Dach1 is highly expressed in normal podocytes, but this expression rapidly disappears after podocyte injury. To investigate the role of Dach1 in podocytes in vivo, we analyzed global, podocyte-specific, and inducible Dach1 knockout mice. Global Dach1 knockout (Dach1-/-) mice were assessed immediately after birth because they die within a day. The kidneys of Dach1-/- mice were slightly smaller than those of control mice but maintained a normal structure and normal podocyte phenotypes, including ultrastructure. To study the role of Dach1 in mature podocytes, we generated Dach1 knockout mice by mating Dach1fl/fl mice with Nphs1-Cre or ROSA-CreERT2 mice. Due to inefficient Cre recombination, only a small number of podocytes lacked Dach1 staining in these mice. However, all eleven Nphs1-Cre/Dach1fl/fl mice displayed abnormal albuminuria, and seven (63%) of them developed focal segmental glomerulosclerosis. Among 13 ROSA-CreERT2/Dach1fl/fl mice, eight (61%) exhibited abnormal albuminuria after treatment with tamoxifen, and five (38%) developed early sclerotic lesions. These results indicate that while Dach1 does not determine the fate of differentiation into podocytes, it is indispensable for maintaining the normal integrity of mature podocytes.

The tumor microenvironment is an extremely complex composed of cancer cells and various non-cancer cells, including lymphatic endothelial cells. Lysophosphatidic acid (LPA) receptors (LPA1 to LPA6) activate a variety of malignant properties in human malignancies. In the present study, we examined the roles of LPA receptor-mediated signaling in biological responses of lymphatic endothelial SVEC4-10 cells induced by hypoxia. Lpar1, Lpar2 and Lpar3 expressions were decreased in SVEC4-10 cells cultured at hypoxic conditions (1 % O2). LPA had no impact on the cell growth activity of SVEC4-10 cells in 21 % O2 culture conditions. Conversely, the cell growth activity of SVEC4-10 cells in 1 % O2 culture conditions was reduced by LPA. The cell motile activity of SVEC4-10 cells was elevated by 1 % O2 culture conditions. GRI-977143 (LPA2 agonist) and (2S)-OMPT (LPA3 agonist) stimulated SVEC4-10 cell motility as well as AM966 (LPA1 antagonist). In tube formation assay, the tube formation of SVEC4-10 cells in 1 % O2 culture conditions was markedly increased, in comparison with 21 % O2. GRI-977143 and (2S)-OMPT elevated the tube formation of SVEC4-10 cells. Furthermore, the tube formation of SVEC4-10 cells was increased by AM966. These results suggest that LPA receptor-mediated signaling contributes to the modulation of hypoxic-induced biological functions of lymphatic endothelial cells.

Abstract CCL21‐Ser, a chemokine encoded by the Ccl21a gene, is constitutively expressed in the thymic epithelial cells and stromal cells of secondary lymphoid organs. It regulates immune cell migration and survival through its receptor CCR7. Herein, using CCL21‐Ser‐expressing melanoma cells and the Ccl21a‐deficient mice, we demonstrated the functional role of cancer cell‐derived CCL21‐Ser in melanoma growth in vivo. The B16‐F10 tumor growth was significantly decreased in Ccl21a‐deficient mice compared with that in wild‐type mice, indicating that host‐derived CCL21‐Ser contributes to melanoma proliferation in vivo. In Ccl21a‐deficient mice, tumor growth of melanoma cells expressing CCL21‐Ser was significantly enhanced, suggesting that CCL21‐Ser from melanoma cells promotes tumor growth in the absence of host‐derived CCL21‐Ser. The increase in tumor growth was associated with an increase in the CCR7⁺ CD62L⁺ T cell frequency in the tumor tissue but was inversely correlated with Treg frequency, suggesting that naïve T cells primarily promote tumor growth. Adoptive transfer experiments demonstrated that naïve T cells are preferentially recruited from the blood into tumors with melanoma cell‐derived CCL21‐Ser expression. These results suggest that CCL21‐Ser from melanoma cells promotes the infiltration of CCR7⁺ naïve T cells into the tumor tissues and creates a tumor microenvironment favorable for melanoma growth.

CCR4 is a major trafficking receptor for Th2 cells and Th17 cells and is considered as a potential therapeutic target for atopic dermatitis (AD). The CCR4 ligands CCL17 and CCL22 have been reported to be upregulated in the skin lesions of AD patients. Of note, Thymic stromal lymphopoietin (TSLP), a master regulator of the Th2 immune response, promotes the expression of CCL17 and CCL22 in AD skin lesions. Here, we investigated the role of CCR4 in an AD mouse model induced by MC903, a TSLP inducer. Topical application of MC903 to ear skin increased the expression of not only TSLP but also CCL17, CCL22, the Th2 cytokine IL-4, and the Th17 cytokine IL-17A. Consistently, MC903 induced AD-like skin lesions as shown by increased epidermal thickness; increased infiltration of eosinophils, mast cells, type 2 innate lymphoid cells, Th2 cells, and Th17 cells; and elevated serum levels of total IgE. We also found increased expansion of Th2 cells and Th17 cells in the regional lymph nodes (LNs) of AD mice. Compound 22, a CCR4 inhibitor, ameliorated AD-like skin lesions with reduction of Th2 cells and Th17 cells in the skin lesions and regional LNs. We further confirmed that compound 22 diminished the expansion of Th2 cells and Th17 cells in the coculture of CD11c + dendritic cells and CD4 + T cells derived from the regional LNs of AD mice. Collectively, CCR4 antagonists may exhibit anti-allergic effects by inhibiting both the recruitment and expansion of Th2 cells and Th17 cells in AD.

Numerous studies have investigated the various cellular responses against genotoxic stress, including those mediated by focal adhesions. We here identified a novel type of focal adhesion remodelling that occurs under genotoxic stress conditions, which involves the replacement of active focal adhesion kinase (FAK) with FAK-related non-kinase (FRNK). FRNK stabilized focal adhesions, leading to strong cell-matrix adhesion, and FRNK-depleted cells were easily detached from extracellular matrix upon genotoxic stress. This remodelling occurred in a wide variety of cells. In vivo, the stomachs of Frnk-knockout mice were severely damaged by genotoxic stress, highlighting the protective role of FRNK against genotoxic stress. FRNK was also found to play a vital role in cancer progression, because FRNK depletion significantly inhibited cancer dissemination and progression in a mouse cancer model. Furthermore, in human cancers, FRNK was predominantly expressed in metastatic tissues and not in primary tissues. We hence conclude that this novel type of focal adhesion remodelling reinforces cell adhesion and acts against genotoxic stress, which results in the protection of normal tissues, but in turn facilitates cancer progression.

Chemokines are a family of cytokines that mediate leukocyte trafficking and are involved in tumor cell migration, growth, and progression. Although there is emerging evidence that multiple chemokines are expressed in tumor tissues and that each chemokine induces receptor-mediated signaling, their collaboration to regulate tumor invasion and lymph node metastasis has not been fully elucidated. In this study, we examined the effect of CXCL12 on the CCR7-dependent signaling in MDA-MB-231 human breast cancer cells to determine the role of CXCL12 and CCR7 ligand chemokines in breast cancer metastasis to lymph nodes. CXCL12 enhanced the CCR7-dependent in vitro chemotaxis and cell invasion into collagen gels at suboptimal concentrations of CCL21. CXCL12 promoted CCR7 homodimer formation, ligand binding, CCR7 accumulation into membrane ruffles, and cell response at lower concentrations of CCL19. Immunohistochemistry of MDA-MB-231-derived xenograft tumors revealed that CXCL12 is primarily located in the pericellular matrix surrounding tumor cells, whereas the CCR7 ligand, CCL21, mainly associates with LYVE-1+ intratumoral and peritumoral lymphatic vessels. In the three-dimensional tumor invasion model with lymph networks, CXCL12 stimulation facilitates breast cancer cell migration to CCL21-reconstituted lymphatic networks. These results indicate that CXCL12/CXCR4 signaling promotes breast cancer cell migration and invasion toward CCR7 ligand-expressing intratumoral lymphatic vessels and supports CCR7 signaling associated with lymph node metastasis.

Whereas adenosine 5'-triphosphate (ATP) is the major energy source in cells, extracellular ATP (eATP) released from activated/damaged cells is widely thought to represent a potent damage-associated molecular pattern that promotes inflammatory responses. Here, we provide suggestive evidence that eATP is constitutively produced in the uninflamed lymph node (LN) paracortex by naïve T cells responding to C-C chemokine receptor type 7 (CCR7) ligand chemokines. Consistently, eATP was markedly reduced in naïve T cell-depleted LNs, including those of nude mice, CCR7-deficient mice, and mice subjected to the interruption of the afferent lymphatics in local LNs. Stimulation with a CCR7 ligand chemokine, CCL19, induced ATP release from LN cells, which inhibited CCR7-dependent lymphocyte migration in vitro by a mechanism dependent on the purinoreceptor P2X7 (P2X7R), and P2X7R inhibition enhanced T cell retention in LNs in vivo. These results collectively indicate that paracortical eATP is produced by naïve T cells in response to constitutively expressed chemokines, and that eATP negatively regulates CCR7-mediated lymphocyte migration within LNs via a specific subtype of ATP receptor, demonstrating its fine-tuning role in homeostatic cell migration within LNs.

Cytokine and growth factor receptors localized on cell membranes play key roles in many biological events. Because the dynamic behaviors of receptors are deeply involved in cytokine- and growth-factor-dependent signaling pathways, reliable techniques for real-time imaging of receptors in live cells are strongly desired. Here, we describe a method of covalently labeling and imaging membrane receptors in live cells by using chemically reactive cytokines and growth factors coupled with 4-dimethylaminopyridine (DMAP), an organocatalyst that facilitates acyl transfer. Construction of the DMAP cytokines and growth factors relies on non-covalent coordination chemistry between an oligo-histidine tag and dimeric Ni(II)-nitrilotriacetate in an in situ manner. This supramolecular approach is relatively simple and flexible and can be applied to labeling not only transiently but also endogenously expressed receptors. Moreover, given the benefit of this traceless labeling of the receptors, our technique allows real-time imaging of the ligand-induced dynamics of endogenously expressed receptors in live cells. Cytokine and growth factor receptors play crucial roles in signaling cascades that modulate cell survival, adhesion, proliferation, and migration. Because the dynamic behaviors of these receptors are highly related to their functions and to changes in ligand-dependent signals, reliable techniques for visualization of these receptors are strongly desired. Here, we have developed a method of covalently and tracelessly labeling membrane receptors in live cells by using chemically reactive cytokines and growth factors coupled with an organocatalyst that facilitates a selective acyl transfer reaction on the target receptors. Construction of the reactive cytokines and growth factors relies on non-covalent coordination chemistry in an in situ manner, and this supramolecular approach is relatively simple and flexible. It can be applied to not only the labeling of various receptors but also real-time imaging of ligand-induced dynamics of endogenously expressed receptors in live cells. With the use of supramolecule-based chemically reactive cytokines and growth factors, membrane receptor proteins can be covalently labeled with fluorescent small molecules. This supramolecular approach is quite simple and flexible, and various endogenous receptors can be visualized specifically real-time imaging of the ligand-induced dynamics of the receptors is achieved in living mammalian cells.

The chemokine receptor CCR7 contributes to various physiological and pathological processes including T cell maturation, T cell migration from the blood into secondary lymphoid tissues, and tumor cell metastasis to lymph nodes. Although a previous study suggested that the efficacy of CCR7 ligand-dependent T cell migration correlates with CCR7 homo- and heterodimer formation, the exact extent of contribution of the CCR7 dimerization remains unclear. Here, by inducing or disrupting CCR7 dimers, we demonstrated a direct contribution of CCR7 homodimerization to CCR7-dependent cell migration and signaling. Induction of stable CCR7 homodimerization resulted in enhanced CCR7-dependent cell migration and CCL19 binding, whereas induction of CXCR4/CCR7 heterodimerization did not. In contrast, dissociation of CCR7 homodimerization by a novel CCR7-derived synthetic peptide attenuated CCR7-dependent cell migration, ligand-dependent CCR7 internalization, ligand-induced actin rearrangement, and Akt and Erk signaling in CCR7-expressing cells. Our study indicates that CCR7 homodimerization critically regulates CCR7 ligand-dependent cell migration and intracellular signaling in multiple cell types.

Despite their functional similarities, peripheral lymphoid tissues are remarkably different according to their developmental properties and structural characteristics, including their specified vasculature. Access of leukocytes to these organs critically depends on their interactions with the local endothelium, where endothelial cells are patterned to display a restricted set of adhesion molecules and other regulatory compounds necessary for extravasation. Recent advances in high throughput analyses of highly purified endothelial subsets in various lymphoid tissues as well as the expansion of various transgenic animal models have shed new light on the transcriptional complexities of lymphoid tissue vascular endothelium. This review is aimed at providing a comprehensive analysis linking the functional competence of spleen and intestinal lymphoid tissues with the developmental programming and functional divergence of their vascular specification, with particular emphasis on the transcriptional control of endothelial cells exerted by Nkx2.3 homeodomain transcription factor.

Naive lymphocytes continuously migrate from the blood into lymph nodes (LNs) via high endothelial venules (HEVs). To extravasate from the HEVs, lymphocytes undergo multiple adhesion steps, including tethering, rolling, firm adhesion and transmigration. We previously showed that autotaxin (ATX), an enzyme that generates lysophosphatidic acid (LPA), is highly expressed in HEVs, and that the ATX/LPA axis plays an important role in the lymphocyte transmigration across HEVs. However, the detailed mechanism underlying this axis's involvement in lymphocyte transmigration has remained ill-defined. Here, we show that two LPA receptors, LPA4 and LPA6, are selectively expressed on HEV endothelial cells (ECs) and that LPA4 plays a major role in the lymphocyte transmigration across HEVs in mice. In the absence of LPA4 expression, lymphocytes accumulated heavily within the HEV EC layer, compared to wild-type (WT) mice. This accumulation was also observed in the absence of LPA6 expression, but it was less pronounced. Adoptive transfer experiments using WT lymphocytes revealed that the LPA4 deficiency in ECs specifically compromised the lymphocyte transmigration process, whereas the effect of LPA6 deficiency was not significant. These results indicate that the signals evoked in HEV ECs via the LPA4 and LPA6 differentially regulate lymphocyte extravasation from HEVs in the peripheral LNs.

Lymph nodes (LNs) are highly confined environments with a cell-dense three-dimensional meshwork, in which lymphocyte migration is regulated by intracellular contractile proteins. However, the molecular cues directing intranodal cell migration remain poorly characterized. Here we demonstrate that lysophosphatidic acid (LPA) produced by LN fibroblastic reticular cells (FRCs) acts locally to LPA2 to induce T-cell motility. In vivo, either specific ablation of LPA-producing ectoenzyme autotaxin in FRCs or LPA2 deficiency in T cells markedly decreased intranodal T cell motility, and FRC-derived LPA critically affected the LPA2-dependent T-cell motility. In vitro, LPA activated the small GTPase RhoA in T cells and limited T-cell adhesion to the underlying substrate via LPA2. The LPA-LPA2 axis also enhanced T-cell migration through narrow pores in a three-dimensional environment, in a ROCK-myosin II-dependent manner. These results strongly suggest that FRC-derived LPA serves as a cell-extrinsic factor that optimizes T-cell movement through the densely packed LN reticular network.

During human immunodeficiency virus (HIV) infection, enhanced migration of infected cells to lymph nodes leads to efficient propagation of HIV-1. The selective chemokine receptors, including CXCR4 and CCR7, may play a role in this process, yet the viral factors regulating chemokine-dependent T cell migration remain relatively unclear. The functional cooperation between the CXCR4 ligand chemokine CXCL12 and the CCR7 ligand chemokines CCL19 and CCL21 enhances CCR7-dependent T cell motility in vitro as well as cell trafficking into the lymph nodes in vivo. In this study, we report that a recombinant form of a viral CXCR4 ligand, X4-tropic HIV-1 gp120, enhanced the CD4 T cell response to CCR7 ligands in a manner dependent on CXCR4 and CD4, and that this effect was recapitulated by HIV-1 virions. HIV-1 gp120 significantly enhanced CCR7-dependent CD4 T cell migration from the footpad of mice to the draining lymph nodes in in vivo transfer experiments. We also demonstrated that CXCR4 expression is required for stable CCR7 expression on the CD4 T cell surface, whereas CXCR4 signaling facilitated CCR7 ligand binding to the cell surface and increased the level of CCR7 homo- as well as CXCR4/CCR7 hetero-oligomers without affecting CCR7 expression levels. Our findings indicate that HIV-evoked CXCR4 signaling promotes CCR7-dependent CD4 T cell migration by up-regulating CCR7 function, which is likely to be induced by increased formation of CCR7 homo- and CXCR4/CCR7 hetero-oligomers on the surface of CD4 T cells.

Lymphocyte extravasation from the high endothelial venules (HEVs) of lymph nodes is crucial for the maintenance of immune homeostasis, but its molecular mechanism remains largely unknown. In this article, we report that lymphocyte transmigration across the basal lamina of the HEVs is regulated, at least in part, by autotaxin (ATX) and its end-product, lysophosphatidic acid (LPA). ATX is an HEV-associated ectoenzyme that produces LPA from lysophosphatidylcholine (LPC), which is abundant in the systemic circulation. In agreement with selective expression of ATX in HEVs, LPA was constitutively and specifically detected on HEVs. In vivo, inhibition of ATX impaired the lymphocyte extravasation from HEVs, inducing lymphocyte accumulation within the endothelial cells (ECs) and sub-EC compartment; this impairment was abrogated by LPA. In vitro, both LPA and LPC induced a marked increase in the motility of HEV ECs; LPC's effect was abrogated by ATX inhibition, whereas LPA's effect was abrogated by ATX/LPA receptor inhibition. In an in vitro transmigration assay, ATX inhibition impaired the release of lymphocytes that had migrated underneath HEV ECs, and these defects were abrogated by LPA. This effect of LPA was dependent on myosin II activity in the HEV ECs. Collectively, these results strongly suggest that HEV-associated ATX generates LPA locally; LPA, in turn, acts on HEV ECs to increase their motility, promoting dynamic lymphocyte-HEV interactions and subsequent lymphocyte transmigration across the basal lamina of HEVs at steady state.

Vascular endothelial cells often change their phenotype to adapt to their local microenvironment. Here we report that the vascular endothelial adhesion molecule nepmucin/CD300LG, which is implicated in lymphocyte binding and transmigration, shows unique expression patterns in the microvascular endothelial cells of different tissues. Under physiological conditions, nepmucin/CD300LG was constitutively and selectively expressed at the luminal surface of the small arterioles, venules, and capillaries of most tissues, but it was only weakly expressed in the microvessels of the splenic red pulp and thymic medulla. Furthermore, it was barely detectable in immunologically privileged sites such as the brain, testis, and uterus. The nepmucin/CD300LG expression rapidly decreased in lymph nodes receiving acute inflammatory signals, and this loss was mediated at least in part by TNF-α. It was also down-regulated in tumors and tumor-draining lymph nodes, indicating that nepmucin/CD300LG expression is negatively regulated by locally produced signals under these circumstances. In contrast, nepmucin/CD300LG was induced in the high endothelial venule-like blood vessels of chronically inflamed pancreatic islets in an animal model of non-obese diabetes. Interestingly, the activated CD4(+) T cells infiltrating the inflamed pancreas expressed high levels of the nepmucin/CD300LG ligand(s), supporting the idea that nepmucin/CD300LG and its ligand interactions are locally involved in pathological T cell trafficking. Taken together, these observations indicate that the nepmucin/CD300LG expression in microvascular endothelial cells is influenced by factor(s) that are locally produced in tissues, and that its expression is closely correlated with the level of leukocyte infiltration in certain tissues.

Although the spleen plays an important role in host defense against infection, the mechanism underlying the migration of the innate immune cells, plasmacytoid dendritic cells (pDCs), into the spleen remains ill defined. In this article, we report that pDCs constitutively migrate into the splenic white pulp (WP) in a manner dependent on the chemokine receptors CCR7 and CXCR4. In CCR7-deficient mice and CCR7 ligand-deficient mice, compared with wild-type (WT) mice, substantially fewer pDCs were found in the periarteriolar lymphoid sheath of the splenic WP under steady-state conditions. In addition, the migration of adoptively transferred CCR7-deficient pDCs into the WP was significantly worse than that of WT pDCs, supporting the idea that pDC trafficking to the splenic WP requires CCR7 signaling. WT pDCs responded to a CCR7 ligand with modest chemotaxis and ICAM-1 binding in vitro, and priming with the CCR7 ligand enabled the pDCs to migrate efficiently toward low concentrations of CXCL12 in a CXCR4-dependent manner, raising the possibility that CCR7 signaling enhances CXCR4-mediated pDC migration. In agreement with this hypothesis, CCL21 and CXCL12 were colocalized on fibroblastic reticular cells in the T cell zone and in the marginal zone bridging channels, through which pDCs appeared to enter the WP. Furthermore, functional blockage of CCR7 and CXCR4 abrogated pDC trafficking into the WP. Collectively, these results strongly suggest that pDCs employ both CCR7 and CXCR4 as critical chemokine receptors to migrate into the WP under steady-state conditions.

The physiological recruitment of circulating lymphocytes from the blood into secondary lymphoid tissues is an essential homeostatic mechanism for the immune system because it allows lymphocytes to encounter efficiently both their specific cognate antigen and the regulatory cells with which they need to interact, to initiate, maintain, and terminate immune responses appropriately. This constitutive lymphocyte trafficking is mediated by high endothelial venules (HEVs), which are present in secondary lymphoid tissues other than the spleen. There is growing evidence that lymphocyte trafficking across HEVs involves at least three steps, namely, (i) tethering/rolling, (ii) arrest/firm adhesion/intraluminal crawling, and (iii) transendothelial migration (TEM). Although the mechanisms underlying the first two steps have been determined relatively well, the mechanism regulating TEM is only partially understood. In particular, the molecular mechanism driving lymphocyte movement from the apical to the basolateral surface of the endothelial cells (ECs) of HEVs remains ill defined. This step is crucial for successful lymphocyte extravasation, and is thus an important target for therapeutic intervention in various immunological diseases. Here, we review the molecular mechanisms governing lymphocyte-HEV interactions, and highlight possible roles for two HEV proteins, i.e., nepmucin/CD300g and autotaxin, in lymphocyte TEM.

Physiological recruitment of lymphocytes from the blood into lymph nodes and Peyer's patches is mediated by high endothelial venules (HEV), specialized blood vessels found in secondary lymphoid tissues except for the spleen. The HEV are distinguished from other types of blood vessels by their tall and plump endothelial cells, and by their expression of specific chemokines and adhesion molecules, which all contribute to the selective lymphocyte trafficking across these blood vessels. The development of HEV is ontogenically regulated, and they appear perinatally in the mouse. High endothelial venules can appear ectopically, for instance in chronically inflamed tissues. Given that HEV enable the efficient trafficking of lymphocytes into tissues, the induction of HEV at a tumor site could potentiate tumor-specific immune responses, and the artificial manipulation of HEV neogenesis might thus provide a new tool for cancer immunotherapy. However, the process of HEV development and the mechanisms by which the unique features of HEV are maintained are incompletely understood. In this review, we discuss the process of HEV neogenesis and development during ontogeny, and their molecular requirements for maintaining their unique characteristics under physiological conditions.

The hyaluronan (HA) receptor CD44 mediates cell adhesion in leukocyte trafficking and tumor metastasis. Our previous nuclear magnetic resonance (NMR) studies revealed that the CD44 hyaluronan-binding domain (HABD) alters its conformation upon HA binding, from the ordered (O) to the partially disordered (PD) conformation. Here, we demonstrate that the HABD undergoes an equilibrium between the O and PD conformations, in either the presence or absence of HA, which explains the seemingly contradictory X-ray and NMR structures of the HA-bound HABD. An HABD mutant that exclusively adopts the PD conformation displayed a higher HA affinity than the wild-type. Rolling of the cells expressing the mutant CD44 was less efficient than those expressing the wild-type, due to the decreased tether frequency and the slow cellular off rate. Considering that the mutant CD44, devoid of the low-affinity state, exhibited impaired rolling, we conclude that the coexistence of the high- and low-affinity states of the HABD is essential for the CD44-mediated rolling.

A number of chemokines, including CCL21, CCL19, CXCL12, and CXCL13, are coexpressed on the lumen or basal lamina of high endothelial venules (HEVs) in lymph nodes (LNs) and Peyer's patches (PPs), consistent with the idea that they might cooperate to regulate lymphocyte trafficking into these lymphoid tissues. In this study we report that CXCL12, acting through its receptor, CXCR4, cooperates with CCR7 ligands to promote T cell trafficking across HEVs. CXCL12 enhanced the CCR7-induced chemotaxis of wild-type but not CXCR4-deficient T cells in vitro at suboptimal concentrations of a CCR7 ligand, but without affecting the expression level or ligand-binding ability of CCR7. Real-time chemotaxis analysis showed that CXCL12 substantially shortened the lag time before cell migration began in vitro, but not the migration speed of T cells responding to suboptimal CCR7 ligand concentrations. In addition, CXCL12 augmented the CCR7 ligand-driven ERK phosphorylation and actin polymerization in T cells under the same conditions. In adoptive transfer experiments, CXCL12 promoted naive T cell trafficking to LNs and PPs in wild-type but not CCR7 ligand-deficient plt/plt recipient mice; this increased T cell trafficking was associated with enhanced binding of the T cells to HEVs and their subsequent migration into the LN parenchyma. Thus, CXCL12 synergizes with CCR7 ligands to promote T cell migration by sensitizing T cells through CXCR4, thus enabling them to respond to lower concentrations of CCR7 ligands. Such concerted action of chemokines provides an additional, previously unknown mechanism for efficient lymphocyte trafficking across HEVs into LNs and PPs.

OBJECTIVE: Smooth muscle cell (SMC) differentiation is a dynamic process that must be tightly regulated for proper vascular development and to control the onset of vascular disease. Our laboratory previously reported that a specific focal adhesion kinase (FAK) inhibitor termed FRNK (FAK Related Non-Kinase) is selectively expressed in large arterioles when SMCs are transitioning from a synthetic to contractile phenotype and that FRNK inhibits FAK-dependent SMC proliferation and migration. Herein, we sought to determine whether FRNK expression modulates SMC phenotypes in vivo. METHODS AND RESULTS: We present evidence that FRNK(-/-) mice exhibit attenuated SM marker gene expression during postnatal vessel growth and after vascular injury. We also show that FRNK expression is regulated by transforming growth factor (TGF)-beta and that forced expression of FRNK in cultured cells induces serum- and TGF-beta-stimulated SM marker gene expression, whereas FRNK deletion or expression of a constitutively activated FAK variant attenuated SM gene transcription. CONCLUSIONS: These data highlight the possibility that extrinsic signals regulate the SMC gene profile, at least in part, by modulating the expression of FRNK and that tight regulation of FAK activity by FRNK is important for proper SMC differentiation during development and after vascular injury.

Autotaxin (ATX) is a secreted protein with lysophospholipase D activity that generates lysophosphatidic acid (LPA) from lysophosphatidylcholine. Here we report that functional ATX is selectively expressed in high endothelial venules (HEVs) of both lymph nodes and Peyer's patches. ATX expression was developmentally regulated and coincided with lymphocyte recruitment to the lymph nodes. In adults, ATX expression was independent of HEV-expressed chemokines such as CCL21 and CXCL13, innate immunity signals including those via TLR4 or MyD88, and of the extent of lymphocyte trafficking across the HEVs. ATX expression was induced in venules at sites of chronic inflammation. Receptors for the ATX enzyme product LPA were constitutively expressed in HEV endothelial cells (ECs). In vitro, LPA induced strong morphological changes in HEV ECs. Forced ATX expression caused cultured ECs to respond to lysophosphatidylcholine, up-regulating lymphocyte binding to the ECs in a LPA receptor-dependent manner under both static and flow conditions. Although in vivo depletion of circulating ATX did not affect lymphocyte trafficking into the lymph nodes, we surmise, based on the above data, that ATX expressed by HEVs acts on HEVs in situ to facilitate lymphocyte binding to ECs and that ATX in the general circulation does not play a major role in this process. Tissue-specific inactivation of ATX will verify this hypothesis in future studies of its mechanism of action.

FRNK, a non-catalytic variant of focal adhesion kinase (FAK), is expressed in major blood vessels throughout mouse development and is postulated to play a role in regulating cell adhesion and signaling in vascular smooth muscle cells (VSMCs). The FRNK transcriptional start site lies within an intron of the FAK gene, suggesting that the FRNK gene is a "gene within a gene". Here, we identified a 1 kb intronic sequence of the FAK gene that is necessary for endogenous FRNK expression. Deletion of this sequence in gene-targeted mice abolished FRNK expression, showing the direct involvement of the FAK intron in the regulation of FRNK expression. The level of FAK expression was normal in the FRNK-deficient mice, indicating that FAK and FRNK are transcriptionally regulated by distinct promoters. The FRNK-deficient mice were viable, fertile, and displayed no obvious histological abnormalities in any of the major blood vessels. Western blot analysis showed that FRNK-deficient and wild-type (WT) cells had comparable levels of steady-state and adhesion-dependent FAK autophosphorylation. Despite the fact that ectopic expression of FRNK suppresses focal adhesion formation in cultured cells, these results suggest that endogenous FRNK is not essential for development or the formation of the mouse vasculature.

Certain lymphoid chemokines are selectively and constitutively expressed in the high endothelial venules (HEV) of lymph nodes and Peyer's patches, where they play critical roles in the directional migration of extravasating lymphocytes into the lymphoid tissue parenchyma. How these chemokines are selectively localized and act in situ, however, remains unclear. In the present study, we examined the possibility that basal lamina-associated extracellular matrix proteins in the HEVs are responsible for retaining the lymphoid chemokines locally. Here we show that collagen IV (Col IV) bound certain lymphoid chemokines, including CCL21, CXCL13, and CXCL12, more potently than did fibronectin or laminin-1, but it bound CCL19 and CCL5 only weakly, if at all. Surface plasmon resonance analysis indicated that Col IV bound CCL21 with a low nanomolar K-D, which required the C-terminal region of CCL21. Col IV can apparently hold these chemokines in their active form upon binding, because the Col IV-bound chemokines induced lymphocyte migration efficiently in vitro. We found by immunohistochemistry that Col IV and CCL21, CXCL13, and CXCL12 were colocalized in the basal lamina of HEVs. When injected s.c. into plt/plt mice, CCL21 colocalized at least partially with Col IV on the basal lamina of HEVs in draining lymph nodes. Collectively, our results suggest that Col IV contributes to the creation of a lymphoid chemokine-rich environment in the basal lamina of HEVs by binding an array of locally produced lymphoid chemokines that promote directional lymphocyte trafficking from HEVs into the lymphoid tissue parenchyma.

Plasmacytoid dendritic cells (pDCs) are natural type I IFN-producing cells found in lymphoid tissues, where they support both innate and adaptive immune responses. They emigrate from the blood to lymph nodes, apparently through high endothelial venules (HEVs), but little is known about the mechanism. We have investigated the molecular mechanisms of pDC migration using freshly isolated DCs and HEV cells. We found that pDCs bound avidly to HEV cells and then transmigrated underneath them. Two observations suggested that these binding and migration steps are differentially regulated. First, treatment of pDCs with pertussis toxin blocked transmigration but not binding. Second, pDCs were able to bind but not to transmigrate under non-HEV endothelial cells, although the binding was observed to both HEV and non-HEV endothelial cells. Antibody inhibition studies indicated that the binding process was mediated by alphaL and alpha4 integrins on pDCs and by intercellular adhesion molecule (ICAM)-1, ICAM-2 and vascular cell adhesion molecule-1 on HEVs. The transmigration process was also mediated by alphaL and alpha4 integrins on pDCs, with junctional adhesion molecule-A on HEV cells apparently serving as an additional ligand for alphaL integrin. These data show for the first time that pDCs employ multiple adhesion molecules sequentially in the processes of adhesion to and transmigration through HEVs.

CD44, a major cell surface receptor for hyaluronan (HA), contains a functional domain responsible for HA binding at its N terminus (residues 21-178). Accumulating evidence indicates that proteolytic cleavage of CD44 in its extracellular region (residues 21-268) leads to enhanced tumor cell migration and invasion. Hence, understanding the mechanisms underlying the CD44 proteolytic cleavage is important for understanding the mechanism of CD44-mediated tumor progression. Here we present the NMR structure of the HA-binding domain of CD44 in its HA-bound state. The structure is composed of the Link module (residues 32-124) and an extended lobe (residues 21-31 and 125-152). Interestingly, a comparison of its unbound and HA-bound structures revealed that rearrangement of the beta-strands in the extended lobe (residues 143-148) and disorder of the structure in the following C-terminal region (residues 153-169) occurred upon HA binding, which is consistent with the results of trypsin proteolysis studies of the CD44 HA-binding domain. The order-to-disorder transition of the C-terminal region by HA binding may be involved in the CD44-mediated cell migration.

Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that interacts with cell-surface receptors, including CD44. Although HA usually exists as a high molecular mass polymer, HA of a much lower molecular mass that shows a variety of biological activities can be detected under certain pathological conditions, particularly in tumors. We previously reported that low molecular weight HAs (LMW-HAs) of a certain size range induce the proteolytic cleavage of CD44 from the surface of tumor cells and promote tumor cell migration in a CD44-dependent manner. Here, we show that MIA PaCa-2, a human pancreatic carcinoma cell line, secreted hyaluronidases abundantly and generated readily detectable levels of LMW-HAs ranging from approximately 10- to 40-mers. This occurred in the absence of any exogenous stimulation. The tumor-derived HA oligosaccharides were able to enhance CD44 cleavage and tumor cell motility. Inhibition of the CD44-HA interaction resulted in the complete abrogation of these cellular events. These results are consistent with the concept that tumor cells generate HA oligosaccha-rides that bind to tumor cell CD44 through the expression of their own constitutive hyaluronidases. This enhances their own CD44 cleavage and cell motility, which would subsequently promote tumor progression. Such an autocrine/paracrine-like process may represent a novel activation mechanism that would facilitate and promote the malignant potential of tumor cells.

Chemokine receptor signaling is critical for lymphocyte trafficking across high endothelial venules (HEVs), but the exact mode of action of individual chemokines expressed in the HEVs is unclear. Here we report that CXCL13, expressed in a substantial proportion of HEVs in both lymph nodes (LNs) and Peyer patches (PPs), serves as an arrest chemokine for B cells. Whole-mount analysis of mesenteric LNs (MLNs) showed that, unlike T cells, B cellsa dhere poorly to the HEVs of CXCL13-/- mice and that B-cell adhesion is substantially restored in CXCL13-/- HEVs when CXCL13 is added to the MLNs by superfusion, as we have previously observed in PP HEVs by intravital microscopy. In vitro, CXCL13 activated the small guanosine triphosphatase (GTPase) Rap1 in B cells, and corroborating this observation, a deficiency of RAPL, the Rap1 effector molecule, caused a significant reduction in shear-resistant B-cell adhesion to intercellular adhesion molecule 1 (ICAM-1). In addition, CXCL13 induced B-cell adhesion to mucosal addressin cell adhesion molecule 1 (MAdCAM-1) by activating alpha4 integrin. These data identify CXCL13 as an arrest chemokine for B cells in HEVs and show that CXCL13 plays an important role in B-cell entry into not only PPs but also MLNs.

FRNK, the autonomously expressed carboxyl-terminal region of focal adhesion kinase(FAK), is expressed in tissues that are rich in vascular smooth muscle cells (VSMCs). Here we report the generation of transgenic mice harboring the putative FRNK promoter fused to LacZ and examine the promoter activity in situ via expression of beta-galactosidase. The transgenic mice exhibited expression of beta-galactosidase predominantly in arterial VSMCs in large and small blood vessels of major organs. Upregulation of beta-galactosidase activity was observed in tunica media following carotid injury, indicating that the FRNK promoter is activated in VSMCs in response to injury. Robust expression of beta-galactosidase in blood vessels was also detected in the developing embryo. However, expression was also observed in the midline, the nose and skin epidermis, indicating distinct transcriptional regulation of the FRNK promoter in embryogenesis. To analyze FRNK expression in vitro, we identified a 116 bp sequence in the FRNK promoter that was sufficient to function as an enhancer when fused to the minimal actin promoter and expressed in cultured smooth muscle cells. Mutation of AP-1 and NF-E2 binding consensus sequences within this element abrogated enhancer activity, supporting the involvement of this promoter element in VSMC expression of FRNK.

Although chemokines have been thought of primarily as leukocyte attractants, a growing body of evidence indicates that they also contribute to a number of tumor-related processes, such as tumor cell growth, angiogenesis/angiostasis, local invasion, and metastasis. The current knowledge of the possible involvement of chemokines and their receptors in these cellular events are reviewed here. The operating mechanism of chemokines in relation to metastatic processes in vivo are also discussed.

Hsp90 is a molecular chaperone that binds and assists refolding of non-native and/or labile polypeptides and also bind various peptides. However, the rules of how Hsp90 recognizes substrates have not been well characterized. By surface plasmon resonance measurements, a physiologically active peptide, neuropeptide Y (NPY), with a strong binding property to Hsp90 was identified from screening of 38 randomly selected peptide candidates. We showed that the carboxy-terminal fragment of NPY (NPY13-36), which forms an amphipathic alpha-helix structure, preserved the strong binding to Hsp90. Immunoprecipitation and immunoblotting using HeLa cell extracts revealed that newly synthesized NPY precursors bound to Hsp90, suggesting that the in vitro binding experiments identified an interactive peptide in vivo. Proteolytic cleavage of the NPY13-36/Hsp90 complex, as well as binding site analysis using deletion mutants of Hsp90, revealed the NPY binding locus on Hsp90alpha as the 192 amino acid region following the N-terminal domain. By electron microscopic analysis using an anti-Hsp90 antibody against the sequence proximal to the highly charged region, we showed that the Hsp90 dimer bound to NPY13-36 at both ends. Mutation of arginine residues in NPY13-36 to alanine abrogated binding to Hsp90. Our studies indicate that the hinge region after the N-terminal domain of Hsp90 and the positive charges on NPY are important for this interaction.

The 90 kDa heat shock protein (Hsp90) induces the condensation of the chromatin structure [Csermely, P., Kajtar, J., Hollosi, M., Oikarinen, J., and Somogyi, J. (1994) Biochem. Biophys. Res. Commun. 202, 1657-1663]. In our present studies we used surface plasmon resonance measurements to demonstrate that Hsp90 binds histones H1, H2A, H2B, H3 and H4 with high affinity having dissociation constants in the submicromolar range. Strong binding of the C-terminal peptide of histone H1 containing the SPKK-motif and a pentaeicosa-peptide including the Hsp90 bipartite nuclear localization signal sequence was also observed. However, a lysine/arginine-rich peptide of casein, and the lysine-rich platelet factor 4 did not display a significant interaction with Hsp90. Histones and positively charged peptides modulated the Hsp90-associated kinase activity. Interactions between Hsp90, histones, and high mobility group (HMG) protein-derived peptides raise the possibility of the involvement of Hsp90 in chromatin reorganization during steroid action, mitosis, or after cellular stress.

CD44 is a transmembrane glycoprotein involved in various cell adhesion events, including lymphocyte migration, early hemopoiesis, and tumor metastasis, To examine the requirements of CD44 for signal delivery through the extracellular domain, we constructed a chimeric CD44 protein fused to the intracellular domain of Fas on its C-terminus, In cells expressing the CD44-Fas fusion protein, apoptosis could be induced by treatment with certain anti-CD44 mAbs alone, especially those recognizing the epitope group d, which has been previously shown to play a role in ligand binding, indicating that ligation of a specific region of the CD44 extracellular domain results in signal delivery. Of note was that appropriate ligation of the epitope h also resulted in the generation of apoptotic signal, although this region was not thought to be involved in ligand binding, In contrast, the so-called blocking anti-CD44 mAbs (epitope group f) that can abrogate the binding of hyaluronate (HA) failed to induce apoptosis even after further cross-linking with the secondary Ab, indicating that a mere mAb-induced oligomerization of the chimeric proteins is insufficient for signal generation. However, these blocking mAbs were instead capable of inhibiting apoptosis induced by nonblocking mAb (epitope group h), Furthermore, a chimeric protein bearing a mutation in the HA binding domain and hence lacking the ability to recognize HA was incapable of mediating the mAb-induced apoptosis, suggesting that the functional integrity of the HA binding domain is crucial to the signal generation in CD44.

By introducing a cDNA library derived from rat embryonic fibroblast cells, we isolated several morphologically flat revertants of rat 3Y1 cells transformed by the E6 and E7 genes of human papillomavirus type 18 (HPV18). From one of the revertants, we recovered a 0.2-kb cDNA, N56, that suppresses the tumor growth of the transformed 3Y1 cells irrespective of the expression of the E6 and E7 genes. The nucleotide sequence of the cDNA was shown to be identical to that of the 3' untranslated region of a putative mammalian polycomb group gene, mel-18. (C) 1997 Elsevier Science Ireland Ltd.

In the present study, we have established a system where engagement of an adhesion molecule triggers a death signal into cells, L-selectin, which is a well characterized adhesion receptor involved in the initial adhesion between lymphocyte and endothelium, was fused to the intracellular domain of an apoptosis-inducing molecule, Fas. Ligation of the chimeric receptors with a carbohydrate ligand for L-selectin, fucoidin or a mAb that recognizes the lectin domain of L-selectin, induced apoptosis in receptor-expressing cells. However, ligation with an anti-L-selectin mAb reactive with a non-ligand binding site did not induce apoptosis, indicating that stimulation through the lectin domain of L-selectin in the chimeric receptor leads to signal delivery. Upon activation L-selectin shows a unique proteolytic cleavage at the membrane proximal site on the extracellular (EC) domain, of which the significance is also unclear. We found that truncations in the EC domain which abrogate the proteolytic cleavage of L-selectin did not influence induction of apoptosis, suggesting that the cleavage on the EC domain itself is not important for the signaling function of the chimeric receptor. This is the first demonstration that an adhesion signal can be converted to a signal that leads to apoptotic cell death.

Certain types of human papillomavirus (HPV), such as types 16 and 18, are thought to be responsible for the development of cervical carcinomas. The E6 and E7 genes of these viruses have transforming activities in various cultured cells and their mRNAs and proteins are expressed in almost all cervical carcinoma cells. Inactivation of the tumor suppressor p53 protein by the E6 gene is believed to be critical for transformation by these oncogenic HPVs. To determine whether degradation of the p53 protein is, in fact, sufficient for cellular transformation by the E6 gene, the E6 gene of HPV16 was introduced into human embryonic fibroblasts (HEF) using recombinant murine retrovirus and examined whether reduction of the p53 protein could substitute for the E6 function. It was found that HEF cells transfected with the E6 gene showed an increased saturation density and degraded the p53 protein. However, when expression of the p53 protein in normal HEF cells was suppressed by the antisense oligonucleotide of the p53 gene, growth stimulation was not observed. These results show that the E6 gene stimulates growth of HEF cells, but that this activity involves some other E6 gene-mediated functions than degradation of the p53 protein. (C) 1994 Wiley-Liss, Inc.

For determination of the correlation between tumorigenicity and the expression levels or splicing patterns of E6 mRNAs of the human papillomavirus type 16 in established cells, a vector containing the intact E6 open reading frame which expresses both unspliced and spliced mRNAs, one expressing only unspliced E6 mRNA, and one expressing both unspliced and spliced mRNAs but producing only truncated E6 proteins were constructed. In transformation assays and analyses of E6 mRNAs, a higher expression level of unspliced E6 mRNA was found to be closely associated with tumorigenicity. Furthermore, it was also related with anchorage-independent growth and a decreased serum requirement of the cells.

A rat embryo fibroblast (REF) cDNA expression library was transfected into 3Y1 cells transformed by human papillomavirus type 18 E6 and E7 genes and 10 flat revertants were isolated. These revertants expressed the same levels of E6 and E7 mRNA as the parent cells, but had greatly reduced ability to form colonies in soft agar. Suppression of transformation was dominant in cell hybrids generated by fusing each revertant with the parental transformed cells. Furthermore, loss of transfected cDNA was observed in re-transformed cell hybrids derived from one flat revertant. Overexpression of the cDNA suppresses the colony-forming efficiency of the cells transformed by E6 and E7 genes.

It has been suggested that the two acidic regions around residue 70 and residue 170 in yeast cytochrome c(1) a subunit of ubiquinol-cytochrome c reductase (complex III), interact with cytochrome c in the electron transfer reaction and that the QCR6 protein, the acidic subunit of yeast complex III, enhances this interaction. In order to determine the roles of the acidic regions of cytochrome c(1) more precisely, we introduced several mutations in the two acidic regions and examined their effects on the ability of modified cytochrome c(1) to complement the respiration deficiency of yeast cells lacking only cytochrome c(1) or both cytochrome c(1) and the QCR6 protein. The mutant cytochrome c(1) with the deletion of the first acidic region (Delta 68-80) was still functional in the cytochrome c(1)-deficient strain. Mutant cytochrome c(1) with the deletion of the second acidic region (Delta 168-179) caused a decrease in the complementing ability, but this is probably due to failure in its proteolytic maturation and/or correct assembly into complex III. Mutant cytochrome c(1) with altered charge distribution in the acidic regions (Asp(170)Asp(171)-->Asn(170)Asn(171) or Asp(170)Asp(171)-->Asn(170)Lys(171)) made the cytochrome c(1)-deficient cells respiration-competent. On the other hand, mutant cytochrome c(1) with the deletion of the first acidic region (Delta 68-80) or altered charge distribution in the second region (Asp(170)Asp(171)-->Asn(170)Lys(171)) did not restore the respiration deficiency of the cells lacking not only cytochrome c(1) but also the QCR6 protein. These results indicate that the acidic regions in cytochrome c(1) are essential for the ubiquinol-cytochrome c reductase activity in yeast cells in the absence of the QCR6 protein, and suggest the acidic regions of cytochrome c(1) may promote binding of complex III to cytochrome c in cooperation with the QCR6 protein.

CD44の細胞外N末端領域に存在するヒアルロン酸(HA)結合ドメインについてNMRを用いた構造生物学的解析を行った。CD44 HA結合ドメインは、他のHA結合タンパク質とのアミノ酸相同性が高いリンクモジュールの立体構造を保持し、さらに付加配列領域においてβシートを形成していた。ヒアルロン酸結合ドメインのC末端βシート領域は、マトリックスメタロプロテアーゼによってCD44が切断を受ける部位の近傍に位置し、糖鎖の結合によるCD44の構造変化とCD44の切断に伴う細胞の移動・浸潤との相関が示唆された。

フラボノイドは代表的な機能性食品成分であり、その生体利用性を明らかにすることは作用機序解明に重要である。本研究においては、リンパカニュレーションラットを用いて、どのような構造を持つフラボノイドがリンパ系に輸送されやすいのかを明らかにし、またフラボノイドのリンパ輸送の生理的意義を解明することを目的としている。フラボノイドの構造とリンパ輸送の関係性については、血漿／リンパ液の分配比がフラボノイドの基本骨格により異なることが示唆されている。この時、通常脂質吸収経路としての役割を果たすリンパ液への輸送であるが、食品中に存在するフラボノイドの極性はリンパ輸送の要因とはならないと考えられた。現在さらに種類を増やして構造輸送相関を検討している。また、一部の配糖体が直接リンパ液に出現することを明らかにした。リンパ系へ輸送されたフラボノイドの生理的役割については、既にリンパ液へ輸送されることを明らかにしているケルセチンとアピゲニンを中心に影響を調べたところ、リンパ管内皮細胞における炎症時の応答に対する抑制効果はフラボノイドの構造により異なることが示唆された。しかし現時点では、どのような分子内構造が活性発揮に重要かは明確でなく、特にフラボノイド基本骨格と抱合代謝による活性変化に着目しながら、引き続き検討している。また、リンパ液へ輸送されるフラボノイドの標的と考えられるリンパ節におけるフラボノイドの作用を探索するため、樹状細胞を用いた実験系の確立を開始した。

特定の二次リンパ組織にのみ存在する高内皮細静脈 (HEV) は特殊な血管であり、リンパ球のみを免疫組織実質へ選択的に動員する機能をもつ。研究代表者らの研究から、高内皮細静脈形成を制御する転写因子候補である Dach1 が、リンパ組織HEV形成を調節することが示唆されている。これまでに、Dach1遺伝子恒常的発現マウスに形成される腫瘍において組織内血管が増加し、腫瘍形成が増大することを示す予備的結果を得ている。また昨年度おこなったDach1遺伝子恒常的発現マウス（Dach1-Tg ）リンパ節の組織解析研究結果から、 Dach1-Tg成体リンパ節血管内皮細胞がHEV 前駆細胞と類似の分子発現パターンを示すことが明らかになった。本年度は、RNA-Seqを用いて、リンパ組織高内皮細静脈内皮細胞においてDach1が発現調節する遺伝子を同定した。その結果、Dach1-Tg HEV内皮細胞では野生型の約6倍のDach1発現が確認できた。若齢Dach1-Tgと野生型間の遺伝子発現比較から、Dach1-TgのHEV内皮細胞では、HEVに特徴的な接着分子群発現に必要な糖鎖修飾酵素が約60倍発現増加することが明らかになった。またこれらの接着分子群コアタンパク質として知られる分子のひとつが50倍高発現することがわかった。これらの分子はDach1ターゲット候補であり、今後Dach1による転写活性化の可能性を検討する必要がある。 一方、腫瘍血管に対するDach1の関与については、血管特異的Dach1-欠損マウス（cKO ）とコントロールの間に統計学的有意差にはいたらなかったものの、Dach1-cKOで腫瘍内血管割合に減少傾向がみられた。腫瘍重量の比較では、Dach1-cKOで有意な減少が見られた 。これらのことから、血管でのDach1 発現は腫瘍内血管形成と腫瘍増殖に促進的に作用する可能性が示唆された。

高内皮細静脈 (HEV) は二次リンパ組織特異的に形成され、定常状態における免疫組織への選択的なリンパ球移行に不可欠なる特殊血管である。近年、癌組織においてもHEVの形態学的特徴や遺伝子発現様式を示す類似の血管が発見され、このHEV様血管 (HEV-LV)が癌組織内に免疫細胞を動員することが示唆されている。私たちは腫瘍組織で形成されるHEV-LVを、二次リンパ組織HEV内皮細胞に発現することが知られているケモカインCCL21 発現を介して検出することに成功した。Ccl21a プロモーターの制御下に蛍光タンパク質TdTomatoを発現するマウスに、メラノーマB16F10を皮内移植し組織解析をおこなった結果、HEV-LVの特徴をもつ腫瘍内血管が観察された。HEV-LV形成が抗腫瘍免疫に関与する可能性を検討するため、私たちが既に同定した新規HEV形成誘導因子Dach1の過剰発現マウスにB16F10を移植し、血管形成の変化を免疫組織解析により比較した。その結果、Dach1恒常発現マウスに形成させた腫瘍組織において、腫瘍細胞全体における I-B4 陽性細胞の割合に有意な増加がみられた。また腫瘍サイズを比較したところ、野生型 マウスに比べ Dach1恒常発現マウスの方が大きい傾向がみられた。以上のことから、腫瘍内HEV様血管形成がメラノーマ組織の増殖に重要である可能性が示唆された。今後、HEV-LVを介して腫瘍組織内部へどのような免疫細胞サブセットが動員され、腫瘍増殖に関与するかについて解析する必要がある。

研究代表者の宮坂、研究分担者の梅本は、フィンランド・Turku大学の竹田、S. Jalkanen、M. Salmi博士の協力を得て、ストローマ細胞が産生する複数の生理活性物質がリンパ球の動態を制御すること、さらに、特定のストローマ細胞がリンパ節への低分子量物質移動を司ることを明らかにした。研究分担者の早坂はケモカイン受容体重合によるケモカイン応答性調節機構を明らかにした。研究分担者の片貝はリンパ節ストローマ細胞を詳細に解析し、新たなストローマ細胞サブセットを発見した。

二次リンパ組織への選択的なリンパ球動員は、高内皮細静脈 (High endothelial venules: HEV) と呼ばれる特殊な血管で媒介される。これまでHEV形成の分子メカニズムは不明であったが、私たちの先行研究から、遺伝子XがHEV成熟が進む新生仔期においてHEV内皮細胞に選択的に発現することが明らかになった。遺伝子Xを欠損するマウスを解析したところ、HEV選択的に発現する接着分子の発現が低下していた。さらに遺伝子Xを恒常的に高発現するトランスジェニックマウスを作製したところ、HEV選択的に発現する接着分子発現上昇がみられた。

領域まとめのシンポジウムを主催した。平成27年2月4日と5日の2日間にわたって、理化学研究所横浜事業所交流棟ホールにて開催した。 海外からの招待講演者として、Peter Wright 博士(Scripps, USA)、Angela M. Gronenborn博士 (Univ. of Pittsburgh, USA)、Tai-huang Huang博士 (Academia Sinica, Taiwan)、 Michael Sattler 博士(Technische Universitat Munchen, Germany)、Wolfgang Jahnke博士 (Novartis, Switzerland)、Mitsuhiko Ikura 博士(Univ. Toronto, Canada)、Christian Griesinger博士 (MPI, Germany)、Juli Feigon博士 (UCLA, USA)、 Bong-Jin Lee博士 (Seoul National Univ. Korea)、Philipp Selenko博士 (Leibniz Institute, Germany)を招待した。 開催に際してはHPやポスターを配布するなどして広く告知を行った。また、参加者への便宜を図るため、講演スライドのハンドアウトを参加者へ配布した。 また計画班から嶋田と白川が、公募班員の片平と児嶋がそれぞれ講演を行い、本新学術領域の成果を公表した。2日間にわたるシンポジウムを行い、134名が出席した。

癌の進展、転移には、癌細胞自身の特性に加えて癌細胞をとりまく微小環境が重要である。最近、癌細胞自身あるいは転移先組織に発現する接着分子やケモカインが癌細胞の遠隔転移に関与する可能性が示唆されている。接着分子CD44は特定の癌細胞に発現し、ヒアルロン酸に結合することで癌転移に関与する可能性がある。一方ケモカインは間質細胞、血管内皮細胞、神経細胞など種々の細胞から産生され、特定のケモカインが特定の受容体に結合することで細胞応答性が調節される。本研究により、リガンドで誘導されるCD44の一過的構造変化、ケモカイン受容体の細胞膜での一過的局在変化が、各分子機能に重要であることが明らかになった。

リンパ節に存在する内皮細胞は主にリンパ管系内皮細胞、血管内皮細胞に大別され、血管内皮細胞はさらに形態的特徴や機能から HEV (高内皮細静脈)内皮細胞、非 HEV 内皮細胞に分類することができる。HEV 内皮細胞は、特異的な接着分子やケモカインを発現しリンパ球のホーミングを媒介するという点で、免疫系において重要な機能をもつ細胞である。 HEV 内皮細胞のホーミングにおける機能的解析は多数行われている一方、 HEV 内皮細胞がどこから発生し、機能的な細胞に分化するのかという点については未だ不明である。 HEV 形成期における HEV 内皮細胞分離技術の開発を行い、初めて胎生期 HEV 内皮細胞の遺伝子発現解析に成功し、選択的に発現する複数の転写因子を同定した。 これらの中の一つについて詳細な解析を行ったところ、 HEV の成熟が進む胎生後期から新生仔期において HEV 内皮細胞に選択的に発現することが明らかとなった。 また、この遺伝子を欠損するマウスは、野生型と比較して新生仔リンパ節が小さく、免疫組織化学的解析から HEV 内皮細胞に特異的に発現する複数の遺伝子発現が低下していた。これらのことから、この転写因子が HEV 内皮細胞分化に機能的に関与する可能性が示唆された。

リンパ節へのリンパ球トラフィキングを制御する血管である高内皮細静脈HEVの内皮細胞に高発現するリゾリン脂質産生酵素autotaxin(ATX)の機能をin vitro, in vivoで解析した。ATXはリンパ球の血管外移動と高い相関を示し、ATXおよびリゾホスファチジン酸(LPA)受容体の阻害実験から、ATXはLPAの産生を介して、リンパ球の血管外移動を正に制御していることが強く示唆された。

癌組織間質に由来するCD44結合性分子として低分子量ヒアルロン酸、低分子量コンドロイチン硫酸Eを同定し、これらの分子が癌細胞自身が産生する分解酵素によって癌細胞周囲に産生され、膜上のCD44を介して癌細胞の浸潤を亢進させるというオートクリン的、パラクリン的作用機構が明らかになった。また、癌細胞の運動性を制御する分子として複数のケモカインが協調的に働く可能性が示唆された。

【1】腫瘍組織へ向かう免疫細胞の動員制御機構の解析:免疫細胞の腫瘍組織への動員制御機構を明らかにするために、リンパ球を効率的に動員する高内皮細静脈(HEV)、非HEVおよび腫瘍血管に由来する血管内皮細胞の遺伝子発現の比較解析をマイクロアレイ法で行った。HEVあるいは腫瘍血管に選択的に発現する遺伝子に注目して解析をすすめ、腫瘍血管で高発現するがHEVで発現が強く抑制されている30種の遺伝子群を特定した。この遺伝子群は細胞接着分子や特定のサイトカインを含み、その発現パターンより、免疫細胞動員に対して抑制的に働く可能性が示唆された。【2】血管系を介する免疫細胞動員を促進する細胞接着制御系の機能的な意義:nepmucinはIgドメインとムチンドメインをもつ新しい細胞接着分子である。nepmucinは血管内皮細胞の管腔面ばかりでなく、ジャンクション部位にも分布する。野生型および変異nepmcuinを発現する細胞を用いてnepmucinの細胞接着制御における意義について解析したところ、nepmucinがホモフィリックな細胞接着を媒介することが示された。またこの細胞接着にはnepmucinのIgドメインが必須であり、Ca^<2+>要求性であることが示された。さらにnepmucinを発現させた血管内皮細胞とリンパ球との相互作用を解析した結果、nepmucinはリンパ球のtransmigrationを促進することが示された。リンパ球はnepmucinを発現しないことから、リンパ球にはnepmucinと結合してtransmigrationを促進するnepmucinとは異なる受容体が発現することが示唆された。一方、種々の腫瘍細胞とnepmucinの結合を解析した結果、特定の白血病細胞がnepmucinのIgドメインに結合することが示された。これら結果は、nepmucinがホモフィリックあるいはヘテロフィリックな細胞接着を制御し、リンパ球の動員とともに特定の白血病細胞の血行性浸潤に関与する可能性を示唆している。

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