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PubMed2004

Expression of {beta}1 integrin receptors during rat pancreas development--sites and dynamics.

The integrin receptors link to extracellular matrix proteins and exert a dynamic role in development by providing the physical basis for cell adhesion and controlling cell growth. In the present study, we examined changes in the expression of beta1 integrins and its associated alpha-subunits to islet cell development in the rat pancreas. A significant increase in protein expression of integrin alpha3, alpha6, and beta1 was observed from fetal to postnatal life. High mRNA levels of these integrin subunits was detected at embryonic d 18 and dropped significantly after birth with relatively low expression throughout postnatal life. Integrins alpha3, alpha5, alpha6, and beta1 were expressed in a cell-specific manner in the pancreas with high integrin immunoreactivity in duct and islet regions during fetal life, and a progressive increase later into postnatal life. The coexpression with islet and putative islet precursor markers during fetal and postnatal development suggest a role for these integrin subunits in differentiation and maturation of islets. Functional studies in vitro showed that anti-beta1 antibody treatment inhibited islet cell adhesion to extracellular matrices and disrupted islet architecture. Blockade of beta1 integrin receptor and knockdown beta1 mRNA resulted in a decrease in the expression of insulin mRNA and increased islet cell death. These results suggest that progression in islet cell development is accompanied by and dependent upon cell adhesion via beta1 integrin and its respective alpha-subunits and suggest that the beta1 family of integrins may play a critical role in islet cell architecture, development, integrity, and function.

Animals↗
PubMed1999

Fine mapping of inhibitory anti-alpha5 monoclonal antibody epitopes that differentially affect integrin-ligand binding.

The high-affinity interaction of integrin alpha5beta1 with the central cell-binding domain of fibronectin requires both the Arg-Gly-Asp (RGD) sequence (in the tenth type III repeat) and a second site Pro-His-Ser-Arg-Asn (PHSRN) in the adjacent ninth type III repeat, which synergizes with RGD. Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA) is a novel peptidic ligand for alpha5beta1, identified by phage display, which blocks alpha5beta1-mediated cell adhesion to fibronectin. A key question is the location of the binding sites for these ligand sequences within the integrin. In this study we have identified residues that form part of the epitopes of three inhibitory anti-alpha5 monoclonal antibodies (mAbs): 16, P1D6 and SNAKA52. These mAbs have distinct functional properties. mAb 16 blocks the recognition of RGD and RRETAWA, whereas P1D6 blocks binding to the synergy sequence. The binding of SNAKA52 is inhibited by anti-beta1 mAbs, indicating that its epitope is close to the interface between the alpha and beta subunits. Residues in human alpha5 were replaced with the corresponding residues in mouse alpha5 by site-directed mutagenesis; wild-type or mutant human alpha5 was expressed on the surface of alpha5-deficient Chinese hamster ovary cells. mAb binding was assessed by flow cytometry and by adhesion to the central cell-binding domain of fibronectin or RRETAWA by cell attachment assay. All three epitopes were located to different putative loops in the N-terminal domain of alpha5. As expected, disruption of these epitopes had no effect on ligand recognition by alpha5beta1. The locations of these epitopes are consistent with the beta-propeller model for integrin alpha-subunit structure and allow us to propose a topological image of the integrin-ligand complex.

Amino Acid Sequence↗
PubMed2005

Extracellular matrix regulates endothelial functions through interaction of VEGFR-3 and integrin alpha5beta1.

Endothelium extracellular matrix (ECM) interactions can provide distinct spatial and molecular signals which control cellular proliferation, migration, and differentiation. Here, we investigated the role of fibronectin (FN), a major ECM protein, on the functions of lymphatic endothelial cells (LEC). We observed that FN, the ligand for integrin alpha5beta1, selectively promoted the growth of LEC as compared with vitronectin (VN) in the presence of the ligand for vascular endothelial growth factor receptor 3 [VEGFR-3 (VEGF-C156S)]. Upon investigating the mechanisms whereby ECM components regulate VEGFR-3 signaling, we found that FN transactivated VEGFR-3 and significantly enhanced the phosphorylation of VEGFR-3 induced by VEGF-C156S as compared to VN. An enhanced association of the integrin subunit alpha5 or beta1 with VEGFR-3, after stimulation with VEGF-C156S, was observed by co-immunoprecipitation. While blockade of integrin alpha5beta1 inhibited the VEGF-C156S-induced phosphorylation of VEGFR-3, no similar effect was obtained by blocking integrin alphavbeta3. FN also protected the endothelial cells from serum deprivation-induced apoptosis. Moreover, while the specific PI3 kinase inhibitor, LY294002, abolished this FN-mediated cell survival, the MAPK kinase inhibitor, PD98059, had no significant effect. Furthermore, a dominant-negative mutant of VEGFR-3 (G857R) reduced VEGF-C156S or FN-mediated cell survival, as well as the activities of PI3 kinase/Akt. Our results indicate that integrin alpha5beta1 participates in the activation of both VEGFR-3 and its downstream PI3 kinase/Akt signaling pathway, which is essential for FN-mediated lymphatic endothelial cell survival and proliferation.

Apoptosis↗
PubMed2003

Temporal and spatial expression of integrins and their extracellular matrix ligands at the maternal-fetal interface in the rhesus monkey during pregnancy.

The integrin and extracellular matrix protein (ECM)-mediated adhesion and invasion of the receptive maternal uterine endometrium by trophoblasts is a critical event in the complex physiological process of pregnancy. Although the process has been largely characterized in mice, the relevant mechanism in primates remains unclear. We investigated the expression patterns and dynamic alterations of integrin subunits (alpha1, alpha5, alpha6, beta1, and beta4) and their ECM ligands, such as laminin (LN), type IV collagen (Col IV), and fibronectin (FN), at the maternal-fetal interface during Gestational Days 15, 25, 50, and 100 and at full term in 20 pregnant rhesus monkeys. Immunohistochemistry and in situ hybridization revealed that a relatively high expression of integrins occurred in trophoblast cells at Gestational Day 15, with the peak level occurring at Day 25. The expression level decreased from Day 50 to term. Along the invasive pathway, expression levels of integrin alpha1, alpha5, and beta1 subunits were gradually elevated from the proximal to distal column, reaching peak level in the trophoblast shell, but were reduced in those invasive extravillous cytotrophoblast (EVCT) cells in contact with the decidua. Integrin alpha1, alpha5, beta1, and beta4 subunits were also highly expressed in decidual stromal cells and moderately expressed in the maternal epithelium and endothelium. Immunoreactive FN, LN, and Col IV were distributed in EVCT and decidual stromal cells and part of the uterine epithelial and endothelial cells. These data suggest that the correlated expression of integrins and their ECM ligands at the maternal-fetal interface might be involved in regulation of cell proliferation and differentiation and the counterbalanced invasion-accelerating and invasion-restraining processes in trophoblast cells during the early stage of pregnancy.

Animals↗
PubMed1996

Identification of receptors binding fibronectin and laminin on fetal rat lung cells.

Fibronectin and laminin have been implicated in regulating lung morphogenesis. In the present study, the cell surface receptors involved in fetal lung cell binding to laminin and fibronectin were identified. Messages for alpha5- and beta1-integrin subunits were detected in both fetal lung epithelial cells and fibroblasts. The presence of alpha5 beta1 -integrin on both cell types was demonstrated by immunocytochemistry and confirmed by cell adhesion experiments with fibronectin and RGD-containing peptides. Epithelial cells adhered more readily to laminin than fibroblasts. The alpha4 beta1-integrin, and RGD-independent fibronectin receptor, was weakly expressed on either cell type. Both cell types expressed alpha6-integrin subunit mRNA and stained immunopositive for the alpha6-subunit. Although either cell type expressed nonintegrin 67-kDa laminin-elastin receptor mRNA, no positive immunoreactivity for this laminin-elastin binding protein was detected. None of these findings explain the enhanced attachment of distal fetal lung epithelial cells to laminin compared with fibroblasts. Previously, we have reported that epithelial cells were enriched in alpha3-integrin subunit mRNA and protein expression. Herein, we found that epithelial cell attachment to laminin was nearly completely inhibited by alpha3- but only partially by alpha6 -monoclonal antibodies. A peptide near the globular region at the long arm of the laminin A-chain, which contained the IKVAV sequence, and the laminin A-chain amino acid sequence representing the alpha3 beta1 -integrin binding site, inhibited the adherence of epithelial cells to laminin. Fetal lung epithelial cells attached to substrata coated with the alpha3 beta1-integrin binding site peptide and the peptide containing the IKVAV sequence. These data suggest that both fetal lung cell types bind to fibronectin via the fibronectin receptor, alpha5 beta1, and fetal lung epithelial cells interact with laminin via alpha3 beta1 and proteins that recognize the IKVAV-containing sequence on the laminin A-chain.

Amino Acid Sequence↗
PubMed2006

In vitro analysis of differential expression of collagens, integrins, and growth factors in cultured human chondrocytes.

OBJECTIVES: Tissue engineering represents a promising method for the construction of autologous chondrogenic grafts for reconstructive surgery. In cultured chondrocytes, the dedifferentiation and proliferation of the cells are critical factors that influence the generation of transplants. The aim of our study was to find and characterize markers for cell proliferation and dedifferentiation in cultured chondrocytes. STUDY DESIGN AND SETTING: Human chondrocytes were isolated from septal cartilage and held in primary cell culture. Cells were harvested after 1, 6, and 21 days. The differentiation of the cells was investigated with bright-field microscopy, the expression patterns of various proteins using immunohistochemistry, and the expression of distinct genes with the microarray technique. RESULTS: The chondrocytes showed a strong proliferation. After 6 and 21 days, collagen 9 and 10 were downregulated; collagen 11 was activated. Collagen 1 and 2 were downregulated after 6 days but were reactivated after 21 days. Tumor growth factor beta (TGF-beta)1 was strongly expressed on days 1, 6, and 21, TGF-beta2 was never expressed, and TGF-beta3 and -beta4 were upregulated from day 1 to day 21. The TGF-beta receptor III was expressed on days 1, 6, and 21. Integrin beta1, beta5, and alpha5 were upregulated from day 1 to day 21; integrin beta3 was downregulated. CONCLUSION AND SIGNIFICANCE: Collagens 3, 4, 8, 9, and 11 might be new markers for the dedifferentiation of chondrocytes. Collagen 2 might be a marker for the synthetic activity of the cells rather than the dedifferentiation. TGF-beta3 and -beta4 might influence the dedifferentiation, which is fortified by the expression of TGF-beta receptor III. Integrin beta1, beta5, and alpha5 might be involved in signal transmission for the dedifferentiation.

Biomarkers↗
PubMed1999

Factors limiting adenovirus-mediated gene transfer into human lung and pancreatic cancer cell lines.

Adenoviral vectors are a widely used means of gene transfer. However, transgene expression after adenoviral administration varies among different carcinoma cell lines. We hypothesized that this variation is attributable, in part, to the presence of cell surface molecules involved in adenoviral infection. To test this, we first assessed adenovirus-mediated transgene expression in four human lung carcinoma cell lines and four human pancreatic carcinoma cell lines in terms of luciferase activities and found it to vary from 4.8 x 10(4) to 6.1 x 10(7) relative light units/microg of protein. Then, to determine whether the molecules involved in the entry of adenovirus into host cells were responsible for this variation, we evaluated the expression of alpha(v)beta5, alpha(v), beta3, alpha5, and beta1 integrins and that of coxsackievirus and adenovirus receptor (CAR) in these cell lines. Statistical analysis revealed that the levels of beta3 were associated with the levels of transgene expression. Blocking analysis showed that adenovirus-mediated gene transfer could be blocked by antibodies against these six molecules but not by the antibodies against alpha2 or alpha3 integrins, thus suggesting that the integrins alphavbeta5, alpha(v), beta3, alpha5, and beta1 and CAR molecules could limit adenovirus-mediated gene transfer when their levels fell below a certain threshold. Furthermore, cells expressing low levels of beta3 and resistant to conventional adenoviral vectors were susceptible to a vector containing the heparin-binding domain in its fiber, thus suggesting that redirecting vectors to receptors other than CAR may bypass the integrin pathway. These findings may have implications for improving the efficiency of adenovirus-mediated gene transfer and developing novel adenoviral vectors.

Adenoviruses, Human↗
PubMed2004

TGF-beta1 (transforming growth factor-beta1)-mediated adhesion of gastric carcinoma cells involves a decrease in Ras/ERKs (extracellular-signal-regulated kinases) cascade activity dependent on c-Src activity.

Signalling by integrin-mediated cell anchorage to extracellular matrix proteins is co-operative with other receptor-mediated signalling pathways to regulate cell adhesion, spreading, proliferation, survival, migration, differentiation and gene expression. It was observed that an anchorage-independent gastric carcinoma cell line (SNU16) became adherent on TGF-beta1 (transforming growth factor beta1) treatment. To understand how a signal cross-talk between integrin and TGF-beta1 pathways forms the basis for TGF-beta1 effects, cell adhesion and signalling activities were studied using an adherent subline (SNU16Ad, an adherent variant cell line derived from SNU16) derived from the SNU16 cells. SNU16 and SNU16Ad cells, but not integrin alpha5-expressing SNU16 cells, showed an increase in adhesion on extracellular matrix proteins after TGF-beta1 treatment. This increase was shown to be mediated by an integrin alpha3 subunit, which was up-regulated in adherent SNU16Ad cells and in TGF-beta1-treated SNU16 cells, compared with the parental SNU16 cells. After TGF-beta1 treatment of SNU16Ad cells on fibronectin, Tyr-416 phosphorylation of c-Src was increased, but Ras-GTP loading and ERK1/ERK2 (extracellular-signal-regulated kinases 1 and 2) activity were decreased, which showed a dependence on c-Src family kinase activity. Studies on adhesion and signalling activities using pharmacological inhibitors or by transient-transfection approaches showed that inhibition of ERK1/ERK2 activity increased TGF-beta1-mediated cell adhesion slightly, but not the basal cell adhesion significantly, and that c-Src family kinase activity and decrease in Ras/ERKs cascade activity were required for the TGF-beta1 effects. Altogether, the present study indicates that TGF-beta1 treatment causes anchorage-independent gastric carcinoma cells to adhere by an increase in integrin alpha3 level and a c-Src family kinase activity-dependent decrease in Ras/ERKs cascade activity.

Animals↗
PubMed1998

maspin suppresses the invasive phenotype of human breast carcinoma.

The recently discovered tumor suppressor gene maspin has been shown to inhibit tumor cell motility, invasion, and metastasis in breast cancer by our laboratories. Nonetheless, the exploitation of maspin as a potential diagnostic and/or therapeutic tool has remained limited due to the lack of knowledge concerning its molecular and biological mechanism(s) of action. The work reported here demonstrates that recombinant maspin (rMaspin) has the ability to induce higher cell surface levels of alpha5- and alpha3-containing integrins and reduced levels of alpha2-, alpha4-, alpha6-, alpha(v)-, and some beta1-containing integrins in the metastatic human breast carcinoma cell line MDA-MB-435 concomitant with its ability to inhibit the invasive process in vitro. Furthermore, treatment of MDA-MB-435 cells with rMaspin results in the selective adhesion of the cell to a fibronectin matrix and conversion from a fibroblastic to a more epithelial-like phenotype. In addition, the ability of rMaspin to inhibit the invasive process can be abrogated with a blocking antibody to the alpha5beta1 integrin, which diminishes the ability of the cells to invade through a fibronectin matrix-containing barrier in vitro. Taken together, these data address the hypothesis that rMaspin reduces the invasive phenotype of MDA-MB-435 cells by altering their integrin profile, particularly alpha5, which in turn converts these cells to a more benign epithelial phenotype, with less invasive ability. These data provide new insights into the biological significance of this tumor suppressor gene found in normal mammary epithelium and may form the basis of novel therapeutic strategies in the management of breast carcinoma.

Antibodies, Blocking↗
PubMed2003

Immunohistochemical expression of integrins and extracellular matrix proteins in non-small cell lung cancer: correlation with lymph node metastasis.

PURPOSE: For patients with non-small cell lung cancer (NSCLC), the biggest threat to survival is metastasis. During metastatic cascade, tumor cells interact with extracellular matrix (ECM) through certain adhesion molecules such as integrins. The aim of this study was to analyze the distribution of the main integrins and ECM in a series of patients with NSCLC to assess their distribution and correlate with lymph node (LN) metastasis of NSCLC. METHODS: Formalin-fixed and paraffin-embedded tissues of NSCLC with (n=45) or without (n=23) regional LN metastasis were obtained form 68 surgically treated patients. The expression of fibronectin, collagen type IV, tenascin and the integrin subunits (alpha2, alpha3, alpha4, alpha5 and beta1) was studied by immunohistochemistry. Chi-square and Fisher's exact tests were used to compare groups and parameters. RESULTS: Extensive (>50% of section area) fibronectin and collagen type IV staining were seen in 22 and 55% of tumors, respectively, with focal areas of immunoreactivity seen in another 75 and 38% of tumors, respectively. Tenascin staining showed just focal areas of immunoreactivity in 21% of tumors. Interstitial collagen matrices were more frequently lost in LN metastasis (P=0.007). Integrins alpha2, alpha5 and beta1 expressions were present in 9, 12 and 26% of tumors, respectively. The expression of integrins alpha5 and beta1 was significantly associated with LN metastasis (P=0.04 and 0.005, respectively). CONCLUSIONS: Increased expression of integrins alpha5 and beta1, and lost expression of collagen matrices significantly correlated with LN metastasis of NSCLC. These findings suggested that enhanced expression of integrins and disrupted collagen stroma in NSCLC might promote tumor cell survival and invasiveness.

Adenocarcinoma↗
PubMed2001

Effects of leptin, interleukin-1alpha, interleukin-6, and transforming growth factor-beta on markers of trophoblast invasive phenotype: integrins and metalloproteinases.

Phenotypic changes of integrin and metalloproteinase secretion of the invasive human cytotrophoblast are regulated by cytokines and growth factors, but how this occurs is not completely understood. We used 24-h cytotrophoblast cultures from first trimester pregnancies to investigate the effects of leptin and cytokines on the expression of the alpha2, alpha5, and alpha6 integrin subunits and on the activity of metalloproteinase-2 (gelatinase A) and metalloproteinase-9 (gelatinase B). The alpha2 subunit was marginally upregulated by leptin and interleukin-1alpha (IL-1alpha). All compounds tested upregulated, in some degree, the alpha5 expression. The a6 integrin subunit was massively upregulated, by leptin, interleukins, and transforming growth factor-beta. None of the factors tested affected metalloproteinase-2 activity, but the activity of metalloproteinase-9 was upregulated by leptin and IL-1alpha. In conclusion, leptin and IL-1alpha actively induce some of the changes that cytotrophoblasts undergo to achieve a more invasive phenotype. A novel role for leptin is proposed during early pregnancy: leptin might be an autocrine/paracrine regulator of cytotrophoblast invasiveness during implantation and placentation.

Antigens, CD↗
PubMedDate not supplied

Cadherins and integrins in renal cell carcinoma: an immunohistochemical study.

AIMS AND BACKGROUND: The aim of this study was to determine the expression of cadherins and integrins in renal cell carcinoma (RCC) and their relationship with tumor morphology and TNM status. METHODS: Cadherin and integrin expression was investigated using an indirect immunoperoxidase technique, applying antibodies to E-, N-, P- and VE-cadherin and to alpha1, alpha2, alpha3, alpha4, alpha5, alpha6, and alpha(v) integrin subunits. Correlation of semiquantitatively scored adhesion molecule levels with histopathological parameters (cytology, growth pattern, nuclear grade) and TNM status was performed for 24 RCCs (17 clear cell, 3 granular, 3 spindle cell and 1 chromophobe cell type according to the WHO classification). RESULTS: E-cadherin and N-cadherin were present in most cases (88% and 67%, respectively) and were usually coexpressed. T3 RCCs displayed higher E-cadherin and N-cadherin levels than T1/T2 tumors regardless of tumor grade, suggesting that impairment of their function might exist without actual loss from tumor cells. P-cadherin was found focally in two RCCs only, while VE-cadherin was present on stromal vessel endothelium in five tumors, showing no differences with regard to cell type, growth pattern, tumor grade or TNM status. All integrins were present in the studied RCCs (ranging from 12% for alpha5 to 79% for alpha3), including those that are normally absent from adult kidney tissue (alpha4 and alpha5). Tumors of higher grade showed increased alpha(v) and decreased alpha6 levels, while RCCs with metastases less often showed diffuse alpha3 presence and never expressed alpha5 integrin. CONCLUSIONS: Our results suggest that the level of expression of N-cadherin and some integrins (most notably alpha3, alpha6 and alpha5) is associated with the capacity of RCC for local and distant spread, regardless of tumor grade.

Antigens, CD↗
PubMed1999

Expression of Epstein-Barr virus latent membrane proteins leads to changes in keratinocyte cell adhesion.

Epstein-Barr virus (EBV) has 3 latent membrane proteins (LMPs)--LMP1, LMP2a, and LMP2b--which are expressed in nasopharyngeal carcinoma. Using keratinocyte cell lines expressing LMP2a and LMP2b and coexpressing LMP1/LMP2a, we grew organotypic raft cultures to analyze changes in morphology and expression of the cell adhesion molecule ICAM-1; alpha2, alpha3, alpha5, beta1, and alpha6beta4 integrins; laminin 5; E-cadherin; and desmoplakin. Cells expressing LMP2a or LMP2b were defective in their ability to mature and progress through normal squamous stratification when compared to the parental cell lines. Cells coexpressing LMP1/LMP2a additionally demonstrated "pseudoinvasion" into the raft dermal equivalent. There was a consistent and dramatic up-regulation in the suprabasal expression of laminin 5 and alpha6beta4 and beta1 integrins in the LMP-expressing cell lines. ICAM-1, not expressed in the control cell lines, was up-regulated in the LMP-expressing cell lines. Expression of alpha3 and alpha5 integrins was also up-regulated in the LMP-expressing cell lines, while alpha2 demonstrated a loss of the normal basal layer expression. E-cadherin and desmoplakin expression patterns were essentially unchanged. We conclude that LMP2a and LMP2b singly, and LMP1/LMP2a coexpressed, are capable of altering keratinocyte cell adhesion molecule expression consistent with nasopharyngeal carcinoma.

Carcinoma, Squamous Cell↗
PubMed2004

The role of fibroblasts in the modulation of integrin-dependent interactions between the gastric cell line HGT-1 and fibronectin.

Fibronectin plays an important role in gastric cancer progression. However, little is known about the microenvironmental factors modulating integrin-dependent interactions between gastric cancer cells and fibronectin. We therefore studied the regulation by fibroblasts of the integrin-dependent adhesion and migration of the gastric cancer cell line HGT-1 onto fibronectin. We first determined, by immunofluorescence, immunoblotting and flow cytometry, that HGT-1 cells expressed alpha3, alpha5, alpha6, alphaV and beta1 integrin chains, and the alphaVbeta3 and alphaVbeta5 dimers. We verified that HGT-1 cells xenografted to the immunosuppressed newborn rat retained the integrin repertoire detected in vitro and were able to induce the formation of tumors rich in fibronectin. By using an in vitro assay in the presence of neutralizing antibodies, we verified that HGT-1 adhesion and migration onto fibronectin involved beta1, alphaV and alpha5 integrin chains; we verified, by using an in situ adhesion test to rat gastric wall frozen sections, that in situ HGT-1 adhesion to fibronectin was integrin dependent. In coculture experiments, we showed that organ-specific fibroblasts from stomach, lung and dermis were able to induce, in a site-specific manner, the expression of beta1, alpha5 and alphaV integrin chains in HGT-1 cells, their integrin-dependent adhesion and migration on fibronectin and their capacity to secrete oncofetal fibronectin. In conclusion, our results show the capacity for tissue-derived fibroblasts to modulate the integrin-dependent interactions between the gastric cell line HGT-1 and fibronectin. They strongly suggest that, in gastric cancer, stromal fibroblasts contribute to promote fibronectin-mediated local invasion by tumor cells.

Animals↗
PubMed1998

Synthetic integrin-binding peptides promote adhesion and proliferation of human periodontal ligament cells in vitro.

Periodontal ligament (PDL) cells have been shown to express several integrins (alphav, alpha5, beta1, beta3) that use RGD (arginine-glycine-aspartic Acid)-dependent mechanisms for the recognition and binding of their ligands. The objective of this study was to evaluate the effects of certain integrin-binding cyclic and linear synthetic RGD-containing peptides on PDL cells' adhesion, proliferation, and de novo protein synthesis in vitro. Fifth passages of normal human PDL cells established from teeth extracted from patients (ages 12 to 14) for orthodontic reasons were used for all experiments. Synthetic peptides containing the EPRGDNYR sequence in two different spatial conformations (linear and cyclic) were covalently attached to bovine serum albumin (BSA). Type I collagen, EPRGDNYR-BSA conjugates, 1:1 mixtures of type I collagen and conjugates, as well as BSA (a negative control) were coated on bacteriological plastic and evaluated for their attachment-promoting activities. In addition, the effects of these substrates on cell proliferation were evaluated by [3H]thymidine incorporation by the PDL cells. For attachment and spreading, the cyclic forms of EPRGDNYR-BSA conjugate and type I collagen were most potent, followed by linear EPRGDNYR-BSA conjugate. The effects of all collagen/conjugate mixtures were equivalent to that of type I collagen except for the collagen/linear EPRGDNYR-BSA mixture, which was less potent. The cyclic EPRGDNYR-BSA conjugate was the most effective substrate to stimulate cell proliferation, and it was followed in potency by the linear peptide-BSA conjugate. Collagen alone did not stimulate [3H]thymidine incorporation above the control level. Mixtures of collagen with all of the conjugates showed stimulatory effects similar to that of the cyclic peptide-BSA conjugate. No significant differences in de novo protein synthesis were detected. These results suggest that the synthetic RGD-containing peptides attached to a carrier are potent ligands for the human PDL cells, and that they could provide a basis for the development of new strategies aimed at the regeneration of the periodontium.

Adolescent↗
PubMed1999

Macrophage inflammatory protein 1alpha enhances in a different manner adhesion of hematopoietic progenitor cells from bone marrow, cord blood, and mobilized peripheral blood.

Regulatory mechanisms governing adhesion of hematopoietic progenitor cells to the stromal nische are poorly understood. Growth factors such as stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor, and thrombopoietin were reported to upregulate the adhesion of hematopoietic progenitors to immobilized fibronectin through activation of integrin alpha4beta1 and alpha5beta1. Macrophage inflammatory protein (MIP)-1alpha is a C-C chemokine that suppresses colony formation by stem/progenitor cells in vitro. We asked if MIP-1alpha would modulate the adhesive phenotype of colony-forming cells (CFCs) obtained from healthy donor bone marrow (BM), cord blood (CB), and mobilized peripheral blood (mPB) CD34+ cells, in comparison with SCF, using immobilized fibronectin. SCF significantly increased the level of adhesion of CFCs from BM, CB, and mPB. On the other hand, MIP-1alpha significantly increased the level of adhesion of CFCs from BM and CB, but less so from mPB. The effects of MIP-1alpha were inhibited by blocking antibodies to integrin alpha4, alpha5, or beta1, and polymerization plus rearrangement of F-actin were observed in affected cells by labeling with rhodamine-conjugated phalloidine. These data indicate that the effect of MIP-1alpha on the adhesive phenotype of CFCs is mediated by modulation of the organization of integrin. The amount of MIP-1alpha receptor on mPB was less than for BM or CB, which may explain the distinct characteristics in the adhesive response induced by MIP-1alpha. We suggest that hematopoietic progenitor cells from different sources may be heterogeneous with respect to maturation, integrin affinity, MIP-1alpha receptor expression, and regulation of MIP-1alpha signaling. Our data indicate that MIP-1alpha may affect migration, homing, and mobilization of hematopoietic progenitors by modulating the adhesive phenotype of these cells.

Actins↗
PubMed2004

Functional differences between integrin alpha4 and integrins alpha5/alphav in modulating the motility of human oral squamous carcinoma cells in response to the V region and heparin-binding domain of fibronectin.

The high-affinity heparin-binding domain and the V region of fibronectin (FN) mediate invasion and migration of human oral squamous cell carcinoma (SCC) cells. We investigated the role of the alpha4, alpha5, and alphav integrin receptors--which are central to mediating interactions with these domains of FN--in regulating SCC cell migration. SCC cells expressed the alpha4, alpha5, and alphav integrin subunits on their surface, although alpha4 expression was low. Treatment with recombinant FN proteins containing an alternatively spliced V region (V+) and either an unmutated (H+) or a mutated, nonfunctional high-affinity heparin-binding domain (H-) increased expression of alpha5 and alphav and cell motility. Antisense alpha5 or alphav oligonucleotides inhibited cell motility stimulated by FN proteins, as did blocking antibodies to alpha5 or alphav. Blocking antibodies to alpha5 increased alphav and alpha4 levels, and blocking antibodies to alphav increased the levels of alpha5 and alpha4, without increasing cell motility. In contrast, an antisense alpha4 oligonucleotide and alpha4-blocking antibodies increased cell motility, especially migration stimulated by V+H+ and V+H- FN proteins. alpha4-Blocking antibodies alone increased motility, probably by inducing alpha5 and alphav expression. Transfection with alpha4 cDNA decreased cell motility and alpha5 and alphav expression. Thus, the increased motility induced by the FN protein is probably mediated by alphav and alpha5, whereas alpha4 downregulates this process in a transdominant fashion.

Base Sequence↗
PubMed2000

Expression of beta1-integrins and N-cadherin in bladder cancer and melanoma cell lines.

Changes in the expression of integrins and cadherins might contribute to the progression, invasion and metastasis of transitional cell cancer of the bladder and of melanomas. The expression of alpha5 (P < 0.001), alpha2 and beta1 (P < 0.05 - P < 0.001) integrin subunits in melanoma cells from noncutaneous metastatic sites (WM9, A375) were significantly increased as compared to cutaneous primary tumor (WM35) and metastatic (WM239) cell lines. These differences might be ascribed to the invasive character of melanoma cells and their metastasis to the noncutaneous locations. The significantly heterogeneous expression of beta1 integrin subunit in two malignant bladder cancer cell lines (T24 and Hu456) and nonsignificant differences in the expression of alpha2, alpha3, and alpha5 subunits between malignant and non-malignant human bladder cell lines do not allow an unanimous conclusion on the role of these intergrin subunits in the progression of transitional cancer of bladder. The adhesion molecule, expressed in all studied melanoma and bladder cell lines, that reacted with anti-Pan cadherin monoclonal antibodies was identified as N-cadherin except in the HCV29 non-malignant ureter cell line. However, neither this nor any other bladder or melanoma cell line expressed E-cadherin. The obtained results imply that the replacement of E-cadherin by N-cadherin accompanied by a simultaneous increase in expression of alpha2, alpha3 and alpha5 integrin subunits clearly indicates an increase of invasiveness of melanoma and, to a lesser extent, of transitional cell cancer of bladder. High expression of N-cadherin and alpha5 integrin subunit seems to be associated with the most invasive melanoma phenotype.

Antibodies, Monoclonal↗