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PubMed2004

In vitro exposure to paclitaxel modulates integrin expression by human T lymphocytes and inhibits T cell adhesion to breast carcinoma cells.

Paclitaxel (Taxol) is a drug that is commonly used in the treatment of metastatic breast cancer. In this study, we investigated the effect of prior exposure to submaximal cytotoxic concentrations (EC(25) and EC(50)) of paclitaxel on the subsequent ability of human Jurkat T lymphocytes to adhere to monolayers of MDA-MB-435 human breast carcinoma cells. Jurkat T cells that survived culture for 24 h in the presence of paclitaxel (0.1 or 3 micro g/ml) exhibited a diminished ability to adhere to MDA-MB-435 breast cancer cell monolayers. Flow cytometric analysis of paclitaxel-treated Jurkat T cells revealed reduced surface expression of alphaL, alpha4, alpha5, and beta7 integrins, but normal beta1 integrin expression. These data suggest that paclitaxel interferes with the expression of alphaLbeta2 (LFA-1), alpha4beta1 (VLA-4), alpha5beta1 (VLA-5), and alpha4beta7 (LPAM-1) adhesion molecules by surviving T cells. Blocking experiments with antibodies against alphaL, alpha4, alpha5, beta1, and beta7 integrins revealed that adhesion of Jurkat T cells to MDA-MB-435 breast cancer cell monolayers was dependent upon alphaL, alpha4, and beta7 but not alpha5 or beta1 integrins. We conclude that prior exposure to paclitaxel caused a reduction in Jurkat T cell adhesion to MDA-MB-435 breast carcinoma cells by preventing optimal alphaLbeta2 and alpha4beta7 interactions with their corresponding ligands on tumor cells. A similar effect by paclitaxel on T lymphocytes of breast cancer patients may compromise cell-mediated anti-tumor immune responses.

Antineoplastic Agents, Phytogenic↗

PubMed2001

Gelsolin functions as a metastasis suppressor in B16-BL6 mouse melanoma cells and requirement of the carboxyl-terminus for its effect.

Gelsolin, an actin-binding protein, is implicated as a critical regulator in cell motility. In addition, we have reported that cellular levels of gelsolin are decreased in various tumor cells, and overexpression of gelsolin by gene transfer suppresses tumorigenicity. We sought to assess the effects of gelsolin overexpression on metastasis and to determine the importance of a carboxyl-terminus that confers Ca(2+) dependency on gelsolin for effects of its overexpression. Expression vectors with cDNA encoding either full-length wild-type or His321 mutant form, isolated from a flat revertant of Ras-transformed cells and a carboxyl-terminal truncate, C-del of gelsolin, were transfected into a highly metastatic murine melanoma cell line, B16-BL6. Expression of introduced cDNA in transfectants was confirmed using Western blotting, 2-dimensional gel electrophoresis and reverse transcription-polymerase chain reaction (RT-PCR). We characterized phenotypes of transfectants, such as growth rate, colony formation in soft agar, cell motility and metastasis formation in vivo. Transfectants expressing the wild-type, His321 mutant and C-del gelsolin exhibited reduced growth ability in soft agar. Although expression of integrin beta1 or alpha4 on the cell surface of transfectants was not changed, wild-type and His321 mutant gelsolin, except for C-del gelsolin, exhibited retardation of cell spreading, reduced chemotatic migration to fibronectin and suppressed lung colonization in spontaneous metastasis assay. Gelsolin may function as a metastasis suppressor as well as a tumor suppressor gene. The carboxyl-terminus of gelsolin is important for retardation of cell spreading, reduced chemotasis and metastasis suppression.

Animals↗

PubMed2000

Prevention of a chronic progressive form of experimental autoimmune encephalomyelitis by an antibody against mucosal addressin cell adhesion molecule-1, given early in the course of disease progression.

A role for alpha4 and beta7 integrins in mediating leucocyte entry into the central nervous system in the multiple sclerosis (MS)-like disease experimental autoimmune encephalomyelitis (EAE) has been demonstrated. However, the individual contributions of their respective ligands mucosal addressin cell adhesion molecule-1 (MAdCAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-cadherin expressed on the blood-brain barrier has not been determined. In the present paper, it is shown that an antibody directed against MAdCAM-1, the preferential ligand for alpha4beta7, effectively prevented the development of a progressive, non-remitting, form of EAE, actively induced by injection of myelin oligodendrocyte glycoprotein peptide (MOG(35-55)) autoantigen. Combinational treatment with both anti-MAdCAM-1, VCAM-1, and intercellular adhesion molecule-1 (ICAM-1) (ligand for integrin lymphocyte function-associated antigen (LFA)-1) mAbs led to more rapid remission than that obtained with anti-MAdCAM-1 antibody alone. However, neither MAdCAM-1 monotherapy, nor combinational antibody blockade was preventative when administered late in the course of disease progression. In conclusion, MAdCAM-1 plays a major contributory role in the progression of chronic EAE and is a potential therapeutic target for the treatment of MS. Critically, antivascular addressin therapy must be given early in the course of disease prior to the establishment of irreversible damage if it is to be effective, as a single treatment modality.

Animals↗

PubMed2001

Interactions of human prostatic epithelial cells with bone marrow endothelium: binding and invasion.

Prostate cancer shows a propensity to form secondary tumours within the bone marrow. Such tumours are the major cause of mortality in this disease. We have developed an in vitro system to study the binding of prostate epithelial cells to bone marrow endothelium (BME) and stroma (BMS). The metastatic prostate cancer cell line, PC3 (derived from a bone metastasis), was seeded onto confluent layers of BME and its binding characteristics compared to human umbilical vein endothelial cells (HUVEC), lung endothelium (Hs888Lu) and BMS. The PC3 cell line showed significantly increased binding to BME (P< 0.05) compared to endothelium derived from HUVEC and lung or BMS with maximal binding occurring at 1 h. Following pre-incubation with a beta1 integrin antibody PC3 binding to BME was inhibited by 64% (P< 0.001). Antibodies directed against the integrins beta4, alpha2, alpha4, alpha5 and the cellular adhesion molecules P-selectin, CD31, VCAM-1 and sialy Lewis X showed no effect on blocking PC3 binding. Primary prostatic epithelial cells from both malignant (n = 11) and non-malignant tissue (n = 11) also demonstrated equivalent levels of increased adhesion to BME and BMS compared to HUVEC, peaking at 24 h. Further studies examined the invasive ability of prostate epithelial cells in response to bone marrow endothelium using Matrigel invasion chamber assays. In contrast to the previous results, malignant cells showed an increase (1000 fold) in invasive ability, whilst non-malignant prostate epithelia did not respond. We have shown that both malignant and non-malignant prostate epithelial cells can bind at equivalent levels and preferentially to primary human bone marrow endothelium in comparison to controls. However, only malignant prostate epithelia show increased invasive ability in response to BME.

Adenocarcinoma↗

PubMed1999

Macrophage and lymphocyte homing in experimental diabetes.

Diabetes mellitus was induced in C57BL/6 mice by multiple low doses of streptozotozin (STZ). By transferring lymphocytes from these diabetic animals to healthy recipient mice insulitis can be induced in healthy recipient mice. On day 21 after the start of STZ-treatment splenic lymphocytes were separated in vitro and stained with the fluorochrom acridine red for adoptive transfer. The recipient mouse had been pretreated with a subdiabetogenic dose of STZ (5 mg/kg) i.p. 24 h prior to the lymphocyte transfer. With in vivo microscopy we measured the lymphocyte adherence to the endothelium of islets of the recipient mouse. After the administration of mAbs directed against LFA-1, ICAM-1, murine VCAM-1, VLA-4, MadCAM or alpha4,beta7-integrin prior to the cell transfer we could demonstrate a significant decrease of donor lymphocyte adherence in islets (P<0.01). The pretreatment of the recipient mice with STZ 24 h before the transfer of lymphocytes might attract macrophages to the islets. Therefore we pretreated the recipients with antibodies to cytokines or silica. mAb IFN-gamma, pAb TNF-alpha, pAb IL-1alpha or silica reduced lymphocyte adherence to islet endothelium significantly (P<0.01). The presented results support the following interpretation: The pretreatment of the recipient mice with the islet specific toxin STZ in subdiabetogenic doses attracts macrophages to the islets. The macrophages release cytokines leading to an increased expression of adhesion molecules on the endothelium of the islets. The consequence is lymphocyte homing selectively to the islets. The cascade of lymphocyte homing is complex and various pairs of adhesion molecules are involved. The discussion on the importance of the single pairs of adhesion molecules is irrelevant, since the cascade of lymphocyte homing can be disturbed by the application of a mAb to any one adhesion molecule involved in the cascade of lymphocyte homing.

Animals↗

PubMed2002

Assessment of G-CSF stimulated BM hematopoietic stem cells in normal donors.

BACKGROUND: The clinical use of G-CSF has recently been expanded to include mobilization of stem cells for both autologous and allogeneic transplantation. Most of the published studies have focused on stem cells released into the peripheral blood (PB) after G-CSF treatment. However, little is known about the effects of G-CSF on BM. This study evaluated the concurrent effects of short-term G-CSF on both BM and PB stem and progenitor cells in normal individuals. METHODS: Volunteers received 5 or 10 microg/kg of G-CSF for 5 consecutive days (Days 1-5). On Days 0, 3, 6, 9 and 15, BM and PB samples were obtained. Flow cytometry and functional assay were performed to analyze stem cells, subpopulations, adhesion molecules, colony-forming units and LTCIC. RESULTS: The total nucleated cells and absolute numbers of CD34(+)/mL showed a similar response pattern in both BM and PB, with a peak around Day 6 that returned to baseline levels by Day 15. However, there was a reciprocal change in the percentage of CD34(+) cells between BM and PB compartments. The expressions of adhesion molecule showed an up- and down-regulation of alpha4 and alpha5 integrin subunits, respectively, also correlated with the CD34(+) mobilization patterns. DISCUSSIONS: The functional characterization of integrins, and further clinical examination of G-CSF-stimulated BM is warranted. G-CSF-stimulated BM maybe considered as an alternative source of stem cells in transplantation.

Adult↗

PubMed2001

Involvement of T-lymphocytes in periodontal disease and in direct and indirect induction of bone resorption.

Periodontal disease is a peripheral infection involving species of gram-negative organisms. T-lymphocytes can be found in the dense inflammatory infiltrate in this disease. CD4+ and CD8+ T-cells are present in periodontal lesions, as are memory/activated T-lymphocytes. In addition, Th1- and Th2-type T-lymphocytes and their associated cytokines with a subtle polarization to Th1 may be present. Th1-type T-cells up-regulate the production of pro-inflammatory cytokines IL-1 and TNF-alpha, which can induce bone resorption indirectly by promoting differentiation of osteoclast precursors and subsequently by activating osteoclasts. Such osteoclast differentiation is dependent on stimulation of osteoprotegerin ligand (OPG-L) production by osteoblastic cells. By contrast, activated T-cells, by virtue of direct production and expression of OPG-L, can directly promote osteoclast differentiation. OPG-L appears to be predominantly expressed on Th1-type cells. The direct and indirect T-cell involvement in periodontal bone resorption appears to be dependent on the degree of Th1-type T-cell recruitment into inflamed gingival tissues. This T-cell recruitment is regulated by adhesion molecules and chemokines/chemokine receptors. The adhesion molecules involved include alpha4 and alpha6 integrins, LFA-1, and ICAM-1. The Th1-type T-cells preferentially express CCR5 and CXCR3, which are found prominently in diseased gingivae. By contrast, little CCR4, expressed by Th2-type T-cells, can be detected. Also, the chemokine ligands RANTES, MIP1-alpha (both CCR5), and IP-10 (CXCR3 ligand) were elevated in inflamed periodontal tissues. The T-cell features in diseased periodontal tissues can be compared with those in rheumatoid arthritis, wherein bone resorption often attributed to Th1-type T-cell involvement has also been demonstrated.

Alveolar Bone Loss↗

PubMed2006

Human lung mast cells adhere to human airway smooth muscle, in part, via tumor suppressor in lung cancer-1.

Mast cells infiltrate the airway smooth muscle (ASM) of patients with asthma, an event which is likely to be a key factor in the development of this disease. Adhesion is a fundamental mechanism facilitating cellular cross-talk. We have examined whether human lung mast cells (HLMC) and ASM adhere, and have also examined the mechanism involved. Primary cultures of HLMC and confluent human ASM were cocultured for 30 min, then nonadherent HLMC were removed by centrifugation. HLMC adhered avidly to ASM monolayers (mean +/- SEM adhesion 43.2 +/- 1.2%, n = 41). Adhesion was increased to 58.8 +/- 2.7% by 1 mM Mn2+ (p = 0.015), and was reduced by EDTA and EGTA to 20.5 +/- 1.5% and 21.0 +/- 1.3%, respectively (p < 0.0001). Adhesion-blocking Abs for ICAM-1, VCAM-1, CD18, and the alpha4 and beta1 integrins had no effect on HLMC adhesion. HLMC expressed tumor suppressor in lung cancer-1 (TSLC-1) and blocking this reduced adhesion from 38.5 +/- 4.8% to 28.3 +/- 3.7% (p = 0.004, n = 7). ASM did not express TSLC-1, indicating that TSLC-1 acts as a heterophilic adhesion molecule. In summary, HLMC adhere avidly to ASM in part via TSLC-1 and in part via an as-yet-undefined Ca2+-dependent pathway. This supports the hypothesis that adhesion is important in the recruitment and retention of HLMC by the ASM in asthma, and for the functional interaction of these cells.

Asthma↗

PubMed1997

Anti-CD43 inhibits monocyte-endothelial adhesion in inflammation and atherogenesis.

Recruitment of blood monocytes into tissues is a central event in the inflammatory response and in atherogenesis. The mechanisms leading to monocyte adhesion and migration through endothelium are not completely defined. We recently reported that MAb L11, against the leukocyte sialomucin CD43, blocks T-lymphocyte binding to lymph node and Peyer's patch high endothelial venules (HEV) and inhibits T-cell extravasation from the blood into organized secondary lymphoid tissues. We have now assessed the ability of L11 to inhibit monocyte-endothelial (EC) interactions and trafficking. L11 blocks binding of WEHI78/24 cells, a murine monocytoid cell line, to inflamed lymph node HEV and inhibits recruitment of monocytes and neutrophils to thioglycollate-inflamed peritoneum. Because monocyte adhesion to the endothelium and diapedesis in lesion-prone regions of the vasculature is among the earliest events in atherogenesis, leading to formation of lipid-laden foam cells, the ability of L11 to block monocyte recognition of aortic endothelial cells was assessed in a novel ex vivo assay of monocyte binding to intact rabbit aortic endothelium. Cholesterol feeding of rabbits induces enhanced aortic adhesiveness for monocytes and WEHI78/24 monocytoid cells, and this adhesion is inhibited by L11. The inhibitory effect of L11 is additive with that of a cocktail of anti-L-selectin and anti-alpha4 and beta2 integrin monoclonal antibodies. Thus, CD43 represents a novel target for manipulation of monocyte recruitment in inflammation and atherogenesis.

Animals↗

PubMed1999

Vascular endothelial cell expression of ICAM-1 and VCAM-1 at the onset of eliciting contact hypersensitivity in mice: evidence for a dominant role of TNF-alpha.

We have studied vascular endothelial activation and increased expression of ICAM-1 and VCAM-1 at the onset of the elicitation phase of oxazolone contact hypersensitivity in mice. By measuring the local uptake of i.v. administered radiolabeled anti-ICAM-1 and anti-VCAM-1 mAb, we found that endothelial ICAM-1 and VCAM-1 was increased by 4 h after challenge, 2 h later than the first peak of ear swelling and 125I-labeled human serum albumen uptake. Increased expression of endothelial ICAM-1 and VCAM-1 was significantly greater in sensitized animals than in naive animals. Anti-TNF-alpha antiserum significantly inhibited both the increase in ear thickness (p < 0.01), and the up-regulation of ICAM-1 and VCAM-1 expression (p < 0.01 for both) at 4 h. In contrast, the combination of anti-IL-1alpha and IL-1beta had only a small inhibitory effect on ICAM-1 expression (p < 0.05) and no significant effect on increased ear thickness or on VCAM-1 expression. A mixture of anti-TNF-alpha, anti-IL-1alpha, and IL-1beta was no more inhibitory for endothelial ICAM-1 and VCAM-1 expression than anti-TNF-alpha alone. ICAM-1 and VCAM-1 expression at 4 h was unaffected by a combination of mAb against alpha4 and beta2 integrins, whereas expression at 24 h was significantly inhibited (p < 0.05), suggesting that the release of TNF-alpha and other cytokines involved in the initiation of the response may not require leukocyte traffic or other leukocyte functions involving these integrins. We conclude that the early up-regulation of endothelial ICAM-1 and VCAM-1 during the elicitation of contact hypersensitivity is primarily due to the immune-dependent local release of TNF-alpha.

Animals↗

PubMed2002

Integrin expression by human osteoblasts cultured on degradable polymeric materials applicable for tissue engineered bone.

The use of biodegradable polymers in the field of orthopaedic surgery has gained increased popularity, as surgical pins and screws, and as potential biological scaffolds for repairing cartilage and bone defects. One such group of polymers that has gained considerable attention are the polyesters, poly(lactide-co-glycolide) (PLAGA) and polylactic acid (PLA), because of their minimal tissue inflammatory response, favorable biocompatibility and degradation characteristics. The objective of this study was to evaluate human osteoblastic cell adherence and growth on PLAGA and PLA scaffolds by examining integrin receptor (alpha2, alpha3, alpha4, alpha5, alpha6 and beta1) expression. Primary human osteoblastic cells isolated from trabecular bone adhered efficiently to both PLAGA and PLA, with the rate of adherence on PLAGA comparable to that of control tissue culture polystyrene (TCPS), and significantly higher than on PLA polymers at 3, 6 and 12 h. Human osteoblastic phenotypic expression, alkaline phosphatase (ALP) activity was positive on both degradable matrices, whereas osteocalcin levels were significantly higher on cells grown on PLAGA than on PLA composites. Interestingly, the integrin subunits, alpha2, alpha3, alpha4, alpha5, alpha6 and beta1 were all expressed at higher levels by osteoblasts cultured on PLAGA than those on PLA as analyzed by westerns blots and by flow cytometry. Among the integrins, alpha2, beta5 and beta1 showed the greatest difference in levels between the two surfaces. Thus, both PLA and PLAGA support osteoblastic adhesion and its accompanying engagement of integrin receptor and expression of osteocalcin and ALP. However PLAGA consistently appeared to be a better substrate for osteoblastic cells based on these parameters. This study is one of the first to investigate the ability of primary human osteoblastic cells isolated from trabecular bone to adhere to the biodegradable polymers PLAGA and PLA, and to examine the expression of their key adhesion receptors (integrins) on these substrates.

Absorbable Implants↗

PubMed2004

Bone marrow microenvironment facilitating dendritic cell: CD4 T cell interactions and maintenance of CD4 memory.

This study shows that bone marrow (BM) stroma expresses constitutively multiple adhesion molecules (ICAM-1, VCAM-1, MadCAM-1, P-selectin) relevant for the homing and infiltration of BM by blood derived T lymphocytes, and also the co-stimulatory molecule CD80, relevant for T cell activation. T cells were capable of homing to BM but not to thymus. Homing to BM involved the integrins LFA-1alpha and alpha4 which interact with the above constitutively expressed cell adhesion molecules (CAMs). CD3 T cells were detected together with BM resident CD11c dendritic cells (DCs), often enriched in follicle-like structures in BM parenchyma. Cognate interactions between transferred antigen specific transgenic CD4 T cells and antigen laden BM-DCs led to formation of multicellular clusters in situ in BM, to generation of lymphoblasts and to clonal T cell expansion within such clusters. The great majority of BM-CD4 T cells had a memory phenotype suggesting that the BM microenvironment facilitates maintenance of CD4 memory. These results extend and corroborate our previous findings on BM-CD8 T cell mediated immune responses. Together these findings suggest that DC-T cell interactions in BM play an important role in immune responses to blood-borne antigen and in the establishment of systemic immunity and long-term memory.

Animals↗

PubMed2005

Mechanical properties of the interaction between fibronectin and alpha5beta1-integrin on vascular smooth muscle cells studied using atomic force microscopy.

The mechanical properties of integrin-extracellular matrix (ECM) interactions are important for the mechanotransduction of vascular smooth muscle cells (VSMC), a process that is associated with focal adhesions, and can be of particular significance in cardiovascular disease. In this study, we characterized the unbinding force and binding activity of the initial fibronectin (FN)-alpha5beta1 interaction on the surface of VSMC using atomic force microscopy (AFM). It is postulated that these initial binding events are important to the subsequent focal adhesion assembly. FN-VSMC adhesions were selectively blocked by antibodies against alpha5- and beta1-integrins as well as RGD-containing peptides but not by antibodies against alpha4- and beta3-integrins, indicating that FN primarily bound to alpha5beta1. A characteristic unbinding force of 39 +/- 8 pN was observed and interpreted to represent the FN-alpha5beta1 single-bond strength. The ability of FN to adhere to VSMC (binding probability) was significantly reduced by integrin antagonists, serum starvation, and platelet-derived growth factor (PDGF)-BB, whereas lysophosphatidic acid (LPA) increased FN binding. However, no significant change in the resolved unbinding force was observed. After engagement, the force required to dislodge the FN-coated bead from VSMC increased with increasing of contact time, suggesting a time-dependent increase in number of adhesions and/or altered binding affinity. LPA enhanced this process, whereas PDGF reduced it, suggesting that these factors also affect the multimolecular process of focal contact assembly. Thus AFM is a powerful tool for the characterization of the mechanical properties of integrin-ECM interactions and their regulation. Our results indicate that the functional activity of alpha5beta1 and focal contact assembly can be rapidly regulated.

Animals↗

PubMed2004

The alpha4 laminin subunit regulates endothelial cell survival.

The alpha4 laminin subunit is a major structural component of assembling basement membranes of endothelial cells. We have been investigating its functions with regard to endothelial cell survival. An anti-laminin alpha4 antibody (2A3), directed against the G domain of the alpha4 laminin subunit of laminins-8 and -9, inhibits proliferation and enhances apoptosis of endothelial cells when cells are maintained in vitro. Activation of caspases-9 and -3 plays a role in 2A3 antibody-induced apoptosis, since inhibitors specific for these caspases and overexpression of the anti-apoptotic protein Bcl-X(L), but not c-FLIP, inhibit 2A3 antibody-triggered endothelial cell death. Extracellular matrix is known to play a role in regulating programmed cell death in an integrin-dependent fashion. The alpha4 laminin subunit conforms to this idea since activation of beta1 integrin subunits on endothelial cells blocks the ability of 2A3 antibody to induce endothelial cell death. In summary, our data indicate that complexes composed of alpha4 laminin/beta1 subunit-containing integrins at the cell surface support endothelial cell survival. Furthermore, we propose that antagonists of alpha4 laminin function, including antibody 2A3, have value as angiogenesis inhibitors in a clinical setting where blocking aberrant growth of blood vessel by triggering apoptosis of endothelial cells may be therapeutic.

Antibodies↗

PubMed2003

Beta1 integrin/focal adhesion kinase-mediated signaling induces intercellular adhesion molecule 1 and receptor activator of nuclear factor kappaB ligand on osteoblasts and osteoclast maturation.

We have assessed characteristics of primary human osteoblasts, shedding light on signaling mediated by beta1 integrin. beta1 integrins are major receptors for these matrix glycoproteins. 1) Integrins beta1, alpha2, alpha3, alpha4, alpha5, alpha6, and alphav were highly expressed on primary osteoblasts. 2) Engagement of beta1 integrins on osteoblasts by cross-linking with specific antibody or ligand matrices, such as fibronectin or collagen, augmented expression of intercellular adhesion molecule 1 (ICAM-1) and receptor activator of nuclear factor kappaB ligand (RANKL) on the surface. 3) Up-regulation of ICAM-1 and RANKL on osteoblasts by beta1 stimulation was completely abrogated by pretreatment with herbimycin A and genistein, tyrosine kinase inhibitors, or transfection of dominant negative truncations of focal adhesion kinase (FAK). 4) Engagement of beta1 integrins on osteoblasts induced tartrate-resistant acid phosphatase-positive multinuclear cell formation in the coculture system of osteoblasts and peripheral monocytes. 5) Up-regulation of tartrate-resistant acid phosphatase-positive multinuclear cell formation by beta1 stimulation was completely abrogated by transfection of dominant negative truncations of FAK. Our results indicate that beta1 integrin-dependent adhesion of osteoblasts to bone matrices induces ICAM-1 and RANKL expression and osteoclast formation via tyrosine kinase, especially FAK. We here propose that beta1 integrin/FAK-mediated signaling on osteoblasts could be involved in ICAM-1- and RANKL-dependent osteoclast maturation.

Antibodies, Monoclonal↗

PubMed2005

CXCR4 chemokine receptor and integrin signaling co-operate in mediating adhesion and chemoresistance in small cell lung cancer (SCLC) cells.

Small cell lung cancer (SCLC) is an aggressive, rapidly metastazising neoplasm with a high propensity for marrow involvement. SCLC cells express high levels of functional CXCR4 receptors for the chemokine stromal-cell-derived factor-1 (SDF-1/CXCL12). Adhesion of SCLC cells to extracellular matrix or accessory cells within the tumor microenvironment confers resistance to chemotherapy via integrin signaling and thus may be responsible for residual disease and relapses commonly seen in SCLC. We examined the signaling mechanisms that regulate CXCL12-induced adhesion of SCLC cells to fibronectin, collagen, and stromal cells and the effects on SCLC cell chemoresistance. We found that CXCL12-induced integrin activation which resulted in an increased adhesion of SCLC cells to fibronectin and collagen. This was mediated by alpha2, alpha4, alpha5, and beta1 integrins along with CXCR4 activation, which could be inhibited by CXCR4 antagonists. Stromal cells protected SCLC cells from chemotherapy-induced apoptosis, and this protection could also be antagonized by CXCR4 inhibitors. We conclude that activation of integrins and CXCR4 chemokine receptors co-operate in mediating adhesion and survival signals from the tumor microenvironment to SCLC cells. Therefore, CXCR4 antagonists in combination with cytotoxic drugs should be explored in SCLC to overcome CXCL12-mediated adhesion and survival signals in the tumor microenvironment.

Apoptosis↗

PubMed2006

Integrin ligands mobilize Ca2+ from ryanodine receptor-gated stores and lysosome-related acidic organelles in pulmonary arterial smooth muscle cells.

Extracellular matrix (ECM) protein receptors, or integrins, participate in vascular remodeling and the systemic myogenic response. Synthetic ligands and ECM fragments regulate the vascular smooth muscle cell contractile state by altering intracellular Ca2+ levels ([Ca2+]i). Information on the Ca2+ effect of integrins in vascular smooth muscle cells is limited, but nonexistent in pulmonary arterial smooth muscle cells (PASMCs). We therefore characterized integrin expression in endothelium-denuded pulmonary arteries, and explored [Ca2+]i mobilization pathways induced by soluble ligands in rat PASMCs. Reverse transcriptase-PCR showed mRNA expression of integrins alpha1, alpha2, alpha3, alpha4, alpha5, alpha7, alpha8, alpha(v), beta1, beta3, and beta4, and immunoblots of alpha5, alpha(v), beta1, and beta3 confirmed protein expression. Exposure of PASMCs to integrin-binding peptides (0.5 mM) containing the arginine-glycine-aspartate (RGD) motif elicited [Ca2+]i responses with an order of potency of GRGDNP > GRGDSP > GRGDTP = cyclo-RGD. Pharmacological analysis revealed that the GRGDSP-induced Ca2+ response was unrelated to Ca2+ influx and the inositol triphosphate receptor-gated Ca2+ store, but partially blocked by ryanodine or inhibition of lysosome-related acidic organelles with bafilomycin A1. Simultaneous inhibition of both pathways was necessary to abolish the response. GRGDSP treatment increased cyclic ADP-ribose, the endogenous activator of ryanodine receptors, by 70%. GRGDSP also rapidly reduced Lysotracker Red accumulation, confirming direct modulation of acidic organelles. These data are the first demonstration of integrin-mediated Ca2+ regulation in PASMCs. The presence of an array of integrins, and activation of ryanodine-sensitive Ca2+ stores and lysosome-like organelles by GRGDSP suggest important roles for integrin-dependent Ca2+ signaling in regulating PASMC function.

Animals↗

PubMed1998

The human WD repeat protein WAIT-1 specifically interacts with the cytoplasmic tails of beta7-integrins.

Integrins of the beta7 subfamily, alpha4 beta7 and alphaE beta7, contribute to lymphocyte homing and to the development of protective or autoreactive immune responses at mucosal sites. The beta subunits of integrins are considered important for regulation of stimulated cell adhesion and adhesion-dependent signal transduction. Using a yeast interaction trap screen, a human WD repeat protein, termed WAIT-1, was isolated that interacts with the integrin beta7 cytoplasmic tail and is homologous to mouse EED and Drosophila ESC proteins. WAIT-1 also binds to the cytoplasmic domains of alpha4 and alphaE but not to those of integrin beta1, beta2, and alphaL subunits. Association of WAIT-1 and beta7-integrin was confirmed by coprecipitation from transiently transfected 293 cells. The binding site for WAIT-1 was mapped to a short membrane-proximal region of the beta7 cytoplasmic tail with Tyr-735 being of critical importance. Northern blot analysis revealed multiple WAIT-1-related transcripts with differential expression in circulating leukocytes, tissue-resident cells of diverse origin, and lymphoid malignancies. These results suggest that WAIT-1, together with the recently identified RACK1, may define a novel subfamily of WD repeat proteins that interact with distinct subsets of integrin cytoplasmic tails and may act as specific regulators of integrin function.

Amino Acid Sequence↗