Chd4 choreographs self-antigen expression for central immune tolerance.

Autoreactive T cells are eliminated in the thymus to prevent autoimmunity by promiscuous expression of tissue-restricted self-antigens in medullary thymic epithelial cells. This expression is dependent on the transcription factor Fezf2, as well as the transcriptional regulator Aire, but the entire picture of the transcriptional program has been obscure. Here, we found that the chromatin remodeler Chd4, also called Mi-2ß, plays a key role in the self-antigen expression in medullary thymic epithelial cells. To maximize the diversity of self-antigen expression, Fezf2 and Aire utilized completely distinct transcriptional mechanisms, both of which were under the control of Chd4. Chd4 organized the promoter regions of Fezf2-dependent genes, while contributing to the Aire-mediated induction of self-antigens via super-enhancers. Mice deficient in Chd4 specifically in thymic epithelial cells exhibited autoimmune phenotypes, including T cell infiltration. Thus, Chd4 plays a critical role in integrating Fezf2- and Aire-mediated gene induction to establish central immune tolerance.

Fezf2 Orchestrates a Thymic Program of Self-Antigen Expression for Immune Tolerance.

Self-tolerance to immune reactions is established via promiscuous expression of tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs), leading to the elimination of T cells that respond to self-antigens. The transcriptional regulator Aire has been thought to be sufficient for the induction of TRAs, despite some indications that other factors may promote TRA expression in the thymus. Here, we show that the transcription factor Fezf2 directly regulates various TRA genes in mTECs independently of Aire. Mice lacking Fezf2 in mTECs displayed severe autoimmune symptoms, including the production of autoantibodies and inflammatory cell infiltration targeted to peripheral organs. These responses differed from those detected in Aire-deficient mice. Furthermore, Fezf2 expression and Aire expression are regulated by distinct signaling pathways and promote the expression of different classes of proteins. Thus, two independent factors, Fezf2 and Aire, permit the expression of TRAs in the thymus to ensure immune tolerance.

Blocking VCAM-1 inhibits pancreatic tumour progression and cancer-associated thrombosis/thromboembolism.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is the deadliest cancer. Cancer-associated thrombosis/thromboembolism (CAT), frequently observed in PDAC, is known as a poor prognostic factor. Here, we investigated the underlying mechanisms between PDAC and CAT, and performed a trial of therapeutic approach for PDAC using a genetically engineered mouse model, PKF (Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox ). DESIGN: Presence of CAT in PKF mice was detected by systemic autopsy. Plasma cytokines were screened by cytokine antibody array. Murine and human plasma atrial natriuretic peptide (ANP) and soluble vascular cell adhesion molecule 1 (sVCAM-1) were determined by ELISA. Distribution of VCAM-1 in PKF mice and human autopsy samples was detected by immunohistochemistry. PKF mice were treated with anti-VCAM-1 antibody and the effects on survival, distribution of CAT and the tumour histology were analysed. RESULTS: We found spontaneous CAT with cardiomegaly in 68.4% PKF mice. Increase of plasma ANP and sVCAM-1 was observed in PKF mice and PDAC patients with CAT. VCAM-1 was detected in the activated endothelium and thrombi. Administration of anti-VCAM-1 antibody to PKF mice inhibited tumour growth, neutrophil/macrophage infiltration, tumour angiogenesis and progression of CAT; moreover, it dramatically extended survival (from 61 to 253 days, p<0.01). CONCLUSION: Blocking VCAM-1/sVCAM-1 might be a potent therapeutic approach for PDAC as well as CAT, which can contribute to the prognosis. Increase of plasma ANP and sVCAM-1 might be a diagnostic approach for CAT in PDAC.

Visualization of the cancer cell cycle by tissue-clearing technology using the Fucci reporter system.

Tissue-clearing technology is an emerging imaging technique currently utilized not only in neuroscience research but also in cancer research. In our previous reports, tissue-clearing methods were used for the detection of metastatic tumors. Here, we showed that the cell cycles of primary and metastatic tumors were visualized by tissue-clearing methods using a reporter system. First, we established cancer cell lines stably expressing fluorescent ubiquitination-based cell cycle indicator (Fucci) reporter with widely used cancer cell lines A549 and 4T1. Fluorescence patterns of the Fucci reporter were investigated in various tumor inoculation models in mice. Interestingly, fluorescence patterns of the Fucci reporter of tumor colonies were different between various organs, and even among colonies in the same organs. The effects of antitumor drugs were also evaluated using these Fucci reporter cells. Of the three antitumor drugs studied, 5-fluorouracil treatment on 4T1-Fucci cells resulted in characteristic fluorescent patterns by the induction of G2 /M arrest both in vitro and in vivo. Thus, the combination of a tissue-clearing method with the Fucci reporter is useful for analyzing the mechanisms of cancer metastasis and drug resistance.

Long-term Risk of Malignancy in Branch-Duct Intraductal Papillary Mucinous Neoplasms.

BACKGROUND & AIMS: Long-term outcomes of patients with branch-duct intraductal papillary mucinous neoplasms (IPMNs), particularly those after 5 years of surveillance, have not been fully evaluated in large studies. We analyzed incidences of IPMN-derived carcinoma and concomitant ductal adenocarcinoma (pancreatic ductal adenocarcinoma [PDAC]) over 20 years in a large population of patients. METHODS: We identified 1404 consecutive patients (52% women; mean age, 67.5 years) with a diagnosis of branch-duct IPMN, from 1994 through 2017, at the University of Tokyo in Japan. Using a competing risk analysis, we estimated cumulative incidence of pancreatic carcinoma, overall and by carcinoma type. We used competing risks proportional hazards models to estimate subdistribution hazard ratios (SHRs) for incidences of carcinomas. To differentiate IPMN-derived and concomitant carcinomas, we collected genomic DNA from available paired samples of IPMNs and carcinomas and detected mutations in GNAS and KRAS by polymerase chain reaction and pyrosequencing. RESULTS: During 9231 person-years of follow-up, we identified 68 patients with pancreatic carcinomas (38 patients with IPMN-derived carcinomas and 30 patients with concomitant PDACs); the overall incidence rates were 3.3%, 6.6%, and 15.0% at 5, 10, and 15 years, respectively. Among 804 patients followed more than 5 years, overall cumulative incidence rates of pancreatic carcinoma were 3.5% at 10 years and 12.0% at 15 years from the initial diagnosis. The size of the IPMN and the diameter of the main pancreatic duct associated with incidence of IPMN-derived carcinoma (SHR 1.85; 95% confidence interval 1.38-2.48 for a 10-mm increase in the IPMN size and SHR 1.56; 95% confidence interval 1.33-1.83 for a 1-mm increase in the main pancreatic duct diameter) but not with incidence of concomitant PDAC. CONCLUSIONS: In a large long-term study of patients with branch-duct IPMNs, we found the 5-year incidence rate of pancreatic malignancy to be 3.3%, reaching 15.0% at 15 years after IPMN diagnosis. We observed heterogeneous risk factor profiles between IPMN-derived and concomitant carcinomas.

The contribution of transcription factor IRF1 to the interferon-gamma-interleukin 12 signaling axis and TH1 versus TH-17 differentiation of CD4+ T cells.

Interleukin-12 (IL-12) and interferon-gamma (IFN-gamma) drive T helper type 1 (T(H)1) differentiation, but the mechanisms underlying the regulation of the complicated gene networks involved in this differentiation are not fully understood. Here we show that the IFN-gamma-induced transcription factor IRF1 was essential in T(H)1 differentiation by acting on Il12rb1, the gene encoding the IL-12 receptor beta1 subunit (IL-12Rbeta1). IRF1 directly interacted with and activated the Il12rb1 promoter in CD4+ T cells. Notably, the IRF1-dependent induction of IL-12Rbeta1 was essential for IFN-gamma-IL-12 signaling but was dispensable for IL-23-IL-17 signaling. Because both IL-12 and IL-23 bind to and transmit signals through IL-12Rbeta1, our data suggest that distinct thresholds of IL-12Rbeta1 expression are required for T(H)1 versus T(H)-17 differentiation.

SIRT2-mediated inactivation of p73 is required for glioblastoma tumorigenicity.

Glioblastoma is one of the most aggressive forms of cancers and has a poor prognosis. Genomewide analyses have revealed that a set of core signaling pathways, the p53, RB, and RTK pathways, are commonly deregulated in glioblastomas. However, the molecular mechanisms underlying the tumorigenicity of glioblastoma are not fully understood. Here, we show that the lysine deacetylase SIRT2 is required for the proliferation and tumorigenicity of glioblastoma cells, including glioblastoma stem cells. Furthermore, we demonstrate that SIRT2 regulates p73 transcriptional activity by deacetylation of its C-terminal lysine residues. Our results suggest that SIRT2-mediated inactivation of p73 is critical for the proliferation and tumorigenicity of glioblastoma cells and that SIRT2 may be a promising molecular target for the therapy of glioblastoma.

c-Ski accelerates renal cancer progression by attenuating transforming growth factor ß signaling.

Although transforming growth factor beta (TGF-ß) is known to be involved in the pathogenesis and progression of many cancers, its role in renal cancer has not been fully investigated. In the present study, we examined the role of TGF-ß in clear cell renal carcinoma (ccRCC) progression in vitro and in vivo. First, expression levels of TGF-ß signaling pathway components were examined. Microarray and immunohistochemical analyses showed that the expression of c-Ski, a transcriptional corepressor of Smad-dependent TGF-ß and bone morphogenetic protein (BMP) signaling, was higher in ccRCC tissues than in normal renal tissues. Next, a functional analysis of c-Ski effects was carried out. Bioluminescence imaging of renal orthotopic tumor models demonstrated that overexpression of c-Ski in human ccRCC cells promoted in vivo tumor formation. Enhancement of tumor formation was also reproduced by the introduction of a dominant-negative mutant TGF-ß type II receptor into ccRCC cells. In contrast, introduction of the BMP signaling inhibitor Noggin failed to accelerate tumor formation, suggesting that the tumor-promoting effect of c-Ski depends on the inhibition of TGF-ß signaling rather than of BMP signaling. Finally, the molecular mechanism of the tumor-suppressive role of TGF-ß was assessed. Although TGF-ß signaling did not affect tumor angiogenesis, apoptosis of ccRCC cells was induced by TGF-ß. Taken together, these findings suggest that c-Ski suppresses TGF-ß signaling in ccRCC cells, which, in turn, attenuates the tumor-suppressive effect of TGF-ß.

Loss of lymph node fibroblastic reticular cells and high endothelial cells is associated with humoral immunodeficiency in mouse graft-versus-host disease.

Graft-versus-host disease (GVHD) is a major risk factor for prolonged humoral immunodeficiency and vaccine unresponsiveness after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the underlying mechanisms for this immunodeficiency are poorly understood. In this article, we describe previously overlooked impacts of GVHD on lymph node (LN) stromal cells involved in humoral immune responses. In major- and minor-mismatched mouse allo-HSCT models, recipients with CD8(+) T cell-mediated GVHD suffered severe and irreversible damage to LN structure. These mice were susceptible to pathogenic infection and failed to mount humoral immune responses despite the presence of peripheral T and B cells. These humoral immune defects were associated with the early loss of fibroblastic reticular cells, most notably the CD157(+) cell subset, as well as structural defects in high endothelial venules. The disruption to these LN stromal cells was dependent on alloantigens expressed by nonhematopoietic cells. Blockade of the Fas-FasL pathway prevented damage to CD157(+) fibroblastic reticular cells and ameliorated LN GVHD. However, blockade of CD62L- or CCR7-dependent migration of CD8(+) T cells to the LN was insufficient to prevent stromal cell injury. Overall, our results highlight GVHD-associated loss of functional stromal cells and LN GVHD as a possible explanation for the prolonged susceptibility to infectious disease that is experienced by allo-HSCT patients.

The niche component periostin is produced by cancer-associated fibroblasts, supporting growth of gastric cancer through ERK activation.

Overexpression of periostin (POSTN), an extracellular matrix protein, has been observed in several cancers. We investigated the importance of POSTN in gastric cancer. Genome-wide gene expression analysis using publicly available microarray data sets revealed significantly high POSTN expression in cancer tissues from stage II-IV gastric cancer, compared with background normal tissues. The POSTN/vimentin mRNA expression ratio was highly associated with gene groups that regulate the cell cycle and cell proliferation. IHC showed that periglandular POSTN deposition, comprising linear deposition abutting the glandular epithelial cells in normal mucosa, disappeared during intestinal gastric cancer progression. Stromal POSTN deposition was also detected at the invasive front of intestinal-type and diffuse-type cancers. In situ hybridization confirmed POSTN mRNA in cancer-associated fibroblasts, but not in tumor cells themselves. POSTN enhanced the in vitro growth of OCUM-2MLN and OCUM-12 diffuse-type gastric cancer cell lines, accompanied by the activation of ERK. Furthermore, coinoculation of gastric cancer cells with POSTN-expressing NIH3T3 mouse fibroblast cells facilitated tumor formation. The OCUM-2MLN orthotopic inoculation model demonstrated that tumors of the gastric wall in Postn(-/-) mice were significantly smaller than those in wild-type mice. Ki-67 and p-ERK positive rates were both lower in Postn(-/-) mice. These findings suggest that POSTN produced by cancer-associated fibroblasts constitutes a growth-supportive microenvironment for gastric cancer.

PCDH10 is required for the tumorigenicity of glioblastoma cells.

Protocadherin10 (PCDH10)/OL-protocadherin is a cadherin-related transmembrane protein that has multiple roles in the brain, including facilitating specific cell-cell connections, cell migration and axon guidance. It has recently been reported that PCDH10 functions as a tumor suppressor and that its overexpression inhibits proliferation or invasion of multiple tumor cells. However, the function of PCDH10 in glioblastoma cells has not been elucidated. In contrast to previous reports on other tumors, we show here that suppression of the expression of PCDH10 by RNA interference (RNAi) induces the growth arrest and apoptosis of glioblastoma cells in vitro. Furthermore, we demonstrate that knockdown of PCDH10 inhibits the growth of glioblastoma cells xenografted into immunocompromised mice. These results suggest that PCDH10 is required for the proliferation and tumorigenicity of glioblastoma cells. We speculate that PCDH10 may be a promising target for the therapy of glioblastoma.

Differential knockdown of TGF-ß ligands in a three-dimensional co-culture tumor- stromal interaction model of lung cancer.

BACKGROUND: Transforming growth factor (TGF)-ß plays a pivotal role in cancer progression through regulating cancer cell proliferation, invasion, and remodeling of the tumor microenvironment. Cancer-associated fibroblasts (CAFs) are the predominant type of stromal cell, in which TGF-ß signaling is activated. Among the strategies for TGF-ß signaling inhibition, RNA interference (RNAi) targeting of TGF-ß ligands is emerging as a promising tool. Although preclinical studies support the efficacy of this therapeutic strategy, its effect on the tumor microenvironment in vivo remains unknown. In addition, differential effects due to knockdown of various TGF-ß ligand isoforms have not been examined. Therefore, an experimental model that recapitulates tumor-stromal interaction is required for validation of therapeutic agents. METHODS: We have previously established a three-dimensional co-culture model of lung cancer, and demonstrated the functional role of co-cultured fibroblasts in enhancing cancer cell invasion and differentiation. Here, we employed this model to examine how knockdown of TGF-ß ligands affects the behavior of different cell types. We developed lentivirus vectors carrying artificial microRNAs against human TGF-ß1 and TGF-ß2, and tested their effects in lung cancer cells and fibroblasts. RESULTS: Lentiviral vectors potently and selectively suppressed the expression of TGF-ß ligands, and showed anti-proliferative effects on these cells. Furthermore, knockdown of TGF-ß ligands attenuated fibroblast-mediated collagen gel contraction, and diminished lung cancer cell invasion in three-dimensional co-culture. We also observed differential effects by targeting different TGF-ß isoforms in lung cancer cells and fibroblasts. CONCLUSIONS: Our findings support the notion that RNAi-mediated targeting of TGF-ß ligands may be beneficial for lung cancer treatment via its action on both cancer and stromal cells. This study further demonstrates the usefulness of this three-dimensional co-culture model to examine the effect of therapeutic agents on tumor-stromal interaction.

Ets family members induce lymphangiogenesis through physical and functional interaction with Prox1.

Prox1 plays pivotal roles during embryonic lymphatic development and maintenance of adult lymphatic systems by modulating the expression of various lymphatic endothelial cell (LEC) markers, such as vascular endothelial growth factor receptor 3 (VEGFR3). However, the molecular mechanisms by which Prox1 transactivates its target genes remain largely unknown. Here, we identified Ets-2 as a candidate molecule that regulates the functions of Prox1. Whereas Ets-2 has been implicated in angiogenesis, its roles during lymphangiogenesis have not yet been elucidated. We found that endogenous Ets-2 interacts with Prox1 in LECs. Using an in vivo model of chronic aseptic peritonitis, we found that Ets-2 enhanced inflammatory lymphangiogenesis, whereas a dominant-negative mutant of Ets-1 suppressed it. Ets-2 also enhanced endothelial migration towards VEGF-C through induction of expression of VEGFR3 in collaboration with Prox1. Furthermore, we found that both Prox1 and Ets-2 bind to the VEGFR3 promoter in intact chromatin. These findings suggest that Ets family members function as transcriptional cofactors that enhance Prox1-induced lymphangiogenesis.

Notch2 signaling is required for proper mast cell distribution and mucosal immunity in the intestine.

Notch receptor-mediated signaling is involved in the developmental process and functional modulation of lymphocytes, as well as in mast cell differentiation. Here, we investigated whether Notch signaling is required for antipathogen host defense regulated by mast cells. Mast cells were rarely found in the small intestine of wild-type C57BL/6 mice but accumulated abnormally in the lamina propria of the small-intestinal mucosa of the Notch2-conditional knockout mice in naive status. When transplanted into mast cell-deficient W(sh)/W(sh) mice, Notch2-null bone marrow-derived mast cells were rarely found within the epithelial layer but abnormally localized to the lamina propria, whereas control bone marrow-derived mast cells were mainly found within the epithelial layer. After the infection of Notch2 knockout and control mice with L3 larvae of Strongyloides venezuelensis, the abundant number of mast cells was rapidly mobilized to the epithelial layer in the control mice. In contrast, mast cells were massively accumulated in the lamina propria of the small intestinal mucosa in Notch2-conditional knockout mice, accompanied by impaired eradication of Strongyloides venezuelensis. These findings indicate that cell-autonomous Notch2 signaling in mast cells is required for proper localization of intestinal mast cells and further imply a critical role of Notch signaling in the host-pathogen interface in the small intestine.

Regulation of osteoclast differentiation and function by the CaMK-CREB pathway.

Calcium (Ca(2+)) signaling is essential for a variety of cellular responses and higher biological functions. Ca(2+)/calmodulin-dependent kinases (CaMKs) and the phosphatase calcineurin activate distinct downstream pathways that are mediated by the transcription factors cAMP response element (CRE)-binding protein (CREB) and nuclear factor of activated T cells (NFAT), respectively. The importance of the calcineurin-NFAT pathway in bone metabolism has been demonstrated in osteoclasts, osteoblasts and chondrocytes. However, the contribution of the CaMK-CREB pathway is poorly understood, partly because of the difficulty of dissecting the functions of homologous family members. Here we show that the CaMKIV-CREB pathway is crucial for osteoclast differentiation and function. Pharmacological inhibition of CaMKs as well as the genetic ablation of Camk4 reduced CREB phosphorylation and downregulated the expression of c-Fos, which is required for the induction of NFATc1 (the master transcription factor for osteoclastogenesis) that is activated by receptor activator of NF-kappaB ligand (RANKL). Furthermore, CREB together with NFATc1 induced the expression of specific genes expressed by differentiated osteoclasts. Thus, the CaMK-CREB pathway biphasically functions to regulate the transcriptional program of osteoclastic bone resorption, by not only enhancing induction of NFATc1 but also facilitating NFATc1-dependent gene regulation once its expression is induced. This provides a molecular basis for a new therapeutic strategy for bone diseases.

The G protein-coupled receptor T-cell death-associated gene 8 (TDAG8) facilitates tumor development by serving as an extracellular pH sensor.

Tumors often are associated with a low extracellular pH, which induces a variety of cellular events. However, the mechanisms by which tumor cells recognize and react to the acidic environment have not been fully elucidated. T-cell death-associated gene 8 (TDAG8) is an extracellular pH-sensing G protein-coupled receptor that is overexpressed in various tumors and tumor cell lines. In this report, we show that TDAG8 on the surface of tumor cells facilitates tumor development by sensing the acidic environment. Overexpression of TDAG8 in mouse Lewis lung carcinoma (LLC) cells enhanced tumor development in animal models and rendered LLC cells resistant to acidic culture conditions by increasing activation of protein kinase A and extracellular signal-regulated kinase in vitro. Moreover, shRNA-mediated knockdown of endogenous TDAG8 in NCI-H460 human non-small cell lung cancer cells reduced cell survival in an acidic environment in vitro as well as tumor development in vivo. Microarray analyses of tumor-containing lung tissues of mice injected with TDAG8-expressing LLC cells revealed up-regulation of genes related to cell growth and glycolysis. These results support the hypothesis that TDAG8 enhances tumor development by promoting adaptation to the acidic environment to enhance cell survival/proliferation. TDAG8 may represent a therapeutic target for arresting tumor growth.

Th17 functions as an osteoclastogenic helper T cell subset that links T cell activation and bone destruction.

In autoimmune arthritis, traditionally classified as a T helper (Th) type 1 disease, the activation of T cells results in bone destruction mediated by osteoclasts, but how T cells enhance osteoclastogenesis despite the anti-osteoclastogenic effect of interferon (IFN)-gamma remains to be elucidated. Here, we examine the effect of various Th cell subsets on osteoclastogenesis and identify Th17, a specialized inflammatory subset, as an osteoclastogenic Th cell subset that links T cell activation and bone resorption. The interleukin (IL)-23-IL-17 axis, rather than the IL-12-IFN-gamma axis, is critical not only for the onset phase, but also for the bone destruction phase of autoimmune arthritis. Thus, Th17 is a powerful therapeutic target for the bone destruction associated with T cell activation.

BMP-9 induces proliferation of multiple types of endothelial cells in vitro and in vivo.

Members of the bone morphogenetic protein (BMP) family have been implicated in the development and maintenance of vascular systems. Whereas members of the BMP-2/4 and osteogenic protein-1 groups signal via activin receptor-like kinase (ALK)-2, ALK-3 and ALK-6, BMP-9 and BMP-10 have been reported to bind to ALK-1 in endothelial cells. However, the roles of BMP-9-ALK-1 signaling in the regulation of endothelial cells have not yet been fully elucidated. Here, using various systems, we examined the effects of BMP-9 on the proliferation of endothelial cells. Vascular-tube formation from ex vivo allantoic explants of mouse embryos was promoted by BMP-9. BMP-9, as well as BMP-4 and BMP-6, also induced the proliferation of in-vitro-cultured mouse embryonic-stem-cell-derived endothelial cells (MESECs) by inducing the expression of vascular endothelial growth factor receptor 2 and Tie2, a receptor for angiopoietin-1. A decrease in ALK-1 expression or expression of constitutively active ALK-1 in MESECs abrogated and mimicked the effects of BMP-9 on the proliferation of MESECs, respectively, suggesting that BMP-9 promotes the proliferation of these cells via ALK-1. Furthermore, in vivo angiogenesis was promoted by BMP-9 in a Matrigel plug assay and in a BxPC3 xenograft model of human pancreatic cancer. Consistent with these in vivo findings, BMP-9 enhanced the proliferation of in-vitro-cultured normal endothelial cells from dermal tissues of adult mice and of tumor-associated endothelial cells isolated from tumor xenografts in host mice. These findings suggest that BMP-9 signaling activates the endothelium tested in the present study via ALK-1.

Characterization of human lung cancer-associated fibroblasts in three-dimensional in vitro co-culture model.

Lung cancer is the most common cause of cancer-related death worldwide. Stromal cancer-associated fibroblasts (CAFs) play crucial roles in carcinogenesis, proliferation, invasion, and metastasis of non-small cell lung carcinoma, and targeting of CAFs could be a novel strategy for cancer treatment. However, the characteristics of human CAFs still remain to be better defined. In this study, we established patient-matched CAFs and normal fibroblasts (NFs), from tumoral and non-tumoral portions of resected lung tissue from lung cancer patients. CAFs showed higher α-smooth muscle actin (α-SMA) expression than NFs, and CAFs clearly enhanced collagen gel contraction. Furthermore, we employed three-dimensional co-culture assay with A549 lung cancer cells, where CAFs were more potent in inducing collagen gel contraction. Hematoxylin and eosin staining of co-cultured collagen gel revealed that CAFs had the potential to increase invasion of A549 cells compared to NFs. These observations provide evidence that lung CAFs have the tumor-promoting capacity distinct from NFs.

LPA4 regulates blood and lymphatic vessel formation during mouse embryogenesis.

Lysophosphatidic acid (LPA) is a potent lipid mediator with a wide variety of biological actions mediated through G protein-coupled receptors (LPA(1-6)). LPA(4) has been identified as a G(13) protein-coupled receptor, but its physiological role is unknown. Here we show that a subset of LPA(4)-deficient embryos did not survive gestation and displayed hemorrhages and/or edema in many organs at multiple embryonic stages. The blood vessels of bleeding LPA(4)-deficient embryos were often dilated. The recruitment of mural cells, namely smooth muscle cells and pericytes, was impaired. Consistently, Matrigel plug assays showed decreased mural cell coverage of endothelial cells in the neovessels of LPA(4)-deficient adult mice. In situ hybridization detected Lpa4 mRNA in the endothelium of some vasculatures. Similarly, the lymphatic vessels of edematous embryos were dilated. These results suggest that LPA(4) regulates establishment of the structure and function of blood and lymphatic vessels during mouse embryogenesis. Considering the critical role of autotaxin (an enzyme involved in LPA production) and Gα(13) in vascular development, we suggest that LPA(4) provides a link between these 2 molecules.