Molecular mechanisms of avian neural crest cell migration on fibronectin and laminin

We have examined the molecular interactions of avian neural crest cells with fibronectin and laminin in vitro during their initial migration from the neural tube. A 105-kDa proteolytic fragment of fibronectin encompassing the defined cell-binding domain (65 kDa) promoted migration of neural crest cells to the same extent as the intact molecule. Neural crest cell migration on both intact fibronectin and the 105-kDa fragment was reversibly inhibited by RGD-containing peptides. The 11.5-kDa fragment containing the RGDS cell attachment site was also able to support migration, whereas a 50-kDa fragment corresponding to the adjacent N-terminal portion of the defined cell-binding domain was unfavorable for neural crest cell movement. In addition to the putative "cell-binding domain," neural crest cells were able to migrate on a 31-kDa fragment corresponding to the C-terminal heparin-binding (II) region of fibronectin, and were inhibited in their migration by exogenous heparin, but not by RGDS peptides. Heparin potentiated the inhibitory effect of RGDS peptides on intact fibronectin, but not on the 105-kDa fragment. On substrates of purified laminin, the extent of avian neural crest cell migration was maximal at relatively low substrate concentrations and was reduced at higher concentrations. The efficiency of laminin as a migratory substrate was enhanced when the glycoprotein occurred complexed with nidogen. Moreover, coupling of the laminin-nidogen complex to collagen type IV or the low density heparan sulfate proteoglycan further increased cell dispersion, whereas isolated nidogen or the proteoglycan alone were unable to stimulate migration and collagen type IV was a significantly less efficient migratory substrate than laminin-nidogen. Neural crest cell migration on laminin-nidogen was not affected by RGDS nor by YIGSR-containing peptides, but was reduced by 35% after addition of heparin. The predominant motility-promoting activity of laminin was localized to the E8 domain, possessing heparin-binding activity distinct from that of the N-terminal E3 domain. Migration on the E8 fragment was reduced by greater than 70% after addition of heparin. The E1' fragment supported a minimal degree of migration that was RGD-sensitive and heparin-insensitive, whereas the primary heparin-binding E3 fragment and the cell-adhesive P1 fragment were entirely nonpermissive for cell movement.(ABSTRACT TRUNCATED AT 400 WORDS)     

Two distinct cell-binding domains in laminin can independently promote nonneuronal cell adhesion and spreading [published erratum appears in J Cell Biol 1989 Jun;108(6):following 2546]

Two subfragments of laminin, E8, a major part of the long arm, and E1-4, the three short arms, promote cell adhesion and spreading. Three distinct types of adhesive behavior are seen in short term (1 h) assays, typified by secondary murine fibroblasts, adherent only on fibronectin; secondary murine myoblasts, adherent on fibronectin, laminin, and the E8 fragment; and Rugli human glioblastoma cells, adherent on fibronectin, laminin, E8, and E1-4. E8-specific polyclonal antibodies block myoblast adhesion to E8 and to laminin with identical concentration dependence; Rugli binding to E8 but not to laminin is also totally blocked by these antibodies. Heating of E8 and laminin to approximately 60 degrees C abolishes cell attachment-promoting activity for myoblasts. Adhesion of Rugli cells to E8 is also lost, but on laminin the attachment-promoting activity remains constant. This is due to an increase in the activity of E1-4 fragment as it is heated. Thus, major sites for initial cell adhesion to and spreading on laminin lie within the E8 and E1-4 fragments, but not all cells binding to laminin will bind to both fragments. These data may tentatively be explained by the existence of more than one type of receptor for laminin at the cell surface; one is needed for each fragment.     

Diverse effects of fibronectin and laminin on phenotypic properties of cultured arterial smooth muscle cells

Plasma fibronectin promotes modulation of rat arterial smooth muscle cells from a contractile to a synthetic phenotype during the first few days in primary culture. This process includes cell adhesion and spreading, loss of myofilaments, and formation of a widespread rough endoplasmic reticulum and a prominent Golgi complex. The structural reorganization is accompanied by activation of overall RNA and protein synthesis. Moreover, the cells gain the ability to replicate their DNA and divide in response to platelet-derived growth factor. Here, it is demonstrated that the power of fibronectin to bring about this change in the differentiated properties of the smooth muscle cells resides in a 105-kD cell-binding fragment, whereas a 70-kD collagen-binding fragment and a 31-kD heparin-binding fragment are inactive in this respect. Laminin, another adhesive glycoprotein and a component of the basement membrane that normally surrounds arterial smooth muscle, was contrarily found to maintain the cells in a contractile phenotype. However, with increasing time more and more cells went through the modulation into a synthetic phenotype. This "catch-up" was counteracted by a peptide that contained the cell-attachment sequence of fibronectin (Arg-Gly-Asp-Ser). Hence, it is possible that the delayed modulation on laminin was due to production of fibronectin by the cells themselves. In support of this notion, fibronectin isolated from smooth muscle cultures was found to be as effective as plasma fibronectin in stimulating the phenotypic modulation. Moreover, using a combination of chemical, immunochemical, and immunocytochemical methods, it was demonstrated that the cells secreted fibronectin as well as laminin at an increasing rate during the first 4 d in primary culture and, notably, cells cultured on laminin produced more fibronectin than cells cultured on fibronectin. Newly synthesized fibronectin was incorporated into a network of pericellular and intercellular fibrils, whereas laminin formed a more diffuse layer covering the cells in a basement membrane-like manner. Taken together, the findings suggest diverse roles for fibronectin and laminin in the control of the differentiated properties of arterial smooth muscle cells. They further indicate that the ability of arterial smooth muscle cells to produce fibronectin and laminin early in primary culture is not directly related to the phenotypic state as determined morphologically and by measurement of overall rates of RNA and protein synthesis. This may be due to the cells being able to sense the macromolecular composition of the pericellular matrix and to modify their secretory activity accordingly.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of human C-reactive protein on the cell-attachment activity of fibronectin and laminin

We have previously reported that purified human C-reactive protein (CRP) specifically binds to the cell-binding region of plasma fibronectin (Fn) in a Ca2+-dependent reaction that is saturable at a molar ratio of CRP/Fn of approximately 9. In this study, the binding of CRP to Fn was found to interfere with the cell-attachment promoting activity of Fn. The inhibition of cell attachment was dependent on the concentration of the CRP and involved the phosphorylcholine (PC) binding site of CRP since inhibition was prevented by allowing the CRP to react with either PC (or closely related monophosphate compounds) or a mAb specific for the PC-binding site of CRP. Binding of CRP to laminin was also Ca2+-dependent; however, this binding did not alter the cell-attachment promoting activity of laminin. CRP by itself does not mediate cell attachment. Since CRP is selectively deposited at sites of tissue damage along with plasma Fn and has the ability to bind to Fn and alter its cell-binding activity, CRP may modulate early events in tissue repair.     

Evaluation of extracellular matrix proteins and tissue inhibitor of matrix metalloproteinases-2 on bovine inner cell mass outgrowth in vitro

Effects of extracellular matrix proteins and tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) on bovine inner cell mass (ICM) outgrowth and proteinase production in vitro were determined. Inner cell masses were isolated immunosurgically from day 7 embryos (day 0 = onset of estrus) and cultured for 96 h. In experiment 1, cellular outgrowth and gelatinase production were evaluated for ICM cultured on collagen IV, fibronectin, or laminin. More (P < 0.05) ICM generated cellular outgrowth on fibronectin (71%). compared with collagen IV (0%) or laminin (15%). Inner cell mass and outgrowth areas were greatest (P < 0.05) on fibronectin after 96 h of culture, compared with laminin. Although the incidence of cellular outgrowth on laminin was limited, numbers of cells in outgrowths supported by laminin were similar (P > 0.10) to fibronectin except at 72 h of culture, where more (P < 0.05) cells were in laminin than in fibronectin outgrowths. Gelatinase activity was not detected in conditioned medium. In experiment 2, cellular outgrowth and plasminogen activator production by ICM cultured on fibronectin in medium containing 0 or 10 microg/ml TIMP-2 were evaluated. Inner cell mass and outgrowth areas, and numbers of cells in outgrowths were greater (P < 0.05) in 10 compared with 0 microg/ml TIMP-2 at 96 h of culture. Mean plasminogen activator activity in conditioned medium from ICM cultured in 10 microg/ml TIMP-2 was greater (P < 0.05) compared with 0 microg/ml TIMP-2 (16.2 +/- 4.8 versus 6.7 +/- 1.4 x 10(-3) IU/ml, respectively). These results demonstrate that cellular outgrowth from bovine ICM is supported by fibronectin and is stimulated by TIMP-2.     

Adhesion of platelets to laminin in the absence of activation

The binding of platelets to components in the subendothelial matrix is an initial event in hemostasis and thrombosis. The glycoprotein components of the matrix are considered important in this interaction. Of these, collagen binds and activates platelets and induces their aggregation. In this study we demonstrate that substrate-bound laminin causes time- and concentration-dependent adherence of human platelets to the substrate. The binding of platelets to laminin was found to be similar in some respects, but different in others, to their binding to surfaces coated with fibronectin or collagen. The binding of platelets to laminin or fibronectin was not associated with their activation under conditions in which type I collagen activates the platelets as measured by [14C]serotonin secretion. Platelets bound to laminin and fibronectin differed in their appearance; they remained rounded on laminin whereas they flattened completely on fibronectin. Binding of platelets to fibronectin, but not laminin, is inhibited by a recently described peptide (Pierschbacher, M., and E. Ruoslahti, 1984, Nature (Lond.), 309:30-33) containing the cell-attachment tetrapeptide sequence of fibronectin, which suggests that separate receptors exist for laminin and fibronectin. These studies establish laminin as a platelet-binding protein and suggest that laminin can contribute to the adhesiveness of exposed tissue matrices to platelets. Since laminin and fibronectin do not activate platelets, whereas collagen does, and laminin differs from fibronectin in that it does not induce spreading of the attached platelets, all three proteins appear to confer different signals to the platelets. Some of these may be related to platelet functions other than those necessary for the formation of a hemostatic plug.     

A cell attachment peptide from human C-reactive protein.

The serum acute phase reactant, C-reactive protein (CRP), is selectively deposited at sites of tissue damage and degraded by neutrophils into biologically active peptides. A synthetic peptide corresponding to residues 27-38 present in each of the five identical subunits of CRP mediated cell attachment activity in vitro. Although the CRP-derived peptide contains a Tuftsin (TKPR)-like sequence at its amino-terminus, the Tuftsin tetrapeptide itself, as well as several synthetic peptides of CRP, failed to inhibit the cell-attachment activity to the CRP-derived peptide. Peptides containing the sequences responsible for the cell attachment activity of the extracellular matrix proteins, fibronectin (Fn) and laminin, failed to inhibit the CRP-derived peptide cell attachment activity. However, the addition of the RGDS and RGDSPASSLP cell-binding peptides of Fn to cells enhanced attachment to the active peptide from CRP. In the converse experiment, the cell-binding peptide of CRP did not influence cell attachment to Fn or laminin. A peptide corresponding to the same stretch of amino acid residues within the homologous Pentraxin, serum amyloid P-component (SAP), displayed nearly identical cell-attachment activity. Several monoclonal antibodies (mAb) specific for the CRP-derived cell-binding peptide neutralized its cell-attachment activity. These mAbs reacted with intact CRP and neutralized the cell-binding activity of CRP itself. The findings suggest that a peptide with cell-binding activity could be generated from the breakdown of CRP and then contribute directly to cellular events leading to tissue repair.     

锂对小鼠高增殖潜能集落形成细胞和粒巨噬系祖细胞体外增殖…

本文观察了锂对ＢＡＬＢ／Ｃ小鼠骨髓高增殖潜能集落形成细胞和粒巨噬系祖细胞ＣＦＵ－ＧＭ体外增殖的影响。ＨＰＰ－ＣＦＣ集落由ＩＬ－１,ＩＬ－６,ＷＥＨＩ３条件培养液及Ｌ９２９条件培养液所支持,而ＣＦＵ－ＧＭ由ＷＥＨＩ３－ＣＭ所支持。结果显示,ＬｉＣｌ浓度在０．４－２ｍｍｏｌ／Ｌ时呈现剂量依赖性抑制ＨＰＰ－ＣＦＣ增殖;而在０．４－１ｍｍｏｌ／Ｌ的浓度范围内,则对ＣＦＵ－ＧＭ的增殖起剂量依赖性促进作用。     

Evidence for involvement of more than one class of glycoprotein in cell interactions with fibronectin

The receptor mechanism by which cells attach to fibronectin has been investigated by a combined immunologic and electrophoretic approach. One particular antiserum directed against 3T3 cell plasma membranes was found to contain antibodies that blocked spreading of these murine cells on fibronectin but not on laminin or serum spreading factor (vitronectin). Proteolysis experiments confirmed that this cell line has calcium-protected polypeptides necessary for cell spreading on fibronectin. Consequently, protein antigens were fractionated according to size by SDS gel electrophoresis, and antigens that could block the inhibitory activity of the polyclonal antibody were identified. One class of blocking antigen appeared to correspond to the 140,000-dalton complexes favored by several laboratories as fibronectin receptor candidates, but a second class of 45,000 daltons was also apparent. This 45,000-dalton antigen was a major absorbing activity from 3T3 cell membranes and the predominant activity from L929 membranes. By isoelectric focusing and two-dimensional gel electrophoresis, it was found to exist as a set of isoelectric point variants with pK = 5.3 to 6.2. Our results indicate that current models postulating a simple, unimolecular receptor mechanism for fibronectin may be oversimplified and that fibronectin may instead interact with more than one protein receptor component on the fibroblast cell surface.     

Subunit structure of a laminin-binding integrin and localization of its binding site on laminin

A laminin receptor was isolated from human MG-63 osteosarcoma cells by affinity chromatography on human laminin. The isolated receptor was defined as the alpha 3 beta 1 integrin by immunoprecipitation with subunit-specific antibodies. A previously unclassified laminin-binding integrin from rat cells was shown also to contain the alpha 3 subunit. Both receptors bound to human and mouse laminin in a radioreceptor assay. They also both bound to some extent to fibronectin in this assay, but only the MG-63 cell receptor showed binding to type IV collagen. The binding of the radiolabeled receptor to insoluble laminin was inhibited by unlabeled receptor, by soluble laminin, and by chymotryptic fragments of laminin that have previously been shown to contain neurite-promoting and cell attachment-promoting activities. Moreover, the receptor binding was also inhibited by monoclonal antibodies capable of inhibiting the neurite-promoting activity of laminin and known to bind to laminin near the junction of the long arm and its terminal globule. One of these antibodies was reactive with fusion proteins expressed from laminin cDNA clones. The immunoreactive clones corresponded to the COOH-terminal end of the B1 subunit. These results identify the integrin-type laminin receptor isolated from the osteosarcoma cells as the alpha 3 beta 1 integrin and localize its binding site in close proximity of the B1 subunit COOH terminus.     

Characterization of a second protein (CM2) encoded by RNA segment 6 of influenza C virus.

The biochemical properties of a second protein (CM2) encoded by RNA segment 6 of influenza C virus were investigated. Three forms of CM2 with different electrophoretic mobilities (CM2(0), CM2a, and CM2b) were detected in infected cells by immunoprecipitation with antiserum to the glutathione S-transferase (GST)-CM2 fusion protein. Treatment of infected cells with tunicamycin and digestion of immunoprecipitated proteins with endoglycosidase H or peptide-N-glycosidase F suggested that a mannose-rich oligosaccharide core is added to unglycosylated CM2(0) (Mr, approximately 16,000) to form CM2a (Mr, approximately 18,000) and that the processing of the carbohydrate chain from the high-mannose type to the complex type converts CM2a into CM2b, which is heterogeneous in electrophoretic mobility (Mr, approximately 22,000 to 30,000). Labeling of infected cells with [3H]palmitic acid showed that CM2 is fatty acylated. The fatty acid bond was sensitive to treatment with hydroxylamine and mercaptoethanol, which indicates a labile thioester-type linkage. The CM2 protein was also found to form disulfide-linked dimers and tetramers on sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions. Trypsin treatment of infected cell surfaces as well as of microsome vesicles from infected cells followed by immunoprecipitation with antiserum to the GST fusion protein containing the 56 C-terminal amino acid residues of CM2 suggested that this C-terminal domain is intracellular and exposed to the cytoplasms of microsomes. Furthermore, evidence that a small amount of CM2 is incorporated into progeny virus particles was obtained by Western blot analysis. These results, altogether, suggest that CM2 is an integral membrane protein with biochemical properties similar to those of influenza A virus M2 and influenza B virus NB proteins.     

Localization and synthesis of entactin in seminiferous tubules of the mouse.

Localization and synthesis of entactin in seminiferous tubules of mouse testis was studied by immunocytochemistry. Frozen sections from adult mice testes were subjected to anti-entactin and anti-laminin immunofluorescence. Both entactin and laminin were localized within the seminiferous tubule basement membrane and intertubular region of the testis. The addition of excess amount of entactin (but not fibronectin), premixed with anti-entactin antiserum, abolished the immunostain. Western blotting showed that a protein extract from a seminiferous tubule basement membrane preparation was recognized by anti-entactin anti-serum and comigrated with recombinant entactin. Enriched fractions of isolated primary Sertoli cells and peritubular myoid cells cultured for 6 days on a glass coverslip were able to synthesize and secrete entactin as detected by immunofluorescence microscopy. Entactin was also produced by TM3 (Leydig-like) and TM4 (Sertoli-like) cell lines as detected by both immunofluorescence and Western blotting. The distribution of entactin vs. laminin within both the cultured primary cells and the TM3 and TM4 cell lines differed. Entactin appeared mainly localized extracellularly. In contrast, laminin was mainly localized intracellularly. The above findings suggested that 1) entactin existed in the seminiferous tubule basement membrane and intertubular region of adult mice testis, co-localized with laminin; 2) entactin was synthesized by the cultured primary Sertoli cells and peritubular myoid cells and the TM3 and TM4 cell lines; 3) entactin was exocytosed with little intracellular accumulation, in contrast to an intracellular accumulation of laminin.     

Identification of Meningeal Cell Released Neurite Promoting Activities for Embryonic Hippocampal Neurons

Primary cultures of meningeal cells from embryonic rat cerebra secrete neurite growth-inducing components into serum-free culture medium. This conditioned medium (CM) was analyzed by FPLC and immunochemical and enzymatic treatments and tested for neurite promoting activity (NPA) in a quantitative bioassay using hippocampal neurons from embryonic rat. By immunoprecipitation or specific adsorption we identified laminin (LN)-proteoglycan complexes and fibronectin (FN), respectively, as the major neurite promoting components within meningeal cell CM. The LN-proteoglycan complexes and their NPA were sensitive to chondroitinase (chondroitin ABC lyase, EC 4.2.2.4) and to a smaller extent to heparitinase (heparitin sulfate lyase, EC 4.2.2.8). Minor fractions of the total NPA in CM correlated with free LN and a putative but not yet characterized FN-proteoglycan complex.     

Production and characterization of functional domains of human fibronectin expressed in Escherichia coli.

An efficient expression system was constructed in Escherichia coli that produced a 33-kDa fragment, C-274, of human fibronectin with a strong cell-adhesive activity. The entire sequence of the heparin-binding domain with 271 amino acids, H-271, was also expressed. Deletion analysis of the type III repeats showed that the heparin-binding site was at type III-13. The cell-adhesive activity of a fusion protein, CH-271, containing the cell- and the heparin-binding domains was twice that of C-274 when BHK but not B16-F10 melanoma cells were tested; H-271 alone was inactive. Recombinant proteins containing the CS1 sequence of the IIICS region were more active than C-274 and CH-271 with B16-F10. However, H-296, which contained both H-271 and CS1, was almost inactive with BHK. CH-296, which contained CS1 at the C-terminus of CH-271, was more active with B16-F10 than H-296 and C-CS1, which was produced by the deletion of H-271 from CH-296. Thus, the cell-binding domain was active with both kinds of cells. The heparin-binding domain promoted the adhesion of both kinds of cells only when linked to the cell-binding domain or CS1. CS1 was specific for the adhesion of B16-F10 but was not essential.     

Production of laminin and fibronectin by Schwannoma cells: cell-protein interactions in vitro and protein localization in peripheral nerve in vivo

We studied a rat Schwannoma cell line (RN22F) to determine if it produced the basement membrane glycoproteins laminin and fibronectin, and how it interacted with these proteins in vitro. We used antisera to laminin and fibronectin for immunoprecipitation experiments and immunocytochemical localization at the electron microscope level. Polyacrylamide gels of antilaminin immunoprecipitates of conditioned medium and solubilized Schwannoma cells contained bands of reduced Mr 200,000 and 150,000. Antilaminin immunoprecipitates of conditioned medium contained nonreduced bands of 850,000 daltons and 150,000, and immunoprecipitates of solubilized cells contained nonreduced bands of 850,000, 400,000, 200,000, and 150,000 daltons. Antifibronectin immunoprecipitates of conditioned medium contained a reduced band of 220,000 daltons, and nonreduced bands of 440,000 and 220,000 daltons. Radio-labeled protein was not detected in antifibronectin immunoprecipitates of solubilized cells. By immunocytochemistry, laminin was found along the cell surface in a continuous band, whereas fibronectin was only sparsely distributed along the cell surface. In cell adhesion assays, Schwannoma cells bound preferentially to laminin- coated substrates as compared to fibronectin or noncoated substrates. A number of Schwannoma cells displayed a curved and elongated morphology on laminin substrates, as compared to a uniformly spread morphology on fibronectin, and a round, nonspread morphology on noncoated substrates. Immunofluorescent staining showed laminin in the endoneurium and perineurium and fibronectin predominantly in the perineurium of mouse sciatic nerve in vivo. The production of laminin and fibronectin by Schwann cells may be important in the development and myelination of peripheral nerves, and the proper regeneration of axons following nerve injury.     

Identification of fibronectin receptors on T lymphocytes

We report the identification of fibronectin receptors on thymocytes and T lymphoma cells. Affinity chromatography of extracts of the T cell lymphoma, WR16.1, on a fibronectin-Sepharose column combined with specific elution using a synthetic peptide containing the cell attachment-promoting sequence, arginine-glycine-aspartic acid, yielded two polypeptide components having apparent molecular masses of approximately 160 kD reduced and 175 and 150 kD nonreduced. Immunoprecipitations from surface-iodinated WR16.1 cells or fibronectin-adherent thymocytes using a rabbit antiserum raised against the fibronectin receptor that is present on human fibroblasts revealed, in each case, the same two radiolabeled components. In contrast, immunoprecipitation from fibronectin-nonadherent T lymphoma cells, designated WR2.3, revealed the presence of only the smaller subunit. Although the lymphocyte receptor and the fibronectin receptor identified on fibroblasts share immunologic determinants, they differ in that the molecular mass of the lymphocyte protein is larger. Moreover, trypsinization of either thymocytes or the WR16.1 T lymphoma cells resulted in a subsequent loss of their ability to adhere to fibronectin-coated substrates and a reduction in the electrophoretic mobility of each of the polypeptide chains of the fibronectin receptor present on their surfaces. These changes, however, were not observed with normal rat kidney fibroblasts or mouse 3T3 fibroblasts in response to trypsinization. The data establish the existence on normal lymphocytes of fibronectin receptors that are quite similar to those found on fibroblasts. The possible function of this molecule on thymocytes is discussed.     

PKC/MAPK signaling suppression by retinal pericyte conditioned medium prevents retinal endothelial cell proliferation

Little is known about the regulation mechanism of endothelial cell proliferation by retinal pericytes. The purpose of this study was to elucidate the suppression mechanism of retinal capillary endothelial cell growth by soluble factors derived from retinal pericytes. Conditioned medium of retinal pericytes (rPCT1-CM) suppressed ischemia-induced retinal neovascularization. The growth and DNA synthesis of TR-iBRB2 cells, a conditionally immortalized rat retinal capillary endothelial cell line, were suppressed in a concentration-dependent manner by concentrated rPCT1-CM. The number of human cultured endothelial cells was also reduced by rPCT1-CM. These results provide the first evidence that CM from the cultivation of pericytes alone can inhibit retinal neovascularization in vivo and in vitro. Although the growth reduction of TR-iBRB2 cells was only partly reversed by treatment of rPCT1-CM with antibodies to transforming growth factor-beta1, it was completely lost by heat-treatment of rPCT1-CM, suggesting that anti-angiogenic factors are soluble proteins. The levels of expression of G1/S-phase-related proteins, such as cyclin D1, cyclin-dependent kinase (cdk)4, cdk6, and proliferating cell nuclear antigen, were reduced and a cdk inhibitor, p21(Cip1), was induced in rPCT1-CM-treated TR-iBRB2 cells. Moreover, phosphorylated p44/42 mitogen-activated protein kinase (p44/42 MAPK) in TR-iBRB2 cells was reduced by rPCT1-CM treatment and phosphorylated protein kinase C (PKC)alpha/betaII, which is upstream of p44/42 MAPK, was also suppressed. In conclusion, CM from retinal pericytes suppresses PKC-p44/42 MAPK signaling, inhibits endothelial cell growth, and prevents retinal neovascularization. Anti-angiogenic factors derived from retinal pericytes are likely to play a critical role in the regulation of retinal endothelial cell growth.     

Effect of serum,fibronectin, and laminin on adhesion of rabbit intestinal epithelial cells in culture

Rabbit intestinal epithelial cells, obtained after a limited hyaluronidase digestion, were incubated in medium with or without calf serum, on bacteriological plastic dishes. The dishes, either plain or coated with an air-dried type I collagen film, were pretreated with medium alone or with medium containing purified laminin or purified fibronectin. Cells did not attach in significant numbers to untreated bacteriological plastic, even in the presence of serum. Cells did attach to collagen-coated dishes, and were judged viable on the basis of their incorporation of radiolabeled leucine into cell protein. Cell adhesion to the collagen substrate increased in proportion to the concentration of serum in the medium, with maximal attachment at 5% serum or greater. Pretreatment of plain or collagen-coated dishes with increasing amounts of fibronectin enhanced cell adhesion in a concentration-dependent manner. Either serum, or fibronectin-free serum in the medium enhanced cell attachment to substrates pretreated with cither fibronectin or laminin. Thus, intestinal epithelial cells appear to possess surface receptors for both laminin and fibronectin. The evidence further suggests that calf serum may contain factors, other than fibronectin, capable of enhancing intestinal epithelial cell attachment to collagen substrates.     

Embryonal carcinoma cells adhere preferentially to fibronectin and laminin but their endodermal differentiation leads to a reduced adherence to laminin

F9 and PC13 embryonal carcinoma (EC) cells adhered rapidly to growth substrata coated with fibronectin or laminin. When F9 cells were induced to differentiate into visceral or parietal endoderm-like cells, their ability to adhere to laminin diminished, but their adherence to fibronectin remained unchanged. Correspondingly, permanently differentiated teratocarcinoma-derived endoderm cells (PYS-2 and PSA-5e) adhered markedly less efficiently to laminin than to fibronectin. F9 cells adhered to proteolytic fibronectin fragments containing the cell-binding site but not to fragments containing gelatin- or heparin-binding sites. They also adhered slowly to gelatin, but this adhesion was completely blocked by cycloheximide. The results show that the teratocarcinoma stem cells may have specific mechanisms mediating adhesion to fibronectin and laminin and that endodermal differentiation leads to a reduction in their capacity to adhere to laminin but not to fibronectin.     

Laminin-10/11 and fibronectin differentially regulate integrin-dependent Rho and Rac activation via p130(Cas)-CrkII-DOCK180 pathway

The alpha(5) chain-containing laminin isoforms, laminins-10 and -11 (laminin-10/11), are the major components of the basement membrane, having potent cell-adhesive activity. We examined the cell-adhesive and integrin-mediated signaling activities of laminin-10/11 in comparison to fibronectin, the best characterized extracellular adhesive ligand. We found that laminin-10/11 are more active than fibronectin in promoting cell migration and preferentially activate Rac, not Rho, via the p130(Cas)-CrkII-DOCK180 pathway. Cells adhering to fibronectin develop stress fibers and focal contacts, whereas cells adhering to laminin-10/11 do not, consistent with the high cell migration-promoting activity of laminin-10/11. Pull-down assays of GTP-loaded Rac and Rho demonstrated the preferential activation of Rac on laminin-10/11, in contrast to the activation of Rho on fibronectin. Activation of Rac by laminin-10/11 was associated with the phosphorylation of p130(Cas) and an increased formation of a p130(Cas)-CrkII-DOCK180 complex. Cell migration on laminin-10/11 was suppressed by the expression of either a dominant-negative Rac or CrkII mutants defective in p130(Cas) or DOCK180 binding. This is the first report demonstrating a distinct activation of Rho family GTPases resulting from adhesion to different extracellular ligands.