The long non-coding RNA PVT1/miR-145-5p/ITGB8 axis regulates cell proliferation, apoptosis, migration and invasion in non-small cell lung cancer cells.

Lung cancer is one of the leading causes of death worldwide and non-small cell lung cancer (NSCLC) accounts for approximately 80% of lung cancer. Long noncoding RNAs (lncRNAs) are closely associated with the development and progression of various cancers, including lung cancer. The purpose of this study was to explore the potential role and molecular mechanism of lncRNA plasmacytoma variant translocation 1 (PVT1) in regulating the proliferation, apoptosis, migration, and invasion of NSCLC cells. The expressions of PVT1, integrin ß-8 (ITGB8), and miR-145-5p were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels of ITGB8, MEK, p-MEK, ERK, and p-ERK were measured by western blot analysis. Cell proliferation, apoptosis, migration, and invasion were determined by MTT assay, flow cytometry, and transwell assay, respectively. The potential binding sites between miR-145-5p and PVT1 or ITGB8 were predicted by online software and verified by luciferase reporter assay. A xenograft tumor model was established to confirm the effect of PVT1 on NSCLC in vivo. We found out that the expression levels of PVT1 and ITGB8 were upregulated in NSCLC tissues and cells. Knockdown of PVT1 or ITGB8 suppressed cell proliferation, migration, invasion and promoted apoptosis in NSCLC cells, which could be reversed by ITGB8 overexpression in NSCLC cells. Moreover, PVT1 could regulate ITGB8 expression via direct binding to miR-145-5p. Furthermore, PVT1 regulated the MEK/ERK pathway by affecting ITGB8 expression. In addition, knockdown of PVT1 inhibited tumor growth, ITGB8 expression, MEK/ERK signaling pathway, and increased miR-145-5p expression in vivo. In conclusion, the knockdown of PVT1 inhibited proliferation, migration, and invasion but induced apoptosis of NSCLC cells by regulating miR-145-5p/ITGB8 axis and inhibiting MEK/ERK signaling pathway, providing a novel avenue for the treatment of NSCLC.

Identification of integrin heterodimers functioning on the surface of undifferentiated porcine primed embryonic stem cells.

In vitro expansion of undifferentiated porcine primed embryonic stem (ES) cells is facilitated by use of non-cellular niches that mimic three-dimensional (3D) microenvironments enclosing an inner cell mass of porcine blastocysts. Therefore, we investigated the integrin heterodimers on the surface of undifferentiated porcine primed ES cells for the purpose of developing a non-cellular niche to support in vitro maintenance of the self-renewal ability of porcine primed ES cells. Immunocytochemistry and a fluorescence immunoassay were performed to assess integrin α and ß subunit levels, and attachment and antibody inhibition assays were used to evaluate the function of integrin heterodimers. The integrin α3 , α5 , α6 , α9 , αV , and ß1 subunits, but not the α1 , α2 , α4 , α7 , and α8 subunits, were identified on the surface of undifferentiated porcine primed ES cells. Subsequently, significant increase of their adhesion to fibronectin, tenascin C, and vitronectin were observed and functional blocking of integrin heterodimer α5 ß1 , α9 ß1 , or αV ß1 showed significantly inhibited adhesion to fibronectin, tenascin C, or vitronectin. No integrin α6 ß1 heterodimer-mediated adhesion to laminin was detected. These results demonstrate that active α5 ß1 , α9 ß1 , and αV ß1 integrin heterodimers are present on the surface of undifferentiated porcine primed ES cells, together with inactive integrin α3 (presumed) and α6 subunits.

Integrin αvß3 expression in tongue squamous carcinoma cells Cal27 confers anticancer drug resistance through loss of pSrc(Y418).

Integrins play key roles in the regulation of tumor cell adhesion, migration, invasion and sensitivity to anticancer drugs. In the present study we investigate the mechanism of resistance of tongue squamous carcinoma cells Cal27 with de novo integrin αvß3 expression to anticancer drugs. Cal27-derived cell clones, obtained by transfection of plasmid containing integrin subunit ß3 cDNA, as compared to control cells demonstrate: expression of integrin αvß3; increased expression of integrin αvß5; increased adhesion to fibronectin and vitronectin; resistance to cisplatin, mitomycin C, doxorubicin and 5-fluorouracil; increased migration and invasion, increased amount of integrin-linked kinase (ILK) and decreased amounts of non-receptor tyrosine kinase (Src) and pSrc(Y418). Knockdown of ILK and integrin ß5 in cells expressing integrin αvß3 ruled out their involvement in drug resistance. Opposite, Src knockdown in Cal27 cells which led to a reduction in pSrc(Y418), as well as treatment with the pSrc(Y418) inhibitors dasatinib and PP2, conferred resistance to all four anticancer drugs, indicating that the loss of pSrc(Y418) is responsible for the observed effect. We identified differential integrin signaling between Cal27 and integrin αvß3-expressing cells. In Cal27 cells integrin αv heterodimers signal through pSrc(Y418) while this is not the case in integrin αvß3-expressing cells. Finally, we show that dasatinib counteracts the effect of cisplatin in two additional head and neck squamous cell carcinoma (HNSCC) cell lines Cal33 and Detroit562. Our results suggest that pSrc(Y418) inhibitors, potential drugs for cancer therapy, may reduce therapeutic efficacy if combined with chemotherapeutics, and might not be recommended for HNSCC treatment.

Minimal amounts of kindlin-3 suffice for basal platelet and leukocyte functions in mice.

Hematopoietic cells depend on integrin-mediated adhesion and signaling, which is induced by kindlin-3 and talin-1. To determine whether platelet and polymorphonuclear neutrophil (PMN) functions require specific thresholds of kindlin-3, we generated mouse strains expressing 50%, 10%, or 5% of normal kindlin-3 levels. We report that in contrast to kindlin-3-null mice, which die perinatally of severe bleeding and leukocyte adhesion deficiency, mice expressing as little as 5% of kindlin-3 were viable and protected from spontaneous bleeding and infections. However, platelet adhesion and aggregation were reduced in vitro and bleeding times extended. Similarly, leukocyte adhesion, extravasation, and bacterial clearance were diminished. Quantification of protein copy numbers revealed stoichiometric quantities of kindlin-3 and talin-1 in platelets and neutrophils, indicating that reduction of kindlin-3 in our mouse strains progressively impairs the cooperation with talin-1. Our findings show that very low levels of kindlin-3 enable basal platelet and neutrophil functions, whereas in stress situations such as injury and infection, platelets and neutrophils require a maximum of functional integrins that is achieved with high and stoichiometric quantities of kindlin-3 and talin-1.

[Expression of ß-integrin family members in children with T-cell acute lymphoblastic leukemia].

OBJECTIVE: To study the expression of ß-integrin family members in children with T-cell acute lymphoblastic leukemia (T-ALL) and their significance. METHODS: Quantitative real-time PCR analyses were performed to assess the expression levels of ß-integrin family members in bone marrow samples from 22 children with newly-diagnosed T-ALL and 21 controls (16 children with non-malignant hematologic disease and 5 healthy donors with bone marrow transplantation). Jurkat cells were treated with integrin inhibitor arginine-glycine-aspartate (Arg-Gly-Asp, RGD) peptide. The cell viability and apoptosis rate were determined by CCK8 assay and flow cytometry respectively. RESULTS: The mRNA levels of integrins ß2, ß3, and ß5 were significantly lower in children with T-ALL than in controls (P<0.05). In T-ALL patients, high integrin ß3 expression was associated with lower white blood cell counts (<100×109/L), minimal residual disease (MRD) positivity, and day 33 bone marrow negative remission (P<0.05). In T-ALL patients, higher integrin ß5 expression was associated with relapse of T-ALL (P<0.05). Based on survival curve analysis, higher integrin ß3 expression was related to lower event-free survival and overall survival rates. RGD peptide treatment inhibited the proliferation of Jurkat cells and increased their apoptosis rate (P<0.05). CONCLUSIONS: ß-Integrin may play a role in the occurrence and development of T-ALL by affecting cell proliferation and apoptosis. The expression of integrin ß5 is closely related to the risk of relapse of T-ALL. The expression of integrin ß3 is closely related the treatment response and prognosis of T-ALL.

A missense mutation in ITGB6 causes pitted hypomineralized amelogenesis imperfecta.

We identified a family in which pitted hypomineralized amelogenesis imperfecta (AI) with premature enamel failure segregated in an autosomal recessive fashion. Whole-exome sequencing revealed a missense mutation (c.586C>A, p.P196T) in the I-domain of integrin-ß6 (ITGB6), which is consistently predicted to be pathogenic by all available programmes and is the only variant that segregates with the disease phenotype. Furthermore, a recent study revealed that mice lacking a functional allele of Itgb6 display a hypomaturation AI phenotype. Phenotypic characterization of affected human teeth in this study showed areas of abnormal prismatic organization, areas of low mineral density and severe abnormal surface pitting in the tooth's coronal portion. We suggest that the pathogenesis of this form of AI may be due to ineffective ligand binding of ITGB6 resulting in either compromised cell-matrix interaction or compromised ITGB6 activation of transforming growth factor-ß (TGF-ß) impacting indirectly on ameloblast-ameloblast interactions and proteolytic processing of extracellular matrix proteins via MMP20. This study adds to the list of genes mutated in AI and further highlights the importance of cell-matrix interactions during enamel formation.

Integrin beta 8 (ITGB8) regulates embryo implantation potentially via controlling the activity of TGF-B1 in mice.

Integrins (ITGs) are mediators of cell-cell and cell-matrix interactions, which are also associated with embryo implantation processes by controlling the interaction of blastocyst with endometrium. During early pregnancy, ITGbeta8 (ITGB8) has been shown to interact with latent transforming growth factor (TGF) beta 1 (TGFB1) at the fetomaternal interface. However, the precise role of ITGB8 in the uterus and its association with embryo implantation has not been elucidated. Therefore, we attempted to ascertain the role of ITGB8 during the window of embryo implantation process by inhibiting its function or protein expression. Uterine plasma membrane-anchored ITGB8 was augmented at peri-implantation and postimplantation stages. A similar pattern of mRNA expression was also found during the embryo implantation period. An immunolocalization study revealed the presence of ITGB8 on luminal epithelial cells along with mild expression on the stromal cells throughout the implantation period studied; however, an intense fluorescence was noted only during the peri- and postimplantation stages. Bioneutralization and mRNA silencing of the uterine Itgb8 at preimplantation stage reduced the rate/frequency of embryo implantation and subsequent pregnancy, suggesting its indispensable role during the embryo implantation period. ITGB8 can also regulate the liberation of active TGFB1 from its latent complex, which, in turn, acts on SMAD2/3 phosphorylation (activation) in the uterus during embryo implantation. This indicates involvement of ITGB8 in the embryo implantation process through regulation of activation of TGFB1.

Integrin-ß1, not integrin-ß5, mediates osteoblastic differentiation and ECM formation promoted by mechanical tensile strain.

BACKGROUND: Mechanical strain plays a great role in growth and differentiation of osteoblast. A previous study indicated that integrin-ß (ß1, ß5) mediated osteoblast proliferation promoted by mechanical tensile strain. However, the involvement of integrin-ß in osteoblastic differentiation and extracellular matrix (ECM) formation induced by mechanical tensile strain, remains unclear. RESULTS: After transfection with integrin-ß1 siRNA or integrin-ß5 siRNA, mouse MC3T3-E1 preosteoblasts were cultured in cell culture dishes and stimulated with mechanical tensile strain of 2500 microstrain (µÎµ) at 0.5 Hz applied once a day for 1 h over 3 or 5 consecutive days. The cyclic tensile strain promoted osteoblastic differentiation of MC3T3-E1 cells. Transfection with integrin-ß1 siRNA attenuated the osteoblastic diffenentiation induced by the tensile strain. By contrast, transfection with integrin-ß5 siRNA had little effect on the osteoblastic differentiation induced by the strain. At the same time, the result of ECM formation promoted by the strain, was similar to the osteoblastic differentiation. CONCLUSION: Integrin-ß1 mediates osteoblast differentiation and osteoblastic ECM formation promoted by cyclic tensile strain, and integrin-ß5 is not involved in the osteoblasts response to the tensile strain.

[Effect of integrin ß8 on TGF-ß1 activation in astrocytes with oxygen glucose deprivation].

OBJECTIVE: To study the effect of ß8 expression on transforming growth factor ß1(TGF-ß1) activation in astrocytes with oxygen glucose deprivation (OGD). METHODS: Astrocytes were cultured and then subjected to OGD to generate hypoxia-ischemia (HI) model in vitro. Immunocytochemistry was used to detect the expression and distribution of ß8 in nomoxia cultured cells. ß8 protein expression was quantified by Western blot at 12 hours, 1 day and 2 days after OGD. Astrocytes and luciferase reporter cells (TMLC) were co-cultured. ß8 RNA interference system was established to specifically inhibit ß8 expression in cultured astrocytes. TGF-ß1 activation was then detected in the co-culture system. RESULTS: ß8 was mainly located in the cytoplasm and neurites of astrocytes. OGD resulted in increase of ß8 protein expression at 12 hours after reoxygenation in astrocytes, which was peaked at 1 day after reoxygenation. TGF-ß1 activation was in accordance with ß8 expression in astrocyte-TMLC co-culture system after reoxygenation. After the inhibition of ß8, TGF-ß1 activation was significantly reduced in all time points. CONCLUSIONS: The highly expressed ß8 plays important roles in the regulation of TGF-ß1 activation in neonatal rats with hypoxic-ischemic brain damage.

Loss of T cell CD98 H chain specifically ablates T cell clonal expansion and protects from autoimmunity.

CD98 H chain (4F2 Ag, Slc3a2) was discovered as a lymphocyte-activation Ag. Deletion of CD98 H chain in B cells leads to complete failure of B cell proliferation, plasma cell formation, and Ab secretion. In this study, we examined the role of T cell CD98 in cell-mediated immunity and autoimmune disease pathogenesis by specifically deleting it in murine T cells. Deletion of T cell CD98 prevented experimental autoimmune diabetes associated with dramatically reduced T cell clonal expansion. Nevertheless, initial T cell homing to pancreatic islets was unimpaired. In sharp contrast to B cells, CD98-null T cells showed only modestly impaired Ag-driven proliferation and nearly normal homeostatic proliferation. Furthermore, these cells were activated by Ag, leading to cytokine production (CD4) and efficient cytolytic killing of targets (CD8). The integrin-binding domain of CD98 was necessary and sufficient for full clonal expansion, pointing to a role for adhesive signaling in T cell proliferation and autoimmune disease. When we expanded CD98-null T cells in vitro, they adoptively transferred diabetes, establishing that impaired clonal expansion was responsible for protection from disease. Thus, the integrin-binding domain of CD98 is required for Ag-driven T cell clonal expansion in the pathogenesis of an autoimmune disease and may represent a useful therapeutic target.

ß5 integrin is the major contributor to the αVintegrin-mediated blockade of HIV-1 replication.

Monocytes and macrophages are targets of HIV-1 infection and play critical roles in multiple aspects of viral pathogenesis. During the differentiation of monocytes to macrophages, adhesion molecules such as integrins are upregulated; therefore, they provide signals that control the process and subsequently may render macrophages more susceptible to HIV-1 infection. Previous work demonstrated that blocking α(v)-containing integrins triggered a signal transduction pathway leading to the inhibition of NF-κB-dependent HIV-1 transcription. In this paper, we show the influence of the different α(v)-coupled ß integrins in HIV-1 replication in macrophages. Inhibition of ß integrins, either by specific mAbs, small arginine-glycine-aspartic acid (RGD) mimetic compounds, or RNA interference, showed that integrin ß(5) was the major contributor to the integrin-mediated blockade of HIV-1 replication. Importantly, such inhibition did not induce changes in cell adhesion to the substrate. In conclusion, our results reveal a significant role of the integrin dimer α(v)ß(5) in HIV-1 infection of macrophages.

Distinct regulatory mechanisms control integrin adhesive processes during tissue morphogenesis.

Cell adhesion must be precisely regulated to enable both dynamic morphogenetic processes and the subsequent transition to stable tissue maintenance. Integrins link the intracellular cytoskeleton and extracellular matrix, relaying bidirectional signals across the plasma membrane. In vitro studies have demonstrated that multiple mechanisms control integrin-mediated adhesion; however, their roles during development are poorly understood. We used mutations that activate or deactivate specific functions of vertebrate ß-integrins in vitro to investigate how perturbing Drosophila ßPS-integrin regulation in developing embryos regulation affects tissue morphogenesis and maintenance. We found that morphogenetic processes use various ß-integrin regulatory mechanisms to differing degrees and that conformational changes associated with outside-in activation are essential for developmental integrin functions. Long-term adhesion is also sensitive to integrin dysregulation, suggesting integrins must be continuously regulated to support stable tissue maintenance. Altogether, in vivo phenotypic analyses allowed us to identify the importance of various ß-integrin regulatory mechanisms during different morphogenetic processes.

ß8 integrin and band 4.1B cooperatively regulate morphogenesis of the embryonic heart.

Morphogenesis of the heart is regulated by various cues, including growth factors and extracellular matrix (ECM) proteins. The mechanisms by which cardiac cells properly integrate these cues to regulate growth, differentiation, and migration remain poorly understood. Here we have used genetic strategies in mice to identify αvß8 integrin and its cytoskeletal adaptor protein, Band 4.1B, as essential regulators of cardiac morphogenesis. We demonstrate that approximately 60% of mouse embryos genetically null for ß8 integrin and Band 4.1B display cardiovascular phenotypes and die by E11.5. This premature death is due, in part, to defective development of the cardiac outflow tract (OFT), with reduced expression of smooth muscle α-actin (SMAα-actin) in OFT cells derived from the cardiac neural crest. These data are the first to identify cell adhesion and signaling pathways regulated by αvß8 integrin and Band 4.1B as essential for normal formation and function of the heart during embryogenesis.

ß8 integrin is essential for neuroblast migration in the rostral migratory stream.

Neurogenesis in the post-natal brain occurs in two primary locations: the subgranular layer of the hippocampal dentate gyrus and the subventricular zone (SVZ) of the lateral ventricles. Following differentiation, neuroblasts within the SVZ migrate several millimeters to the olfactory bulbs (OBs) via a distinct anatomic route, or rostral migratory stream (RMS). The genes that govern neuroblast directional migration, and particularly those encoding cell adhesion and signaling factors, remain largely uncharacterized. Here, we report that the extracellular matrix adhesion receptor, ß8 integrin, is essential for proper neuroblast chain formation and directional navigation in the RMS. Primary neuroblasts isolated from the mouse brain express robust levels of ß8 integrin protein, and selective ablation of ß8 integrin gene expression in neuroblasts leads to aberrant chain migration and size-reduced OBs. These integrin-dependent defects can be recapitulated ex vivo using isolated neurospheres or SVZ explants. Collectively, these data identify essential cell-intrinsic functions for ß8 integrin in regulating neuroblast polarity and directional navigation in the mouse forebrain.

Alpha4beta7/MAdCAM-1 interactions play an essential role in transitioning cryptopatches into isolated lymphoid follicles and a nonessential role in cryptopatch formation.

The alpha(4) integrins alpha(4)beta(7) and alpha(4)beta(1), and their ligands mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) and VCAM-1, have diverse functions, including roles in the formation of secondary lymphoid tissues at early time points during the colonization and clustering of the fetal lymphoid tissue inducer (LTi) cells and at later time points during the recruitment of lymphocytes. In this study, we evaluated the role of alpha(4) integrins in the development of a recently appreciated class of intestinal lymphoid tissues, isolated lymphoid follicles (ILFs). We observed that diverse ILF cellular populations express alpha(4)beta(7) and alpha(4)beta(1), including the LTi-like cells and lymphocytes, while ILF stromal cells and vessels within ILFs express VCAM-1 and MAdCAM-1, respectively. Evaluation of adult and neonatal beta(7)(-/-) mice and adult and neonatal mice given blocking Abs to alpha(4)beta(7), MAdCAM-1, or VCAM-1 did not identify a role for alpha(4) integrins in cryptopatch (CP) development; however, these studies demonstrated that alpha(4)beta(7) and MAdCAM-1 are required for the transitioning of CP into lymphoid tissues containing lymphocytes or ILFs. Competitive bone marrow transfers demonstrated that beta(7)(-/-) LTi-like cells had a reduced but not significantly impaired ability to localize to CP. Bone marrow transfers and adoptive transfers of B lymphocytes revealed that beta(7) expression by B lymphocytes was essential for their entry into the developing ILFs. These findings demonstrate an essential role for alpha(4)beta(7)/MAdCAM-1 in ILF development corresponding to the influx of beta(7)-expressing lymphocytes and a nonessential role for beta(7)-localizing LTi-like cells to the small intestine.

Cross-talk between integrins and oncogenes modulates chemosensitivity.

Chemotherapy often relies on cancer cell death resulting from DNA damage. The p53 tumor suppressor pathway that is an important player in DNA damage response is frequently inactivated in cancer. Genotoxicants also activate DNA damage-independent stress pathways and activity of oncogenic signaling and adhesive interactions with the cancer microenvironment can have a strong impact on chemosensitivity. Here, we have investigated how two different oncogenes modulate the response to genotoxicants in the context of two classes of integrin adhesion receptors. Epithelial cells expressing either beta1 or beta3 integrins, in which p53 activity is suppressed, undergo G(2) arrest but show little apoptosis after treatment with cisplatin or other genotoxicants. The apoptotic response is strongly enhanced by the c-Src[Y530F] oncogene in cells expressing beta1 integrins, whereas such sensitization is reduced when these cells are engineered to express beta3 integrins instead. The H-Ras[G12V] oncogene fails to sensitize, regardless of the integrin expression profile. The enhanced sensitivity induced by c-Src[Y530F] in the context of beta1 integrins does not rely on p53-mediated DNA damage signaling but instead involves increased endoplasmic reticulum stress and caspase-3 activation. Our data implicate that the expression profiles of oncogenes and integrins strongly affect the response to chemotherapeutics and may thus determine the efficacy of chemotherapy.

Identification of integrins alpha6 and beta7 as c-Jun- and transformation-relevant genes in highly invasive fibrosarcoma cells.

Understanding the mechanisms of tumor cell invasion is essential for our attempts to prevent cancer deaths. We screened by DNAmicroarrays the c-Jun- and transformation-related gene expression changes in S-adenosylmethionine decarboxylase (AdoMetDC)-overexpressing mouse fibroblasts that are highly invasive in vivo, and their derivatives expressing a tetracycline-inducible dominant-negative mutant of c-Jun (TAM67) or c-Jun shRNA. Among the small set of target genes detected were integrins alpha6 and beta7, cathepsin L and thymosin beta4, all upregulated in the AdoMetDC-transformed cells and downregulated upon reversal of transformation by TAM67 or c-Jun shRNA. The upregulation of integrin alpha6 subunit, pairing with integrin beta1, endowed the transformed cells with the capability to attach to basement membrane laminin and to spread. Further, inhibition of integrin alpha6 or beta1 function with neutralizing antibodies blocked the invasiveness of AdoMetDC-transformants and human HT-1080 fibrosarcoma cells in three-dimensional Matrigel. Moreover, immunohistochemical analyses showed strong integrin alpha6 staining in high-grade human fibrosarcomas. Our data show that c-Jun can regulate all three key steps of invasion: cell adhesion (integrin alpha6), basement membrane/extracellular matrix degradation (cathepsin L) and cell migration (thymosin beta4). In addition, this is the first study to associate integrin beta7, known as a leukocyte-specific integrin binding to endothelial/epithelial cell adhesion molecules, with the transformed phenotype in cells of nonleukocyte origin. As tumor cell invasion is a prerequisite for metastasis, the observed critical role of integrin alpha6beta1 in fibrosarcoma cell invasion/spreading allures testing antagonists to integrin alpha6beta1, alone or combined with inhibitors of cathepsin L and thymosin beta4, as chemotherapeutic agents.

Harnessing region-specific neurovascular signaling to promote germinal matrix vessel maturation and hemorrhage prevention.

Germinal matrix hemorrhage (GMH), affecting about 1 in 300 births, is a major perinatal disease with lifelong neurological consequences. Yet despite advances in neonatal medicine, there is no effective intervention. GMH is characterized by localized bleeding in the germinal matrix (GM), due to inherent vessel fragility unique to this developing brain region. Studies have shown that reduced TGFß signaling contributes to this vascular immaturity. We have previously shown that a region-specific G-protein-coupled receptor pathway in GM neural progenitor cells regulates integrin ß8, a limiting activator of pro-TGFß. In this study, we use mice to test whether this regional pathway can be harnessed for GMH intervention. We first examined the endogenous dynamics of this pathway and found that it displays specific patterns of activation. We then investigated the functional effects of altering these dynamics by chemogenetics and found that there is a narrow developmental window during which this pathway is amenable to manipulation. Although high-level activity in this time window interferes with vessel growth, moderate enhancement promotes vessel maturation without compromising growth. Furthermore, we found that enhancing the activity of this pathway in a mouse model rescues all GMH phenotypes. Altogether, these results demonstrate that enhancing neurovascular signaling through pharmacological targeting of this pathway may be a viable approach for tissue-specific GMH intervention. They also demonstrate that timing and level are likely two major factors crucial for success. These findings thus provide critical new insights into both brain neurovascular biology and the intervention of GMH.

Lysophosphatidic acid induces integrin ß6 expression in human oral squamous cell carcinomas cells via LPAR1 coupling to Gαi and downstream SMAD3 and ETS-1 activation.

Integrin ß6 (ITGB6), an epithelial-specific integrin, is upregulated in oral squamous cell carcinomas (OSCC) and is associated with progression and metastasis of OSCC. Lysophosphatidic acid (LPA), an important bioactive phospholipid present in saliva, has also been related to OSCC cell migration and invasiveness. LPA exerts its biological effects through signal transduction pathways that ultimately regulate gene expression. However, it is unclear whether LPA signaling is involved in ITGB6 upregulation in OSCC. Therefore, the aim of the current study was to investigate the role of LPA in the regulation of ITGB6 expression in OSCC cells, and to delineate the molecular signaling pathways involved. Using SAS and HSC-3 OSCC cell lines, we found that LPA increases ITGB6 mRNA expression without affecting mRNA stability, suggesting that LPA acts by regulating ITGB6 gene transcription. In addition, we show that LPA stimulation increases phosphorylation and binding of the transcription factors SMAD3 and ETS-1 to the ITGB6 promoter resulting in ITGB6 active transcription. Finally, we demonstrate that LPA-induced ITGB6 expression is mediated via the LPA receptors 1 (LPAR1) coupling to Gαi. Our findings provide insights into the molecular mechanism underlying ITGB6 overexpression in OSCC and may have important implications for therapeutic purposes.

The role of adhesion molecules, alpha v beta 3, alpha v beta 5 and their ligands in the tumor cell and endothelial cell adhesion.

Tumor metastasis is a complex process involving the interaction between tumor cells and endothelial cells in which some adhesion molecules play an important role. It was our aim to investigate the role of the adhesion molecules, alpha v beta 3 and alpha v beta 5 and their ligands, developmental endothelial locus-1 (Del-1) and L1, in tumor cell adhesion to endothelial cells in vitro. In this study, the expression and regulation of alpha v beta 3, alpha v beta 5 and intercellular adhesion molecule -1 on liver sinusoidal endothelial cells and liver cancer endothelial cells (T3A) were analyzed by real-time PCR and fluorescent-activated cell sorter. The expression and regulation of the integrin ligands, Del-1 and L1, in six tumor cell lines were analyzed by real-time PCR and western blot. We found the expressions of alpha v beta 3 and alpha v beta 5 were higher on T3A than that on liver sinusoidal endothelial cells, whereas expression of intercellular adhesion molecule-1 was lower on T3A than that on liver sinusoidal endothelial cells. After 24 h hypoxia, the expressions of alpha v beta 3 and alpha v beta 5 were upregulated on T3A and liver sinusoidal endothelial cells; the expression of intercellular adhesion molecule-1 was increased on liver sinusoidal endothelial cells, but remained unchanged on T3A. Del-1 and L1 expression levels were obviously diverse in various tumor cell lines and differentially modulated after 12 h hypoxia. The adhesion of tumor cells with Del-1 and L1 expression was higher in T3A than that in liver sinusoidal endothelial cells, and was significantly increased under hypoxic conditions. Interestingly, the tumor cell adherence could be inhibited by antibodies against alpha v beta 5 and alpha v beta 5, but not by an antibody against intercellular adhesion molecule-1. The adhesion of tumor cells without Del-1 and L1 expression was also higher on T3A than that on liver sinusoidal endothelial cells, but the adhesion could not be inhibited by antibodies against alpha v beta 5, alpha v beta 5 or intercellular adhesion molecule-1, suggesting that other receptors are involved. In conclusion, alpha v beta 5, alpha v beta 5 and their ligands Del-1 and L1 play an important role in the process of tumor cells moving from the original place.