Expression and characterization of αvβ5 integrin on intestinal macrophages

Macrophages play a crucial role in maintaining homeostasis in the intestine, but the underlying mechanisms have not yet been elucidated fully. Here, we show for the first time that mature intestinal macrophages in mouse intestine express high levels of αvβ5 integrin, which acts as a receptor for the uptake of apoptotic cells and can activate molecules involved in several aspects of tissue homeostasis such as angiogenesis and remodeling of the ECM. αvβ5 is not expressed by other immune cells in the intestine, is already present on intestinal macrophages soon after birth, and its expression is not dependent on the microbiota. In adults, αvβ5 is induced during the differentiation of monocytes in response to the local environment and it confers intestinal macrophages with the ability to promote engulfment of apoptotic cells via engagement of the bridging molecule milk fat globule EGF‐like molecule 8. In the absence of αvβ5, there are fewer monocytes in the mucosa and mature intestinal macrophages have decreased expression of metalloproteases and IL 10. Mice lacking αvβ5 on haematopoietic cells show increased susceptibility to chemical colitis and we conclude that αvβ5 contributes to the tissue repair by regulating the homeostatic properties of intestinal macrophages.     

High‐resolution in vivo imaging of breast cancer by targeting the pro‐invasive integrin αvβ6

The integrin αvβ6 is expressed only on epithelia and then usually only during processes of tissue remodelling including cancer, where its high expression correlates with reduced survival. Thus, αvβ6 represents an important target for imaging and therapy of cancer and new molecular‐specific targeting agents are required. We have developed A20FMDV2, a peptide derived from the VP1 coat protein of foot‐and‐mouth‐disease virus that binds specifically and stably to αvβ6. Using a newly generated pair of isogenic human cell lines that differ only in αvβ6 expression, it was shown, using biodistribution and SPECT imaging, that indium‐111‐labelled A20FMDV2 locates specifically to αvβ6‐expressing tissues in vivo, achieving at least seven‐times higher retention in αvβ6‐positive than in αvβ6‐negative tumours. In further studies with MCF10.DCIS.COM and MCF10A.CA1a breast carcinoma cell lines, which express αvβ6 endogenously, the radiopeptide achieved similar levels of tumour retention and permitted excellent discriminatory imaging of tumours. Thus, A20FMDV2 can be used for molecular‐specific targeting of αvβ6 for imaging in vivo the often more aggressive, αvβ6‐positive cancers. In the future, A20FMDV2 could serve also to deliver therapy to these same cancers. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Pro‐migratory and TGF‐β‐activating functions of αvβ6 integrin in pancreatic cancer are differentially regulated via an Eps8‐dependent GTPase switch

The integrin αvβ6 is up‐regulated in numerous carcinomas, where expression commonly correlates with poor prognosis. αvβ6 promotes tumour invasion, partly through regulation of proteases and cell migration, and is also the principal mechanism by which epithelial cells activate TGF‐β1; this latter function complicates therapeutic targeting of αvβ6, since TGF‐β1 has both tumour‐promoting and ‐suppressive effects. It is unclear how these different αvβ6 functions are linked; both require actin cytoskeletal reorganization, and it is suggested that tractive forces generated during cell migration activate TGF‐β1 by exerting mechanical tension on the ECM‐bound latent complex. We examined the functional relationship between cell invasion and TGF‐β1 activation in pancreatic ductal adenocarcinoma (PDAC) cells, and confirmed that both processes are αvβ6‐dependent. Surprisingly, we found that cellular functions could be biased towards either motility or TGF‐β1 activation depending on the presence or absence of epidermal growth factor receptor pathway substrate 8 (Eps8), a regulator of actin remodelling, endocytosis, and GTPase activation. Similar to αvβ6, we found that Eps8 was up‐regulated in >70% of PDACs. In complex with Abi1/Sos1, Eps8 regulated αvβ6‐dependent cell migration through activation of Rac1. Down‐regulation of Eps8, Sos1 or Rac1 suppressed cell movement, while simultaneously increasing αvβ6‐dependent TGF‐β1 activation. This latter effect was modulated through increased cell tension, regulated by Rho activation. Thus, the Eps8/Abi1/Sos1 tricomplex acts as a key molecular switch altering the balance between Rac1 and Rho activation; its presence or absence in PDAC cells modulates αvβ6‐dependent functions, resulting in a pro‐migratory (Rac1‐dependent) or a pro‐TGF‐β1 activation (Rho‐dependent) functional phenotype, respectively. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Hypoxic inactivation of glycogen synthase kinase‐3β promotes gastric tumor growth and angiogenesis by facilitating hypoxia‐inducible factor‐1 signaling

Since the molecular mechanism of hypoxic adaptation in cancer cells is cell‐type specific, we investigated whether glycogen synthase kinase‐3β (GSK‐3β) activation is involved in hypoxia‐induced gastric tumor promotion. Stable gastric cancer cell lines (SNU‐638, SNU‐484, MKN1, and MKN45) were cultured under hypoxic conditions. Cells overexpressing wild‐type GSK‐3β (WT‐GSK‐3β) or kinase‐dead mutant of GSK‐3β (KD‐GSK‐3β) were generated and used for cell culture and animal studies. In cell culture experiments, hypoxia decreased GSK‐3β activation in gastric cancer cells. Cell viability and the expressions of HIF‐1α protein and VEGF mRNA in gastric cancer cells were higher in KD‐GSK‐3β transfectants than in WT‐GSK‐3β transfectants under hypoxic conditions, but not under normoxic conditions. Gastric cancer xenografts showed that tumor growth, microvessel area, HIF‐1α activation, and VEGF expression were higher in KD‐GSK‐3β tumors than in WT‐GSK‐3β tumors in vivo. In addition, the expression of hypoxia‐induced HIF‐1α protein was regulated by GSK‐3β at the translational level. Our data suggest that GSK‐3β is involved in hypoxic adaptation of gastric cancer cells as an inhibitory upstream regulator of the HIF‐1α/VEGF signaling pathway.     

Involvement of α‐ and β‐catenins and E‐cadherin in the development of mammary phyllodes tumours

Tsang J Y S, Mendoza P, Lam C C F, Yu A M C, Putti T C, Karim R Z, Scolyer R A, Lee C S, Tan P H & Tse G M
(2012) Histopathology  61, 667–674 Involvement of α‐ and β‐catenins and E‐cadherin in the development of mammary phyllodes tumours Aims: Phyllodes tumours (PT) are rare but clinically important fibroepithelial tumours of the breast. β‐Catenin, a key component in Wnt signalling, has been shown to be important in the development of PT. It also functions as a component of the cadherin complex, which may therefore be implicated in PT pathogenesis. By assessing stromal α‐catenin, β‐catenin and E‐cadherin expression in 158 PT cases using immunohistochemistry and examining associations with clinicopathological features, we aimed to determine the role of these proteins in PT pathogenesis. Methods and results: Cytoplasmic β‐catenin correlated with α‐catenin expression. A significantly higher expression of both markers was observed in borderline than in benign PT (P = 0.003 and <0.001, respectively), but a lower level was found in malignant PT. Cytoplasmic E‐cadherin expression was significantly higher in borderline and malignant than in benign PT (P = 0.001 and 0.012, respectively), but was not correlated with other markers. Both E‐cadherin and α‐catenin showed stronger correlations with histological parameters than β‐catenin. α‐Catenin showed a significant correlation with recurrence (P = 0.005 and 0.016, respectively). Conclusions: α‐ and β‐catenins may be important in the early stages of PT development, while E‐cadherin may be required for malignant development. The correlation of α‐catenin expression with tumour recurrence may be relevant in predicting PT behaviour.     

Integrin α4 Induces Lymphangiogenesis and Metastasis via Upregulation of VEGF‐C in Human Colon Cancer

Vascular endothelial growth factor‐C (VEGF‐C) is a key regulator in lymphangiogenesis, and is overexpressed in various malignancies. Integrin α4β1, a new member of the VEGF‐C/VEGF receptor pathway, was found to be overexpressed in melanoma tumors. However, little is known regarding the potential role of integrin α4β1 in lymphangiogenesis and other solid tumors. The aim of this study was to investigate the expression patterns of integrin α4 and VEGF‐C in relation to lymphangiogenesis and clinicopathological parameters in human colon cancer. The expression of integrin α4, VEGF‐C, and VEGFR‐3 was assessed in 71 human colon cancer tissues and 30 paracancerous normal tissues by immunohistochemical staining. Lymphatic microvessel density (LMVD) was measured after D2‐40‐labeling, and the correlations among different factors were statistically analyzed. The expression of integrin α4, VEGF‐C, VEGFR‐3, and LMVD was higher in colon cancer tissues compared with the normal paracancerous colon tissues. There was a positive correlation between the expression of integrin α4 and VEGF‐C. Integrin α4 and VEGF‐C were significantly associated with the clinicopathological parameters (LMVD, Duke's stage, and lymph node metastasis). Kaplan–Meier analyses indicated that patients with high integrin α4 or VEGF‐C expression had significantly shorter overall survival and tumor‐free survival time. Multivariate analyses suggested that integrin α4 and VEGF‐C may serve as independent prognostic factors for human colon cancer. Both integrin α4 and VEGF‐C are involved in lymphangiogenesis and lymphatic metastasis. Our results demonstrated that integrin α4 is a novel prognostic indicator for human colon cancer. Anat Rec, 299:741–747, 2016. © 2016 Wiley Periodicals, Inc.     

α4β1 integrin promotes accumulation of tissue‐resident memory CD8+ T cells in salivary glands

The salivary glands (SGs) of virus‐immune mice contain substantial numbers of tissue‐resident memory CD8⁺ T cells (T_RM cells) that can provide immunity to local infections. Integrins regulate entry of activated T cells into nonlymphoid tissues but the molecules that mediate migration of virus‐specific CD8⁺ T cells to the SGs have not yet been defined. Here, we found that polyinosinic‐polycytidylic acid (poly(I:C)) strongly promoted the accumulation of P14 TCR‐transgenic CD8⁺ T_RM cells in SGs in an α₄β₁ integrin‐dependent manner. After infection with lymphocytic choriomeningitis virus, accumulation of P14 T_RM cells in SGs and intestine but not in kidney was also α₄ integrin dependent. Blockade of α₄β₇ by monoclonal antibodies (mAbs) inhibited lymphocytic choriomeningitis virus‐induced accumulation of P14 T_RM cells in the intestine but not in SGs. In conclusion, our data reveal that α₄β₁ integrin mediates CD8⁺ T_RM accumulation in SGs and that poly(I:C) can be used to direct activated CD8⁺ T cells to this organ.     

1α,25‐dihydroxyvitamin D3 acts via transforming growth factor‐β to up‐regulate expression of immunosuppressive CD73 on human CD4+ Foxp3– T cells

Vitamin D deficiency is associated with increased incidence and severity of various immune‐mediated diseases. Active vitamin D (1α,25‐dihydroxyvitamin D3; 1,25(OH)₂D3) up‐regulates CD4⁺ T‐cell expression of the purine ectonucleotidase CD39, a molecule that is associated with the generation of anti‐inflammatory adenosine. Here we aimed to investigate the direct impact of 1,25(OH)₂D3 on expression of the downstream ecto‐5′‐nucleotidase CD73 by human CD4 T cells, and components of the transforming growth factor‐β (TGF‐β) pathway, which have been implicated in the modulation of CD73 by murine T cells. At 10^?8 to 10^?7 m , 1,25(OH)₂D3 significantly increased expression of CD73 on peripheral human CD4⁺ T cells. Although 1,25(OH)₂D3 did not affect the mRNA expression of latent TGF‐β₁, 1,25(OH)₂D3 did up‐regulate expression of TGF‐β‐associated molecules [latency‐associated peptide (LAP), glycophorin A repetitions predominant (GARP), GP96, neuropilin‐1, thrombospondin‐1 and α_v integrin] which is likely to have contributed to the observed enhancement in TGF‐β bioactivity. CD73 was highly co‐expressed with LAP and GARP following 1,25(OH)₂D3 treatment, but unexpectedly, each of these cell surface molecules was expressed primarily on CD4⁺ Foxp3^– T cells, rather than CD4⁺ Foxp3⁺ T cells. Notably, neutralization of TGF‐β significantly impaired 1,25(OH)₂D3‐mediated induction of CD73. Collectively, we show that 1,25(OH)₂D3 enhances expression of CD73 on CD4⁺ Foxp3^– T cells in a process that is at least partially TGF‐β‐dependent. These data reveal an additional contributing mechanism by which vitamin D may be protective in immune‐mediated disease.     

Interleukin‐17‐producing γδ+ T cells protect NOD mice from type 1 diabetes through a mechanism involving transforming growth factor‐β

Whether interleukin (IL)‐17 promotes a diabetogenic response remains unclear. Here we examined the effects of neutralization of IL‐17 on the progress of adoptively transferred diabetes. IL‐17‐producing cells in non‐obese diabetic (NOD) mice were identified and their role in the pathogenesis of diabetes examined using transfer and co‐transfer assays. Unexpectedly, we found that in vivo neutralization of IL‐17 did not protect NOD–severe combined immunodeficiency (SCID) mice against diabetes transferred by diabetic splenocytes. In NOD mice, γδ⁺ T cells were dominated by IL‐17‐producing cells and were found to be the major source of IL‐17. Interestingly, these IL‐17‐producing γδ T cells did not exacerbate diabetes in an adoptive transfer model, but had a regulatory effect, protecting NOD mice from diabetes by up‐regulating transforming growth factor (TGF)‐β production. Our data suggest that the presence of IL‐17 did not increase the chance of the development of diabetes; γδ T cells protected NOD mice from diabetes in a TGF‐β‐dependent manner, irrespective of their role as major IL‐17 producers.     

αβ Lineage-committed thymocytes can be rescued by the γδ T cell receptor (TCR) in the absence of TCR β chain

Commitment of the αβ and γδ T cell lineages within the thymus has been studied in T cell receptor (TCR)-transgenic and TCR mutant murine strains. TCRγδ-transgenic or TCRβ knockout mice, both of which are unable to generate TCRαβ-positive T cells, develop phenotypically αβ-like thymocytes in significant proportions. We provide evidence that in the absence of functional TCRβ protein, the γδTCR can promote the development of αβ-like thymocytes, which, however, do not expand significantly and do not mature into γδ T cells. These results show that commitment to the αβ lineage can be determined independently of the isotype of the TCR, and suggest that αβ versus γδ T cell lineage commitment is principally regulated by mechanisms distinct from TCR-mediated selection. To accommodate our data and those reported previously on the effect of TCRγ and δ gene rearrangements on αβ T cell development, we propose a model in which lineage commitment occurs independently of TCR gene rearrangement.     

ERRα augments HIF‐1 signalling by directly interacting with HIF‐1α in normoxic and hypoxic prostate cancer cells

Adaptation of cancer cells to a hypoxic microenvironment is important for their facilitated malignant growth and advanced development. One major mechanism mediating the hypoxic response involves up‐regulation of hypoxia‐inducible factor 1 (HIF‐1) expression, which controls reprogramming of energy metabolism and angiogenesis. Oestrogen‐related receptor‐α (ERRα) is a pivotal regulator of cellular energy metabolism and many biosynthetic pathways, and has also been proposed to be an important factor promoting the Warburg effect in advanced cancer. We and others have previously shown that ERRα expression is increased in prostate cancer and is also a prognostic marker. Here we show that ERRα is oncogenic in prostate cancer and also a key hypoxic growth regulator. ERRα‐over‐expressing prostate cancer cells were more resistant to hypoxia and showed enhanced HIF‐1α protein expression and HIF‐1 signalling. These effects could also be observed in ERRα‐over‐expressing cells grown under normoxia, suggesting that ERRα could function to pre‐adapt cancer cells to meet hypoxia stress. Immunoprecipitation and FRET assays indicated that ERRα could physically interact with HIF‐1α via its AF‐2 domain. A ubiquitination assay showed that this ERRα–HIF‐1α interaction could inhibit ubiquitination of HIF‐1α and thus reduce its degradation. Such ERRα–HIF‐1α interaction could be attenuated by XCT790, an ERRα‐specific inverse agonist, resulting in reduced HIF‐1α levels. In summary, we show that ERRα can promote the hypoxic growth adaptation of prostate cancer cells via a protective interaction with HIF‐1α, suggesting ERRα as a potential therapeutic target for cancer treatment. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

MD‐2 is involved in the stimulation of matrix metalloproteinase‐1 expression by interferon‐γ and high glucose in mononuclear cells – a potential role of MD‐2 in Toll‐like receptor 4‐independent signalling

We reported recently that treatment of diabetic apolipoprotein E‐deficient mice with the Toll‐like receptor 4 (TLR4) antagonist Rs‐LPS, a lipopolysaccharide isolated from Rhodobacter sphaeroides, inhibited atherosclerosis. Since it is known that Rs‐LPS antagonizes TLR4 by targeting TLR4 co‐receptor MD‐2, this finding indicates that MD‐2 is a potential target for the treatment of atherosclerosis. In this study, we determined if MD‐2 is involved in the gene expression regulated by signalling pathways independent of TLR4. Given that interferon‐γ (IFNγ) and hyperglycaemia play key roles in atherosclerosis, we determined if MD‐2 is involved in IFN‐γ and high‐glucose‐regulated gene expression in mononuclear cells. Results showed that IFN‐γ and high glucose synergistically stimulated matrix metalloproteinase 1 (MMP‐1), a proteinase essential for vascular tissue remodelling and atherosclerosis, in U937 mononuclear cells, but Rs‐LPS inhibited the MMP‐1 stimulation. To provide more evidence for a role of MD‐2 in IFN‐γ‐stimulated MMP‐1, studies using antibodies and small interfering RNA demonstrated that MD‐2 blockade or knockdown attenuated the effect of IFN‐γ on MMP‐1. Furthermore, studies using PCR arrays showed that MD‐2 blockade had a similar effect as IFN‐γ receptor blockade on the inhibition of IFN‐γ‐stimulated pro‐inflammatory molecules. Although these findings indicate the involvement of MD‐2 in IFN‐γ signalling, we also observed that MD‐2 was up‐regulated by IFN‐γ and high glucose. We found that MD‐2 up‐regulation by IFN‐γ played an essential role in the synergistic effect of IFN‐γ and LPS on MMP‐1 expression. Taken together, these findings indicate that MD‐2 is involved in IFN‐γ signalling and IFN‐γ‐augmented MMP‐1 up‐regulation by LPS.