Changes in glycosylation of vitronectin modulate multimerization and collagen binding during liver regeneration

Elucidating the mechanisms and factors regulating multimerization is biologically important in order to modulate the biological activities of functional proteins, especially adhesive proteins in the extracellular matrix (ECM). Vitronectin (VN) is a multifunctional glycoprotein present in plasma and ECM. Linkage of cellular adhesion and fibrinolysis by VN plays an essential role during tissue remodeling. Our previous study determined that the collagen-binding activity of VN was markedly enhanced with the decreased glycosylation during liver regeneration. This study demonstrated how alternations of glycans modulate the biological activity of VN. Human and rat VNs were used because of their similarities in structure and activities. The binding affinity of human VN to immobilized collagen was shown to be higher at pH 4.5 than at 7.5, at 37 degrees C than at 4 degrees C. Sedimentation velocity studies indicated that the greater the multimerization of human VN, the better it bound to collagen. The results indicate that the collagen binding of VN was modulated through its multimerization. Stepwise trimming of glycan with various exoglycosidases increased both the multimer size and the collagen binding of human VN, indicating that they are modulated by changes in glycosylation. The multimer sizes of VN purified from plasma of partially hepatectomized (PH) rats and sham-operated (SH) rats increased by about 45 and 31%, respectively, compared with those of nonoperated (NO) rats. In accordance with this, PH-VN exhibited remarkably enhanced collagen binding than SH-VN and NO-VN on surface plasmon resonance. In the PH rat sera, the multimer VN was increased in both amount and size compared with those in SH- and NO-sera. The results demonstrate that glycan alterations during tissue remodeling induce increased multimerization state to enhance the biological activity of VN.     

Survival Signals of Hepatic Stellate Cells in Liver Regeneration Are Regulated by Glycosylation Changes in Rat Vitronectin,Especially Decreased Sialylation

The extracellular matrix (ECM) molecules play important roles in many biological and pathological processes. During tissue remodeling, the ECM molecules that are glycosylated are different from those of normal tissue owing to changes in the expression of many proteins that are responsible for glycan synthesis. Vitronectin (VN) is a major ECM molecule that recognizes integrin on hepatic stellate cells (HSCs). The present study attempted to elucidate how changes in VN glycans modulate the survival of HSCs, which play a critical role in liver regeneration. Plasma VN was purified from partially hepatectomized (PH) and sham-operated (SH) rats at 24 h after operation and non-operated (NO) rats. Adhesion of rat HSCs (rHSCs), together with phosphorylation of focal adhesion kinase, in PH-VN was decreased to one-half of that in NO- or SH-VN. Spreading of rHSCs on desialylated NO-VN was decreased to one-half of that of control VN, indicating the importance of sialylation of VN for activation of HSCs. Liquid chromatography/multiple-stage mass spectrometry analysis of Glu-C glycopeptides of each VN determined the site-specific glycosylation. In addition to the major biantennary complex-type N-glycans, hybrid-type N-glycans were site-specifically present at Asn¹⁶⁷. Highly sialylated O-glycans were found to be present in the Thr¹¹⁰–Thr¹²⁴ region. In PH-VN, the disialyl O-glycans and complex-type N-glycans were decreased while core-fucosylated N-glycans were increased. In addition, immunodetection after two-dimensional PAGE indicated the presence of hyper- and hyposialylated molecules in each VN and showed that hypersialylation was markedly attenuated in PH-VN. This study proposes that the alteration of VN glycosylation modulates the substrate adhesion to rat HSCs, which is responsible for matrix restructuring.     

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.     

The extracellular matrix ligands fibronectin and tenascin collaborate in regulating collagenase gene expression in fibroblasts.

Tenascin (TN) is a large oligomeric glycoprotein that is present transiently in the extracellular matrix (ECM) of cells and is involved in morphogenetic movements, tissue patterning, and tissue repair. It has multiple domains, both adhesive and anti-adhesive, that interact with cells and with fibronectin (FN) and other ECM macromolecules. We have studied the consequences of the interaction of TN with a FN matrix on gene expression in rabbit synovial fibroblasts. Fibroblasts plated on a mixed substrate of FN and TN, but not on FN alone, upregulated synthesis of four genes: collagenase, stromelysin, the 92-kDa gelatinase, and c-fos. Although the fibroblasts spread well on both FN and FN/TN substrates, nuclear c-Fos increased within 1 h only in cells that were plated on FN/TN. TN did not induce the expression of collagenase in cells plated on substrates of type I collagen or vitronectin (VN). Moreover, soluble TN added to cells adhering to a FN substrate or to serum proteins had no effect, suggesting that TN has an effect only in the context of mixed substrates of FN and TN. Collagenase increased within 4 h of plating on a FN/TN substrate and exhibited kinetics similar to those for induction of collagenase gene expression by signaling through the integrin FN receptor. Arg-Gly-Asp peptide ligands that recognize either the FN receptor or the VN receptor and function-perturbing anti-integrin monoclonal antibodies diminished the interaction of fibroblasts with a mixed substrate of FN, TN, and VN, but had no effect on the adhesion of fibroblasts to a substrate of FN and VN, suggesting that both receptors recognize the complex. Anti-TN68, an antibody that recognizes an epitope in the carboxyl-terminal type III repeats involved in the interaction of TN with both FN and cells, blocked the inductive effect of the FN/TN substrate, whereas anti-TNM1, an antibody that recognizes an epitope in the amino-terminal anti-adhesive region of epidermal growth factor-like repeats, had no effect. These data suggest that transient alteration of the composition of ECM by addition of proteins like TN may regulate the expression of genes involved in cell migration, tissue remodeling, and tissue invasion, in regions of tissue undergoing phenotypic changes.     

Differential binding of factor XII and activated factor XII to soluble and immobilized fibronectin--localization of the Hep-1/Fib-1 binding site for activated factor XII

Fibronectins (FNs) are dimeric glycoproteins that adopt a globular conformation when present in plasma and solution and an extended conformation in the extracellular matrix. Factor XII (FXII) is a zymogen of the proteolytically active FXIIa that plays a role in thrombus stabilization by enhancing clot formation and in inflammation by enhancing bradykinin formation. To investigate whether the extracellular matrix could play a role in these events, we have recently shown that FXIIa, but not FXII, binds to the extracellular matrix (ECM), and suggested that FN may be the target for the binding. Immunofluorescence microscopy has in the present investigation confirmed that FXIIa added to the ECM colocalizes with FN deposited during growth of human umbilical vein endothelial cells. The aim of the present study, therefore, was to further elucidate the interaction between FXIIa and FN by the use of a solid face binding assay. This showed, like the binding to the ECM, that FXIIa, but not FXII, binds in a Zn2+-independent manner to immobilized FN. The K(D) for the binding was 8.5 +/- 0.9 nM (n = 3). The binding was specific for the immobilized FN, as the binding could not be inhibited by soluble FN. Furthermore, soluble FN did not bind to immobilized FXIIa. However, soluble FN could bind to FXII, and this binding inhibited the surface-induced autoactivation of FXII and subsequent binding of the generated FXIIa to immobilized FN. The presence of FXII in an anti-FN immunoprecipitate of plasma indicated that some FXII in plasma circulates bound to FN. The binding of FXIIa to FN was inhibited by gelatine and fibrin but not by heparin, indicating that FXIIa binds to immobilized FN through the type I repeat modules. Accordingly, FXIIa was found to bind to immobilized fragments of FN containing the type I repeat modules in the N-terminal domain to which fibrin and gelatine bind.     

The Effect of Extracellular Matrix Molecules on the in Vitro Behavior of Bovine Endothelial Cells

Extracellular matrix (ECM) is an important mediator of endothelial functions such as adhesion, spreading, migration, proliferation, and maintenance of differentiated functions. Attachment of cultured cells to tissue culture polystyrene (TCPS) is dependent on vitronectin which adsorbs onto the surface from the serum in the culture medium. Vitronectin (VN) will adsorb efficiently to TCPS even if the latter has been coated with another matrix molecule and blocked with albumin. This means that studies of the interactions of cells with individual coated ECM molecules will be confounded by the presence of adsorbed VN if serum is present in the culture medium. In this study, the adhesion, spreading, growth, and output of endogenous matrix molecules by bovine corneal endothelial (BCE) cells were measured on five different matrix substrates using medium which had been depleted of vitronectin to avoid such confounding effects. The same cell adhesion and spreading maxima were achieved on vitronectin, fibronectin (FN), laminin (LM), and types I and IV collagen (col I, col IV). The coating concentrations required to achieve these maxima, however, differed among the substrates, LM needing considerably higher concentrations than the other substrates for both maximal adhesion and spreading and FN needing higher concentrations for cell spreading. When cells were continuously passaged on each of the five substrates coated at concentrations optimal for cell spreading, no differences in cell proliferation rates or cell morphology were observed. Significant differences, however, were observed in the subcellular output of endogenous matrix molecules (FN, LM, col IV, and thrombospondin) between the different substrates. Col I was a poor substrate for the production of all ECM molecules tested over the 10 passages of the experiment, whereas col IV was a consistently good substrate. LM and FN substrates displayed differential effects on the output of different ECM molecules. VN was unique in that BCE cells at early passage on this substrate produced high levels of endogenous matrix molecules, whereas with continued passage on this substrate, a progressive decline in ECM secretion was observed. These results show that incorporation of individual molecules into the ECM by BCE cells in culture is significantly affected by the nature of the substratum. They further suggest that passage of endothelial cells in media containing serum (which results in coating of VN onto the substrate) may result in a progressive reduction of ECM output.     

Integrin-mediated neurite outgrowth in neuroblastoma cells depends on the activation of potassium channels

Electrical signals elicited by integrin interaction with ECM components and their role in neurite outgrowth were studied in two clones (N1 and N7) isolated from 41A3 murine neuroblastoma cell line. Although the two clones similarly adhered to fibronectin (FN) and vitronectin (VN), this adhesion induced neurite outgrowth in N1 but not in N7 cells. Patch clamp recordings in whole cell configuration showed that, upon adhesion to FN or VN but not to platelet factor 4 (PF4), N1 cells undergo a marked (approximately equal to 20 mV) hyperpolarization of the resting potential (Vrest) that occurred within the first 20 min after cell contact with ECM, and persisted for approximately 1 h before reverting to the time zero values. This hyperpolarization was totally absent in N7 cells. A detailed analysis of the molecular mechanisms involved in N1 and N7 cell adhesion to ECM substrata was performed by using antibodies raised against the FN receptor and synthetic peptides variously competing with the FN or VN binding to integrin receptor (GRGDSP and GRGESP). Antibodies, as well as GRGDSP, abolished adhesion of N1 and N7 clones to FN and VN, revealing a similar implication of integrins in the adhesion of these clones to the ECM proteins. However, these anti-adhesive treatments, while ineffective on Vrest of N7 cells, abolished in N1 cells the FN- or VN-induced hyperpolarization and neurite outgrowth, that appeared therefore strictly associated and integrin-mediated phenomena. The nature of this association was deepened through a comparative analysis of the integrin profiles and the ion channels of N1 and N7 cells. The integrin immunoprecipitation profile resulted very similarly in the two clones, with only minor differences concerning the alpha V containing complexes. Both clones possessed Ca2+ and K+ delayed rectifier (KDR) channels, while only N1 cells were endowed with inward rectifier K+ (KIR) channels. The latter governed the Vrest, and, unlike KDR channels, were blocked by Ba2+ and Cs+. By moving patched cells in contact with FN-coated beads, it was shown that KIR channel activation was responsible for the FN-mediated hyperpolarization of Vrest. Treatment with Pertuxis toxin (PTX) abolished this hyperpolarization and neurite outgrowth, indicating that a G protein is interposed between integrins and KIR channels and that the activation of these channels is required for neuritogenesis. In fact, the block of KIR channels by Cs+ abolished both hyperpolarization and neurite outgrowth, provided that the cation was supplied during the first two hours after N1 cell contact with FN.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characteristics of false-negative smears tested in the normal screening situation.

It is common practice to rescreen false-negative (FN) smears. However, it is inevitable that this is done with some foreknowledge; at least it is known that the test smears contain one or more special cases. Therefore, we decided to test smears in the normal screening situation, when cytotechnologists are completely unaware of being tested. This experiment was done with five FNs and five true-positive (TP) smears. In a third experiment the FNs were tested with the cytotechnologists aware of their presence. Finally, 10 qualities of FNs and TPs were analyzed. In the normal screening situation, only in 1 of 25 tests was the FN recognized as malignant, while all the TPs were detected at the first testing. However, when the cytotechnologists were aware of being tested, the FN was detected in seven of eight tests. The FNs differed from the TPs in 5 of the 10 analyzed characteristics. FNs contained few (if any) large neoplastic epithelial fragments. Detached malignant cells were nearly lacking. The cancer cells had small nuclei that presented little anisokaryosis. The FNs possessed generally hypochromatic nuclei. These features explain why the malignant cells were almost never identified in the normal screening situation. We conclude that FNs and TPs differ and that it seems virtually impossible to avoid all false-negative diagnoses.     

Concurrent changes in sinusoidal expression of laminin and affinity of hepatocytes to laminin during rat liver regeneration.

Distribution of fibronectin, laminin, and collagens type I, III, IV, and V in the lobular regions of regenerating rat liver was studied by indirect immunofluorescence. Little or no laminin was detected in sham-operated controls throughout the experimental period, while it was detected in sinusoids of regenerating liver as early as 6 h after partial hepatectomy (PH). After reaching a maximum at 24 h, it decreased and was barely detectable 6 days after PH. Changes in the other extracellular matrix (ECM) proteins were evident 3 days after PH, but not earlier than 24 h. Hepatocytes isolated from regenerating rat livers were tested in a short term assay for attachment to the substrates coated with the ECM proteins. The attachment of hepatocytes to laminin substrates increased 12 h after PH, reached a maximum at 24 h, and decreased to the control level 6 days after PH, while that of the control remained constant. The attachment to fibronectin substrates was not different between regenerating livers and controls at any time point. The attachment to collagen did not change earlier than 24 h after PH, but increased slightly 3 days after PH. Primary rat hepatocytes cultured on the substrates coated with the ECM proteins were determined for replicative DNA synthesis in response to epidermal growth factor. Both in normal liver and in regenerating liver 24 h after PH, laminin was one of the most effective substrates in supporting the responsiveness of hepatocytes to the growth stimulus. Taken together, these results suggest the importance of hepatocyte-laminin interaction during the early stage of liver regeneration possibly in growth stimulation of hepatocytes and/or maintenance of hepatocyte-specific functions.     

Retroviral expression of alternatively spliced forms of rat fibronectin

We describe the construction in retroviral vectors and the expression of recombinant rat fibronectin (FN) cDNAs corresponding with the various alternatively spliced forms of FN. In NIH 3T3 cells, the exogenous rat FN subunits are efficiently secreted as heterodimers with endogenous mouse subunits. In contrast, in lymphoid WEHI231 cells, there is no endogenous FN synthesis and the recombinant FNs are secreted and can be purified as homogeneous proteins. We show that the purified recombinant FNs are biochemically and biologically functional. In basic assays for adhesion, spreading, cytoskeletal organization, and migration using various established adherent cell lines, different forms of FNs containing the different alternatively spliced segments show no marked differences in activity. We have used these recombinant FNs to investigate three systems in which earlier results had suggested potential differences between different forms of FN. First, all forms tested appear equally active in restoring normal morphology to a transformed cell line. Second, we detect minor differences in their ability to assemble into preexisting extracellular matrices. Finally, we report that only those forms of FN that contain the V segment will promote the spreading of a lymphoid cell line indicating that this segment confers additional biological functions for some cell types, a result that confirms and extends earlier data. These homogeneous, biologically active recombinant FNs will allow further studies of the role of the alternatively spliced segments of FN.     

ECM regulates MT1-MMP localization with beta1 or alphavbeta3 integrins at distinct cell compartments modulating its internalization and activity on human endothelial cells

Regulation of membrane-type 1 matrix metalloproteinase (MT1-MMP) by different extracellular matrices (ECMs) on human endothelial cells (ECs) has been investigated. First, MT1-MMP is found at the intercellular contacts of confluent ECs grown on beta1 integrin-dependent matrix such as type 1 collagen (COL I), fibronectin (FN), or fibrinogen (FG), but not on gelatin (GEL) or vitronectin (VN). The novel localization of MT1-MMP at cell-cell contacts is assessed by confocal videomicroscopy of MT1-MMP-GFP-transfected ECs. Moreover, MT1-MMP colocalizes with beta1 integrins at the intercellular contacts, whereas it is preferentially found with alphavbeta3 integrin at motility-associated structures on migrating ECs. In addition, clustered integrins recruit MT1-MMP and neutralizing anti-beta1 or anti-alphav integrin mAb displace MT1-MMP from its specific sites, pointing to a biochemical association that is finally demonstrated by coimmunoprecipitation assays. On the other hand, COL I, FN, or FG up-regulate cell surface MT1-MMP on confluent ECs by an impairment of its internalization, whereas expression and internalization are not modified on GEL or VN. In addition, MT1-MMP activity is diminished in confluent ECs on COL I, FN, or FG. Finally, MT1-MMP participates and cooperates with beta1 and alphavbeta3 integrins in the migration of ECs on different ECM. These data show a novel mechanism by which ECM regulates MT1-MMP association with beta1 or alphavbeta3 integrins at distinct cellular compartments, thus modulating its internalization, activity, and function on human ECs.     

The changes in glycosylation after partial hepatectomy enhance collagen binding of vitronectin in plasma

Vitronectin is a multifunctional glycoprotein present in the extracellular matrix and plasma. Changes in rat vitronectin were studied during liver regeneration after partial hepatectomy. Carbohydrate concentrations of vitronectin decreased to 2/3 of sham-operated rats at 24 h after partial hepatectomy. Carbohydrate composition and lectin reactivity indicated that N-glycosylation and sialylation of vitronectin changed markedly after partial hepatectomy, while amino acid composition did not change significantly. We previously showed that deN-glycosylation of vitronectin in vitro affects collagen binding among various ligands (Yoneda et al., Biochemistry (1998) 37, 6351-6360). Vitronectins from partially hepatectomized rats at 24 h were found to exhibit markedly enhanced binding to type I collagen. The effect of sialylation on collagen binding was further examined using enzymatically deglycosylated vitronectin of nonoperated rats. Collagen binding increased by 1.2 times after deN-glycosylation of vitronectin, while it increased more than 2.9 times after desialylation. Various glycosyltransferases in liver are known to change after partial hepatectomy, including the attenuation of N-oligosaccharide transferase. The findings therefore suggest that the collagen binding of vitronectin is modulated by the alteration of peptide glycosylation caused by postoperative physiological changes of glycosyltransferases and that the change may contribute to tissue remodeling processes.     

Expression of alphav, alpha4, alpha5 and beta3 integrin subunits, fibronectin and vitronectin in goat peri-implantation

Integrins are glycoprotein heterodimers located in the cell membranes that stimulate intercellular adhesion and act as extracellular matrix (ECM) protein receptors. Although integrins have been detected in the implantation sites of various species, little is known about their participation in ruminant non-invasive placentation. The objective of this study was the detection of alphav, alpha4, alpha5, beta1 and beta3 integrin subunits and of two of their ligands, fibronectin and vitronectin, to determine their participation in the caprine peri-implantation process. On Day 21 post-coitum (pc), endometrial epithelium and trophoblastic cells showed an intense alphav and beta3 integrin subunits expression and moderate staining for alpha4 and alpha5. On Day 23 pc, integrin expression decreased noticeably and only a weak staining of alpha4 and beta3 integrin subunits were observed. No beta1 integrin subunit expression was detected on either of the days studied. Fibronectin (FN) expression in trophectodermic and endometrial epithelium was weak or moderate on the days studied while vitronectin (VN) expression in the same tissues was moderate or strong on Day 21 pc but decreased on Day 23 pc. These results suggest that alphavbeta3 integrin, alpha4 and alpha5 subunits, VN and FN are expressed in caprine endometrium and blastocyst and may play a role in the cascade of the implantation process.     

Fibronectin inhibits osteoclastogenesis while enhancing osteoclast activity via nitric oxide and interleukin‐1β‐mediated signaling pathways

Osteoclasts are bone‐resorbing cells formed by fusion of mononuclear precursors. The matrix proteins, fibronectin (FN), vitronectin (VN), and osteopontin (OPN) are implicated in joint destruction and interact with osteoclasts mainly through integrins. To assess the effects of these matrix proteins on osteoclast formation and activity, we used RAW 264.7 (RAW) cells and mouse splenocytes differentiated into osteoclasts on tissue culture polystyrene (TCP) or osteologic? slides pre‐coated with 0.01–20 µg/ml FN, VN, and OPN. At 96 h, osteoclast number and multinucleation were decreased on VN and FN compared to OPN and TCP in both RAW and splenocytes cell cultures. When early differentiation was assessed, VN but not FN decreased cytoplasmic tartrate‐resistant acid phosphatase activity and pre‐osteoclast number at 48 h. OPN had the opposite effect to FN on osteoclast formation. When RAW cells were differentiated on OPN and treated by FN and OPN, osteoclast number only in the FN treated group was 40–60% lower than the control, while the total number of nuclei was unchanged, suggesting that FN delays osteoclast fusion. In contrast to its inhibitory effect on osteoclastogenesis, FN increased resorption by increasing both osteoclast activity and the percentage of resorbing osteoclasts. This was accompanied by an increase in nitric oxide (NO) levels and interleukin‐1β (IL‐1β). IL‐1β production was inhibited using the NO‐synthase inhibitor only on FN indicating a FN‐specific cross‐talk between NO and IL‐1β signaling pathways. We conclude that FN upregulates osteoclast activity despite inhibiting osteoclast formation and that these effects involve NO and IL‐1β signaling. J. Cell. Biochem. 111: 1020–1034, 2010. © 2010 Wiley‐Liss, Inc.     

Selective adhesion of osteoblastic cells to different integrin ligands induces osteopontin gene expression.

Skeletal homeostasis is partly regulated by the mechanical environment and specific signals generated by a cell's adhesion to the matrix. Previous studies demonstrated that osteopontin (OPN) expression is stimulated in response to both cellular adhesion and mechanical stimulation. The present studies examine if specific integrin ligands mediate osteoblast selective adhesion and whether opn mRNA expression is induced in response to these same ligands. Embryonic chicken calvaria osteoblastic cells were plated on bacteriological dishes coated with fibronectin (FN), collagen type I (Col1), denatured collagen/gelatin (G), OPN, vitronectin (VN), laminin (LN) or albumin (BSA). Osteoblastic cells were shown to selectively adhere to FN, Col1, G and LN, yet not to VN, OPN or BSA. Opn mRNA expression was induced by adhesion to Col1, FN, LN and G, but neither OPN nor VN induced this expression. Examination of the activation of the protein kinases A and C second signaling systems showed that only adhesion to FN induced protein kinase A and protein kinase C (PKC) activity while adherence to Col1 induced PKC. Evaluation of the intracellular distribution of focal adhesion kinase (FAK) and p-tyrosine within cells after adherence to FN, VN or BSA demonstrated that adherence to FN stimulated FAK translocation from the nucleus to the cytoplasm and high levels of p-tyrosine localization at the cell surface. However, cell adherence to VN or BSA did not show these morphological changes. These data illustrate that osteoblast selective adhesion is mediated by specific integrin ligands, and induction of intracellular second signal kinase activity is related to the nature of the ligand.     

Effects of matrix proteins on the expression of matrix metalloproteinase-2, -9, and -14 and tissue inhibitors of metalloproteinases in human cytotrophoblast cells during the first trimester.

The activity of matrix metalloproteinases (MMPs) specifies the ability of the trophoblast cell to degrade extracellular matrix (ECM) substrates. Usually the process of normal human placentation involves a coordinated interaction between the fetal-derived trophoblast cells and their microenvironment in the uterus. In this study, the effects of ECM proteins on the expression of MMP-2, -9, and -14 (membrane-type MMP-1); and the production of tissue inhibitors of metalloproteinase (TIMP) types -1, -2, and -3 have been investigated. Cytotrophoblast cells at 9 or 10 wk of gestation were cultured on various ECM coated dishes under serum-free conditions. Gelatin zymography analysis showed that cells grown on fibronectin (FN), laminin (LN), and vitronectin (VN) secreted more MMP-9 (about 1.5- to 3-fold more) than cells cultured on collagen I (Col I), whereas the secretion of MMP-9 by cells cultured on collagen IV (Col IV) was only half that by the cells on Col I. Northern Blot analysis gave the same results as zymography, indicating that expression of the MMP-9 gene in cytotrophoblast cells can be affected by matrix proteins. There was no significant difference in the expression of MMP-2 either at protein or mRNA levels among the cells cultured on the different matrix substrates. The expression of MMP-14 was regulated in a manner similar to that of MMP-2. Using ELISA, we detected higher levels of TIMP-1 in the culture medium of cells grown on VN, LN, and FN compared with that grown on Col I. But the expression of TIMP-3 mRNA was remarkably inhibited by VN, and ECM proteins had no effect on TIMP-1 and TIMP-2 mRNA expression. It was also observed that cultured cytotrophoblast cells expressed the corresponding receptors for the tested matrix proteins, such as integrins alpha(1), alpha(5), alpha(6), beta(1), and beta(4). Furthermore, the adhesiveness of cytotrophoblast cells on Col I, Col IV, FN, and LN was increased by 62%, 45%, 21%, and 22%, respectively, when compared with adhesiveness on VN. Isolated cytotrophoblast cells remained stationary when cultured on dishes coated with Col I and Col IV, but they assumed a more motile morphology and aggregated into a network when cultured on LN and VN. These data indicate that human trophoblast cells interact with their microenvironment to control their behavior and function.     

Spatial expression of the alternatively spliced EIIIB and EIIIA segments of fibronectin in the early chicken embryo

Using domain-specific antibodies, we have analyzed the tissue distribution of fibronectins (FNs) containing the alternatively spliced EIIIB and EIIIA segments relative to total FN in early chicken embryos. The results show a selective loss of EIIIA+ FN staining in the notochordal sheath and in cartilaginous structures between 4.5 and 7.0 days of development. In other regions, EIIIB+ and EIIIA+ FNs are extensively codistributed in and around mesoderm-derived structures (somites, notochord, heart, and blood vessels), in basal laminae of endoderm and ectoderm-derived structures, as well as within the vicinity of neural crest formation and migration. We also noted that EIIIA staining overlaps with spatial patterns of distribution that have previously been described for the alpha4 integrin subunit, a component of the EIIIA receptor alpha4beta1.     

Bone matrix proteins and extracellular acidification: Potential co‐regulators of osteoclast morphology

Osteoclasts are signaled by the bone matrix proteins fibronectin (FN), vitronectin (VN), and osteopontin (OPN) via integrins. To perform their resorptive function, osteoclasts cycle between compact (polarized), spread (non‐resorbing) and migratory morphologies. Here we investigate the effects of matrix proteins on osteoclast morphology and how those effects are mediated using RAW 264.7 cells differentiated into osteoclasts on FN, VN, and OPN‐coated culture dishes. After 96 h, 80% of osteoclasts on FN were compact while 25% and 16% on VN were in compact and migratory states respectively. In contrast, OPN induced osteoclast spreading. Furthermore, osteoclasts formed on VN and FN were two‐ to fourfold smaller than those formed on OPN in the 21–30 nuclei/osteoclast group. These effects were not due to defects in cytoskeletal reorganization of osteoclasts on VN and FN, demonstrated by the ability of these cells to spread in response to 35 ng/ml macrophage colony stimulating factor (M‐CSF). Conversely, osteoclasts on OPN failed to spread when induced by M‐CSF. Moreover, the extracellular pH on FN and VN (7.25 and 7.3, respectively) was significantly lower than that on OPN (～7.4). We further investigated the role of extracellular pH and found that at pH 7.5 the duration of an osteoclast's compact phase was 25.6 min and that of the spread phase was 62.5 min. Reducing the pH to 7.0 increased the frequency of osteoclast cycling by threefold. These results show that matrix proteins play a role in regulating osteoclast morphology, possibly via altering extracellular and intracellular pH. J. Cell. Biochem. 111: 350–361, 2010. © 2010 Wiley‐Liss, Inc.