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Results for “Integrin alpha2”

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PubMed1999

Induction of collagenase-3 (MMP-13) expression in human skin fibroblasts by three-dimensional collagen is mediated by p38 mitogen-activated protein kinase.

Collagenase-3 (matrix metalloproteinase-13, MMP-13) is a recently identified human MMP with an exceptionally wide substrate specificity and restricted tissue-specific expression. Here we show that MMP-13 expression is induced in normal human skin fibroblasts cultured within three-dimensional collagen gel resulting in production and proteolytic activation of MMP-13. Induction of MMP-13 mRNAs by collagen gel was potently inhibited by blocking antibodies against alpha1 and alpha2 integrin subunits and augmented by activating antibody against beta1 integrin subunit, indicating that both alpha1 beta1 and alpha2 beta1 integrins mediate the MMP-13-inducing cellular signal generated by three-dimensional collagen. Collagen-related induction of MMP-13 expression was dependent on tyrosine kinase activity, as it was abolished by treatment of fibroblasts with tyrosine kinase inhibitors genistein and herbimycin A. Contact of fibroblasts to three-dimensional collagen resulted in simultaneous activation of mitogen-activated protein kinases (MAPKs) in three distinct subgroups: extracellular signal-regulated kinase (ERK)1 and ERK2, Jun N-terminal kinase/stress-activated protein kinase, and p38. Induction of MMP-13 expression was inhibited by treatment of fibroblasts with a specific p38 inhibitor, SB 203580, whereas blocking the ERK1,2 pathway (Raf/MEK1,2/ERK1,2) by PD 98059, a selective inhibitor of MEK1,2 activation potently augmented MMP-13 expression. Furthermore, specific activation of ERK1,2 pathway by 12-O-tetradecanoylphorbol-13-acetate markedly suppressed MMP-13 expression in dermal fibroblasts in collagen gel. These results show that collagen-dependent induction of MMP-13 in dermal fibroblasts requires p38 activity, and is inhibited by activation of ERK1,2. Therefore, the balance between the activity of ERK1,2 and p38 MAPK pathways appears to be crucial in regulation of MMP-13 expression in dermal fibroblasts, suggesting that p38 MAPK may serve as a target for selective inhibition of collagen degradation, e.g. in chronic dermal ulcers.

Base Sequence↗

PubMed2001

A new peritoneal dissemination model established from the human pancreatic cancer cell line.

We established a new cell line, HPC-3P4a, with high peritoneal disseminated potential in nude mice. HPC-3P4a was derived from a human pancreatic carcinoma cell line (HPC-3) that had low capacity for peritoneal dissemination. HPC-3P4a developed peritoneal dissemination in 10 of 11 (90.9%) cases, whereas parental HPC-3 developed peritoneal dissemination in one of six (16.7%) cases. The metastatic foci in the peritoneum showed essentially the same histologic appearance of parental involvement. The tumorigenicity, motility, and adhesive activity of HPC-3P4a to the extracellular matrix were stronger than were those of the HPC-3. In FACS analysis, HPC-3P4a significantly increased the expression of alpha6 and alpha(v)beta5 integrins, while it decreased alpha2 integrin, hCD44H, and hCD44v 10, as compared with HPC-3. The VEGF production of HPC-3P4a was significantly lower than that of HPC-3. Analysis of gene macroarrays showed a variety of cytokines, interleukin, and other immunomodulatory, and their receptors were up-regulated and down-regulated on an mRNA level in HPC-3P4a cells, compared with HPC-3 cells. Intrasplenic injection of HPC-3P4a produced no liver metastasis. We named our original highly liver metastatic cell line HPC-3H4 (previously reported). This HPC-3H4 cell was established by repeated intrasplenic injection from parental cell HPC-3; thus, it developed high liver metastasis. Moreover, HPC-3H4 developed peritoneal dissemination by intra-abdominal injection. In contrast, HPC-3P4a did not develop liver metastasis by intrasplenic injection. These findings are very interesting and might suggest that the process of hematogenous metastasis differed from that of peritoneal dissemination. Thus, this cell line may be useful for investigating the mechanism of peritoneal dissemination in human pancreatic cancer.

Adenocarcinoma↗

PubMed1999

Binding of the renal epithelial cell line LLC-PK1 to laminin is regulated by protein kinase C.

The alpha6beta1 integrin heterodimer has been implicated in the mediation of renal epithelial cell binding to laminin, and it has been suggested that this binding is important for renal morphogenesis and development. Studies of nonrenal cells have suggested that the functional activity of alpha6beta1 integrin is regulated by protein kinase C (PKC) activity. In this study, the binding of a renal epithelial cell line, LLC-PK1, to laminin was characterized and the role of PKC activity in the modulation of binding was investigated. LLC-PK1 cells bound to laminin-coated surfaces in a time- and laminin concentration-dependent manner. Binding was strongly inhibited by anti-beta1 integrin antibodies and by anti-alpha6 integrin antibodies. Antibodies against alpha2 integrin and a3 integrin had little inhibitory effect. Cells bound to both whole laminin and laminin fragment E8, i.e., the fragment to which the alpha6beta1 integrin heterodimer binds. Exposure of cells to PKC activators for as little as 2 h enhanced cell binding to laminin approximately twofold, in a protein synthesis-dependent manner. PKC inhibitors antagonized this effect. PKC-stimulated binding was also inhibited by anti-beta1 integrin and anti-alpha6 integrin antibodies. PKC activation did not alter expression of beta1 integrin subunits at the cell surface after short time periods (2 to 4 h), but expression was increased after longer time periods (24 h). These results indicate that the renal epithelial cell line LLC-PK1 binds to laminin via the alpha6betal integrin heterodimer and binding is enhanced by PKC activation. The PKC-mediated enhancement of binding requires protein synthesis and is mediated in part by activation of surface alpha6beta1 integrin.

Animals↗

PubMed2000

Expression of integrin subunits correlates with differentiation of epithelial cell lineages in developing human gastric mucosa.

Laminins may play important roles in gastric gland development due to the differential localization of their alpha chains in human fetal fundic (oxyntic) mucosa. To extend this hypothesis, the current investigation was undertaken to compare the anatomical and cellular distribution of epithelial integrin subunits with those of laminin alpha chains in the human stomach at different ages (8-22 weeks of gestation) using indirect immunofluorescence. In the body, fundus and antrum regions, the beta1 and alpha6 subunits were associated with the entire epithelium at all developmental stages in the same way as laminin chains (alpha1/alpha5) detected with 4C7 monoclonal antibody. By contrast, the alpha3 and beta4 subunits of alpha3beta1 and alpha6beta4 integrins together with the laminin alpha3-chain were concentrated in the surface and foveolus compartments composed of differentiating mucous cells. Most importantly, the alpha2 integrin subunit was expressed in a rather complex pattern: (1) it was located at the base and at cell-cell boundaries of surface/foveolar epithelium, (2) was specifically repressed in differentiating parietal cells, and (3) its expression increased in maturing glands, where it became concentrated at the basal pole of epithelial cells simultaneously exhibiting a strong reactivity for laminin-2 (alpha2-chain). Taken altogether, our observations provide new evidence for the implication of different laminins and their receptors in the development of all human gastric epithelial lineages, surface mucous cells or glandular cells. The coordinated expression of alpha2 and alpha3 integrin subunits as well as the cellular re-distribution of alpha2beta1 integrin likely represent key events for the differentiation of glandular secretory cell types, especially maturing chief cells responsible for the synthesis/secretion of gastric digestive enzymes in fundic-type glands.

Antigens, CD↗

PubMed2004

Immuno-histochemical expression of alpha1, alpha2 and alpha3 integrin subunits during angiogenesis in vitro.

Aortic explants were obtained from mouse fetuses and cultured in collagen gels. Immuno-fluorescence microscopy, antibodies (anti alpha1, alpha2 and alpha3 integrin subunits) were used. Fibroblastic cells migrated from the aortic explant after one day of cultivation. The migrating cells located in the peripheral part of the aortic explant were positive for alpha1 and alpha2 integrin subunit antibodies. Immuno-fluorescence-positive staining for the alpha3 integrin subunit antibody was clearly seen in the migrating cells located near the aortic explant and surrounding tube-like structures. In an immuno-electron microscope study performed by pre-embedding immuno labeling, gold particles associated with the alpha3 integrin subunit were found to reside on the membranes of the cells surrounding the capillary-like tubes. Two synthetic peptides, GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) and KDGEA (Lys-Asp-Gly-Glu-Ala), were added to the growth medium to study their effects on cell migration. KDGEA, a compound containing the recognition sequence for alpha2beta1 integrin, decreased cell migration, while GRGDSP exhibited no effect. The migration of fibroblastic cells is an important phenomenon for tube formation. The present study suggested that the alpha1 and alpha2 integrin subunits are both involved in the cell migration, and more specifically, that the alpha2 integrin subunit participates in cell migration through the KDGEA sequence. The alpha3 integrin subunit played a role in tube formation.

Animals↗

PubMed2003

Monkey rotavirus binding to alpha2beta1 integrin requires the alpha2 I domain and is facilitated by the homologous beta1 subunit.

Rotaviruses utilize integrins during virus-cell interactions that lead to infection. Cell binding and infection by simian rotavirus SA11 were inhibited by antibodies (Abs) to the inserted (I) domain of the alpha2 integrin subunit. To determine directly which integrins or other proteins bind rotaviruses, cell surface proteins precipitated by rotaviruses were compared with those precipitated by anti-alpha2beta1 Abs. Two proteins precipitated by SA11 and rhesus rotavirus RRV from MA104 and Caco-2 cells migrated indistinguishably from alpha2beta1 integrin, and SA11 precipitated beta1 from alpha2beta1-transfected CHO cells. These viruses specifically precipitated two MA104 cell proteins only, but an additional 160- to 165-kDa protein was precipitated by SA11 from Caco-2 cells. The role of the alpha2 I domain in rotavirus binding, infection, and growth was examined using CHO cell lines expressing wild-type or mutated human alpha2 or alpha2beta1. Infectious SA11 and RRV, but not human rotavirus Wa, specifically bound CHO cell-expressed human alpha2beta1 and, to a lesser extent, human alpha2 combined with hamster beta1. Binding was inhibited by anti-alpha2 I domain monoclonal Abs (MAbs), but not by non-I domain MAbs to alpha2, and required the presence of the alpha2 I domain. Amino acid residues 151, 221, and 254 in the metal ion-dependent adhesion site of the alpha2 I domain that are necessary for type I collagen binding to alpha2beta1 were not essential for rotavirus binding. Rotavirus-alpha2beta1 binding led to increased virus infection and RRV growth. SA11 and RRV require the alpha2 I domain for binding to alpha2beta1, and their binding to this integrin is distinguishable from that of collagen.

Animals↗

PubMed2005

Drug-induced gingival overgrowth--a review.

Drug-induced gingival overgrowth is a side effect associated with 3 types of drugs: anticonvulsants (phenytoin), immunosuppressive agents (cyclosporine A), and various calcium channel blockers for cardiovascular diseases. Gingival overgrowth is characterized by the accumulation of extracellular matrix in gingival connective tissues, particularly collagenous components with various degrees of inflammation. Although the mechanisms of these disorders have not been elucidated, recent studies suggest that these disorders seem to be induced by the disruption of homeostasis of collagen synthesis and degradation in gingival connective tissue, predominantly through the inhibition of collagen phagocytosis of gingival fibroblasts. The integrins are a large family of heterodimeric transmembrane receptors for extracellular matrix molecules. alpha2beta1 integrin serves as a specific receptor for type I collagen on fibroblasts, and alpha2 integrin has been shown to play a crucial role in collagen phagocytosis. Actin filaments, which are assembled from monomers and oligomers, are involved in collagen internalization after binding to integrins. Furthermore, the implication of intracellular calcium in the regulation of integrin-mediated binding activity and gelsolin activity, known as a calcium-dependent actin-severing protein, is also described. In this review, we focus on collagen metabolism in drug-induced gingival overgrowth, focusing on the regulation of collagen phagocytosis in fibroblasts.

Anticonvulsants↗

PubMed1998

Integrin-mediated platelet adhesion.

Adhesion of platelets to the damaged subendothelium is a prerequisite reaction for the initiation of hemostasis in vivo. Platelet membranes contain high concentrations of integrins and other glycoproteins (GPs) that are involved in the platelet adhesion to the extracellular matrix. In the present review, we focus on two platelet integrins, integrin alphaIIb beta3 (GPIIb/IIIa) and integrin alpha2 beta1 (GPIa/IIa) because these integrins are major components of the platelet membrane proteins and are known to contribute to platelet adhesion to fibrin(ogen) and collagen surfaces, respectively. These integrins bind soluble ligands (fibrinogen or collagen) after platelets are activated but only have low affinity towards these ligands when platelets are in the resting state. We describe the binding properties of these integrins and discuss the mechanism for the activation of these integrins. Platelets can adhere to fibrin(ogen) or collagen immobilized on a surface. When platelets adhere to a collagen- or fibrin-coated surface, they become activated and form aggregates; this is especially prominent under flow conditions. We discuss the contribution of these integrins and non-integrin proteins, GPIb and GPVI, to the platelet adhesion on to the collagen surface, especially under flow conditions, a system that most closely approximates platelet adhesion in vivo.

Animals↗

PubMed1999

Expression of integrin messenger ribonucleic acid in human endometrium: a quantitative reverse transcription polymerase chain reaction study.

OBJECTIVE: To assess the expression of selected integrin subunit's messenger RNA (mRNA) in human endometrium during the menstrual cycle. DESIGN: A prospective comparative study. SETTING: Academic research environment. PATIENT(S): Premenopausal women with histologically normal endometrium who were undergoing hysterectomy. INTERVENTION(S): Endometrial tissues were collected. RESULT(S): Human endometrium expresses integrins alpha2, alpha3, alpha4, alpha5, alpha6.1, alpha6.2, alpha v, beta1, beta2, beta3, and beta5, as well as fibronectin mRNA. The levels of endometrial integrins mRNA expression varied significantly, with the lowest levels observed for alpha2, beta3, and fibronectin and the highest for alpha5, beta2, and beta5. The levels of integrins alpha2, alpha3, and alpha5 mRNA expression were significantly higher during the proliferative phase, whereas alpha4, alpha6.2, alpha v, beta1, beta2, beta3, beta5, and fibronectin were higher during the secretory phase of the menstrual cycle. The alpha6.1 mRNA was found to be equally expressed in the endometrium during the menstrual cycle, whereas the most dramatic changes occurred in alpha v and beta3 expression, compared with other integrin subunits. CONCLUSION(S): Human endometrium expresses mRNA for several integrins and fibronectin, with up-regulation of alpha4, alpha v, beta1, and beta3 during the secretory phase of the menstrual cycle, suggesting that their differential expression may be regulated in part by ovarian steroids.

Adult↗

PubMed1997

Cell-adhesion molecules in human meningiomas: correlation with clinical and morphological data.

Integrins form a family of cell adhesion molecules. CD44 glycoproteins are found in a wide variety of isoforms; the most common, CD44s (standard) is widely distributed, and functions as an adhesion molecule. In this study, we have investigated immunohistochemically the distribution of some VLA integrins (alpha2, alpha5 and alpha6 chains of beta1 integrins) and CD44s in 44 meningioma specimens and normal arachnoid villi. Meningiomas were of meningothelial (16), transitional (13) and fibroblastic (15) subtypes. There were 13 grade I, 19 grade II and 12 grade III (27%). Immunoprecipitates were quantified by image analysis and correlated with clinical (age, sex, location) and morphological data (histological subtypes and grades). VLA alpha5 chain was expressed by normal arachnoid villi (mainly cap cells) and by 42 out of 44 meningioma specimens. Expression was lower in fibroblastic meningiomas (P=0.02). VLA alpha2 and alpha6 chains were not observed in normal arachnoid villi. VLA alpha2 was expressed by 15 meningiomas, VLA alpha6 by 10. Interestingly, meningiomas expressing either VLA alpha2 or alpha6 were usually of grade III (P< or =(0.05). CD44s was found on various parts of arachnoid villi and in all meningiomas although expression was higher in meningothelial and transitional than in fibroblastic (P< or =0.001). These results show that VLA alpha5 and CD44s are widely expressed by arachnoid villi and meningiomas, in contrast to VLA alpha2 and VLA alpha6. It was noted that high grade meningiomas (III) express VLA alpha2 and alpha6 suggesting that changes in integrin pattern expression are a feature of these meningiomas. Moreover, strong CD44s expression characterizes meningothelial and transitional meningiomas. Previous studies have shown that high NCAM expression is a feature of fibroblastic meningiomas whereas meningothelial and transitional meningiomas expressed mainly E-Cadherin, and that polysialylated NCAM expression was restricted to high grade meningiomas. Taken together these features suggest that each cell adhesion molecule has a characteristic pattern of expression according to meningioma subtype and grade. No correlation was seen between integrins and CD44s expression and clinical data.

Adolescent↗

PubMed1999

An immunohistochemical examination of the expression of E-cadherin, alpha- and beta/gamma-catenins, and alpha2- and beta1-integrins in invasive breast cancer.

This study examines the expression of the cell-cell adhesion molecules E-cadherin and its associated proteins, the catenins and the matrix-cell adhesion molecules beta1- and alpha2-integrins, in primary invasive breast carcinoma. Expression was assessed immunohistochemically on frozen sections by semi-quantitative scoring of the intensity and proportion of immunoreactivity in 55 cases. Associations with each other and with other histological and prognostic features and survival were sought. There was a significant association between loss of E-cadherin expression and loss of alpha- and beta/gamma-catenin immunostaining. In 20 per cent of cases, membranous immunoreactivity with E-cadherin antibody was absent. Absent cytoplasmic expression of alpha- and beta/gamma-catenins was seen in 24 and 22 per cent of breast cancers, respectively. The intensity of reactivity with E-cadherin showed a significant association with histological grade (p=0.002) and tumour type (p<0.001). Lobular carcinomas frequently showed loss of expression of E-cadherin, as reported elsewhere; loss of catenin expression was also found in these tumours. alpha-catenin intensity also showed a relationship with grade (p=0.008) and with oestrogen receptor (ER) status (p=0.006). beta/gamma-catenin expression was not associated with other known prognostic factors. Forty-nine per cent and 42 per cent of cases showed no membrane immunostaining with beta1- and alpha2-integrin, respectively, and co-ordinated loss of beta1- and alpha2-integrin expression was found. Both beta1- and alpha2-integrin expression were associated with histological grade (p=0.003 and p=0.031, respectively) and beta1 immunoreactivity with tumour type (p=0.010). None of the variables examined showed a statistically significant association with tumour size or lymph node stage, or with overall survival, although a trend was seen (p=0.087) towards poorer survival of patients with tumours with absent or weak expression of beta1-integrin. The expression of these markers is of biological interest, but appears to be of little additional use in predicting clinical behaviour.

Adult↗

PubMed1998

The involvement of the fibronectin type II-like modules of human gelatinase A in cell surface localization and activation.

Recombinant collagen-binding domain (rCBD) comprising the three fibronectin type II-like modules of human gelatinase A was found to compete the zymogen form of this matrix metalloproteinase from the cell surface of normal human fibroblasts in culture. Upon concanavalin A treatment of cells, the induced cellular activation of gelatinase A was markedly elevated in the presence of the rCBD. Therefore, the mechanistic aspects of gelatinase A binding to cells by this domain were further studied using cell attachment assays. Fibroblasts attached to rCBD-coated microplate wells in a manner that was inhibited by soluble rCBD, blocking antibodies to the beta1-integrin subunit but not the alpha2-integrin subunit, and bacterial collagenase treatment. Addition of soluble collagen rescued the attachment of collagenase-treated cells to the rCBD. As a probe on ligand blots of octyl-beta-D-thioglucopyranoside-solubilized cell membrane extracts, the rCBD bound 140- and 160-kDa protein bands. Their identities were likely procollagen chains being both bacterial collagenase-sensitive and also converted upon pepsin digestion to 112- and 126-kDa bands that co-migrated with collagen alpha1(I) and alpha2(I) chains. A rCBD mutant protein (Lys263 --> Ala) with reduced collagen affinity showed less cell attachment, whereas a heparin-binding deficient mutant (Lys357 --> Ala), heparinase treatment, or heparin addition did not alter attachment. Thus, a cell-binding mechanism for gelatinase A is revealed that does not involve the hemopexin COOH domain. Instead, an attachment complex comprising gelatinase A-native type I collagen-beta1-integrin forms as a result of interactions involving the collagen-binding domain of the enzyme. Moreover, this distinct pool of cell collagen-bound proenzyme appears recalcitrant to cellular activation.

Antibodies↗

PubMed1999

Human corneal epithelial cell adhesion to laminins.

PURPOSE: To analyze alpha-integrin mediated adhesion of human corneal epithelial cells to placental and EHS laminin isoforms. METHODS: Western blot analysis was used to partially characterize commercially available preparations of laminin isolated from the mouse EHS sarcoma and from human placenta. Using the human corneal epithelial cell line HCE-T, adhesion to laminin isoforms and fibronectin was determined using a colorimetric adhesion assay. alpha-integrin sub-unit modulation of corneal epithelial cell interaction with laminin isoforms was analyzed using immunofluorescence microscopy and adhesion assays incorporating functional blocking antibodies. RESULTS: In short-term adhesion assays, the preferred substrate for HCE-T attachment is placental laminin. Immunofluorescence microscopy reveals that alpha-integrin protein localization patterns are not significantly different in HCE-T interacting with EHS or placental laminin. However, in short-term assays alpha3 integrin plays a major role, and alpha2 integrin a minor role, in mediating HCE-T adhesion to laminin. alpha6 integrin does not appear to mediate adhesion to either substrate. CONCLUSIONS: These studies demonstrate that human corneal epithelial cells are capable of rapid adhesion to, and enhanced spreading on, laminin isoforms not characteristically resident in the adult corneal basement membrane. This characteristic of human corneal epithelium may explain, at least in part, why amniotic membrane transplantation is proving to be clinically useful for human ocular surface reconstruction.

Animals↗

PubMed1997

Immunohistochemical localization of integrins and basement membrane components in colorectal epithelial tumors correlates with their histologic architecture.

We studied the immunohistochemical localization of integrins (alpha2, alpha6) and basement membrane components (type IV collagen, laminin) in colorectal epithelial tumors (adenomas and adenocarcinomas) with respect to their intramucosal and invasive growth. The integrins showed two patterns of localization: basal polarization and diffuse expression. In intramucosal tumors, the basement membrane components were well preserved, and the staining intensity for the basal polarization of alpha6 increased with increasing size and grade of dysplasia of the tumors. In invasive adenocarcinomas, the basement membrane components were preserved, and the basal polarization of alpha6 was apparent in well-differentiated areas, while the tumor cells showed disorganization or loss of the basement membrane components and the diffuse expression of alpha6 with a reduction or loss of the basal polarization in moderately and poorly differentiated areas. The expression of alpha2 did not correlate with the grade of dysplasia of intramucosal tumors or the pattern of differentiation of invasive adenocarcinomas. These results suggest that the basal polarization of alpha6 and the preservation of the basement membrane components contribute to the glandular architecture of colorectal epithelial tumors, and that the diffuse expression of alpha6 with a reduction or loss of the basal polarization, and the disorganization and loss of the basement membrane components to the dispolarized affinity of tumor cells to the extracellular matrix.

Adenocarcinoma↗

PubMed1997

The role of alpha1beta1 integrin in wound contraction. A quantitative analysis of liver myofibroblasts in vivo and in primary culture.

An unresolved question in wound contraction concerns the identity of integrins mediating the attachment of tissue myofibroblasts to matrix in the injury site. Previous studies with cell lines have focussed on alpha1beta1 and alpha2beta1, the principal collagen-binding integrins, but have yielded conflicting data. We have examined this issue in wound healing in the liver, isolating the myofibroblast population (activated stellate cells) and quantitating expression of the alpha1 and alpha2 integrin subunits during the in vivo injury. Normal stellate cells displayed alpha1 but no detectable alpha2. During injury, alpha1 expression was maintained; alpha2 became detectable at the mRNA level but at all times was <8% of alpha1 mRNA. Contraction of collagen lattices, studied with 24-h cultured cells and initiated by endothelin 1, was blocked 70% by anti-alpha1 and 30% by anti-alpha2 (both significant, p < 0.05). The inhibition by anti-alpha2, which was unexpected, was attributable to culture-induced change in integrin expression; both the mRNA and protein for alpha2 increased strikingly within 24 h of plating stellate cells on a collagen gel. We conclude that alpha1beta1 is the sole integrin utilized by contracting myofibroblasts in vivo. Although alpha2beta1 is capable of mediating contraction, its expression by myofibroblasts occurs largely, if not exclusively, in response to culture.

Animals↗

PubMed2000

Integrin expression by primary and immortalized human chondrocytes: evidence of a differential role for alpha1beta1 and alpha2beta1 integrins in mediating chondrocyte adhesion to types II and VI collagen.

OBJECTIVE: Chondrocytes have been shown to express beta1-containing integrins both in vitro and in situ, but their role in regulating chondrocyte function is poorly understood. The objective of this study was to determine how the relative expression of different integrins may be modulated in relation to the differentiated state and proliferative capacity of the chondrocyte. DESIGN: Integrin expression by four different cell lines of human chondrocytes immortalized with Simian virus 40 large T-antigen (SV40-TAg) was studied and compared to primary chondrocytes. Differences in alpha1 and alpha2 integrin subunit expression were utilized to further study the role of these integrins in mediating adhesion to types II and VI collagen. RESULTS: The overall cell-surface levels of beta1-containing integrins were higher on all four immortalized cell lines which expressed over 10-fold higher levels of alpha2 and alpha3 integrin subunits compared to primary cells. However, primary cells expressed higher levels of the alpha1 integrin subunit which was not expressed by T/C28a4 cells and expressed at variable and lower levels in the other lines. Levels of the alpha3 integrin subunit were significantly greater on the highly proliferative juvenile costal chondrocyte lines (T/C-28a4, C-2812, and C-20a4) compared to primary articular chondrocytes and tsT/AC-62 cells which were derived from adult articular chondrocytes. Expression of alpha5 was similar among primary cells and cell lines except on C-20/A4 cells which had an average of over 4-fold higher levels. None of the primary or immortalized chondrocytes tested expressed significant levels of alpha4. Cell adhesion assays revealed that both alpha1beta1 and alpha2beta1 could serve as chondrocyte adhesion receptors for types II and VI collagen. In cell lines expressing both integrins, alpha1beta1 was the preferential receptor for type VI collagen while alpha2beta1 was the preferential receptor for type II collagen. Rather than inhibiting adhesion, incubation with the alpha3 blocking antibody P1B5 increased adhesion of C-28/12 cells to both fibronectin and type II collagen by 67% and 100% respectively. CONCLUSIONS: Immortalization with SV40-TAg results in altered integrin expression by chondrocytes. Changes in the relative levels of alpha1, alpha2, and alpha3 subunits may significantly alter the manner in which chondrocytes interact with types II and VI collagen in the extracellular matrix.

Adult↗

PubMed2005

Decreased n-6/n-3 fatty acid ratio reduces the invasive potential of human lung cancer cells by downregulation of cell adhesion/invasion-related genes.

Recent studies have shown opposing effects of n-6 and n-3 fatty acids on the development of cancer and suggest a role for the ratio of n-6 to n-3 fatty acids in the control of cancer. However, whether an alteration in the n-6/n-3 fatty acid ratio of cancer cells affects their invasive potential has not been well investigated. We recently developed a genetic approach to modify the n-6/n-3 ratio by expression of the Caenorhabditis elegans fat-1 gene encoding an n-3 desaturase that converts n-6 to n-3 fatty acids in mammalian cells. The objective of this study was to examine the effect of alteration in the n-6/n-3 fatty acid ratio on the invasive potential of human lung cancer A549 cells. Adenovirus-mediated gene transfer of the n-3 desaturase resulted in a marked reduction of the n-6/n-3 fatty acid ratio, particularly the ratio of arachidonic acid to eicosapentaenic acid. Cell adhesion assay showed that the cells expressing fat-1 gene had a delayed adhesion and retarded colonization. Matrigel assay for invasion potential indicated a 2-fold reduction of cell migration in the fat-1 transgenic cells when compared with the control cells. An increased apoptosis was also observed in the fat-1 transgenic cells. Microarray and quantitative polymerase chain reaction revealed a downregulation of several adhesion/invasion-related genes (MMP-1, integrin-alpha2 and nm23-H4) in the fat-1 transgenic cells. These results demonstrate that a decreased n-6/n-3 fatty acid ratio reduces the invasion potential of human lung cancer cells by probably downregulating the cell adhesion/invasion-related molecules, suggesting a role for the ratio of n-6 to n-3 fatty acids in the prevention and treatment of cancer.

Adenoviridae↗

PubMed2002

A novel nude mouse model of liver metastasis and peritoneal dissemination from the same human pancreatic cancer line.

INTRODUCTION: Recently, several mice models have been used for investigating cancer metastasis. However, there are no metastatic and peritoneal dominated variants from the same parental cell line. AIM AND METHODOLOGY: To elucidate the mechanisms of metastasis, we established highly liver metastatic and peritoneal disseminated models in nude mice, and then characterized several factors related to metastasis in these cells. We established a series of well-characterized sublines that showed metastatic potentials to different organ sites of nude mice. Two sublines were selected sequentially from the parental pancreatic cancer cell line, HPC-4, resulting in a highly liver metastatic cell line, HPC-4H4, and a highly peritoneal disseminated cell line, HPC-4P4a. Using these three cell lines, we investigated several biologic properties and mRNA levels of differentially expressed genes involved in cancer metastasis. RESULTS: The tumorigenicity, the motile activity, and the adhesive activity of metastatic sublines were higher than those of parental HPC-4 cells. Macroscopic and microscopic findings and the DNA ploidy pattern were the same among the three cell lines. In addition, HPC-4H4 cells expressed clearly higher levels of vascular endothelial growth factor and IL-8 expression than did HPC-4P4a cells. In fluorescence-activated cell sorter analysis of adhesion molecules, the expression of integrin-alpha2 was enhanced in HPC-4 cells, integrin-alphavbeta5 was enhanced in HPC-4H4 cells, and integrin-alpha3 was enhanced in HPC-4P4a cells. Osteopontin, vascular endothelial growth factor, and hepatocyte growth factor were among the genes that were upregulated in HPC-4H4 cells compared with HPC-4P4a cells. HPC-4P4a cells did not metastasize to the liver by intrasplenic injection. Conversely, HPC-4H4 cells metastasized remarkably to the peritoneum by intraabdominal injection. CONCLUSION: These sublines are the first reported liver metastatic and peritoneal disseminated models derived from the same parental cell lines. The results of our study suggest that the process of hematogenous metastasis is not the same as that of peritoneal dissemination.

Animals↗