Integrin Alpha Subunits Research Articles

Elucidation of the Roles of Tumor Integrin β1 in the Extravasation Stage of the Metastasis Cascade.

Tumor integrin β1 (ITGB1) contributes to primary tumor growth and metastasis, but its specific roles in extravasation have not yet been clearly elucidated. In this study, we engineered a three-dimensional microfluidic model of the human microvasculature to recapitulate the environment wherein extravasation takes place and assess the consequences of β1 depletion in cancer cells. Combined with confocal imaging, these tools allowed us to decipher the detailed morphology of transmigrating tumor cells and associated endothelial cells in vitro at high spatio-temporal resolution not easily achieved in conventional transmigration assays. Dynamic imaging revealed that β1-depleted cells lacked the ability to sustain protrusions into the subendothelial matrix in contrast with control cells. Specifically, adhesion via α3β1 and α6β1 to subendothelial laminin was a critical prerequisite for successful transmigration. β1 was required to invade past the endothelial basement membrane, whereas its attenuation in a syngeneic tumor model resulted in reduced metastatic colonization of the lung, an effect not observed upon depletion of other integrin alpha and beta subunits. Collectively, our findings in this novel model of the extravasation microenvironment revealed a critical requirement for β1 in several steps of extravasation, providing new insights into the mechanisms underlying metastasis. Cancer Res; 76(9); 2513-24. ©2016 AACR.

Mas receptor deficiency exacerbates lipopolysaccharide-induced cerebral and systemic inflammation in mice

Beyond the classical actions of the renin-angiotensin system on the regulation of cardiovascular homeostasis, several studies have shown its involvement in acute and chronic inflammation. The G protein-coupled receptor Mas is a functional binding site for the angiotensin-(1–7); however, its role in the immune system has not been fully elucidated. In this study, we evaluated the effect of genetic deletion of Mas receptor in lipopolysaccharide (LPS)-induced systemic and cerebral inflammation in mice. Inflammatory response was triggered in Mas deficient (Mas−/−) and C57BL/6 wild-type (WT) mice (8–12 weeks-old) by intraperitoneal injection of LPS (5mg/kg). Mas−/− mice presented more intense hypothermia compared to WT mice 24h after LPS injection. Systemically, the bone marrow of Mas−/− mice contained a lower number of neutrophils and monocytes 3h and 24h after LPS injection, respectively. The plasma levels of inflammatory mediators KC, MCP-1 and IL-10 were higher in Mas−/− mice 24h after LPS injection in comparison to WT. In the brain, Mas−/− animals had a significant increase in the number of adherent leukocytes to the brain microvasculature compared to WT mice, as well as, increased number of monocytes and neutrophils recruited to the pia-mater. The elevated number of adherent leukocytes on brain microvasculature in Mas−/− mice was associated with increased expression of CD11b – the alpha-subunit of the Mac-1 integrin – in bone marrow neutrophils 3h after LPS injection, and with increased brain levels of chemoattractants KC, MIP-2 and MCP-1, 24h later. In conclusion, we demonstrated that Mas receptor deficiency results in exacerbated inflammation in LPS-challenged mice, which suggest a potential role for the Mas receptor as a regulator of systemic and brain inflammatory response induced by LPS.

The role of integrins in glioma biology and anti-glioma therapies.

Integrins are a group of molecules expressed by various cells including glioma cells and endothelial cells within the tumor. There are 18 known alpha and beta integrin subunits which form a heterodimer. Integrins regulate different cellular processes such as proliferation, adhesion, motility and survival as shown in numerous preclinical models. Furthermore, integrins control the activity of the transforming growth factor (TGF)-beta pathway and are involved in the process of angiogenesis which is indispensable for continued tumor growth. Because of the high expression levels of some integrins on glioma cells and their numerous functions, inhibition of integrin signaling has been considered a promising strategy for the treatment of glioma patients. Besides blocking antibodies which are currently under clinical investigation in other cancer entities, the integrin inhibitor cilengitide has been tested within several trials in glioblastoma patients over the last years. Cilengitide is a cyclic RDG peptide which targets integrins alphvbeta3 and alphavbeta5. Based on the results of smaller, initial trials suggesting an activity of cilengitide against glioblastoma, 2 larger trials were subsequently performed. However, both trials, which combined temozolomide-based chemoradiation with cilengitide failed to demonstrate an improved outcome with the addition of cilengitide. Ongoing translational analyses suggest that integrin levels in the tumor tissue are neither prognostic nor predict response to cilengitide. While the clinical development of cilengitide has been stopped, integrin inhibition with more effective agents may still be a promising approach in clinical neurooncology.

Abstract 218: Dynamics of Blood Leukocytes Following ST Segment Elevation Myocardial Infarction

Blood leukocyte counts are linked to cardiovascular risk stratification, and excess activation of neutrophil and monocytes has been observed in patients of myocardial infarction (MI). However, the profile of major subsets of leukocyte following ST Segment Elevation MI (STEMI) is still unclear. It was aimed to compare relative change of neutrophil, monocytes, T and B lymphocytes in blood leukocytes in patients of STEMI undergoing percutaneous coronary intervention (PCI). STEMI patients subject to PCI were consecutively recruited. Peripheral blood was collected upon patient admission to hospital (referred to as T0), one day(T1), and/or four days (T4) after PCI, and were measured by flow cytometry (n=12, 18, 34 for T0, T1 and T4, respectively). Compared to T0, percentage of neutrophil (CD45+CD66b+) decreased, whereas T cell (CD3+CD45+) increased, at both T1 and T4. Percentage of monocytes (CD14+CD45+) and B cells (CD19+CD45+) both increased at T4 relative to T0. Interestingly, monocyte- and neutrophil- expression of CD11c, an inducible integrin alpha subunit, increased significantly at T4 compared to T1, despite a dichotomous change in population percentage for these two cell types. A dynamic shift in different leukocyte subsets, as well as in their activation status, occurs in STEMI patients underwent PCI. Such changes might reflect different aspects of biology in patients of STEMI with concurrent standard therapy, and might inform selection of new markers for cardiovascular risk stratification.

Role of integrin subunits in mesenchymal stem cell differentiation and osteoblast maturation on graphitic carbon-coated microstructured surfaces

Surface roughness, topography, chemistry, and energy promote osteoblast differentiation and increase osteogenic local factor production in vitro and bone-to-implant contact in vivo, but the mechanisms involved are not well understood. Knockdown of integrin heterodimer alpha2beta1 (α2β1) blocks the osteogenic effects of the surface, suggesting signaling by this integrin homodimer is required. The purpose of the present study was to separate effects of surface chemistry and surface structure on integrin expression by coating smooth or rough titanium (Ti) substrates with graphitic carbon, retaining surface morphology but altering surface chemistry. Ti surfaces (smooth [Ra < 0.4 μm], rough [Ra ≥ 3.4 μm]) were sputter-coated using a magnetron sputtering system with an ultrapure graphite target, producing a graphitic carbon thin film. Human mesenchymal stem cells and MG63 osteoblast-like cells had higher mRNA for integrin subunits α1, α2, αv, and β1 on rough surfaces in comparison to smooth, and integrin αv on graphitic-carbon-coated rough surfaces in comparison to Ti. Osteogenic differentiation was greater on rough surfaces in comparison to smooth, regardless of chemistry. Silencing integrins β1, α1, or α2 decreased osteoblast maturation on rough surfaces independent of surface chemistry. Silencing integrin αv decreased maturation only on graphitic carbon-coated surfaces, not on Ti. These results suggest a major role of the integrin β1 subunit in roughness recognition, and that integrin alpha subunits play a major role in surface chemistry recognition.

Recently Identified Forms of Epidermolysis Bullosa.

Epidermolysis bullosa (EB) comprises a collection of clinically diverse inherited blistering diseases that affect the skin and, in some subtypes, mucous membranes and other organs. Currently classified into four main subtypes (EB simplex, junctional EB, dystrophic EB, and Kindler syndrome, mainly based on the level of skin cleavage), the spectrum of EB extends to more than 30 clinical subtypes with pathogenic mutations in at least 18 distinct genes. This review focuses on three recent additions to variants of EB: all are autosomal recessive, and result from mutations in either DST-e (coding for epidermal dystonin, also known as the 230 kDa bullous pemphigoid antigen, BP230), EXPH5 (coding for exophilin-5, also known as Slac2-b), or ITGA3 (coding for the integrin alpha-3 subunit). Each of these new forms of EB is reviewed with respect to the initial gene discovery, clinical features, the current mutation database, and skin pathology. Awareness of these recently described forms of EB is helpful in the clinical evaluation of patients with EB and in defining genotype-phenotype correlation for inherited blistering skin diseases.

Developmentally regulated collagen/integrin interactions confer adhesive properties to early postnatal neural stem cells

BackgroundIt is becoming increasingly apparent that the extracellular matrix acts as an important regulator of the neural stem niche. Previously we found that neural stem and progenitor cells (NSPCs) derived from the early postnatal subventricular zone of mice adhere to a collagen/hyaluronan hydrogel, whereas NSPCs from the adult and embryonic brain do not. MethodsTo examine the specific adhesive properties of young stem cells in more detail, NSPCs isolated from embryonic, postnatal day 6 (P6), and adult mouse brains were cultured on collagen I. ResultsEarly postnatal NSPCs formed paxillin-positive focal adhesions on collagen I, and these adhesions could be prevented by an antibody that blocked integrin β1. Furthermore, we found the corresponding integrin alpha subunits α2 and α11 levels to be highest at the postnatal stage. Gene ontology analysis of differentially expressed genes showed higher expression of transcripts involved in vasculature development and morphogenesis in P6 stem cells, compared to adult. ConclusionsThe ability to interact with the extracellular matrix differs between postnatal and adult NSPCs. General significanceOur observations that the specific adhesive properties of early postnatal NSPCs, which are lost in the adult brain, can be ascribed to the integrin subunits expressed by the former furthering our understanding of the developing neurogenic niche. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.

Vascular matrix biology and bioengineering workshop IV and biology of signaling in the cardiovascular system workshop III October 20–24, 2013

Previous work has shown that ECM remodeling is a key component of the inflammatory activation of the endothelium in atherosclerosis, which occurs in regions of arteries subject to disturbed flow. Fibronectin is deposited in these atherosclerosis-prone regions of arteries even in WT mice. Fluid shear-dependent inflammatory signaling mediated by NF-jB or JNK is selectively induced in endothelial cells on fibronectin whereas it is suppressed on basement membrane proteins. Investigation of the mechanism behind these effects showed that PKA is selectively activated in cells on basement membrane and suppresses inflammatory signaling. We therefore investigated the integrin receptors that mediate ECM binding. The major fibronectin receptor, a5b1, and the major collagen/laminin receptor, a2b1, share the common b1 subunit. We therefore analyzed the cytoplasmic domains of the unique integrin alpha subunits. A chimera in which the integrin a5 cytosolic tail was replaced by the a2 cytosolic tail was examined. Endothelial cells expressing the integrin a5/2 chimera adhered and spread normally on fibronectin. However, shear stress activated PKA and inhibited NF-jB, similar to cells on basement membranes. Pharmacological inhibition of PDE4 activity abolished fibronectin-specific NF-jB activation, indicating that cAMP degradation is required for the pro-inflammatory effects of fibronectin. PDE4D5 directly and specifically associates with the integrin a5 cytosolic tail. Knock-down of PDE4D impaired flow-dependent NFjB activation in the cells on fibronectin. These data suggest that the interaction of the integrin a5 cytoplasmic domain with PDE4D mediates the pro-inflammatory effects of fibronectin by inhibiting the anti-inflammatory cAMP/PKA pathway.

Canine Chondrodysplasia Caused by a Truncating Mutation in Collagen-Binding Integrin Alpha Subunit 10

The skeletal dysplasias are disorders of the bone and cartilage tissues. Similarly to humans, several dog breeds have been reported to suffer from different types of genetic skeletal disorders. We have studied the molecular genetic background of an autosomal recessive chondrodysplasia that affects the Norwegian Elkhound and Karelian Bear Dog breeds. The affected dogs suffer from disproportionate short stature dwarfism of varying severity. Through a genome-wide approach, we mapped the chondrodysplasia locus to a 2-Mb region on canine chromosome 17 in nine affected and nine healthy Elkhounds (praw = 7.42×10−6, pgenome-wide = 0.013). The associated locus contained a promising candidate gene, cartilage specific integrin alpha 10 (ITGA10), and mutation screening of its 30 exons revealed a nonsense mutation in exon 16 (c.2083C>T; p.Arg695*) that segregated fully with the disease in both breeds (p = 2.5×10−23). A 24% mutation carrier frequency was indicated in NEs and an 8% frequency in KBDs. The ITGA10 gene product, integrin receptor α10-subunit combines into a collagen-binding α10β1 integrin receptor, which is expressed in cartilage chondrocytes and mediates chondrocyte-matrix interactions during endochondral ossification. As a consequence of the nonsense mutation, the α10-protein was not detected in the affected cartilage tissue. The canine phenotype highlights the importance of the α10β1 integrin in bone growth, and the large animal model could be utilized to further delineate its specific functions. Finally, this study revealed a candidate gene for human chondrodysplasias and enabled the development of a genetic test for breeding purposes to eradicate the disease from the two dog breeds.

Expression of Integrin Alpha-3 and Beta-4 Subunits on the Process of Peri-Implant Epithelium Formation

Integrin, a component of the hemidesmosome, plays a role for epithelial cell migration and adhesion. This study investigated the process of peri-implant epithelium (PIE) formation after implantation, and compared it to the process of oral mucosa healing after tooth extraction. At the healing site of extraction socket without implant, the original junctional epithelium (JE) had disappeared at week 2, and the oral epithelium (OE) with integrin-α3 positive basal cells extending from the sides of the wound, then joined in the middle of the extraction socket. On the other hand in implant group, newly formed epithelium with integrin-α3 positive cells from the OE extended apically 1 week after implantation. After 3 weeks, basal cells of the new epithelium consisted of those with integrin-α3 positive but β4 negative. Finally, after 4 weeks, integrin-β4 was expressed at the implant-PIE interface. These findings suggest that integrin α3β1 plays a role in cell migration during PIE formation from OE. Furthermore, after the completion of PIE constitution, integrin α6β4 contributes to the attachment to titanium.

Natalizumab Treatment Effects Sustained Decrease in Serum Levels of Fibronectin and Soluble Adhesion Molecules sVCAM-1 and sICAM-3 (P02.114)

Objective: To obtain a better understanding of potential additional effector mechanisms of natalizumab we analyzed the influence of short and long-term treatment on soluble adhesion molecules (sAMs) in peripheral blood of MS patients. Background Natalizumab (Tysabri) is a humanized monoclonal antibody used in the treatment of relapsing-remitting multiple sclerosis (MS). It directly binds to the alpha4-integrin subunit of very late activation antigen-4 (VLA-4) and results in decreased detectability of unblocked alpha-4 leading to an inhibition of immune cell extravasation across the blood-brain-barrier. The impressive therapeutic effect of natalizumab is overshadowed by rare but potentially serious side effects such as progressive multifocal leukoencephalopathy. Design/Methods: Serum levels of fibronectin (FN) and 4 different sAMs (soluble intercellular adhesion molecule-1,-2,-3 (sICAM-1,-2,-3) and vascular cell adhesion molecule-1 (sVCAM-1)) were analyzed before (baseline, n=16), 3 months after (short-term, n=16) and 24 months after (long-term, n=14) onset of Natalizumab treatment by fluorescent bead immunoassay and enzyme-linked immunosorbent assay. Results: A significant and sustained decrease in serum concentrations of FN, sICAM-3 and sVCAM-1 was observed during the first 3 months (FN p Conclusions: The observed decreases in serum levels highlight sICAM-3, sVCAM-1, and FN to be affected by natalizumab therapy. Decreases in sVCAM-1 serum levels have already been previously reported. We add sICAM-3 and the soluble form of the extracellular matrix protein FN. Both, sVCAM-1 and FN have been shown to be directly engaged in VLA-4-mediated extravasation of immune cells and sICAM-3 is important in the initiation of immune responses. Decreased serum levels indicate potential therapy-induced interference with fine-tuning and/or feedback mechanisms between endothelium and immune cells with yet unresolved consequences. Supported by: Biogen-Idec Austria. Disclosure: Dr. Kraus has received personal compensation for activities with Biogen Idec, Bayer Pharmaceuticals Corporation, Sanofi-Aventis Pharmaceuticals, Merck-Serono, Novartis, Genzyme Corporation as a consultant. Dr. Kraus has received research support from Biogen Idec, Bayer Pharmaceuticals Corporation, Sanofi-Aventis Pharmaceuticals, Merck Serono, Novartis, Genzyme Corporation. Dr. Oppermann has nothing to disclose. Dr. Pilz has nothing to disclose. Dr. Wipfler has nothing to disclose. Dr. Afazel has nothing to disclose. Dr. Haschke-Becher has nothing to disclose. Dr. Trinka has received personal compensation for activities with UCB Pharma, Eisai Inc., Sunovion, Medtronic, Pfizer Inc, Ever-Neuropharma and GlaxoSmithKline, Inc. as a consultant and/or speaker. Dr. Trinka has received research support from UCB Pharma, Ever-Neuropharma, Medtronic and GlaxoSmithKline, Inc. Dr. Harrer has nothing to disclose.

Characterization of the EMT response in Crybb2Phil mutants

The most abundant protein in the adult lens fiber cells is βB2‐crystallin (Crybb2). Mutations in the Crybb2 gene cause cataracts in mice and humans. A twelve nucleotide deletion in mouse lens epithelial cells (LEC), known as the Crybb2Phil mutation, leads to cataracts associated with lens fiber cell defects as well as epithelial to mesenchymal transition (EMT) of the LEC in adulthood. However, the mechanism behind this phenotype is poorly understood. The aim of this project is to characterize the EMT response caused by the Crybb2Phil mutation.Tissues obtained from Crybb2Phil mutants were immunostained for alpha and beta integrin subunits, phospo‐SMAD3 (p‐SMAD3), transforming growth factor β‐induced (BIGH3), and EMT the markers E‐cadherin, vimentin, follistatin, and α‐smooth muscle actin (α‐SMA).Compared to the wildtype lens, β1, αV, and α5 integrins, vimentin, α‐SMA, and p‐SMAD3 were upregulated in the Crybb2Phil mice. No changes were seen in α2, α3, and α6 integrin subunits or follistatin. Consistent with EMT, E‐cadherin was downregulated in the Crybb2Phil mice.Upregulation of α‐SMA signifies that Crybb2Phil mice undergo EMT around two months of age. Furthermore, over expression of β1 and αV integrins along with p‐SMAD3 in the Crybb2Phil mutant suggests that the EMT seen in Crybb2Phil mice may be occurring via the TGFβ‐SMAD interacting pathway. This project is funded by National Eye Institute grant EY015279.

Molecular evidence of transient therapeutic effectiveness of natalizumab despite high-titre neutralizing antibodies

Natalizumab is a humanized monoclonal antibody directed against the alpha-4 integrin subunit of very late activation antigen-4 (VLA-4). Natalizumab neutralizing antibodies (NAB) have been found to significantly reduce beneficial effects of natalizumab treatment in multiple sclerosis. We investigated interactions of NAB with natalizumab by serial measurements of alpha-4 integrin levels on peripheral blood mononuclear cells using flow cytometry. In addition, serum concentrations of soluble vascular cell adhesion molecule-1 (sVCAM-1), the endothelial ligand of VLA-4, and serum NAB were serially determined. Natalizumab infusion led to a transient reduction in alpha-4 integrin levels on immune cells and serum sVCAM-1 levels along with serum negativity of NAB lasting for a few days post-infusion. Apparently, the high-dose effect of freshly infused natalizumab resulted in a transient neutralization of NAB possibly involving a transient therapeutic effectiveness.

Adult Mesenchymal Stem Cells and Cell Surface Characterization - A Systematic Review of the Literature

Human adult mesenchymal stem cells (MSCs) were first identified by Friedenstein et al. when observing a group of cells that developed into fibroblastic colony forming cells (CFU-F). Ever since, the therapeutic uses and clinical applications of these cells have increased research and interest in this field. MSCs have the potential to be used in tissue engineering, gene therapy, transplants and tissue injuries. However, identifying these cells can be a challenge. Moreover, there are no articles bringing together and summarizing the cell surface markers of MSCs in adults. The purpose of this study is to summarize all the available information about the cell surface characterization of adult human MSCs by identifying and evaluating all the published literature in this field. We have found that the most commonly reported positive markers are CD105, CD90, CD44, CD73, CD29, CD13, CD34, CD146, CD106, CD54 and CD166. The most frequently reported negative markers are CD34, CD14, CD45, CD11b, CD49d, CD106, CD10 and CD31. A number of other cell surface markers including STRO-1, SH2, SH3, SH4, HLA-A, HLA-B, HLA-C, HLA-DR, HLA-I, DP, EMA, DQ (MHC Class II), CDIO5, Oct 4, Oct 4A, Nanog, Sox-2, TERT, Stat-3, fibroblast surface antigen, smooth muscle alpha-actin, vimentin, integrin subunits alpha4, alpha5, beta1, integrins alphavbeta3 and alphavbeta5 and ICAM-1 have also been reported. Nevertheless, there is great discrepancy and inconsistency concerning the information available on the cell surface profile of adult MSCs and we suggest that further research is needed in this field to overcome the problem.

Integrin subunits alpha5 and alpha6 regulate cell cycle by modulating the chk1 and Rb/E2F pathways to affect breast cancer metastasis

BackgroundAlthough integrins have been implicated in the progression of breast cancer, the exact mechanism whereby they exert this regulation is clearly not understood. To understand the role of integrins in breast cancer, we examined the expression levels of several integrins in mouse breast cancer cell lines by flow cytometry and the data were validated by Western and RT-PCR analysis. The importance of integrins in cell migration and cell invasion was examined by in vitro assays. Further the effect of integrins on metastasis was investigated by in vivo experimental metastasis assays using mouse models.ResultsIntegrin α5 subunit is highly expressed in the nonmetastatic cell line 67NR and is significantly low in the highly invasive cell line 4T1. In contrast, expression levels of integrin α6 subunit are high in 4T1 cells and low in 67NR cells. In vitro data indicated that overexpression of α5 subunit and knockdown of α6 integrin subunit inhibited cell proliferation, migration, and invasion. Our in vivo findings indicated that overexpression of integrin α5 subunit and knockdown of α6 subunit decreased the pulmonary metastasis property of 4T1 cells. Our data also indicated that overexpression of alpha 5 integrin subunit and suppression of alpha6 integrin subunit inhibited cells entering into S phase by up-regulating p27, which results in downregulation of cyclinE/CDK2 complexes, This suggests that these integrins regulate cell growth through their effects on cell-cycle-regulated proteins. We also found that modulation of these integrins upregulates E2F, which may induce the expression of chk1 to regulate cdc25A/cyclin E/CDK2/Rb in a feedback loop mechanism.ConclusionThis study indicates that Integrin α5 subunit functions as a potential metastasis suppressor, while α6 subunit functions as a metastasis promoter. The modulation of integrins reduces cdc25 A, another possible mechanism for downregulation of CDK2. Taken together we demonstrate a link between integrins and the chk1-cdc25-cyclin E/CDK2-Rb pathway.

Laminin isoforms in atherosclerotic arteries from mice and man.

The properties of the arterial vasculature depend to a large extent on the activities of smooth muscle cells, which, in turn, are determined by their extracellular environment. During pathological conditions, such as atherosclerosis, this interaction is altered. In close proximity to medial smooth muscle cells are basement membrane components, such as different isoforms of laminin. These proteins can have great impact on cellular function via interaction with cell surface integrins. However, knowledge of laminins in smooth muscle cell basement membranes during normal and pathological conditions is scarce. Therefore, we have analyzed the presence of laminin isoforms in atherosclerotic lesions of apolipoprotein E (ApoE)-deficient mice. Our study revealed that the laminin chain isotype composition within atherosclerotic plaque tissue was different from the chain composition in the media. In addition, obvious differences in laminin chain composition could be observed in areas of the media, which were or were not associated with plaque tissue. Our major findings demonstrate that laminin gamma3 was exclusively present in media associated with plaque tissue. Laminin alpha2 was also enriched in these medial areas. Plaque tissue was predominantly enriched in laminin alpha5 chains. This general distribution applied to lesions both with and without a fibrous cap-like structure. The differential distribution of laminin chains were partially accompanied by changes in the presence of the integrin alpha subunits 7 and V. The distribution of laminin chains in human atherosclerotic arteries, with different size and morphology, grossly resembled their distribution in mouse arteries.

Effect of 17β-estradiol on adhesion of Mytilus galloprovincialis hemocytes to selected substrates. Role of alpha2 integrin subunit

The process of hemocyte adhesion to extracellular matrix (ECM) proteins plays a crucial role in cell immunity. In most of these interactions between ECM proteins and cells, integrins are involved. The results of the present study showed that incubation of Mytilus galloprovincialis hemocytes with 17β-estradiol caused significant increased adhesion of hemocytes to ECM proteins and specifically to laminin-1, collagen IV and oxidized collagen IV, in relation to control cells. The adhesion of hemocytes to oxidized collagen was significantly higher than to either collagen IV or to laminin-1. In accordance with this, inhibition of either NADPH oxidase or nitric oxide (NO) synthase attenuated 17β-estradiol effect on hemocyte adhesion, suggesting that the high levels of free radicals, produced after 17β-estradiol effect, could contribute to the high adhesion of hemocytes to laminin-1 and collagen IV. The implication of ROS was further confirmed by the use of the oxidant rotenone, which caused elevation of cell adhesion in relation to control and by the antioxidant NAC which attenuated 17β-estradiol effect. The mechanism of 17β-estradiol induced adhesion to laminin-1, collagen IV and oxidized collagen IV involves a large number of intracellular components, as Na +/H + exchanger (NHE), all isoforms of protein kinase C (PKC), phosphatidylinositol-3-kinase (PI3K) and c-jun N-terminal kinase (JNK) as well as alpha2 integrin subunit. Maintenance of high cyclic adenosine-3′-5′-monophosphate (cAMP) levels caused non significant higher adhesion of hemocytes to ECM proteins in relation to control cells. Our results showed that 17β-estratiol caused a significant increase in α 2 integrin subunit levels, which was reduced after inhibition of NHE, PI3K, PKC, NO synthase, NADPH oxidase and JNK. In addition, our results showed that apart from 17β-estradiol, high cAMP and high ROS levels caused significantly higher induction of α 2 integrin subunit levels in relation to control. Our results imply a potential involvement of cAMP in immune responses of Mytilus hemocytes, which needs further investigation.

Induction of type XVI collagen expression facilitates proliferation of oral cancer cells

Type XVI collagen belongs to the family of fibril-associated collagens with interrupted triple helices (FACIT). Recently, high affinity to integrin alpha1beta1 has been shown allowing cells expressing those integrins to attach and spread on recombinant type XVI collagen. Here, we show that type XVI collagen is overexpressed in dysplastic areas of mucosal epithelium from oral squamous cell carcinoma (OSCC) patients. Induction of its expression in OSCC cell lines (COLXVI cells) leads to an increased expression of Kindlin-1. Moreover, we demonstrate a significantly increased Kindlin-1/beta1-integrin interaction. Additionally, we detected a higher number of activated beta1-integrins in COLXVI cells and found a neo-expression of alpha1 integrin subunit on these cells. FACS analysis revealed a significantly higher amount of COLXVI cells in S-phase and G2/M-phase 6 h after synchronisation leading to a markedly higher proliferation activity. Blocking beta1-integrins with a specific antibody resulted in reduced proliferation of COLXVI cells. In summary, we demonstrate that overexpression of type XVI collagen in aberrant oral keratinocytes leads to Kindlin-1 induction, increased Kindlin-1/beta1-integrin interaction, integrin activation and subsequently to a proliferative cellular phenotype.

Comparison of satellite cell-derived myoblasts and C2C12 differentiation in two- and three-dimensional cultures: changes in adhesion protein expression

Changes in the expression of adhesion proteins involved in myoblast differentiation were investigated in monolayer (two-dimensional) and 3D (three-dimensional) cell cultures. The expression of integrin alpha3 subunit, integrin beta1 subunit, ADAM12 (a disintegrin and metalloproteinase 12), tetraspanins CD9 and CD81 and M-cadherin were examined in the murine myoblast cell line C2C12 and in a primary culture of rat satellite cells. Myoblasts in monolayer and 3D cultures showed significant differences in their morphology and cytoskeletal organization. All of the studied proteins participated in myoblast fusion in each culture examined, but differences in their levels of expression were observed. Satellite cell-derived myoblasts exhibited higher expression of adhesion protein mRNAs than C2C12 cells. Also, C2C12 cells from a 3D culture showed slightly higher expression of adhesion protein transcripts than the same cells cultured as a monolayer. Significantly, the levels of adhesion protein mRNAs were found to change in parallel in all cell culture types. Despite this finding, it is important that differences between satellite cell-derived myoblasts and cell line C2C12 grown in monolayer and 3D cultures are taken into account when studying processes of myoblast differentiation in vitro.

CD38 Expression Marks CLL Cells with Invasive Properties

AbstractAbstract 2422CLL is characterized by a dynamic balance between proliferating cells in the lymphoid organs and circulating cells resisting programmed cell death. Regulating this equilibrium entails complex interactions between tumor and host, modulated by a set of surface molecules expressed by the CLL cell according to environmental conditions.CD38 is a negative prognostic marker, involved in the homing process. We previously reported that CD38+ cells are significantly more sensitive to CXCL12, a critical chemokine for the recirculation of neoplastic cells, both in vitro and in vivo. Activation of CD38 by means of agonistic mAbs promotes chemotaxis, while block of the molecule impairs it. CD38 is also co-expressed with CD49d, the alpha4 integrin subunit and a further independent negative prognostic marker for CLL. The two molecules appear to be intertwined in a dynamic loop which involves CD31 (the CD38 ligand predominantly expressed by endothelial cells) and VCAM-1 (the CD49d ligand).Attention has now been focused on MMP-9, the main matrix metalloproteinase expressed by CLL cells, due to the relevance of extra-vasation in the homing process. The final aim is to clarify whether CD38 may represent a key player, taking part to all the main steps of CLL cells recirculation.Results obtained analyzing a large cohort of CLL patients indicate that i) CD38+ cases are characterized by a higher expression and activity of MMP-9, as measured by gelatin zymography. Moreover, ii) the analysis of CLL patients with a bimodal expression of CD38 indicate that the CD38+ fraction of the clone is the one expressing higher levels of MMP-9 compared to the negative one. A formal proof of the connection between CD38 and this gelatinase has been obtained using a lentiviral technique that allows genetic manipulation of CLL cells. iii) De novo expression of CD38 is followed by secretion of high amounts of the active form of MMP-9, suggesting that de novo CD38+ cells digest extracellular matrix more readily. Furthermore, iv) the engagement of CD38 by means of an agonistic antibody is followed by an increased MMP-9 activation, while blocking anti-CD38 mAbs are highly effective in the modulation of MMP-9 secretion by CLL cells. Finally, v) CD38 appears to co-localize with MMP-9 in the same membrane areas, as inferred by confocal microscopy analysis.Considered together, these information pinpoint CD38 as a connecting element between chemokines, adhesion molecules and matrix metalloproteinases. The finding of a physical proximity of all these molecules suggests that they form a large supramolecular complex, with the characteristics of the invadosome, a podosome of neoplastic cells that controls diffusion and metastasis. If confirmed, these results would link the ability to migrate and invade tissues with an inferior clinical outcome also in leukemia and not only in solid tumors. Disclosures:No relevant conflicts of interest to declare.