Interactions of thrombospondins with alpha4beta1 integrin and CD47 differentially modulate T cell behavior.

Thrombospondin (TSP)-1 has been reported to modulate T cell behavior both positively and negatively. We found that these opposing responses arise from interactions of TSP1 with two different T cell receptors. The integrin alpha4beta1 recognizes an LDVP sequence in the NH2-terminal domain of TSP1 and was required for stimulation of T cell adhesion, chemotaxis, and matrix metalloproteinase gene expression by TSP1. Recognition of TSP1 by T cells depended on the activation state of alpha4beta1 integrin, and TSP1 inhibited interaction of activated alpha4beta1 integrin on T cells with its counter receptor vascular cell adhesion molecule-1. The alpha4beta1 integrin recognition site is conserved in TSP2. A recombinant piece of TSP2 containing this sequence replicated the alpha4beta1 integrin-dependent activities of TSP1. The beta1 integrin recognition sites in TSP1, however, were neither necessary nor sufficient for inhibition of T cell proliferation and T cell antigen receptor signaling by TSP1. A second TSP1 receptor, CD47, was not required for some stimulatory responses to TSP1 but played a significant role in its T cell antigen receptor antagonist and antiproliferative activities. Modulating the relative expression or function of these two TSP receptors could therefore alter the direction or magnitude of T cell responses to TSPs.

Alpha4beta1 integrin mediates selective endothelial cell responses to thrombospondins 1 and 2 in vitro and modulates angiogenesis in vivo.

We examined the function of alpha4beta1 integrin in angiogenesis and in mediating endothelial cell responses to the angiogenesis modulators, thrombospondin-1 and thrombospondin-2. Alpha4beta1 supports adhesion of venous endothelial cells but not of microvascular endothelial cells on immobilized thrombospondin-1, vascular cell adhesion molecule-1, or recombinant N-terminal regions of thrombospondin-1 and thrombospondin-2. Chemotactic activities of this region of thrombospondin-1 and thrombospondin-2 are also mediated by alpha4beta1, whereas antagonism of fibroblast growth factor-2-stimulated chemotaxis is not mediated by this region. Immobilized N-terminal regions of thrombospondin-1 and thrombospondin-2 promote endothelial cell survival and proliferation in an alpha4beta1-dependent manner. Soluble alpha4beta1 antagonists inhibit angiogenesis in the chick chorioallantoic membrane and neovascularization of mouse muscle explants. The latter inhibition is thrombospondin-1-dependent and not observed in explants from thrombospondin-1-/- mice. Antagonizing alpha4beta1 may in part block proangiogenic activities of thrombospondin-1 and thrombospondin-2, because N-terminal regions of thrombospondin-1 and thrombospondin-2 containing the alpha4beta1 binding sequence stimulate angiogenesis in vivo. Therefore, alpha4beta1 is an important endothelial cell receptor for mediating motility and proliferative responses to thrombospondins and for modulation of angiogenesis.

Proteomic identification of new biomarkers and application in thyroid cytology.

OBJECTIVE: To validate proteins identified by proteomics as potentially usable markers in thyroid pathology. STUDY DESIGN: Frozen sections of thyroid tumors were manually micro-dissected and proteins extracted. Two-dimensional (2D) gel electrophoresis and subsequent liquid chromatography/mass spectroscopy were performed, and differentially expressed proteins were identified. Validation of candidates for tumor markers (galectin-1, galectin-3, S100C and voltage-dependent anion channel 1 [VDAC1]) was done by immunohistochemistry in 21 cell blocks from fine needle aspiration biopsies (FNAB) and corresponding histology specimens (13 cases). RESULTS: Galectin-3 was negative in benign lesions and positive in FNAB from papillary carcinoma (5 of 5), follicular variant of papillary carcinoma (1 of 4) and follicular carcinoma (1 of 2). S100C was positive in some benign lesions: hyperplasia (2 of 4), goiter (1 of 3) and follicular adenoma (1 of 3), with predominantly nuclear pattern of staining. S100C was positive in malignant lesions, showing cytoplasmic location. Galectin-1 was negative in benign lesions and positive in follicular carcinoma (1 of 2), papillary carcinoma (2 of 5) and follicular variant of papillary carcinoma (1 of 4). VDAC1 was detected in benign and malignant lesions, showing a strong positivity in follicular carcinomas. CONCLUSION: Immunohistochemical validation of potential markers is a crucial step before clinical application in diagnosis. Galectin-3, galectin-1 and S100C can be used to help in discriminating benign and malignant thyroid lesions.

Angiogenesis inhibitors target the endothelial cell cytoskeleton through altered regulation of heat shock protein 27 and cofilin.

Inhibition of angiogenesis has emerged as a key focus for the treatment of cancer, necessitating a better understanding of the downstream molecular targets of angiogenesis inhibitors. Endostatin, thrombospondin-1, fumagillin, and its synthetic derivative, TNP-470, are potent inhibitors of endothelial cell proliferation and migration in culture and of angiogenesis in vivo. To identify targets that mediate the effects of these inhibitors, we compared two-dimensional gel electrophoresis patterns from lysates of treated and untreated human endothelial cells. Among the proteins identified were cofilin and hsp27, two proteins involved in actin dynamics. Western blotting and immunofluorescence experiments confirmed that the phosphorylation states and subcellular localization of these two proteins were affected by all of the inhibitors tested and that treated cells had a more extensive network of actin stress fibers and more numerous focal adhesion plaques compared with untreated cells. Endothelial monocyte activating polypeptide II, another angiogenesis inhibitor, elicited the same response in the actin cytoskeleton and focal adhesions of endothelial cells. This more adherent phenotype may explain the shared ability of these inhibitors to block endothelial migratory signals. Starting with a proteomics approach, we have identified common effector molecules used by a panel of angiogenesis inhibitors that perturb the cytoskeleton to prevent endothelial migration.

Identification of heat shock protein 60 as a molecular mediator of alpha 3 beta 1 integrin activation.

The alpha 3 beta 1 integrin is involved in the adhesion of metastatic breast cancer cells to the lymph nodes and to osteoblasts in the bone. Regulation of the affinity or avidity of integrins for their ligands may result from conformational changes induced by changes in the microenvironment of the integrin. Two surface proteins, 55 and 32 kDa, coimmunoprecipitated with the alpha 3 beta 1 integrin from breast carcinoma cells. The 55-kDa protein preferentially associated with the active form of the alpha 3 beta 1 integrin. The protein was identified as HSP60 using two-dimensional electrophoresis and mass spectrometry and confirmed by reimmunoprecipitation of the integrin immune complex with an anti-HSP60 antibody. In cell spreading assays on a thrombospondin-1 substrate, addition of exogenous-recombinant HSP60 was sufficient to specifically activate alpha 3 beta 1 integrin but not to activate function of alpha 2 beta 1, alpha v beta 3, alpha 4 beta 1, or alpha 5 beta 1 integrins. Furthermore, mizoribine, an HSP60-binding drug, blocked activation of the alpha 3 beta 1 integrin induced by insulin-like growth factor 1 (IGF1) or exogenous recombinant HSP60 and inhibited the association of HSP60 with the integrin. Additionally, inhibiting the surface expression of endogenous HSP60 by nonactin inhibited activation of the alpha 3 beta 1 integrin by IGF1. These data demonstrate that HSP60 binding is sufficient to activate alpha 3 beta 1 integrin function and suggest that association of endogenous HSP60 with alpha 3 beta 1 integrin is necessary for IGF1-induced activation.

Proteomics of human breast ductal carcinoma in situ.

We report the first proteomic analysis of matched normal ductal/lobular units and ductal carcinoma in situ (DCIS) of the human breast. An understanding of the transition from normal epithelium to the first definable stage of cancer at the functional level of protein expression is hypothesized to contribute to improved detection, prevention, and treatment. Ten sets of two-dimensional gels were evaluated, containing either matched normal ductal/lobular units or DCIS from either whole tissue sections or up to 100,000 laser capture microdissected epithelial cells. Differential protein expression was confirmed by image analysis. Protein spots (315) were excised and subjected to mass spectrometry sequencing. Fifty-seven proteins were differentially expressed between normal ductal/lobular units and DCIS. Differences in overall protein expression levels and posttranslational processing were evident. Ten differentially expressed proteins were validated in independent DCIS specimens, and 14 of 15 proteomic trends from two-dimensional gel analyses were confirmed by standard immunohistochemical analysis using a limited independent tumor cohort. Many of the proteins identified were previously unconnected with breast cancer, including proteins regulating the intracellular trafficking of membranes, vesicles, cancer preventative agents, proteins, ions, and fatty acids. Other proteomic identifications related to cytoskeletal architecture, chaperone function, the microenvironment, apoptosis, and genomic instability. Proteomic analysis of DCIS revealed differential expression patterns distinct from previous nucleic acid-based studies and identified new facets of the earliest stage of breast cancer progression.

Differential expression of S100C in thyroid lesions.

Identification of new potential markers that may help in the diagnosis of benign and malignant thyroid lesions is needed. By comparative 2-dimensional gel electrophoresis of microdissected cells from tumors and normal thyroid tissue, we identified a new protein, S100C, which is highly expressed in papillary carcinomas. In order to validate this finding, we investigated the immunohistochemical expression and the potential role in diagnosis of these markers in 94 specimens representing the spectrum of malignant and benign thyroid lesions. Normal thyroid tissue was evaluated in 57 specimens. Galectin-3, a marker reported as specific for malignant lesions, was also evaluated in the same lesions. S100C protein was expressed in the nuclei of normal tissue, hyperplastic nodules, and follicular adenomas and carcinomas. Papillary carcinomas showed a strong, but cytoplasmic, pattern of staining. Galectin-3 immunostaining was strongly positive in papillary carcinomas, and negative in benign lesions, confirming its value in differential diagnosis. These findings suggest that immunohistochemical staining of S100C could be helpful in the pathological study of thyroid lesions, especially in cases in which follicular variants of papillary carcinoma and follicular carcinoma are considered in the differential diagnosis.

Proteomic analysis and identification of new biomarkers and therapeutic targets for invasive ovarian cancer.

Epithelial ovarian cancer kills almost 16 000 women each year in part due to late stage of presentation and lack of reliable biomarkers for disease detection. CA-125, the currently accepted serum marker, alone lacks the sensitivity for early stage diagnosis, as only 50% of early stage cases are detected with this marker. Although more early stage cases may be detected by lysophosphatidic acid, this marker is also elevated in other cancers. One major objective of the NCI-FDA Tissue Proteomics Initiative has been to combine the technique of laser capture microdissection (LCM) of epithelial tumor cells in human tissue specimens with two-dimensional gel electrophoresis (2-D PAGE) to identify proteins that may serve as invasive ovarian cancer-specific biomarkers for early detection and/or new therapeutic targets. We performed 2-D PAGE on lysates from five microdissected ovarian tumors (three invasive ovarian cancers and two noninvasive, low malignant potential (LMP) ovarian tumors). We then compared silver stained 2-D gels created from microdissected lysates with SYPRO-Ruby stained 2-D PAGE profiles of the patient-matched undissected bulk tumor lysates from all five patients. Twenty-three proteins were consistently differentially expressed between both the LMP and three invasive ovarian tumors in the limited study set. Thirteen were uniquely present in all three of the invasive ovarian cancer cases and absent or underexpressed in the two LMP cases. Ten were uniquely present in the LMP cases but absent or underexpressed in all invasive ovarian cancer cases. Credentialing and preliminary target validation of the mass spectrometry identified proteins cut from the Ruby-red stained gels was performed by LCM coupled Western blot and reverse-phase array technology in a study set of six cases (the aforementioned five cases used in the 2-D PAGE profiling component of the study plus one additional LMP case). The analysis revealed that the 52 kDa FK506 binding protein, Rho G-protein dissociation inhibitor (RhoGDI), and glyoxalase I are found to be uniquely overexpressed in invasive human ovarian cancer when compared to the LMP form of this cancer. The direct comparison of LCM generated proteomic profiles of invasive vs. LMP ovarian cancer may more directly generate important markers for early detection and/or therapeutic targets unique to the invasive phenotype.

Thrombospondin-1 is a major activator of TGF-beta in fibrotic renal disease in the rat in vivo.

BACKGROUND: Transforming growth factor-beta (TGF-beta), a profibrotic cytokine involved in many scarring processes, has to be activated extracellularly before it can bind to its receptors. Thrombospondin 1 (TSP1), a multifunctional matricellular glycoprotein, has been identified as an activator of TGF-beta in in vitro systems and during mouse postnatal development in vivo. TSP1 is expressed de novo in many inflammatory disease processes, including glomerular disease. METHODS: In this study we investigated whether peptides specifically interfering with the activation process of TGF-beta by TSP1 may be able to block activation of TGF-beta in an in vivo model of mesangial proliferative glomerulonephritis. RESULTS: Continuous intravenous infusion of blocking peptide by minipumps significantly reduced expression of active TGF-beta in glomeruli on day 7 of disease as indicated by immunohistochemistry, bioassay, and activation of the TGF-beta signal transduction pathway, while total TGF-beta expression was unchanged. Inhibition of glomerular TGF-beta activation was accompanied by a decrease of glomerular extracellular matrix accumulation and proteinuria, but was without effect on mesangial cell proliferation or influx of monocytes/macrophages. CONCLUSION: TSP1 is a major endogenous activator of TGF-beta in experimental inflammatory glomerular disease. Drugs interfering with the activation of TGF-beta by locally produced TSP1 may be considered as a future specific treatment of scarring kidney disease.

Proteomic analysis of lymph.

This report provides the first proteomic analysis of normal ovine lymph. By establishing the fact that lymph is more than an ultrafiltrate of blood plasma, it documents that the lymph proteome contains an array of proteins that differentiates it from plasma. The protein chip technology, surface-enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS), two-dimensional gel electrophoresis (2-D PAGE) and MS, were employed to examine the protein expression profiles of ovine lymph. Using a weak cation exchange chip surface to assay lymph and plasma samples by SELDI-TOF-MS showed that the analysis of peak maps from lymph contained three protein peaks that were found only in lymph, while analysis of peak maps from plasma samples showed that five protein peaks were found only in plasma. Lymph and plasma samples showed eight peaks that were common to both. There were also more ions present in plasma than in lymph, which is consistent with the 2-D PAGE analysis. MS analysis of a large number of protein spots from 2-D PAGE gels of lymph produced MS/MS sequences for 18 proteins that were identified by searching against a comprehensive protein sequence database. As in plasma, large protein spots of albumin dominated the protein pattern in lymph. Other major proteins identified in 2-D PAGE gels of lymph included, fibrinogen alpha- and beta-chains, immunoglobulin G (IgG) heavy chain, serotransferrin precursor, lactoferrin, and apolipoprotein A-1. Two proteins that were identified and were differentially expressed in lymph were glial fibrillary astrocyte acidic protein and neutrophil cytosol factor-1. By bringing the technologies of proteomics to bear on the analysis of lymph, it is possible to detect proteins in lymph that are quantitatively and qualitatively differentially expressed from those of plasma.

Recognition of the N-terminal modules of thrombospondin-1 and thrombospondin-2 by alpha6beta1 integrin.

In addition to its recognition by alpha3beta1 and alpha4beta1 integrins, the N-terminal pentraxin module of thrombospondin-1 is a ligand for alpha6beta1 integrin. alpha6beta1 integrin mediates adhesion of human microvascular endothelial and HT-1080 fibrosarcoma cells to immobilized thrombospondin-1 and recombinant N-terminal regions of thrombospondin-1 and thrombospondin-2. alpha6beta1 also mediates chemotaxis of microvascular cells to thrombospondin-1 and thrombospondin-2. Using synthetic peptides, LALERKDHSG was identified as an alpha6beta1-binding sequence in thrombospondin-1. This peptide inhibited alpha6beta1-dependent cell adhesion to thrombospondin-1, thrombospondin-2, and the E8 fragment of murine laminin-1. The Glu residue in this peptide was required for activity, and the corresponding residue (Glu90) in the N-terminal module of thrombospondin-1 was required for its recognition by alpha6beta1, but not by alpha4beta1. alpha6beta1 was also expressed in human umbilical vein endothelial cells; but in these cells, only certain agonists could activate the integrin to recognize thrombospondins. Selective activation of alpha6beta1 integrin in microvascular endothelial cells by the anti-beta1 antibody TS2/16 therefore accounts for their adhesion responses to thrombospondins and explains the distinct functions of alpha4beta1 and alpha6beta1 integrins as thrombospondin receptors in microvascular and large vessel endothelial cells.

Regulation of integrin function by CD47 ligands. Differential effects on alpha vbeta 3 and alpha 4beta1 integrin-mediated adhesion.

We examined the regulation of alpha4beta1 integrin function in melanoma cells and T cells by ligands of CD47. A CD47 antibody (B6H12) that inhibited alphavbeta3-mediated adhesion of melanoma cells induced by CD47-binding peptides from thrombospondin-1 directly stimulated alpha4beta1-mediated adhesion of the same cells to vascular cell adhesion molecule-1 and N-terminal regions of thrombospondin-1 or thrombospondin-2. B6H12 also stimulated alpha4beta1- as well as alpha2beta1- and alpha5beta1-mediated adhesion of CD47-expressing T cells but not of CD47-deficient T cells. alpha4beta1 and CD47 co-purified as a detergent-stable complex on a CD47 antibody affinity column. CD47-binding peptides based on C-terminal sequences of thrombospondin-1 also specifically enhanced adhesion of melanoma cells and T cells to alpha4beta1 ligands. Unexpectedly, activation of alpha4beta1 function by the thrombospondin-1 CD47-binding peptides also occurred in CD47-deficient T cells. CD47-independent activation of alpha4beta1 required the Val-Val-Met (VVM) motif of the peptides and was sensitive to inhibition by pertussis toxin. These results indicate that activation of alpha4beta1 by the CD47 antibody B6H12 and by VVM peptides occurs by different mechanisms. The antibody directly activates a CD47-alpha4beta1 complex, whereas VVM peptides may target an unidentified Gi-linked receptor that regulates alpha4beta1.