TAB1-dependent basal and TAB2-mediated stimuli-dependent mechanisms.

The cell adhesion gene PVRL3 is associated with congenital ocular defects.

We describe a male patient (patient DGAP113) with a balanced translocation, 46,XY,t(1;3)(q31.3;q13.13), severe bilateral congenital cataracts, CNS abnormalities and mild developmental delay. Fluorescence in situ hybridization (FISH) and suppression PCR demonstrated that the chromosome 3 breakpoint lies ~515 kb upstream of the PVRL3 gene, while the chromosome 1 breakpoint lies ~50 kb upstream of the NEK7 gene. Despite the fact that NEK7 is closer to a translocation breakpoint than PVRL3, NEK7 transcript levels are unaltered in patient DGAP113 lymphoblastoid cells and Nek7-deficient mice exhibit no detectable ocular phenotype. In contrast, the expression of PVRL3, which encodes the cell adhesion protein Nectin 3, is significantly reduced in patient DGAP113 lymphoblastoid cells, likely due to a position effect caused by the chromosomal translocation. Nectin 3 is expressed in the mouse embryonic ciliary body and lens. Moreover, Pvrl3 knockout mice as well as a spontaneous mouse mutant ari (anterior retinal inversion), that maps to the Pvrl3 locus, exhibit lens and other ocular defects involving the ciliary body. Collectively, these data identify PVRL3 as a critical gene involved in a Nectin-mediated cell-cell adhesion mechanism in human ocular development.

TAK1-binding protein 1, TAB1, mediates osmotic stress-induced TAK1 activation but is dispensable for TAK1-mediated cytokine signaling.

TAK1 kinase is an indispensable intermediate in several cytokine signaling pathways including tumor necrosis factor, interleukin-1, and transforming growth factor-beta signaling pathways. TAK1 also participates in stress-activated intracellular signaling pathways such as osmotic stress signaling pathway. TAK1-binding protein 1 (TAB1) is constitutively associated with TAK1 through its C-terminal region. Although TAB1 is known to augment TAK1 catalytic activity when it is overexpressed, the role of TAB1 under physiological conditions has not yet been identified. In this study, we determined the role of TAB1 in TAK1 signaling by analyzing TAB1-deficient mouse embryonic fibroblasts (MEFs). Tumor necrosis factor- and interleukin-1-induced activation of TAK1 was entirely normal in Tab1-deficient MEFs and could activate both mitogen-activated protein kinases and NF-kappaB. In contrast, we found that osmotic stress-induced activation of TAK1 was largely impaired in Tab1-deficient MEFs. Furthermore, we showed that the C-terminal 68 amino acids of TAB1 were sufficient to mediate osmotic stress-induced TAK1 activation. Finally, we attempted to determine the mechanism by which TAB1 activates TAK1. We found that TAK1 is spontaneously activated when the concentration is increased and that it is totally dependent on TAB1. Cell shrinkage under the osmotic stress condition increases the concentration of TAB1-TAK1 and may oligomerize and activate TAK1 in a TAB1-dependent manner. These results demonstrate that TAB1 mediates TAK1 activation only in a subset of TAK1 pathways that are mediated through spontaneous oligomerization of TAB1-TAK1.

Generation of a conditional mutant allele for Tab1 in mouse.

TAK1 binding protein 1 (TAB1) binds and induces autophosphorylation of TGF-beta activating kinase (TAK1). TAK1, a mitogen-activated kinase kinase kinase, is involved in several distinct signaling pathways including non-Smad pathways for TGF-beta superfamily members and inflammatory responses caused by cytokines. Conventional disruption of the murine Tab1 gene results in late gestational lethality showing intraventricular septum defects and underdeveloped lung alveoli. To gain a better understanding of the roles of TAB1 in different tissues, at different stages of development, and in pathological conditions, we generated Tab1 floxed mice in which the loxP sites flank Exons 9 and 10 to remove the C-terminal region of TAB1 protein necessary for activation of TAK1. We demonstrate that Cre-mediated recombination using Sox2-Cre, a Cre line expressed in the epiblast during early embryogenesis, results in deletion of the gene and protein. These homozygous Cre-recombined null embryos display an identical phenotype to conventional null embryos. This animal model will be useful in revealing distinct roles of TAB1 in different tissues at different stages.

Enterocyte-derived TAK1 signaling prevents epithelium apoptosis and the development of ileitis and colitis.

Recent studies have revealed that TAK1 kinase is an essential intermediate in several innate immune signaling pathways. In this study, we investigated the role of TAK1 signaling in maintaining intestinal homeostasis by generating enterocyte-specific constitutive and inducible gene-deleted TAK1 mice. We found that enterocyte-specific constitutive TAK1-deleted mice spontaneously developed intestinal inflammation as observed by histological analysis and enhanced expression of IL-1beta, MIP-2, and IL-6 around the time of birth, which was accompanied by significant enterocyte apoptosis. When TAK1 was deleted in the intestinal epithelium of 4-wk-old mice using an inducible knockout system, enterocytes underwent apoptosis and intestinal inflammation developed within 2-3 days following the initiation of gene deletion. We found that enterocyte apoptosis and intestinal inflammation were strongly attenuated when enterocyte-specific constitutive TAK1-deleted mice were crossed to TNF receptor 1(-/-) mice. However, these mice later (>14 days) developed ileitis and colitis. Thus, TAK1 signaling in enterocytes is essential for preventing TNF-dependent epithelium apoptosis and the TNF-independent development of ileitis and colitis. We propose that aberration in TAK1 signaling might disrupt intestinal homeostasis and favor the development of inflammatory disease.

Role of cell adhesion molecule nectin-3 in spermatid development.

Seminiferous epithelia of the testes contain two types of intercellular junctions: Sertoli-Sertoli junctions and Sertoli-spermatid junctions. The former junctions are equipped with tight and adherens junctions while the latter junctions are not. Ca2+ -independent immunoglobulin-like cell-cell adhesion molecules, nectin-2 and nectin-3, asymmetrically localize at the Sertoli cell side and at the spermatid side of Sertoli-spermatid junctions, respectively. They heterophilically trans-interact to make contact between the two cells. Nectin-2(-/-) mice have shown male-specific infertility, disrupted Sertoli-spermatid junctions and morphologically impaired spermatid development. Here we report testicular phenotypes of nectin-3(-/-) mice exhibiting male-specific infertility. Nectin-3(-/-) mice had defects in the later steps of sperm morphogenesis including distorted nuclei and abnormal distribution of mitochondria, as well as in localization of nectin-2 at the Sertoli-spermatid junctions. Transplantation of wild-type spermatogenic stem cells into the nectin-3(-/-) testes partially rescued these defects in sperm morphogenesis. These results indicate that the heterophilic trans-interaction between nectin-2 and nectin-3 is essential for the formation and maintenance of Sertoli-spermatid junctions that plays a critical role in spermatid development.

Roles of cell-adhesion molecules nectin 1 and nectin 3 in ciliary body development.

Nectins are Ca2+-independent immunoglobulin-like cell-cell-adhesion molecules consisting of four members. Nectins homophilically and heterophilically trans-interact to form a variety of cell-cell junctions, including cadherin-based adherens junctions in epithelial cells and fibroblasts in culture, synaptic junctions in neurons, and Sertoli cell-spermatid junctions in the testis, in cooperation with, or independently of, cadherins. To further explore the function of nectins, we generated nectin 1-/- and nectin 3-/-)mice. Both nectin 1-/- and nectin 3-/- mice showed a virtually identical ocular phenotype, microphthalmia, accompanied by a separation of the apex-apex contact between the pigment and non-pigment cell layers of the ciliary epithelia. Immunofluorescence and immunoelectron microscopy revealed that nectin 1 and nectin 3, but not nectin 2, localized at the apex-apex junctions between the pigment and non-pigment cell layers of the ciliary epithelia. However, nectin 1-/- and nectin 3-/- mice showed no impairment of the apicolateral junctions between the pigment epithelia where nectin 1, nectin 2 and nectin 3 localized, or of the apicolateral junctions between the non-pigment epithelia where nectin 2 and nectin 3, but not nectin 1, localized. These results indicate that the heterophilic trans-interaction between nectin 1 and nectin 3 plays a sentinel role in establishing the apex-apex adhesion between the pigment and non-pigment cell layers of the ciliary epithelia that is essential for the morphogenesis of the ciliary body.

Nectin-dependent localization of ZO-1 at puncta adhaerentia junctions between the mossy fiber terminals and the dendrites of the pyramidal cells in the CA3 area of adult mouse hippocampus.

Nectin and afadin constitute a novel intercellular adhesion system that organizes adherens junctions in cooperation with the cadherin-catenin system in epithelial cells. Nectin is a Ca(2+)-independent immunoglobulin-like adhesion molecule and afadin is an actin filament (F-actin)-binding protein that connects nectin to the actin cytoskeleton. At the puncta adhaerentia junctions (PAs) between the mossy fiber terminals and the dendrites of the pyramidal cells in the CA3 area of the adult mouse hippocampus, the nectin-afadin system also colocalizes with the cadherin-catenin system and has a role in the formation of synapses. ZO-1 is another F-actin-binding protein that localizes at tight junctions (TJs) and connects claudin to the actin cytoskeleton in epithelial cells. The nectin-afadin system is able to recruit ZO-1 to the nectin-based cell-cell adhesion sites in nonepithelial cells that have no TJs. In the present study, we investigated the localization of ZO-1 in the mouse hippocampus. Immunofluorescence and immunoelectron microscopy revealed that ZO-1 also localized at the PAs between the mossy fiber terminals and the dendrites of the pyramidal cells in the CA3 area of the adult mouse hippocampus, as described for afadin. ZO-1 colocalized with afadin during the development of synaptic junctions and PAs. Microbeads coated with the extracellular fragment of nectin, which interacts with cellular nectin, recruited both afadin and ZO-1 to the bead-cell contact sites in cultured rat hippocampal neurons. These results indicate that ZO-1 colocalizes with nectin and afadin at the PAs and that the nectin-afadin system is involved in the localization of ZO-1.

Role of the second immunoglobulin-like loop of nectin in cell-cell adhesion.

Nectin is a Ca(2+)-independent immunoglobulin (Ig)-like cell-cell adhesion molecule that forms cell-cell adherens junctions cooperatively with E-cadherin in a variety of cells. Nectin has one transmembrane segment and three Ig-like loops in the extracellular region. The first Ig-like loop is essential for the trans-dimer formation of nectin of two neighboring cells, causing cell-cell adhesion. We show here that the second Ig-like loop is essential for the cis-dimer formation of nectin on the same cell, and that the cis-dimer formation is essential for the trans-dimer formation.