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Biomedical subjects

F Grinnell

Publications and source records attributed to F Grinnell.

At least 55 records · Page 3Linked to original sources
PubMed1991

Altered processing of integrin receptors during keratinocyte activation.

We used monoclonal antibodies against specific integrin subunits to examine the role of integrin receptors in keratinocyte activation. We found that before activation, beta 1 subunits in keratinocytes showed a diffuse distribution, whereas after activation, keratinocytes organized beta 1 receptors into marginal adhesion plaques. In immunoprecipitation experiments with antibodies against beta 1 integrin subunits, we found mostly immature subunits synthesized in keratinocytes freshly harvested from skin. Moreover, integrin receptor complexes immunoprecipitated from these cells by monoclonal antibodies against alpha 2, alpha 3, or alpha 5 subunits contained only immature beta 1 subunits. With keratinocytes cultured 4-7 days, anti-beta 1 antibodies immunoprecipitated mostly mature beta 1 subunits, and integrin complexes immunoprecipitated from cultured cells by anti-alpha subunit antibodies contained mostly mature beta 1 subunits. Antibodies directed against beta 1 subunits also inhibited keratinocyte migration. Based on these results, we suggest that up-regulation of migration by activated keratinocytes depends on changes in processing of pre-beta 1 subunits to mature beta 1 subunits. We also studied the distribution of integrin subunits in skin and on keratinocytes migrating out of skin explants. Whereas beta 1, alpha 2, and alpha 3 subunits were detected in keratinocytes in skin and migrating out of explants, alpha 5 subunits were observed only in migrating cells.

Cell Differentiation↗
PubMed1991

Role of integrin receptors in manganese-dependent BHK cell spreading on albumin-coated substrata.

In manganese-containing medium, tissue cells can spread on albumin and other substrata typically nonadhesive for cells in calcium/magnesium-containing medium. To learn whether integrin receptors play a role in Mn-dependent adhesion, we tested the effects of RGD peptides and polyclonal anti-fibronectin receptor antibodies on BHK cell spreading on fibronectin and albumin-coated substrata. In Ca/Mg-containing medium on fibronectin substrata, the RGD-related peptides GRG-DSP and GRGDS but not RGDS inhibited cell spreading. In Mn-containing medium, spreading on albumin was inhibited by GRGDSP and GRGDS and also by RGDS. GRGESP, on the other hand, did not inhibit cell spreading under any condition tested. Antibodies directed against fibronectin receptors also inhibited Mn-dependent cell spreading on albumin substrata, but higher levels of antibody were required than were necessary to inhibit Ca/Mg-dependent spreading on fibronectin. On the basis of these results, we suggest that integrin receptors, but probably not fibronectin receptors, mediate Mn-dependent BHK cell spreading on albumin.

Albumins↗
PubMed1991

Stress relaxation of contracted collagen gels: disruption of actin filament bundles, release of cell surface fibronectin, and down-regulation of DNA and protein synthesis.

Relaxation of stressed collagen gels provides a model system uniquely suited to studying the regulation of cell morphology and biosynthetic function by tissue organization. Stress relaxation results in rapid, synchronous changes in cell morphology without enzymatic or other drug treatments, and makes possible an analysis of the initial cellular events associated with changes in tissue organization. During the first hour after stress relaxation, we observed transient hypercontraction of collagen gels and loss of collagen fibril organization as stress in the system dissipated. Morphological changes in the fibroblasts included retraction of pseudopodia, collapse of cytoplasmic actin filament bundles, and loss of cell surface fibronectin. Accompanying these morphological changes, we observed marked decreases in DNA and protein synthesis, especially of fibronectin and type I procollagens. These results show that changes in tissue organization can exert rapid and profound effects on the morphology and biosynthetic function of cells within the tissue.

Actin Cytoskeleton↗
PubMed1990

Spatial organization of extracellular matrix and fibroblast activity: effects of serum, transforming growth factor beta, and fibronectin.

The goal of our research is to understand reciprocal relationships between cell function and tissue organization. We studied the regulation of fibroblast activity in an in vitro culture model that recapitulates in continuous fashion the cycle of events occurring during connective tissue repair. We present evidence that concomitant with spatial reorganization of the extracellular matrix, there was a dramatic decline in extracellular matrix synthesis and cell proliferation. Therefore, spatial reorganization was a crucial turning point for fibroblast activity. Factors that regulated the timing of spatial reorganization included serum, transforming growth factor beta, and fibronectin. By accelerating spatial reorganization of the cultures, transforming growth factor beta led to a relative decrease in cell proliferation and extracellular matrix synthesis. By retarding spatial reorganization of the cultures, fibronectin led to a relative increase in cell proliferation and extracellular matrix synthesis. The results indicate that spatial information in the three-dimensional cell-matrix interaction permits higher order, tissue-level regulation of fibroblast function.

Blood Proteins↗
PubMed1990

Activation of human keratinocyte migration on type I collagen and fibronectin.

The purpose of our studies was to learn more about the regulation of keratinocyte migration. Human keratinocytes freshly harvested from skin were relatively immotile cells, whereas keratinocytes harvested from cell culture migrated on type I collagen or fibronectin as measured in a phagokinesis assay. Development of migratory competence by keratinocytes varied depending on the culture substratum. Cells cultured on plastic were activated more quickly and to a greater extent than cells cultured on dermis. The effect of the culture substratum on migratory competence was reversible. That is, cells cultured on plastic showed reduced activity after subculture on dermis. Cells cultured on dermis showed increased activity after subculture on plastic. Freshly isolated as well as cultured keratinocytes contained beta 1 integrin subunits, but only cultured cells were able to organize the subunits into focal adhesions. These adhesion sites also contained vinculin. In epidermal explants, beta 1 integrin subunits were mostly in basal cells, often more prominent between lateral cell borders than at the epidermal-dermal interface. In keratinocytes that migrated out of skin explants, there appeared to be an increase in the intensity of beta 1 integrin subunit immunostaining, possibly because of the change in shape of migrating cells. Also, beta 1 integrin subunits were found around and beneath migrating keratinocytes. These results show that changes in the distribution of beta 1 integrin subunits accompany development of migratory competence.

Cell Adhesion↗
PubMed1990

The activated keratinocyte: up regulation of cell adhesion and migration during wound healing.

Keratinocytes play different roles in normal and wounded skin. In normal skin, these cells are specialized for differentiation. In wounded skin, they are specialized for migration. This article discusses the idea that migratory-competent keratinocytes are "activated." Changes that characterize the activation process include increased cell attachment, spreading, focal adhesion formation, and migration. In part, these changes can be explained by enhanced expression of alpha 5 integrin subunits and reorganization of beta 1 integrin subunits.

Cell Adhesion↗
PubMed1990

Fibronectin profiles in normal and chronic wound fluid.

Fibronectin, an adhesion protein found in blood and tissues, is an important factor in wound repair. Recently, we began studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting to compare fibronectin in the fluid of normal and chronic wounds. We found similar fibronectin profiles in suction blister fluid (2 normal volunteers) and mastectomy fluid (5 patients). There was primarily an intact 250 kilodalton fibronectin subunit and in addition some higher molecular mass fibronectin-containing complexes. On the other hand, fibronectin in diabetic ulcer wound fluid (2 patients) was partially degraded, and there was no intact fibronectin in stasis ulcer wound fluid (2 patients). The most prominent degradation products were 93 and 125 kilodalton polypeptides. When intact fibronectin was added back to stasis ulcer wound fluid, fragmentation occurred within 15 minutes at 37 degrees C indicating the presence of protease activity in the wound fluid.

Biopsy↗
PubMed1989

Fibronectin and fibrinolysis are not required for fibrin gel contraction by human skin fibroblasts.

Human skin fibroblasts contracted fibrin gels in a time- and cell-dependent manner. Under optimal conditions, gel contraction amounted to more than 50% in 2 hr. Fibronectin did not promote contraction, and fibrinolysis was not required for contraction, although gels contracted without serum or aprotinin were lysed. Before contraction, fibrin was present in loosely packed, randomly organized fibrils. After contraction, the fibrils were more densely packed and aligned in the plane of cell spreading. Cycloheximide treatment of fibroblasts inhibited gel contraction in serum-free medium but not in serum-containing medium. Fibronectin could not substitute for serum in overcoming the cycloheximide effect. Binding sites for fibrin were distributed randomly over the cells' surfaces based on electron microscopic observations. Often small groups of fibrils were localized in indentations at the cell surface. Finally, peptides containing the arg-gly-asp-ser sequence inhibited gel contraction.

Antigens, Surface↗
PubMed1989

Collagen processing, crosslinking, and fibril bundle assembly in matrix produced by fibroblasts in long-term cultures supplemented with ascorbic acid.

Human foreskin fibroblasts were cultured for up to 6 weeks in medium supplemented with ascorbic acid. During this time, the cells produced an extensive new connective tissue matrix in which the accumulated collagen (mostly type I) amounted to about 0.25 mg/10(6) cells. The matrix was highly differentiated as shown by complete processing of procollagen to collagen alpha-chains and covalent crosslinking of the collagen. Alignment of collagen fibrils occurred as the fibrils were deposited between cells, and binding of adjacent fibrils to the cell surface appeared to hold the fibrils in register. Groups of aligned fibrils were subdivided into bundles by cell-surface folds. If beta-aminopropionitrile was added to the medium, collagen crosslinking was inhibited, but not collagen synthesis or fibril bundle organization. If ascorbic acid was omitted from the culture medium, the extensive new connective tissue matrix was not produced. Our results indicate that fibroblasts in long-term cultures supplemented with ascorbic acid produce a connective tissue matrix with many in vivo-like properties including supermolecular organization of collagen.

Aminopropionitrile↗
PubMed1989

Extracellular matrix organization modulates fibroblast growth and growth factor responsiveness.

To learn more about the relationship between extracellular matrix organization, cell shape, and cell growth control, we studied DNA synthesis by fibroblasts in collagen gels that were either attached to culture dishes or floating in culture medium during gel contraction. After 4 days of contraction, the collagen density (initially 1.5 mg/ml) reached 22 mg/ml in attached gels and 55 mg/ml in floating gels. After contraction, attached collagen gels were well organized; collagen fibrils were aligned in the plane of cell spreading; and fibroblasts had an elongated, bipolar morphology. Floating collagen gels, however, were unorganized; collagen fibrils were arranged randomly; and fibroblasts had a stellate morphology. DNA synthesis by fibroblasts in contracted collagen gels was suppressed if the gels were floating in medium but not if the gels were attached, and inhibition was independent of the extent of gel contraction. Therefore, growth of fibroblasts in contracted collagen gels could be regulated by differences in extracellular matrix organization and cell shape independently of extracellular matrix density. We also compared the responses of fibroblasts in contracted collagen gels and monolayer culture to peptide growth factors including fibroblast growth factor, platelet-derived growth factor, transforming growth factor-beta, and interleukin 1. Cells in floating collagen gels were generally unresponsive to any of the growth factors. Cells in attached collagen gels and monolayer culture were affected similarly by fibroblast growth factor but not by the others. Our results indicate that extracellular matrix organization influenced not only cell growth, but also fibroblast responsiveness to peptide growth factors.

Cell Adhesion↗
PubMed1989

The collagen recognition sequence for fibroblasts depends on collagen topography.

We found that the peptide Gly-Arg-Gly-Glu-Ser-Pro (GRGESP) inhibited spreading of human fibroblasts inside collagen gels and markedly decreased gel contraction, but this peptide had no effect on cell spreading on collagen-coated surfaces. On the other hand, the peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), which inhibited cell spreading on collagen-coated surfaces, did not inhibit cell spreading within collagen gels and was a less effective inhibitor of collagen gel contraction than GRGESP. Based on these findings, we conclude that human fibroblasts can interact with different collagen cell recognition sequences depending upon topographical organization of the collagen.

Amino Acid Sequence↗
PubMed1989

Enhanced binding of fibronectin-coated latex beads to quiescent 3T3-L1 cells is correlated with escape from growth arrest.

The possible involvement of fibronectin receptors in growth stimulation was investigated by an analysis of fibronectin-coated latex bead binding to 3T3-L1 cells under various conditions. 3T3-L1 cells, growth-arrested in a medium with a low concentration of calf serum, bound few fibronectin-coated beads. After addition of serum at concentrations of 1.0% or higher, there was a rapid and transient increase in the number of cells with bound beads and a subsequent increase in the incorporation of bromodeoxyuridine (BrdU) into cell nuclei. Incorporation of BrdU was observed in about 60% of the cells with bound beads. Fibroblast growth factor and platelet-derived growth factor at concentrations of 5 ng/ml or higher also enhanced binding of fibronectin-coated beads to cells. Stimulation of bead binding by epidermal growth factor and insulin was weak. Fibroblast growth factor, but not epidermal growth factor, increased the incorporation of BrdU into nuclei. These results indicate a relationship between stimulation of cell proliferation in quiescent cells and increased binding by cells of fibronectin-coated latex beads.

Animals↗
PubMed1989

Long-term culture of fibroblasts in contracted collagen gels: effects on cell growth and biosynthetic activity.

The purpose of these studies was to analyze the consequences of long-term collagen gel contraction on fibroblast growth and metabolic activity. After 4 weeks, floating gels were 98% contracted, and attached gels were 94% contracted. During this culture period, fibroblasts in floating gels regressed significantly compared to fibroblasts in attached gels, although the cells remaining in the floating gels were viable. In attached gels, fibroblasts were bipolar; whereas in floating gels, fibroblasts were stellate. Therefore, differences between survival of fibroblasts in attached and floating collagen gels might depend on cell shape. Similarly, extracellular matrix organization and its influence on cell shape might control fibroblast proliferation in granulation tissue. During long-term culture of fibroblasts in contracted collagen gels, 70%-80% of the starting collagen was degraded. Collagen synthesized by cells in 4-d cultures was mostly procollagen secreted into the medium. On the other hand, collagen synthesized in 4-week cultures was processed to alpha (I) chains and incorporated into the matrix. There also were other differences between the proteins synthesized by fibroblasts after short-term and long-term culture in contracted gels. These findings show that fibroblasts in long-term collagen gel cultures express unique growth and biosynthetic characteristics.

Cells, Cultured↗
PubMed1989

Basement membrane and human epidermal differentiation in vitro.

To test the role of basement membrane in differentiation of human epidermis reconstituted on human dermis, we prepared dermis with and without basement membrane and cultured epidermal cells on these two dermal substrata. The absence of basement membrane components was confirmed by immunofluorescence staining for laminin and type IV collagen and by electron microscopy. A high degree of differentiation of reconstituted epidermis did not require basement membrane as shown by the development of basal, spinous, and granular cell layers, and synthesis of 58 and 65-67 kDa keratins when epidermis was attached directly to dermis. On the other hand, we found that the basement membrane regulated the adhesive interaction between the epidermis and dermis. On dermis with basement membrane, attached epidermal cells formed hemidesmosomes and mechanically stable bonding. In the absence of basement membrane, the epidermal cells did not form hemidesmosomes, and bonding between the epidermis and dermis was unstable. Moreover, dermis from which the basement membrane was removed was reorganized by the epidermal cell layer.

Basement Membrane↗
PubMed1988

Wheat-germ-agglutinin and Ricinus communis-agglutinin-binding sites of BHK cells compared with each other and with 140 kDa fibronectin receptors.

We compared the wheat-germ agglutinin (WGA) and Ricinus communis agglutinin (RCA) binding sites of baby-hamster kidney (BHK) cells. There were 1.01 X 10(8) WGA-binding sites per cell (Kd = 0.027 nM) and 6 X 10(6) RCA-binding sites per cell (Kd = 0.014 nM). Binding of WGA or RCA to BHK cells resulted in more than 75% of the cell-surface binding sites becoming associated with the cytoskeleton (i.e. resistant to extraction with detergent), although no more than 10% of these sites were associated with the cytoskeleton before addition of the lectins. After binding of WGA to the cells, the cell surface was cross-linked so extensively that it remained intact even after detergent extraction of the treated cells, and could be observed by electron microscopy. A similar cross-linking effect did not occur after binding of RCA to cells, which may be because there were so many more binding sites for WGA than for RCA. The composition of WGA- and RCA-binding molecules was analysed by lectin affinity chromatography of metabolically radiolabelled BHK cells. We found that in the WGA-binding-molecule preparations there were eight major polypeptides, ranging in molecular mass from 93 to 340 kDa, and that the RCA-binding molecules were a subpopulation of the WGA-binding molecules. A polyclonal antibody against the 140 kDa fibronectin (FN) receptors of Chinese-hamster ovary (CHO) cells immunoblotted a 145 kDa polypeptide component in both WGA- and RCA-binding-molecule preparations. The results indicated that the 145 kDa component was present in at least two FN-receptor complexes that differed in glycosylation, only one of which was able to bind to RCA affinity columns. The oligomeric nature of the FN-receptor complex, which contained three polypeptides with molecular masses of 120-145 kDa, was demonstrated by using anti-(CHO-cell FN receptor) antibodies to immunoprecipitate extracts prepared from radioiodinated BHK cells.

Binding Sites↗
PubMed1988

Cell adhesion and phagocytosis promoted by monoclonal antibodies not directed against fibronectin receptors.

In this report we describe cell adhesion and phagocytosis promoted by two monoclonal antibodies that were selected for immunofluorescence staining of non-permeabilized baby hamster kidney (BHK) cells. Anti-BHK1 staining was heaviest along cell margins, whereas anti-BHK2 staining was continuous along cell margins. Neither antibody stained elongated plaque structures such as were observed when cells were reacted with antibodies to fibronectin (FN) receptors. The monoclonal antibodies functioned as adhesion ligands in four different assays: attachment to culture dishes, spreading, binding of latex beads and phagocytosis. Anti-BHK1 and anti-BHK2 promoted attachment to culture dishes similarly, but anti-BHK2 was more effective at promoting cell spreading. Antibody-promoted cell spreading was inhibited by the peptides Ser-Asp-Gly-Arg and Gly-Arg-Gly-Asp-Ser-Pro but not by other, related, peptides tested. The monoclonal antibodies also promoted binding of latex beads, and the bead binding sites were motile, on the basis of their 'capping' response. Nevertheless, anti-BHK2 beads were phagocytosed by cells 5- to 20-fold more efficiently than anti-BHK1 beads. The binding sites for anti-BHK1 and anti-BHK2 were characterized by immunoprecipitation experiments. Anti-BHK1 binding sites contained 50K (K = 10(3) Mr) and 88K components under non-reducing conditions that migrated as a 51/53K doublet and a 93K component under reducing conditions. On the other hand, anti-BHK2 binding sites contained 88K and 110K components under non-reducing conditions that shifted to apparent 107K and 128K values when measured under reducing conditions.

Animals↗