Quantification of fibronectin 1 (FN1) splice variants, including two novel ones, and analysis of integrins as candidate FN1 receptors in bovine preimplantation embryos.

BACKGROUND: Fibronectin 1 (FN1), a glycoprotein component of the extracellular matrix, exerts different functions during reproductive processes such as fertilisation, gastrulation and implantation. FN1 expression has been described to increase significantly from the morula towards the early blastocyst stage, suggesting that FN1 may also be involved in early blastocyst formation. By alternative splicing at 3 defined regions, different FN1 isoforms are generated, each with a unique biological function. The analysis of the alternative FN1 splicing on the one hand and the search for candidate FN1 receptors on the other hand during early bovine embryo development may reveal more about its function during bovine preimplantation embryo development. RESULTS: RT-qPCR quantification of the FN1 splice isoforms in oocytes, embryos, cumulus cells and adult tissue samples revealed a large variation in overall FN1 expression and in splice variant expression. Moreover, two new FN1 transcript variants were identified, the first one expressed in bovine preimplantation embryos and the second one expressed in cumulus cells. In the search for candidate receptors for the new embryo specific FN1 isoform, RNA expression analysis identified 5 alpha integrin subunits (ITGA2B, ITGA3, ITGA5, ITGA8, ITGAV) and 2 beta integrin subunits (ITGB1 and ITGB3) with a similar or overlapping RNA expression pattern as compared to FN1. But double immunofluorescent stainings could not confirm complete co-localisation between FN1 and one out of 3 selected integrins alpha subunits (ITGA3, ITGA5, ITGAV). CONCLUSION: The existence of a new FN1 transcript variant, specifically expressed in morulae and blastocysts strengthens the idea that FN1 is involved in the process of compaction and blastocyst formation. Analysis of the integrin expression could not identify the binding partner for the embryo specific FN1 transcript variant making further steps necessary for the identification of the FN1 receptor and the downstream effects of FN1-receptor binding.

A novel role for the integrin-binding III-10 module in fibronectin matrix assembly.

Fibronectin matrix assembly is a cell-dependent process which is upregulated in tissues at various times during development and wound repair to support the functions of cell adhesion, migration, and differentiation. Previous studies have demonstrated that the alpha 5 beta 1 integrin and fibronectin's amino terminus and III-1 module are important in fibronectin polymerization. We have recently shown that fibronectin's III-1 module contains a conformationally sensitive binding site for fibronectin's amino terminus (Hocking, D.C., J. Sottile, and P.J. McKeown-Longo. 1994. J. Biol. Chem. 269: 19183-19191). The present study was undertaken to define the relationship between the alpha 5 beta 1 integrin and fibronectin polymerization. Solid phase binding assays using recombinant III-10 and III-1 modules of human plasma fibronectin indicated that the III-10 module contains a conformation-dependent binding site for the III-1 module of fibronectin. Unfolded III-10 could support the formation of a ternary complex containing both III-1 and the amino-terminal 70-kD fragment, suggesting that the III-1 module can support the simultaneous binding of III-10 and 70 kD. Both unfolded III-10 and unfolded III-1 could support fibronectin binding, but only III-10 could promote the formation of disulfide-bonded multimers of fibronectin in the absence of cells. III-10-dependent multimer formation was inhibited by both the anti-III-1 monoclonal antibody, 9D2, and amino-terminal fragments of fibronectin. A fragment of III-10, termed III-10/A, was able to block matrix assembly in fibroblast monolayers. Similar results were obtained using the III-10A/RGE fragment, in which the RGD site had been mutated to RGE, indicating that III-I0/A was blocking matrix assembly by a mechanism distinct from disruption of integrin binding. Texas red-conjugated recombinant III-1,2 localized to beta 1-containing sites of focal adhesions on cells plated on fibronectin or the III-9,10 modules of fibronectin. Monoclonal antibodies against the III-1 or the III-9,10 modules of fibronectin blocked binding of III-1,2 to cells without disrupting focal adhesions. These data suggest that a role of the alpha 5 beta 1 integrin in matrix assembly is to regulate a series of sequential self-interactions which result in the polymerization of fibronectin.

Depletion of intracellular potassium disrupts coated pits and reversibly inhibits cell polarization during fibroblast spreading.

To learn more about the possible role of the coated pits endocytic pathway in cell adhesion, we studied attachment and spreading of fibroblasts whose coated pits were disrupted by depletion of intercellular potassium. Fibroblasts incubated in suspension in potassium-free medium lost 80% of their intracellular potassium within 10 min and showed disrupted coated pits based on fluorescence staining of clathrin. Potassium-depleted cells attached and spread on fibronectin-coated substrata over the same time course (15 min-2 h) as control cells. Unlike controls, however, potassium-depleted fibroblasts attained a radial morphology with circumferentially organized actin filament bundles and were unable to make the transition to a polarized morphology with stress fibers. In the radially spread fibroblasts, fibronectin receptors and vinculin colocalized in focal adhesion sites and appeared to be membrane insertion points for circumferentially arranged actin filament bundles, but these sites were much smaller than the focal adhesion plaques in polarized cells. The effects of potassium depletion on cell adhesion were reversible. Within 1 h after switching K(+)-depleted fibroblasts to medium containing KCl, cells developed a polarized morphology with actin stress fibers inserting into focal adhesion plaques. Coated pits also reformed on the cell surface during this time. Because formation of focal adhesion plaques preceded reappearance of clathrin-coated pits at the cell margins, it seems unlikely that coated pits play a direct role in adhesion plaque assembly. Polarization of fibroblasts upon addition of KCl was inhibited by ouabain showing that intracellular potassium was required for activity. Polarization also was inhibited when potassium-depleted cells were switched to potassium-containing medium under hypertonic or acidified conditions, both of which have been shown to inhibit receptor-mediated endocytosis. Our results suggest that the coated pit endocytic pathway is not required for initial attachment, spreading, and formation of focal adhesions by fibroblasts, but may play a role in cell polarization.

Fibronectin receptor functions in embryonic cells deficient in alpha 5 beta 1 integrin can be replaced by alpha V integrins.

alpha 5 beta 1 integrin mediates cell adhesion to extracellular matrix by interacting with fibronectin (FN). Mouse lines carrying null mutations in genes encoding either the alpha 5 integrin subunit or FN have been generated previously. Both mutations are embryonic lethal with overlapping defects, but the defects of alpha 5-null embryos are less severe. Primary embryonic cells lacking alpha 5 beta 1 are able to adhere to FN, form focal contacts, migrate on FN, and assemble FN matrix. These results suggest the involvement of (an)other FN receptors(s). In this study, we examined functions of alpha 4 beta 1 and alpha V integrins in embryonic cells lacking alpha 5 beta 1. Our analysis of cells lacking both alpha 4 beta 1 and alpha 5 beta 1 showed that alpha 4 beta 1 is also not required for these FN-dependent functions. Using alpha V-specific blocking reagents, we showed that alpha V integrins are required for alpha 5-null cells, but not wild-type cells, to adhere and spread on FN. Our data also showed that, although the expression levels of alpha V integrins on the wild-type and alpha 5-null cells are similar, there is an increase in recruitment of alpha V integrins into focal contacts in alpha 5-null cells plated on FN, indicating that alpha V integrins can compensate functionally for the loss of alpha 5 beta 1 in focal contacts of alpha 5-null cells. Finally, our data suggested possible roles for alpha V integrins in replacing the role of alpha 5 beta 1 in FN matrix assembly in vitro and in FN-dependent embryonic functions in vivo.

Overexpression of alpha1-6 fucosyltransferase in hepatoma cells suppresses intrahepatic metastasis after splenic injection in athymic mice.

Changes in oligosaccharide structures alter the biological functions of cancer cells. Alpha1-6 fucosyltransferase (alpha1-6FucT) catalyzes the transfer of fucose to the innermost GlcNAc in N-glycans. Although alpha1-6FucT is barely detected in normal liver, it is enhanced during rat hepatocarcinogenesis and in human hepatoma. To understand the biological meaning of the alpha1-6FucT in hepatoma, especially in terms of metastasis, we established human hepatoma cell lines, which express high levels of alpha1-6FucT by transfection of the alpha1-6FucT gene and investigated intrahepatic metastasis after splenic injection to athymic mice. Tumor formation in the liver was dramatically suppressed in the alpha1-6FucT transfectants (1 of 9 and 1 of 10 in alpha1-6FucT transfectants versus 6 of 9 and 6 of 9 in controls). Although there were no differences in terms of cell invasiveness to a Matrigel or in terms of cytotoxicity to interleukin 2-treated lymphocytes between alpha1-6FucT transfectants and control cells, cell adhesion to mice hepatocytes and nonparenchymal liver cells in culture was significantly inhibited in alpha1-6FucT transfectants, compared to the controls. Attachment of alpha1-6FucT transfectants to a fibronectin-coated dish was decreased compared to controls because alpha5beta1 integrin was more strongly alpha1-6 fucosylated in the alpha1-6FucT transfectants. Two-dimensional electrophoresis followed by lectin blot showed that certain glycoproteins (Mr 50,000-150,000, pI 4.8-5.5) were alpha1-6 fucosylated and might be linked to suppression of intrahepatic metastasis. This is the first demonstration of the biological significance of alpha1-6 fucosylation on N-glycans in hepatoma cells under in vivo conditions.

Additive effect of recombinant Mycobacterium tuberculosis ESAT-6 protein and ESAT-6/CFP-10 fusion protein in adhesion of macrophages through fibronectin receptors.

BACKGROUND/PURPOSE: Tuberculous granulomas are the sites of interaction between the T cells, macrophages, and extracellular matrix (ECM) to control the infection caused by Mycobacterium tuberculosis (M. tuberculosis). A predominant role of RD-1-encoded secretory proteins, early secreted antigenic target-6 (ESAT-6), and culture filtrate protein-10 (CFP-10) in the formation of granulomas has recently been emphasized. However, the precise molecular events that induce the formation of these granulomatous structures are yet to be elucidated. Macrophages use integrins to adhere to fibronectin (FN) as a major component of the ECM. The major goal of this study was to investigate whether recombinant M. tuberculosis antigens can modulate integrin-mediated macrophage adhesion. METHODS: Differentiated THP-1 cell line was stimulated with recombinant ESAT-6, CFP-10, and ESAT-6/CFP-10 proteins and evaluated for alterations in the expression levels of α5ß1 and α4ß1 by semiquantitative real-time polymerase chain reaction. The role of these recombinant antigens in the cytoskeleton rearrangement was determined by adhesion assay and immunofluorescent microscopy. RESULTS: Our data showed that ESAT-6 and ESAT-6/CFP-10 fusion proteins could induce adhesion of macrophages to FN through α4ß1 integrin. An increased expression level of α4ß1 integrin in comparison with α5ß1 integrin in differentiated THP-1 cells was also observed. Results of immunofluorescence studies showed that recombinant proteins-treated THP-1 cells form well-organized stress fibers and focal contacts containing vinculin compared with untreated THP-1 cells. CONCLUSION: Increased expression level of α4ß1 in differentiated THP-1 cells could suggest the important role of α4ß1 integrin in adhesion and focal contact formation of macrophages exposed to M. tuberculosis antigens.

Altered interaction of human granulation-tissue fibroblasts with fibronectin is regulated by alpha 5 beta 1 integrin.

Granulation-tissue fibroblasts express an unique phenotype distinct from normal fibroblasts. Due to the importance of the cell-matrix interactions in the regulation of cell morphology and behavior, we have compared the cell adhesion apparatus, especially integrin-type receptors, in fibroblasts cultured from healthy human periodontal connective tissues and from chronic and wound granulation tissues. The spreading of granulation-tissue cells on fibronectin, but not on type I collagen or laminin, was slower when compared with the normal fibroblasts. Cell spreading on fibronectin could be inhibited by RGD-containing peptide, suggesting integrin-mediated interaction. Both cell types expressed beta 1 integrin subunit, which associated with several integrin alpha subunits, namely alpha 1, alpha 2, alpha 3, alpha 5 and alpha v. In addition to beta 1 subunit, alpha v chain formed heterodimers with beta 3 and beta 5 subunits. Thus, these cells have multiple putative fibronectin, laminin, collagen, and vitronectin receptors. Cell spreading of both cell types on fibronectin was inhibited with anti-beta 1 and anti-alpha 5 antibodies, but antibodies against other putative FN-binding integrins (alpha 3, alpha v, and alpha v beta 3) had no effects. Furthermore, granulation-tissue fibroblasts showed delayed spreading on substrates coated with anti-beta 1 or anti-alpha 5 integrin antibodies. On substrates coated with anti-alpha 3 antibody, both cell types spread equally well. By FACS analysis, the amount of beta 1 and alpha 5 integrin subunits expressed on the cell surfaces was slightly elevated in GTFs compared with HGFs. Thus, the findings in this study indicate that the weakened interaction of granulation-tissue fibroblasts with fibronectin is regulated by altered function of alpha 5 beta 1 integrin.

Differential surface expression of cell adhesion molecules during granulocyte maturation.

Individual steps of granulocyte maturation, such as lineage commitment, proliferation, maturation, and migration from the marrow to the peripheral blood, may be influenced by distinct interactions with the bone marrow stroma. To identify candidates of membrane components involved in maturational stage-specific interactions, we studied changes in the expression of cell adhesion molecules along the granulocyte maturational pathway. Three-color flow cytometric measurements were used to measure levels of cell adhesion molecules along this pathway. The alpha chains of VLA-4 (CD49d) and VLA-5, the integrin beta 1 chain (CD29), and CD31 (PECAM-1) were expressed in high density on all early myeloid cells but down-modulated during postproliferative maturation. CD44 and L-selectin were expressed on CD34+ myeloid progenitor cells and mature granulocytes but down-modulated during the intermediate stages of maturation. The granulocyte receptor for endothelial selectins, sLex, was specifically expressed by myeloid progenitor cells. sLex was down-modulated during the intermediate stages of granulocyte maturation but up-regulated again during terminal maturation. In contrast, CD67, a putative granulocyte adhesion molecule, was negative on progenitors, transiently up-regulated during the intermediate stages of maturation, and almost absent from the surface of mature granulocytes. These results show that each stage of granulocyte maturation is associated with the expression of a unique combination of cell adhesion molecules. L-selectin, CD44, and beta 1 integrins were regulated as previously described for immature lymphopoietic cells and may therefore play general roles in the compartmentalization and development of leukocytes. In contrast, sLex and CD67 were specifically expressed by myeloid cells and could be specifically important for compartmentalization of distinct phases of granulocyte maturation.

Molecular mechanisms of human melanocyte attachment to fibronectin.

In this report we show that fetal and neonatal melanocyte attachment to fibronectin (FN) is inhibited by antibodies to the beta 1 integrin subunit, suggesting a role for these molecules in melanocyte attachment to FN. The VLA-5 integrin was shown to be the predominant receptor for fetal melanocyte attachment to FN, in contrast with neonatal melanocytes in which the very late antigen (VLA)-5, VLA-3, and alpha v integrins each contributed to melanocyte attachment to FN. Peptides containing the arginyl-glycyl-aspartyl-serine (RGDS) sequence inhibited fetal and neonatal melanocyte attachment to FN by a maximum of 48% and 85%, respectively. The almost complete inhibition of neonatal melanocyte attachment to FN by RGDS-containing peptides suggests that the central cell-binding domain of FN is the primary recognition site for neonatal cell attachment to FN. Fetal and neonatal melanocytes showed a concentration-dependent attachment to two proteolytically derived fragments of the FN molecule: a 75-kD fragment, which contains the central cell-binding domain, and 33/66-kD fragments of the FN molecule, which encompass the heparin-binding domains V and VI. Antibodies to the beta 1 subunit inhibited fetal and neonatal melanocyte attachment to the 33/66-kD fragments by a maximum of only 15% and 24%, respectively, suggesting that other, non-integrin, receptors are involved in melanocyte recognition of this portion of the FN molecule. We propose that human fetal and neonatal melanocytes attach to FN by different complements of receptors and ligand target sequences, and that these differences may direct melanocyte interactions with FN during development.

Expression of the alphavbeta6 integrin promotes migration and invasion in squamous carcinoma cells.

The integrin alphavbeta6 is a fibronectin receptor whose expression is not detectable on normal oral epithelium but is increased significantly in healing and in oral epithelial dysplasia and oral squamous cell carcinoma, suggesting it may promote changes associated with tumor development. To study whether alphavbeta6 may drive invasive behavior we have used transfection and retroviral infection to create a panel of epithelial cell lines expressing various levels of alphavbeta6. We report that increased expression of alphavbeta6 in malignant keratinocytes promotes invasion and leads to an increased capacity for migration towards fibronectin. alphavbeta6 expression may have a significant role in contributing to the malignant behavior of epithelial cells.

Integrin alpha5beta1: a potent inhibitor of experimental lung metastasis.

The integrin alpha5beta1 seems to be the most relevant receptor of tumor cells for binding to fibronectin. Although numerous studies suggest a role of tumor cell fibronectin interaction in tumor metastasis, differential integrin expression on tumor cells has, however, not been correlated with metastatic capabilities. We addressed this question by transfection of the integrin alpha5beta1 cDNA into HT-29 human colon carcinoma cells which led to de novo expression of functional integrin alpha5beta1. Similar to other reports, expression of the integrin alpha5beta1 in HT-29 tumor cells exerted an inhibitory action on cell proliferation as indicated in our study by formation of fewer colonies in soft agar. The tumor growth inhibitory property of the integrin alpha5beta1 was also shown by reduction of subcutaneous xenograft growth in nude mice to approximately 50% of that of control transfectants. For the first time, we found that several clones of integrin alpha5 subunit transfectants displayed dramatically reduced formation of lung colonies and cutaneous metastasis after intravenous injection into nude mice. While most animals inoculated with control transfectant cells formed macroscopically visible lung colonies ranging from 12.6 +/- 2.6 to 22.0 +/- 6.6 (mean colony number +/- SEM), mice inoculated with HT-29 cell clones expressing the integrin alpha5beta1 were almost completely free of lung colonies (ranging from 0.0 +/- 0 to 0.2 +/- 0.1). Our results imply that integrin alpha5beta1 expression inhibits circulating tumor cells in pursuing late steps of the metastatic process as represented by the artificial metastasis (lung colonisation) model.

Inhibition of smooth muscle cell migration by the p21 cyclin-dependent kinase inhibitor (Cip1).

In vascular smooth muscle cells (SMCs), proliferation and migration contribute to lesion formation after arterial injury. In the cell cycle, several cyclin-dependent kinases (cdks) inhibitors are implicated in the regulating of cyclin-cdk activity such as p21Cip1, p16Ink4 and p27Kip1. Although Cip1 inhibits SMC proliferation, its effects on SMC migration are unknown. To test the hypothesis that Cip1 inhibits SMCs migration and proliferation, we transfected the Cip1 gene into a strain of rabbit aortic SMCs (SM3 cells). Both the spreading and the attachment of Cip1-transfected SM3 cells to extracellular matrices (ECMs) were inhibited compared to that of vector-transfected cells. In the modified Boyden's chamber assay the effect of fibronectin on the migratory activity of Cip1-transfected SM3 cells was significantly less than that of vector transfected cells in response to PDGF-BB. These data suggested that Cip1 inhibited both the migration and proliferation of SMC.

Identification of integrins in cultured corneal fibroblasts and in isolated keratocytes.

PURPOSE: The integrins are a family of transmembrane glycoproteins that function in attachment of cells to one another and to the extracellular matrix. When cell--cell and cell--matrix interactions are altered, the population of integrins may change. In particular, removing cells from their normal environment may be used as a model of wounding. The current study reports the identification of the integrins expressed at the cell surface of noncultured keratocytes and of cultured corneal fibroblasts, which are derived from keratocytes grown in primary culture. METHODS: For integrin identification, the surface proteins of keratocytes and cultured corneal fibroblasts were labeled with biotin, and the integrins were immunoprecipitated using anti-integrin antibodies. Attachment assays determined (1) the extracellular matrix preference of the cultured corneal fibroblasts and (2) the effects of function-perturbing antibodies against the fibronectin receptor (alpha 5 beta 1) or against other beta 1-containing integrins. RESULTS: The integrins of noncultured keratocytes were present as heterodimeric alpha, beta surface proteins that were immunoprecipitated by anti-beta 1, anti-alpha v, anti-alpha 6, anti-alpha 3, anti-alpha 1, and anti-beta 3. Furthermore, when the keratocytes were placed in culture, the integrin pattern changed. The classic fibronectin receptor, alpha 5 beta 1, is then expressed along with additional integrins that bind to fibronectin. Using attachment assays, we determined that the cultured corneal fibroblasts prefer fibronectin to collagen, vitronectin, or laminin as extracellular matrix substrate. In addition, function-perturbing antibodies against the fibronectin receptor (alpha 5 beta 1) or against beta 1 inhibit attachment of cultured corneal fibroblasts to fibronectin. CONCLUSIONS: Receptors for fibronectin and other extracellular matrix molecules are expressed at the cell surface in cultured corneal fibroblasts, and are in position to play a significant functional role as seen in attachment to extracellular matrix.

Very late antigen (VLA) expression by normal and neoplastic human plasma cells; including an assessment of antibodies submitted to the Vth International Workshop on Leucocyte Differentiation Antigens using human myeloma cell lines.

The biology of normal plasma cells and the pathophysiology of human multiple myeloma remain poorly understood. Functional assays are scarce and at present cell phenotyping is providing the most information about how human plasma cells may behave. Three different types of human plasma cells: normal, fresh neoplastic myeloma cells and plasma cell lines, have been studied for their reactivity with antibodies to the beta-1 integrins (Very Late Antigens; VLAs), including a panel obtained from the Vth International Workshop on Leucocyte Differentiation Antigens. Most plasma cell targets express VLA-4 (CD49d positive) and the common beta chain recognized by CD29. CD49e (VLA-5) was occasionally positive. Other VLAs were not usually expressed. These data suggest the wide use by plasma cells of VLA-4, possibly as a ligand with fibronectin and high endothelial venules (HEV). Of other adhesion structures expressed by plasma cells, only CD44 is seen as frequently, and this is also a HEV ligand.

Circulating fibronectin and fibronectin receptor in children with pertussis.

AIM: To determine concentrations of fibronectin and fibronectin receptor in children with pertussis. METHODS: Concentrations of circulating fibronectin and serum fibronectin receptor were detected in eight children affected by pertussis, eight children with acute upper or lower respiratory tract infections, and in 14 healthy control children. The single radial immunodiffusion technique and a solid phase enzyme immunoassay were used to detect circulating serum concentrations of fibronectin and fibronectin receptor. RESULTS: On admission, a significant decrease in fibronectin was detected in children with pertussis (p = 0.0006). Significant and decreased concentrations of fibronectin were also observed in children with upper or lower respiratory tract infections (p = 0.0002). On the other hand, serum fibronectin receptor concentrations were significantly increased in patients with pertussis, whereas patients with upper or lower respiratory tract infections had normal circulating fibronectin receptor concentrations. CONCLUSIONS: Fibronectin deficiency in children with pertussis may be related to diffusion and deposition of this protein in bronchial and alveolar spaces to limit infection, while increased fibronectin receptor concentrations are probably the expression of T cell activation and cell-mediated immunity during Bordetella pertussis infection.

A new phenotypic classification of bone marrow plasmacytosis.

Here, we propose a new phenotypic classification of bone marrow plasmacytosis. By 2-color phenotypic analysis with FITC anti-CD38 and PE anti-CD19, -CD56, -VLA-5 or MPC-1 antibody, plasma cells are easily identified on the histogram, even though no more than 1% of plasma cells are found in the bone marrow. Hence, plasma cells are phenotypically classified into polyclonal (reactive) (CD19+CD56-) or monoclonal (neoplastic) plasma cells (mostly CD19-CD56+), and furthermore immature (VLA-5-MPC-1-), intermediate (VLA-5-MPC-1+) and mature plasma cells (VLA-5+MPC-1+). According to these findings, plasmacytosis in the bone marrow can be classified into polyclonal marrow plasmacytosis (POMP) and monoclonal marrow plasmacytosis (MOMP) states. The MOMP state is further subclassified into MOMP-1 and MOMP-2, MOMP-3 and MOMP-4; MOMP-1 is defined by co-existence of monoclonal plasma cells and polyclonal plasma cells, and MOMP-2 to MOMP-4 are dependent on increased proportions of VLA-5-MPC-1- immature myeloma (plasma) cells. We found that the cases of benign monoclonal gammopathy (BMG) according to the conventional classification were in the MOMP-1 state, and myelomas could be classified into the MOMP-2 to MOMP-4 state. Subclassification of the MOMP state may be useful in determining the prognosis of myelomas, where an increase in immature myeloma cells is reported to correlate well with their aggravation during the clinical courses. Therefore, this new phenotypic classification of bone marrow plasmacytosis (POMP and MOMP-1 to MOMP-4) will contribute to differential diagnosis and understanding of therapeutic responses and prognosis in myelomas.

Endometrial integrin expression is independent of estrogen or progestin treatment in vitro.

OBJECTIVE: To examine the regulation of endometrial integrin expression by estrogens and progestins in vitro. DESIGN: Immunocytochemical study. SETTING: Academic research unit. PATIENT(S): Twenty-five regularly cycling women without endometrial pathology, of whom seven had endometriosis. INTERVENTION(S): Endometrial cells obtained by aspiration curettage were treated with diethylstilbestrol, promegestone, and antiprogestin. Immunocytochemistry was performed with antibodies directed against integrins alpha 1 beta 1, alpha 2 beta 1, alpha 4 beta 1, alpha 5 beta 1, alpha v beta 3, and beta 3 integrin subunit. MAIN OUTCOME MEASURE(S): Semiquantitative staining score. RESULT(S): Endometrial cells express several integrins in vitro in a consistent and cell specific pattern. Neither differences between treated and untreated cells nor an effect of treatment duration or dosage were observed. Cells from patients with and without endometriosis showed similar patterns. CONCLUSION(S): The cellular distribution of integrin expression was similar to that described in vivo. In contrast, a steroid regulated expression could not be detected in vitro. Rather, a derepression by a factor not included in our model could be responsible for the cyclic appearance of some integrins. In endometriosis, no fundamental difference of integrin expression was detected.

A beta 1 integrin-like molecule in Entamoeba histolytica.

Human invasive amoebiasis is highly destructive, causing rapid necrosis and liquefaction of all tissues reached by the trophozoites. Degradation of extracellular matrix components (EMC) has been demonstrated during invasion of the basal lamina. Pursuing the idea that trophozoites might behave similarly to other invasive cells with respect to their interaction with EMC, plasma membrane proteins biochemically or functionally related to integrins were looked for. A 140 kDa molecular mass membrane protein from Entamoeba histolytica trophozoites with the characteristics of a beta 1 integrin-like fibronectin receptor was identified.

The response of periodontal ligament cells to fibronectin.

Fibronectin (fn) is an extracellular matrix (ECM) molecule important in cell adhesion and migration and in wound healing. It is also likely important in periodontal ligament (PDL) cell-ECM interactions, and thus in regenerating periodontal tissues. In this study we characterized PDL cells and their interactions with FN, testing different PDL cell isolates taken from healthy and diseased conditions. PDL cells were characterized by their morphology, integrin profile, motility, and bone nodule formation. Cells were then assayed for adhesion, proliferation, and chemotaxis in response to FN or FN fragments. Cell isolates were morphologically heterogeneous and fibroblastic, had a normal-appearing actin cytoskeleton and a wide range of migration potentials, and formed bone-like nodules in vitro. They expressed alpha5, beta1, alpha v, and alpha4 integrin subunits, known receptors for FN, and in fact they bound FN preferentially at 5 and 10 microg/ml. Intact FN induced greater PDL cell proliferation and chemotaxis than did FN fragments (120-kDa cell-binding, 60-kDa heparin-binding, and 45-kDa collagen-binding). PDL cells harvested from diseased and healthy conditions were no different on the basis of these assays. These data demonstrate that PDL cells are a mixed population of fibroblastic cells, capable of forming a mineralized matrix. They also suggest that maximal proliferation and chemotaxis require specific FN domains that are present on the intact molecule but not its fragments.

Co-ordinate expression of alpha 5 beta 1 integrin and fibronectin in Dupuytren's disease.

The expression of alpha 5 beta 1 (alpha 5 beta 1) integrin and its extracellular ligand fibronectin was studied immunohistochemically in 23 cases of Dupuytren's disease using an immunoperoxidase method for light microscopic visualization. All cases consisted of multiple nodules showing a variable degree of cellularity and fibrosis. Depending on the histological appearance of these nodules, each case was assigned to the three following phases: proliferative, involutional and residual. Alpha 5 beta 1 integrin was detected in the highly cellular areas of both proliferative and involutional phases where fibronectin was simultaneously expressed in the extracellular matrix (ECM). Diversely alpha 5 beta 1 and fibronectin disappeared from the hypocellular areas of involutional phase, undergoing fibrotic transformation, and from the fibrotic connective tissue of residual phases. These findings indicate that the expression pattern of alpha 5 beta 1 integrin correlates with the presence in the ECM of the corresponding ligand fibronectin during the different phases of Dupuytren's disease. We suggest that alpha 5 beta 1 integrin, linking fibronectin to stromal cells of both proliferative and involutional phases, may be involved in the contractile processes occurring in Dupuytren's disease.