The β2 integrin Mac-1 but not p150,95 associates with FcγRIIA

In this study we have compared the ligand binding activity of the two closely related β2 integrins, Mac-1 and p150,95, which are expressed separately as receptors permanently transfected into K562 cells. Mac-1 has previously been shown to associate with FcγR, particularly FcγRIII, but K562 cells express only endogenous FcγRIIA. We have, therefore, taken advantage of this situation to examine a possible relationship between FcγRIIA with Mac-1 and p150,95 in the absence of other FcγR. The main finding is that anti-FcγRII mAb have a profound inhibitory effect on cell adhesion mediated by Mac-1, but not on the adhesion mediated by p150,95. Thus, in spite of the fact that Mac-1 and p150,95 bind to the same or at least a very similar selection of ligands, their association with other receptors on the cellular membrane, and therefore their mode of regulation may be different.     

Deficiency of bone marrow β3‐integrin enhances non‐functional neovascularization

β3‐Integrin is a cell surface adhesion and signalling molecule important in the regulation of tumour angiogenesis. Mice with a global deficiency in β3‐integrin show increased pathological angiogenesis, most likely due to increased vascular endothelial growth factor receptor 2 expression on β3‐null endothelial cells. Here we transplanted β3‐null bone marrow (BM) into wild‐type (WT) mice to dissect the role of BM β3‐integrin deficiency in pathological angiogenesis. Mice transplanted with β3‐null bone marrow show significantly enhanced angiogenesis in subcutaneous B16F0 melanoma and Lewis lung carcinoma (LLC) cell models and in B16F0 melanoma lung metastasis when compared with tumours grown in mice transplanted with WT bone marrow. The effect of bone marrow β3‐integrin deficiency was also assessed in the RIPTAg mouse model of pancreatic tumour growth. Again, angiogenesis in mice lacking BM β3‐integrin was enhanced. However, tumour weight between the groups was not significantly altered, suggesting that the enhanced blood vessel density in the mice transplanted with β3‐null bone marrow was not functional. Indeed, we demonstrate that in mice transplanted with β3‐null bone marrow a significant proportion of tumour blood vessels are non‐functional when compared with tumour blood vessels in WT‐transplanted controls. Furthermore, β3‐null‐transplanted mice showed an increased angiogenic response to VEGF in vivo when compared with WT‐transplanted animals. BM β3‐integrin deficiency affects the mobilization of progenitor cells to the peripheral circulation. We show that VEGF‐induced mobilization of endothelial progenitor cells is enhanced in mice transplanted with β3‐null bone marrow when compared with WT‐transplanted controls, suggesting a possible mechanism underlying the increased blood vessel density seen in β3‐null‐transplanted mice. In conclusion, although BM β3‐integrin is not required for pathological angiogenesis, our studies demonstrate a role for BM β3‐integrin in VEGF‐induced mobilization of bone marrow‐derived cells to the peripheral circulation and for the functionality of those vessels in which BM‐derived cells become incorporated. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

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.     

E-cadherin and α-, β-, and γ-catenin protein expression in relation to metastasis in human breast carcinoma

In the metastatic process, various cell–cell adhesion molecules seem to play an important role. E-cadherin, a transmembrane protein with an extracellular and an intracellular domain, is one of the key players involved in cell–cell adhesion. The function of E-cadherin in preventing metastasis in tumour development is believed to be dependent on intracellular catenins. In a previous study, the expression of E-cadherin was examined in a series of human breast carcinomas. In that study, down-regulation of E-cadherin failed to correlate with lymph node and/or distant metastasis. In the present study, the expression of α-, β-, and γ-catenins has been examined in a subset of the same tumours in order to evaluate their possible role in breast cancer metastasis. Tumour tissues from 90 primary breast carcinomas were immunostained for α-, β-, and γ-catenins. Reduced or absent immunoreactivity in the tumour tissue was seen in 63 (70·0 per cent) for α-catenin, in 50 (55·6 per cent) for β-catenin, and in 50 (55·6 per cent) for γ-catenin. Reduced expression of each of the catenins alone failed to correlate to metastasis. However, when all of the four proteins (E-cadherin, α-catenin, β-catenin, and γ-catenin) were analysed as one group, a significant association was seen between reduction in immunoreactivity of at least one of these four proteins and the presence of metastases. These results indicate that if one of these proteins is down-regulated, the function of the others in suppressing metastasis is altered. A significant association was seen between lobular invasive tumours and β-catenin expression. © 1998 John Wiley & Sons, Ltd.     

In silico analysis‐based identification of the target residue of integrin α6 for metastasis inhibition of basal‐like breast cancer

Metastasis causes death in breast cancer patients. To inhibit breast cancer metastasis, we focused on integrin α6, a membrane protein that contributes to cell migration and metastasis. According to in silico analysis, we identified Asp‐358 as an integrin α6‐specific vertebrate‐conserved residue and consequently as a potential therapeutic target. Because Asp‐358 is located on the surface of the β propeller domain that interacts with other molecules for integrin α6 function, we hypothesized that a peptide with the sequence around Asp‐358 competitively inhibits integrin α6 complex formation. We treated basal‐like breast cancer cells with the peptide and observed reductions in cell migration and metastasis. The result of the immunoprecipitation assay showed that the peptide inhibited integrin α6 complex formation. Our immunofluorescence for phosphorylated paxillin, a marker of integrin‐regulated focal adhesion, showed that the peptide reduced the number of focal adhesions. These results indicate that the peptide inhibits integrin α6 function. This study identified the functional residue of integrin α6 and designed the inhibitory peptide. For breast cancer patients, metastasis inhibition therapy may be developed in the future based on this study.     

αE‐catenin is a candidate tumor suppressor for the development of E‐cadherin‐expressing lobular‐type breast cancer

Although mutational inactivation of E‐cadherin (CDH1) is the main driver of invasive lobular breast cancer (ILC), approximately 10–15% of all ILCs retain membrane‐localized E‐cadherin despite the presence of an apparent non‐cohesive and invasive lobular growth pattern. Given that ILC is dependent on constitutive actomyosin contraction for tumor development and progression, we used a combination of cell systems and in vivo experiments to investigate the consequences of α‐catenin (CTNNA1) loss in the regulation of anchorage independence of non‐invasive breast carcinoma. We found that inactivating somatic CTNNA1 mutations in human breast cancer correlated with lobular and mixed ducto‐lobular phenotypes. Further, inducible loss of α‐catenin in mouse and human E‐cadherin‐expressing breast cancer cells led to atypical localization of E‐cadherin, a rounded cell morphology, and anoikis resistance. Pharmacological inhibition experiments subsequently revealed that, similar to E‐cadherin‐mutant ILC, anoikis resistance induced by α‐catenin loss was dependent on Rho/Rock‐dependent actomyosin contractility. Finally, using a transplantation‐based conditional mouse model, we demonstrate that inducible inactivation of α‐catenin instigates acquisition of lobular features and invasive behavior. We therefore suggest that α‐catenin represents a bona fide tumor suppressor for the development of lobular‐type breast cancer and as such provides an alternative event to E‐cadherin inactivation, adherens junction (AJ) dysfunction, and subsequent constitutive actomyosin contraction. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Reduced expression of oestrogen receptor‐β is associated with tumour invasion and metastasis in oestrogen receptor‐α‐negative human papillary thyroid carcinoma

Oestrogens play an important role in the development and progression of papillary thyroid carcinoma (PTC) through oestrogen receptor (ER)‐α and ‐β, which may exert different or even opposing actions in PTC. The roles of ERβ in ERα‐negative PTC are still not clear. This study investigated the expression dynamics of ERβ1 (wild‐type ERβ) and its clinical significance in female ERα‐negative PTC patients. ERβ1 expression was detected in thyroid tissues of 136 female patients diagnosed with PTC. The relationships between ERβ1 expression and clinicopathological/biological factors were also analysed in female ERα‐negative PTC patients. The total score for ERβ1 was significantly lower in female ERα‐negative PTC patients with LNM or ETE when compared to those without LNM or ETE (Z = ?2.923, P = 0.003 and Z = ?3.441, P = 0.001). Accordingly, the total score for ERβ1 was significantly higher in ERα‐negative PTC patients expressing E‐cadherin compared to patients negative for E‐cadherin expression (Z = ?2.636, P = 0.008). The total score was lower in ERα‐negative PTC patients positive for VEGF expression compared to those negative for VEGF expression (Z = ?1.914, P = 0.056). This preliminary study indicates that reduced expression of ERβ1 in female ERα‐negative PTC patients is associated with greater progression of the disease. This may provide insights into the underlying molecular mechanisms of ERβ1 and could help design targeted approaches for treating or even preventing this disease.     

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.     

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.     

A role for gangliosides and β1‐integrin in the motility of olfactory ensheathing glia

Olfactory ensheathing glia (OEG) are found in the olfactory mucosa, nerve and bulb, and provide in vivo ensheathment for the unmyelinated olfactory axons within the central and peripheral nervous system domains. OEG cells are able to migrate long distances within the neuropil of the central nervous system. Because gangliosides such as 9‐O‐acetyl GD3 have crucial regulatory roles in neuronal migration during development, we analyzed whether OEG in organotypical cultures are revealed by anti‐9‐O‐acetyl GD3 and/or gangliosides are recognized by the A2B5 antibody (G‐A2B5), and whether these gangliosides are involved in OEG migration. Our results showed that all OEG migrating out of a section of olfactory bulb onto a laminin substrate bound to the 9‐O‐acetyl GD3 and A2B5 antibodies, and that 2′,3′‐cyclic nucleotide phosphodiesterase (CNPase) colocalized with 9‐O‐acetyl GD3 and with G‐A2B5. Additionally, we showed that the immune blockade of 9‐O‐acetyl GD3 or G‐A2B5 reduced the migration of OEG on laminin, and that 9‐O‐acetyl GD3 and G‐A2B5 colocalized with the β1‐integrin subunit. We also confirmed the phenotype of in‐vitro‐grown OEG cells derived from adult rats, showing that they express CNPase, and also α‐smooth muscle actin, which is not expressed by Schwann cells. Our data showed that the gangliosides 9‐O‐acetyl GD3 and G‐A2B5 participate in the migratory activity of OEG cells, and that the β1‐integrin subunit colocalizes with these gangliosides. These results suggest a new role for β1‐integrin and gangliosides in the polarized migration of OEG cells, and provide new information on the molecules controlling OEG motility and behavior.     

Transforming growth factor-β1-induced expression of the mucosa-related integrin αE on lymphocytes is not associated with mucosa-specific homing

The integrin α_E (HML-1, α_IEL, α_M290) is largely expressed on lymphocytes in epithelial sites, especially the gut mucosa. We investigated whether α_E has any role in homing or delineates a phenotype with distinct migratory behavior. Lymph node T cells were stimulated for 5 days with anti-CD3 in the presence or absence of transforming growth factor (TGF)-β₁ to generate α_E⁺ or α_E^? cells, respectively. The two populations were then tested for their homing properties in mice. Both α_E⁺ (TGF-β-treated) and α_E^? (control) cells of either CD4⁺ or CD8⁺ subset had a low capacity to enter the gut and showed the same homing behavior with respect to a variety of other organs. The same was true for α_E⁺ and α_E^? cells that had been briefly stimulated with anti-CD3 (24 h) and then allowed to return to a resting state before injection, though in this case both populations showed a greater capacity to recirculate through lymphoid tissue than was seen with fully activated cells. The results indicate that α_Eβ₇ does not act as a homing receptor, and that the expression of the site-specific marker α_E does not correlate with a distinct homing behavior.     

β1 and β4 integrin expression in methacarn and formalin-fixed material from in situ ductal carcinoma of the breast

The integrins are αβ heterodimeric transmembrane proteins mediating cell-substratum as well as cell-cell interactions. Previous distribution studies on integrin expression have been limited by the requirement for cryostat sectioned tissues, and consequent poor resolution. We have examined 40 examples of ductal carcinoma in situ (DCIS) for the expression of both β1 and β4 integrin chains. These showed strong polarized membrane staining of residual myoepithelial cells (correlating with expression of smooth muscle specific actin) and of the basement membrane region with β1 and β4 antibodies respectively. In 12 out of 40 cases, the DCIS was negative for the β1 chain and a variable pattern of reactivity was seen in the remaining cases. The β4 chain was detected focally and weakly in the tumour cells of 7/40 DCIS and strongly in one; all of these cases were also positive for the β1 chain. Of the 22 cases where co-existent invasion was present, the infiltrating component showed either a similar degree or a diminution of the extent of immunostaining when compared with the in situ component; only one showed enhanced staining (β1 only). This study demonstrates that two of the main β chains, β1 and β4, can be effectively demonstrated on methacarn and cold (4°C) formalin-fixed tissues by avidin-biotin indirect immunoperoxidase staining and that the results are similar to those achieved using frozen tissue.     

Peroxisome proliferator‐activated receptor‐β/δ modulates mast cell phenotype

The peroxisome proliferator‐activated receptor‐β/δ (PPARβ/δ) is known to have multiple anti‐inflammatory effects, typically observed in endothelial cells, macrophages, T cells and B cells. Despite the fact that mast cells are important mediators of inflammation, to date, the role of PPARβ/δ in mast cells has not been examined. Hence, the present study examined the hypothesis that PPARβ/δ modulates mast cell phenotype. Bone‐marrow‐derived mast cells (BMMCs) and peritoneal mast cells from Pparβ/δ^+/+ mice expressed higher levels of high‐affinity IgE receptor (FcεRI) compared with Pparβ/δ^−/− mice. BMMCs from Pparβ/δ^+/+ mice also exhibited dense granules, associated with higher expression of enzymes and proteases compared with Pparβ/δ^−/− mice. Resting BMMCs from Pparβ/δ^+/+ mice secreted lower levels of inflammatory cytokines, associated with the altered activation of phospholipase Cγ1 and extracellular signal‐regulated kinases compared with Pparβ/δ^−/− mice. Moreover, the production of cytokines by mast cells induced by various stimuli was highly dependent on PPARβ/δ expression. This study demonstrates that PPARβ/δ is an important regulator of mast cell phenotype.     

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.     

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.