Bub1 suppresses inflammatory arthritis-associated bone loss in mice through inhibition of TNFα-mediated osteoclastogenesis.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by synovitis, bone and cartilage destruction, and increased fracture risk with bone loss. Although disease-modifying antirheumatic drugs have dramatically improved clinical outcomes, these therapies are not universally effective in all patients because of the heterogeneity of RA pathogenesis. Therefore, it is necessary to elucidate the molecular mechanisms underlying RA pathogenesis, including associated bone loss, in order to identify novel therapeutic targets. In this study, we found that Budding uninhibited by benzimidazoles 1 (BUB1) was highly expressed in RA patients' synovium and murine ankle tissue with arthritis. As CD45+CD11b+ myeloid cells are a Bub1 highly expressing population among synovial cells in mice, myeloid cell-specific Bub1 conditional knockout (Bub1ΔLysM) mice were generated. Bub1ΔLysM mice exhibited reduced femoral bone mineral density when compared with control (Ctrl) mice under K/BxN serum-transfer arthritis, with no significant differences in joint inflammation or bone erosion based on a semi-quantitative erosion score and histological analysis. Bone histomorphometry revealed that femoral bone mass of Bub1ΔLysM under arthritis was reduced by increased osteoclastic bone resorption. RNA-seq and subsequent Gene Set Enrichment Analysis demonstrated a significantly enriched nuclear factor-kappa B pathway among upregulated genes in receptor activator of nuclear factor kappa B ligand (RANKL)-stimulated bone marrow-derived macrophages (BMMs) obtained from Bub1ΔLysM mice. Indeed, osteoclastogenesis using BMMs derived from Bub1ΔLysM was enhanced by RANKL and tumor necrosis factor-α or RANKL and IL-1ß treatment compared with Ctrl. Finally, osteoclastogenesis was increased by Bub1 inhibitor BAY1816032 treatment in BMMs derived from wildtype mice. These data suggest that Bub1 expressed in macrophages plays a protective role against inflammatory arthritis-associated bone loss through inhibition of inflammation-mediated osteoclastogenesis.

Rheumatoid arthritis (RA) is a disease caused by an abnormal immune system, resulting in inflammation, swelling, and bone destruction in the joints, along with systemic bone loss. While new medications have dramatically improved treatment efficacy, these therapies are not universally effective for all patients. Therefore, we need to understand the regulatory mechanisms behind RA, including associated bone loss, to develop better therapies. In this study, we found that Budding uninhibited by benzimidazoles 1 (Bub1) was highly expressed in inflamed joints, especially in myeloid cells, which are a type of immune cells. To explore its role, we created myeloid cell­specific Bub1 conditional knockout (cKO) mice and induced arthritis to analyze its role during arthritis. The cKO mice exhibited lower bone mineral density when compared with control mice under inflammatory arthritis because of increased osteoclastic bone resorption, without significant differences in joint inflammation or bone erosion. Further investigation showed that Bub1 prevents excessive osteoclast differentiation induced by inflammation in bone marrow macrophages. These data suggest that Bub1 in macrophages protects against bone loss caused by inflammatory arthritis, offering potential insights for developing treatments that focus on bone health.

Macrophage Polarization and Osteoclast Differentiation.

Macrophages are a key player to regulate rheumatoid arthritis pathogenesis from onset to remission. They can alter innate functions under microenvironmental conditions. To understand heterogeneous functions of macrophages in rheumatoid arthritis, several activated statuses of macrophages should be mimicked in vitro. Here, we describe basic protocols for macrophage polarization and osteoclast differentiation.

Crosstalk between synovial macrophages and fibroblasts in rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease associated with chronic inflammation of joints. Abnormally activated cells such as synovial macrophages and synovial fibroblasts induce RA pathogenesis and ultimately joint destruction. Since macrophages can change their own characteristics depending on the microenvironmental condition, it has been suggested that activation and remission of RA are regulated by crosstalk between synovial macrophages and other cells. Moreover, recent findings of heterogeneity of synovial macrophages and fibroblasts support the idea that complex interactions regulate RA from its onset to remission. Importantly, an understanding of the intercellular crosstalk in RA is far from complete. Here, we summarize the molecular mechanisms underlying the pathological development of RA with particular reference to the crosstalk between synovial macrophages and fibroblasts.

Isolation and Culture of Primary Synovial Macrophages and Fibroblasts from Murine Arthritis Tissue.

Rheumatoid arthritis is an autoimmune disease that leads to chronic inflammation of joints. Synovial macrophages and synovial fibroblasts have central roles in the pathogenesis of rheumatoid arthritis. It is important to understand the functions of both cell populations to reveal the mechanisms underlying pathological progression and remission in inflammatory arthritis. In general, in vitro experimental conditions should mimic the in vivo environment as much as possible. Primary tissue-derived cells have been used in experiments characterizing synovial fibroblasts in arthritis. In contrast, in experiments investigating the biological functions of macrophages in inflammatory arthritis, cell lines, bone marrow-derived macrophages, and blood monocyte-derived macrophages have been used. However, it is unclear whether such macrophages actually reflect the functions of tissue-resident macrophages. To obtain resident macrophages, previous protocols were modified to isolate and expand both primary macrophages and fibroblasts from synovial tissue in an inflammatory arthritis mouse model. These primary synovial cells may be useful for in vitro analysis of inflammatory arthritis.

LIM1 contributes to the malignant potential of endometrial cancer.

Introduction: The incidence of endometrial cancer (EC) has been increasing worldwide. However, because there are limited chemotherapeutic options for the treatment of EC, the prognosis of advanced-stage EC is poor. Methods: Gene expression profile datasets for EC cases registered in The Cancer Genome Atlas (TCGA) was reanalyzed. Highly expressed genes in advanced-stage EC (110 cases) compared with early-stage EC (255 cases) were extracted and Gene Ontology (GO) enrichment analysis was performed. Among the enriched genes, Kaplan-Meier (KM) plotter analysis was performed. Candidate genes expression was analyzed in HEC50B cells and Ishikawa cells by RT-qPCR. In HEC50B cells, LIM homeobox1 (LIM1) was knocked down (KD) and cell proliferation, migration, and invasion ability of the cells were evaluated. Xenografts were generated using LIM1-KD cells and tumor growth was evaluated. Ingenuity Pathway Analysis (IPA) of RNA-seq data using LIM-KD cells was performed. Expression of phospho-CREB and CREB-related proteins were evaluated in LIM1-KD cells by western blotting and in xenograft tissue by immunofluorescent staining. Two different CREB inhibitors were treated in HEC50B and cell proliferation was evaluated by MTT assay. Results: Reanalysis of TCGA followed by GO enrichment analysis revealed that homeobox genes were highly expressed in advanced-stage EC. Among the identified genes, KM plotter analysis showed that high LIM1 expression was associated with a significantly poorer prognosis in EC. Additionally, LIM1 expression was significantly higher in high-grade EC cell lines, HEC50B cells than Ishikawa cells. Knockdown of LIM1 showed reduced cell proliferation, migration and invasion in HEC50B cells. Xenograft experiments revealed that tumor growth was significantly suppressed in LIM1-KD cells. IPA of RNA-seq data using LIM-KD cells predicted that the mRNA expression of CREB signaling-related genes was suppressed. Indeed, phosphorylation of CREB was decreased in LIM1-KD cells and LIM1-KD cells derived tumors. HEC50B cells treated by CREB inhibitors showed suppression of cell proliferation. Conclusion and discussion: Collectively, these results suggested that high LIM1 expression contributed to tumor growth via CREB signaling in EC. Inhibition of LIM1 or its downstream molecules would be new therapeutic strategies for EC.

Epigenetic regulator UHRF1 orchestrates proinflammatory gene expression in rheumatoid arthritis in a suppressive manner.

Rheumatoid arthritis (RA) is characterized by chronic synovial inflammation with aberrant epigenetic alterations, eventually leading to joint destruction. However, the epigenetic regulatory mechanisms underlying RA pathogenesis remain largely unknown. Here, we showed that ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) is a central epigenetic regulator that orchestrates multiple pathogeneses in RA in a suppressive manner. UHRF1 expression was remarkably upregulated in synovial fibroblasts (SFs) from arthritis model mice and patients with RA. Mice with SF-specific Uhrf1 conditional knockout showed more severe arthritic phenotypes than littermate controls. Uhrf1-deficient SFs also exhibited enhanced apoptosis resistance and upregulated expression of several cytokines, including Ccl20. In patients with RA, DAS28, CRP, and Th17 accumulation and apoptosis resistance were negatively correlated with UHRF1 expression in synovium. Finally, Ryuvidine administration stabilized UHRF1 ameliorated arthritis pathogeneses in a mouse model of RA. This study demonstrated that UHRF1 expressed in RA SFs can contribute to negative feedback mechanisms that suppress multiple pathogenic events in arthritis, suggesting that targeting UHRF1 could be one of the therapeutic strategies for RA.

Myofiber androgen receptor increases muscle strength mediated by a skeletal muscle splicing variant of Mylk4.

Androgens have a robust effect on skeletal muscles to increase muscle mass and strength. The molecular mechanism of androgen/androgen receptor (AR) action on muscle strength is still not well known, especially for the regulation of sarcomeric genes. In this study, we generated androgen-induced hypertrophic model mice, myofiber-specific androgen receptor knockout (cARKO) mice supplemented with dihydrotestosterone (DHT). DHT treatment increased grip strength in control mice but not in cARKO mice. Transcriptome analysis by RNA-seq, using skeletal muscles obtained from control and cARKO mice treated with or without DHT, identified a fast-type muscle-specific novel splicing variant of Myosin light-chain kinase 4 (Mylk4) as a target of AR in skeletal muscles. Mylk4 knockout mice exhibited decreased maximum isometric torque of plantar flexion and passive stiffness of myofibers due to reduced phosphorylation of Myomesin 1 protein. This study suggests that androgen-induced skeletal muscle strength is mediated with Mylk4 and Myomesin 1 axis.

DNA maintenance methylation enzyme Dnmt1 in satellite cells is essential for muscle regeneration.

Epigenetic transcriptional regulation is essential for the differentiation of various types of cells, including skeletal muscle cells. DNA methyltransferase 1 (Dnmt1) is responsible for maintenance of DNA methylation patterns via cell division. Here, we investigated the relationship between Dnmt1 and skeletal muscle regeneration. We found that Dnmt1 is upregulated in muscles during regeneration. To assess the role of Dnmt1 in satellite cells during regeneration, we performed conditional knockout (cKO) of Dnmt1 specifically in skeletal muscle satellite cells using Pax7CreERT2 mice and Dnmt1 flox mice. Muscle weight and the cross-sectional area after injury were significantly lower in Dnmt1 cKO mice than in control mice. RNA sequencing analysis revealed upregulation of genes involved in cell adhesion and apoptosis in satellite cells from cKO mice. Moreover, satellite cells cultured from cKO mice exhibited a reduced number of cells. These results suggest that Dnmt1 is an essential factor for muscle regeneration and is involved in positive regulation of satellite cell number.

Reprogramming of synovial macrophage metabolism by synovial fibroblasts under inflammatory conditions.

BACKGROUND: Macrophages adapt to microenvironments, and change metabolic status and functions to regulate inflammation and/or maintain homeostasis. In joint cavities, synovial macrophages (SM) and synovial fibroblasts (SF) maintain homeostasis. However, under inflammatory conditions such as rheumatoid arthritis (RA), crosstalk between SM and SF remains largely unclear. METHODS: Immunofluorescent staining was performed to identify localization of SM and SF in synovium of collagen antibody induced arthritis (CAIA) model mice and normal mice. Murine arthritis tissue-derived SM (ADSM), arthritis tissue-derived SF (ADSF) and normal tissue-derived SF (NDSF) were isolated and the purity of isolated cells was examined by RT-qPCR and flow cytometry analysis. RNA-seq was conducted to reveal gene expression profile in ADSM, NDSF and ADSF. Cellular metabolic status and expression levels of metabolic genes and inflammatory genes were analyzed in ADSM treated with ADSM-conditioned medium (ADSM-CM), NDSF-CM and ADSF-CM. RESULTS: SM and SF were dispersed in murine hyperplastic synovium. Isolations of ADSM, NDSF and ADSF to analyze the crosstalk were successful with high purity. From gene expression profiles by RNA-seq, we focused on secretory factors in ADSF-CM, which can affect metabolism and inflammatory activity of ADSM. ADSM exposed to ADSF-CM showed significantly upregulated glycolysis and mitochondrial respiration as well as glucose and glutamine uptake relative to ADSM exposed to ADSM-CM and NDSF-CM. Furthermore, mRNA expression levels of metabolic genes, such as Slc2a1, Slc1a5, CD36, Pfkfb1, Pfkfb3 and Irg1, were significantly upregulated in ADSM treated with ADSF-CM. Inflammation marker genes, including Nos2, Tnf, Il-1b and CD86, and the anti-inflammatory marker gene, Il-10, were also substantially upregulated by ADSF-CM. On the other hand, NDSF-CM did not affect metabolism and gene expression in ADSM. CONCLUSIONS: These findings suggest that crosstalk between SM and SF under inflammatory conditions can induce metabolic reprogramming and extend SM viability that together can contribute to chronic inflammation in RA. Video Abstract.

GPRC5A facilitates cell proliferation through cell cycle regulation and correlates with bone metastasis in prostate cancer.

The prognosis of patients with progressive prostate cancers that are hormone refractory and/or have bone metastasis is poor. Multiple therapeutic targets to improve prostate cancer patient survival have been investigated, including orphan GPCRs. In our study, we identified G Protein-Coupled Receptor Class C Group 5 Member A (GPRC5A) as a candidate therapeutic molecule using integrative gene expression analyses of registered data sets for prostate cancer cell lines. Kaplan-Meier analysis of TCGA data sets revealed that patients who have high GPRC5A expression had significantly shorter overall survival. PC3 prostate cancer cells with CRISPR/Cas9-mediated GPRC5A knockout exhibited significantly reduced cell proliferation both in vitro and in vivo. RNA-seq revealed that GPRC5A KO PC3 cells had dysregulated expression of cell cycle-related genes, leading to cell cycle arrest at the G2/M phase. Furthermore, the registered gene expression profile data set showed that the expression level of GPRC5A in original lesions of prostate cancer patients with bone metastasis was higher than that without bone metastasis. In fact, GPRC5A KO PC3 cells failed to establish bone metastasis in xenograft mice models. In addition, our clinical study revealed that GPRC5A expression levels in prostate cancer patient samples were significantly correlated with bone metastasis as well as the patient's Gleason score (GS). Combined assessment with the immunoreactivity of GPRC5A and GS displayed higher specificity for predicting the occurrence of bone metastasis. Together, our findings indicate that GPRC5A can be a possible therapeutic target and prognostic marker molecule for progressive prostate cancer.

The E3 ubiquitin ligase MIB2 enhances inflammation by degrading the deubiquitinating enzyme CYLD.

The tumor suppressor CYLD is a deubiquitinating enzyme that suppresses polyubiquitin-dependent signaling pathways, including the proinflammatory and cell growth-promoting NF-κB pathway. Missense mutations in the CYLD gene are present in individuals with syndromes such as multiple familial trichoepithelioma (MFT), but the pathogenic roles of these mutations remain unclear. Recent studies have shown that CYLD interacts with a RING finger domain protein, mind bomb homologue 2 (MIB2), in the regulation of NOTCH signaling. However, whether MIB2 is an E3 ubiquitin ligase that acts on CYLD is unknown. Here, using the cell-free-based AlphaScreen and pulldown assays to detect protein-protein interactions, along with immunofluorescence assays and murine Mib2 knockout cells and animals, we demonstrate that MIB2 promotes proteasomal degradation of CYLD and enhances NF-κB signaling. Of note, arthritic inflammation was suppressed in Mib2-deficient mice. We further observed that the ankyrin repeat in MIB2 interacts with the third CAP domain in CYLD and that MIB2 catalyzes Lys-48-linked polyubiquitination of CYLD at Lys-338 and Lys-530. MIB2-dependent CYLD degradation activated NF-κB signaling via tumor necrosis factor alpha (TNFα) stimulation and the linear ubiquitination assembly complex (LUBAC). Mib2-knockout mice had reduced serum interleukin-6 (IL-6) and exhibited suppressed inflammatory responses in the K/BxN serum-transfer arthritis model. Interestingly, MIB2 significantly enhanced the degradation of a CYLDP904L variant identified in an individual with MFT, although the molecular pathogenesis of the disease was not clarified here. Together, these results suggest that MIB2 enhances NF-κB signaling in inflammation by promoting the ubiquitin-dependent degradation of CYLD.

Zscan10 suppresses osteoclast differentiation by regulating expression of Haptoglobin.

Zinc finger and SCAN domain containing 10 (Zscan10) was identified as a novel transcription factor that is involved in osteoclast differentiation in our previous report. However, the biological functions of Zscan10 are not fully understood except its roles in the maintenance of genome stability and pluripotency of embryonic stem cells. Therefore, the purpose of this study was to clarify the function of Zscan10 in somatic cells, especially during osteoclast differentiation. First, Zscan10 KO RAW264 (KO) cells were established by genome editing using CRISPR/Cas9 and single cell sorting. Then, control (Ctrl) and KO cells were differentiated into osteoclasts by RANKL stimulation. We observed that TRAP activity and the expression levels of differentiation marker genes, such as Nfatc1, were significantly increased and the expression of inhibitory factors, such as Irf8, was decreased in KO cells compared to Ctrl cells. These results suggest that Zscan10 might regulate transcription of the genes that negatively control osteoclastogenesis. To understand gene expression profiles controlled by Zscan10, RNA-seq was performed and stringent analyses identified the haptoglobin gene (Hp) as a possible target of Zscan10. In addition, ChIP against Zscan10 revealed that Zscan10 could interact with its binding motif located near the Hp gene locus as well as the transcription start site of Hp, suggesting that Zscan10 can directly regulate transcription of Hp. Finally, to examine the effects of Hp on osteoclastogenesis, KO cells were treated with recombinant Hp (rHp). rHp treatment suppressed TRAP activity of KO cells without affecting cell viability. Furthermore, it has been reported that Hp KO mice exhibit decreased bone mass and increased osteoclast number. Importantly, hemolytic disease patients exhibited decreased serum level of Hp as well as low bone mineral density. Taken together, this study suggests that Zscan10 negatively regulates osteoclast differentiation through transcription of Hp.

Uhrf1 is indispensable for normal limb growth by regulating chondrocyte differentiation through specific gene expression.

Transcriptional regulation can be tightly orchestrated by epigenetic regulators. Among these, ubiquitin-like with PHD and RING finger domains 1 (Uhrf1) is reported to have diverse epigenetic functions, including regulation of DNA methylation. However, the physiological functions of Uhrf1 in skeletal tissues remain unclear. Here, we show that limb mesenchymal cell-specific Uhrf1 conditional knockout mice (Uhrf1ΔLimb/ΔLimb ) exhibit remarkably shortened long bones that have morphological deformities due to dysregulated chondrocyte differentiation and proliferation. RNA-seq performed on primary cultured chondrocytes obtained from Uhrf1ΔLimb/ΔLimb mice showed abnormal chondrocyte differentiation. In addition, integrative analyses using RNA-seq and MBD-seq revealed that Uhrf1 deficiency decreased genome-wide DNA methylation and increased gene expression through reduced DNA methylation in the promoter regions of 28 genes, including Hspb1, which is reported to be an IL1-related gene and to affect chondrocyte differentiation. Hspb1 knockdown in cKO chondrocytes can normalize abnormal expression of genes involved in chondrocyte differentiation, such as Mmp13 These results indicate that Uhrf1 governs cell type-specific transcriptional regulation by controlling the genome-wide DNA methylation status and regulating consequent cell differentiation and skeletal maturation.

EphA receptors and ephrin-A ligands are upregulated by monocytic differentiation/maturation and promote cell adhesion and protrusion formation in HL60 monocytes.

BACKGROUND: Eph signaling is known to induce contrasting cell behaviors such as promoting and inhibiting cell adhesion/spreading by altering F-actin organization and influencing integrin activities. We have previously demonstrated that EphA2 stimulation by ephrin-A1 promotes cell adhesion through interaction with integrins and integrin ligands in two monocyte/macrophage cell lines. Although mature mononuclear leukocytes express several members of the EphA/ephrin-A subclass, their expression has not been examined in monocytes undergoing during differentiation and maturation. RESULTS: Using RT-PCR, we have shown that EphA2, ephrin-A1, and ephrin-A2 expression was upregulated in murine bone marrow mononuclear cells during monocyte maturation. Moreover, EphA2 and EphA4 expression was induced, and ephrin-A4 expression was upregulated, in a human promyelocytic leukemia cell line, HL60, along with monocyte differentiation toward the classical CD14++CD16- monocyte subset. Using RT-PCR and flow cytometry, we have also shown that expression levels of αL, αM, αX, and ß2 integrin subunits were upregulated in HL60 cells along with monocyte differentiation while those of α4, α5, α6, and ß1 subunits were unchanged. Using a cell attachment stripe assay, we have shown that stimulation by EphA as well as ephrin-A, likely promoted adhesion to an integrin ligand-coated surface in HL60 monocytes. Moreover, EphA and ephrin-A stimulation likely promoted the formation of protrusions in HL60 monocytes. CONCLUSIONS: Notably, this study is the first analysis of EphA/ephrin-A expression during monocytic differentiation/maturation and of ephrin-A stimulation affecting monocyte adhesion to an integrin ligand-coated surface. Thus, we propose that monocyte adhesion via integrin activation and the formation of protrusions is likely promoted by stimulation of EphA as well as of ephrin-A.

Truncated EphA2 likely potentiates cell adhesion via integrins as well as infiltration and/or lodgment of a monocyte/macrophage cell line in the red pulp and marginal zone of the mouse spleen, where ephrin-A1 is prominently expressed in the vasculature.

We previously established a J774.1 monocyte/macrophage subline expressing a truncated EphA2 construct lacking the kinase domain. We demonstrated that following ephrin-A1 stimulation, endogenous EphA2 promotes cell adhesion through interaction with integrins and integrin ligands such as ICAM1 and that truncated EphA2 potentiates the adhesion and becomes associated with the integrin/integrin ligand complex. Based on these findings, we hypothesized that the EphA/ephrin-A system, particularly EphA2/ephrin-A1, regulates transendothelial migration/tissue infiltration of monocytes/macrophages, because ephrin-A1 is widely recognized to be upregulated in inflammatory vasculatures. To evaluate whether this hypothesis is applicable in the spleen, we screened for EphA2/ephrin-A1 expression and reexamined the cellular properties of the J774.1 subline. We found that ephrin-A1 was expressed in the vasculature of the marginal zone and the red pulp and that its expression was upregulated in response to phagocyte depletion; further, CD115, F4/80, and CXCR4 were expressed in J774.1 cells, which serve as a usable substitute for monocytes/macrophages. Moreover, following ephrin-A1 stimulation, truncated EphA2 did not detectably interfere with the phosphorylation of endogenous EphA2, and it potentiated cell adhesion possibly through modulation of integrin avidity. Accordingly, by intravenously injecting mice with equal numbers of J774.1 and the subline cells labeled with distinct fluorochromes, we determined that truncated EphA2 markedly potentiated preferential cell infiltration into the red pulp and the marginal zone. Thus, modulation of EphA2 signaling might contribute to effective transplantation of tissue-specific resident macrophages and/or monocytes.

EphA2 promotes cell adhesion and spreading of monocyte and monocyte/macrophage cell lines on integrin ligand-coated surfaces.

Eph signaling, which arises following stimulation by ephrins, is known to induce opposite cell behaviors such as promoting and inhibiting cell adhesion as well as promoting cell-cell adhesion and repulsion by altering the organization of the actin cytoskeleton and influencing the adhesion activities of integrins. However, crosstalk between Eph/ephrin with integrin signaling has not been fully elucidated in leukocytes, including monocytes and their related cells. Using a cell attachment stripe assay, we have shown that, following stimulation with ephrin-A1, kinase-independent EphA2 promoted cell spreading/elongation as well as adhesion to integrin ligand-coated surfaces in cultured U937 (monocyte) and J774.1 (monocyte/macrophage) cells as well as sublines of these cells expressing dominant negative EphA2 that lacks most of the intracellular region. Moreover, a pull-down assay showed that dominant negative EphA2 is recruited to the ß2 integrin/ICAM1 and ß2 integrin/VCAM1 molecular complexes in the subline cells following stimulation with ephrin-A1-Fc. Notably, this study is the first comprehensive analysis of the effects of EphA2 receptors on integrin-mediated cell adhesion in monocytic cells. Based on these findings we propose that EphA2 promotes cell adhesion by an unknown signaling pathway that largely depends on the extracellular region of EphA2 and the activation of outside-in integrin signaling.

Aberrant EphB/ephrin-B expression in experimental gastric lesions and tumor cells.

AIM: To determine whether the expression profiles of EphB receptor and ephrin-B ligand can be used as markers for dysplastic/oncogenic transformation in gastric mucosa. METHODS: The protein expression and localization of EphB and ephrin-B in normal, ulcerated regenerating, and dysplastic gastric mucosa were examined in a rat experimental model by immunolabeling, and mRNA expression was assessed in four human gastric carcinoma cell lines by reverse transcription-polymerase chain reaction. RESULTS: Ephrin-B- and EphB-expressing regions were divided along the pit-gland axis in normal gastric units. EphB2 was transiently upregulated in the experimental ulcer, and its expression domain extended to gastric pits and/or the luminal surface where ephrin-B-expressing pit cells reside. EphB2, B3, and B4 and ephrin-B1 were coexpressed in the experimental gastric dysplasia, and more than one ligand-receptor pair was highly expressed in each of the gastric carcinoma cell lines. CONCLUSION: Robust and stable coexpression of EphB and ephrin-B is a feature common to experimentally induced gastric dysplasia and human gastric carcinoma cell lines as compared to normal gastric and ulcerated regenerating epithelia. Thus, EphB/ephrin-B may be a useful marker combination for dysplastic/oncogenic transformation in gastric cancer.

Complementary expression and repulsive signaling suggest that EphB2 and ephrin-B1 are possibly involved in epithelial boundary formation at the squamocolumnar junction in the rodent stomach.

Eph receptors and ephrin ligands are cell-cell communication molecules with well-defined roles in cell adhesion, migration, and tissue boundary formation. However, their expression levels in the squamocolumnar epithelial junction region at the distal esophagus are completely unknown. We examined EphB2 and ephrin-B1 localization in the squamocolumnar epithelial junction region between the proximal and distal stomach of the rodents. Immunostaining showed complimentary expression patterns along the proximal-to-distal axis of the gastric epithelia across the junction: EphB2 expression was maximal around the epithelial junction and sharply decreased in the stratified squamous epithelium at a short distance from the junction, whereas ephrin-B1 was strongly expressed in the stratified squamous epithelium at a distance from the junction and sharply decreased toward the junction. These expression patterns suggest that EphB2/ephrin-B1 signaling occurs preferentially in the epithelia across the junction, where the receptor and ligand expression highly overlap. We also show that (1) EphB2 preferentially binds ephrin-B1, and (2) cell repulsion/lateral migration was induced in primary cultured gastric keratinocytes on ephrin-B1-Fc- and EphB2-Fc-coated surfaces. On the basis of these findings, we propose that EphB2 and ephrin-B1 are possibly involved in epithelial boundary formation at the squamocolumnar junction.