Lloyd A. Culp

Substratum contacts and cytoskeletal reorganization of BALB/c 3T3 cells on a cell-binding fragment and heparin-binding fragments of plasma fibronectin.

BALB/c 3T3 cells make both close contacts and tight-focal contacts (with associated microfilament stress fibers) on plasma fibronectin (pFN)-coated substrata. To resolve the importance of the heparan sulfate-binding or cell-binding activities of the pFN molecule in these adhesive responses, a cell-binding fragment (120K) (CBF) free of any heparan sulfate-binding activity was prepared from human pFN by chymotrypic digestion and isolated as described by Pierschbacher et al. (Cell 26 (1981) 259). These adhesive responses to CBF were also compared to those of the model heparan sulfate-binding protein, platelet factor-4 (PF4), or heparin-binding fragments (HBF) of pFN. On intact pFN, greater than 70% of the cells formed tight-focal contacts and associated stress fibers by 4 h, the latter staining with NBD-phallacidin. In contrast, cells spread differently on CBF and failed to form tight-focal contacts; staining with NBD-phallacidin was localized to spiky projections at the cell margin with no detectable stress fiber formation. On PF4 or HBF, cells failed to form tight-focal contacts but did spread well and formed long microfilament bundles in peripheral lamellae. Spreading on CBF, HBF, or PF4 was paralleled by formation of close contacts. Spreading and to some extent attachment of cells on CBF was inhibited with a small peptide containing the Arg-Gly-Asp-Ser sequence; responses on HBF were unaffected by this peptide. When mixtures of CBF and PF4 were tested, cells still failed to form tight-focal contacts and stress fibers. These results demonstrate that the binding of CBF to its probable receptor under conditions routinely used to assay spreading activity results in an incomplete adhesive response compared with intact pFN. While this partial response may result from quantitative differences in the density of active cell-binding domains on the substratum, the pattern of microfilament reorganization produced by the binding of PF4 to cell surface heparan sulfate proteoglycans suggests that the ability of pFN to promote formation of tight-focal contacts and stress fibers may reside in the coordinate interaction of two or more binding activities in the intact molecule.

Stimulation of 2-deoxyglucose uptake in growth-inhibited BALB/c 3T3 and revertant SV40-transformed 3T3 cells

Abstract Sugar transport in the contact-inhibited BALB/c 3T3 and revenant SV40-transformed (contact-inhibited variants of transformed cells described previously) 3T3 cells was markedly diminished when these cells became growth-inhibited [22]. Several agents have been tested for their ability to stimulate transport of 2-deoxyglucose in growth-inhibited cells and their mechanism of action has been examined in some detail. Insulin rapidly stimulated transport in both normal 3T3 and revertant cells, when they were contact-inhibited, by increasing the Michaelis-Menton kinetic parameter Vmax—possibly a rapid increase in the number of transport sites on the surface of these cells. Insulin had no effect on sugar transport in SV40-transformed (SVT2) cells, growing 3T3 cells, or growing revertant cells. Increasing concentrations of fresh fetal calf serum caused a stimulation of transport in contact-inhibited 3T3 and revertant cells to levels identical with growing cells (there was no effect on growing cells or on SVT2 cells); this correlated with its ability to ‘turn-on’ DNA synthesis in the vast majority of these cells. This stimulation was maximal at serum concentrations of 35–80%. The mechanisms of stimulation for the two cell types were quite different—stimulation of transport in 3T3 cells was initially rapid with an increase in Vmax followed 1 h later by a second increase resulting from a diminished Km (an increased binding affinity). Stimulation in revertant cells proceeded slowly and linearly and was a result of an increased Vmax with no apparent change in the Km constant. Although revertant cells exhibited sugar transport properties which in general resembled those same properties in normal 3T3 cells and not of their parental SVT2 cells, the mechanisms of transport changes were quite different. The nature of the interaction of fresh fetal calf serum with BALB/c 3T3 cells in terms of stimulation of transport was examined in some detail. The serum was shown to be partially multipotential, while the cells were shown not to be. Serum was modified in that it lost its ability to cause the second, long-term stimulation of transport mentioned above (Km decrease); this correlated with the loss of its ability to induce DNA synthesis in these cells. These experiments suggest a unique approach to the study of the interaction between cells and serum and the importance of this interaction in growth control of normal cells and its loss by virally-transformed cells.

Induction of monocyte expression of tumor necrosis factor alpha by the 30-kD alpha antigen of Mycobacterium tuberculosis and synergism with fibronectin.

Native 30-kD antigen, also known as alpha antigen, is a fibronectin-binding protein that is secreted by live Mycobacterium tuberculosis. This antigen may play an important biological role in the host-parasite interaction since it elicits delayed type hypersensitivity response and protective immunity in vivo and T lymphocyte blastogenesis and IFN-gamma production in vitro. In the present study, we show that, TNF-alpha protein is produced in monocyte culture supernatants in response to 30-kD antigen and the level is as high as that to purified protein derivative of M. tuberculosis. This stimulatory effect was not due to contamination with either bacterial lipopolysaccharide or mycobacterial lipoarabinomannan. The preincubation of monocytes with plasma fibronectin significantly enhanced the release of TNF-alpha into the culture supernatants in response to 30-kD antigen. This effect was blocked by polygonal antibody to plasma fibronectin. In contrast, the monocytic cell line U937 failed to release TNF-alpha protein in the culture supernatants in response to 30-kD antigen with or without preincubation with plasma fibronectin. To determine whether this observation was due to differential binding of the 30-kD to fibronectin on these cells, a cell based ELISA was used. Pretreatment of monocytes with fibronectin enhanced their binding of the 30-kD antigen. U937 cells bound the 30-kD antigen weakly with or without fibronectin pretreatment. These results indicate that 30-kD antigen which is a known secretary antigen of M. tuberculosis is a stimulus for human monocytes to express TNF-alpha and that stimulatory effect may be mediated through plasma fibronectin.

Contact-inhibited revertant cell lines isolated from SV40-transformed cells: V. Contact inhibition of sugar transport☆

Abstract The transport of 2-deoxyglucose in BALB/c 3T3 cells, Simian virus 40-transformed BALB/c 3T3 (SVT2) cells, and concanavalin A-selected revertant cells of SVT2 has been measured. Sparsely-seeded BALB/c 3T3 cells transport the sugar at about one-fourth, and sparsely-seeded revertant cells at three-fourths, the rate of SVT2 cells. BALB/c 3T3 cells undergo a dramatic drop in sugar uptake at confluency, transporting sugar at about one-tenth the rate of subconfluent cells. Revertant cells (contact-inhibited variants of transformed cells) are similar in this respect, but the drop is only 5-fold. SVT2 cells show no such change in uptake over wide cell densities. Subconfluent BALB/c 3T3, SVT2, and revertant cells have similar K m and V max values for 2-deoxyglucose transport; however, confluent 3T3 and confluent revertant cells show a large increase in K m and a 5-fold decrease in V max as compared to their subconfluent counterparts or SVT2 cells—indications of a decreased number of transport sites and a decreased affinity of these sites for sugar when these cells make intimate contacts with each other. These data indicate that extensive changes in the architecture of the cell surface occur when contactinhibited cells are in close apposition with each other, regardless of the persistence of partially expressed SV40 genetic information, and are discussed with regard to the membrane compositions of these cell lines.

Contact formation by fibroblasts adhering to heparan sulfate-binding substrata (fibronectin or platelet factor 4).

The process of cell-substratum adhesion of BALB/c 3T3 fibroblasts on fibronectin (FN)-coated substrata was compared with that of cells adhering to substrata coated with the heparan sulfate (HS)-binding protein, platelet factor four (PF4). FN has binding domains for HS and an unidentified cell surface receptor, whereas PF4 binds to only HS on the surface of the cell. The attachment and early spreading sequences of cells on either substratum were similar as shown by scanning electron microscopy (SEM). Within 2 h of spreading, cells on FN developed typical fibroblastic morphologies, whereas those on PF4 lacked polygonal orientations and formed numerous broadly spread lamellae. Interference reflection microscopic analysis indicated that PF4-adherent cells formed only close adhesive contacts, whereas FN-adherent cells formed both close contacts and tight focal contacts. Cells on either substratum responded to Ca2+ chelation with EGTA by rounding up, but remained adherent to the substratum by relatively EGTA-resistant regions of the cells undersurface, demonstrating that cell surface HS by binding to an appropriate substratum is capable of initiating a Ca2+-dependent spreading response. The EGTA-resistant substratum-attached material on PF4 was morphologically similar to that on FN, the latter of which was derived from both tight focal contacts and discrete specializations within certain close contacts. These studies show that heparan sulfate proteoglycans on the surface of these cells can participate in the formation of close contact adhesions by binding to an appropriate substratum and suggest that sub-specializations within close contact adhesions may evolve into tight focal contacts by the participation of an unidentified cell surface receptor which binds specifically to fibronectin but not to PF4. In addition, the functional role of FN in tight focal contact formation is demonstrated.

Complementary adhesive responses of human skin fibroblasts to the cell-binding domain of fibronectin and the heparan sulfate-binding protein, platelet factor-4

Plasma fibronectin (pFN) contains binding domains for an unidentified receptor on the surface of fibroblasts and for heparan sulfate chains of proteoglycans on these same cells. A series of experiments were designed to assess the relative importance of these activities in mediating substratum adhesion of human skin fibroblasts (strain 4449) grown in the absence of ascorbate (asc-) or in its presence (asc+) to minimize or maximize collagen production-maturation, respectively. The cell-binding fragment (CBF) of pFN was purified from chymotryptic digests free of any heparan sulfate-binding activity. The responses of cells to CBF were then compared with those mediated by the heparan sulfate-binding protein, platelet factor-4 (PF4). At early time points when cells had spread effectively on pFN, both asc- or asc+ cells extended spiky projections on PF4 and long projections on CBF with actively ruffling membranes at their tips. By 4 h, asc+ cells had spread much more effectively on CBF than asc+ cells on PF4 or asc- cells on either binding activity. Mixtures (w/w) of CBF:PF4 between 1:1 and 9:1 generated a more physiologically normal response than to either of the binding proteins alone, particularly for asc+ cells. Examination of cytoskeletal reorganization by fluorescence analysis with an antibody to 7S tubulin (for microtubules) and NBD-phallacidin (for F-actin) revealed condensations of microfilaments at the ruffling edges of asc- cells on CBF or on PF4 and for asc+ cells on PF4; in contrast, asc+ cells on CBF generated long bundles of microfilaments in their spreading lamellae within 4 h. Microtubule networks reorganized very well on CBF but only partially on PF4 with either cell type. Microfilament reorganization was comparable to that on intact pFN with CBF:PF4 mixtures of 1:1 and 9:1 for asc+ cells, whereas asc- cells generated condensations of microfilaments but little bundling. These studies reveal that the adhesive responses to mixtures of these two binding activities are significantly greater than to the individual activities and that the responses of asc+ cells approach the properties of cells on intact pFN, whereas asc- cells remain incapable of forming stress fiber-like bundles of microfilaments under all conditions.

Concomitant down-regulation of expression of integrin subunits by N-myc in human neuroblastoma cells : differential regulation of α2, α3 and β1

Amplification and over-expression of the N-myc oncogene is associated with the progression of neuroblastoma in children and in a nude mouse model system. Neuroblastoma cell lines with widely different levels of N-myc illustrate an inverse relationship between N-myc over-expression and reduced expression of several integrin extracellular matrix receptors. Transfection and over-expression of N-myc in a neuroblastoma cell line not normally expressing the protein resulted in cells that grew loosely associated with tissue culture plates; this correlated with reduced levels of β1 integrin subunit. Evidence is now presented that α2 and α3 integrin subunit levels are also reduced in cells that over-express N-myc, with virtually no association of α2 or α3 subunits with β1. Consequently, maturation of the β1 subunit and cell surface expression of the integrins are greatly reduced in N-myc-transfected cells. A small amount of β1 protein does get to the cell surface, however, suggesting that an as yet unidentified α subunit is produced by the N-myc-expressing cells. Finally, the observed reductions in integrin protein levels are reflections of greatly reduced levels of integrin α2 and α3 mRNAs, as well as a smaller reduction in β1 mRNA (80%, 94% and 52%, respectively). Post-transcriptional mechanisms modulating β1 integrin levels are also operative. These results indicate that over-expression of N-myc from a transfected gene in a neuroblastoma cell line that does not normally produce the protein generates cell lines with many of the characteristics of naturally metastatic cells with amplified N-myc genes. Modulation of N-myc and integrin expressions may play a significant role in progression of human neuroblastoma.

Rat mesangial cell-matrix interactions in culture

The glomerular mesangium contains fibronectin (FN), laminin, and collagen IV, but it remains unclear whether these matrix proteins affect mesangial cellular functions. The present experiments were designed to test whether cell-matrix interactions could affect some functions of mesangial cells. Cultured rat mesangial cells synthesized a cellular form of FN that was both secreted and incorporated into an extensive, fibrillar pericellular matrix. This FN matrix was increased in high-density cultures and was more developed in human mesangial cells. Rat mesangial cells in vitro displayed a marked capacity to incorporate exogenous FN into a pericellular matrix, demonstrating that accumulations of FN in the mesangial matrix could result from endogenous and/or exogenous sources. Rat mesangial cells also expressed RGD-sensitive integrin receptors for FN, laminin, and collagens I and IV that promoted cell adhesion and that directed differential changes in morphology. Indirect evidence suggested the existence of other mesangial binding sites for extracellular matrix proteins. FN and collagen IV also stimulated modest increases in [3H]thymidine uptake and cell number by quiescent cells. Taken together, these results suggest that cultured mesangial cells present a model system for studying the regulation of cell-matrix interactions in the mesangium.

Neurite extension by neuroblastoma cells on substratum-bound fibronectin's cell-binding fragment but not on the heparan sulfate-binding protein, platelet factor-4☆

Human and rat neuroblastoma cells extend neurites over plasma fibronectin (pFN)-coated substrata. For resolution of which fibronectin binding activities (the cell-binding domain (CBD), the heparan sulfate-binding domains, or a combination of the two) are responsible for neurite outgrowth, CBD was prepared free of heparan sulfate-binding activity as described by Pierschbacher et al. (Cell 26 (1981) 259-267). Neuroblastoma cells attached and extended neurites as stably and as effectively on CBD-coated substrata as on intact pFN, while cytoplasmic spreading was more extensive on pFN-coated substrata. The structures of growth cones on CBD or pFN were virtually identical. On substrata coated with the model heparan sulfate-binding protein, platelet factor 4 (PF4), cells attached and spread somewhat but never extended neurites. When cells were challenged with substrata coated with various ratios of CBD and PF4, PF4 was found to be an effective inhibitor of CBD-mediated neurite extension. Similarly, cells grown on substrata coated at different locations with CBD or PF4 in order to evaluate topographical dependence of growth cone formation extended neurites only onto the CBD-coated region or along the interface between these two proteins, but never onto the PF4 side of cells that bridged the interface. These studies indicate that (a) the CBD activity of pFN, and not its heparan sulfate-binding activity, is the critical determinant in neurite extension of these neural tumor cells from the central nervous system; (b) under some circumstances, heparan sulfate-binding activity can be antagonistic to neurite extension; (c) the chemical nature of the substratum controls the direction of neurite extension; (d) these neuroblastoma cells respond to these binding proteins very differently than fibroblasts or neurons from the peripheral nervous system.

CELL TYPE-SPECIFIC MODULATION OF FIBRONECTIN ADHESION FUNCTIONS ON CHEMICALLY-DERIVATIZED SELF-ASSEMBLED MONOLAYERS

Cell type-specific responses (microfilament stress fibers for fibroblasts or neurites for neuroblastoma cells) were evaluated in culture on inert and chemically-derivatized silane substrata adsorbed with fibronectin (Fn). Substrata of self-assembled monolayers contain a 14-17 carbon aliphatic chain terminating with different chemical endgroups -- [CH3], [C=C], [Br], [CN], [Diol], [COOH], [NH2], [SH], [SCOCH3], or [SO3H]. Fn adsorbed effectively to all derivatized surfaces. 3T3 fibroblasts or neuroblastoma cells attached equivalently to all surfaces preadsorbed with Fn, indicating availability of receptor binding sites on Fns. However, transmembrane signaling from Fn(adsorbed): receptor(cell) surface complexes yielded a range of abilities for generating F-actin stress fibers in fibroblasts or neurites in neuroblastoma cells. Efficiency for stress fiber formation was very different from that of neurite extension. The same chemical endgroups on glass, titanium, or germanium yielded the same patterns of cellular physiological responses, indicating that inert substrata do not act at a distance and that only chemical endgroups regulate Fn signaling functions. When adhesion-inert albumin is co-adsorbed with Fn, efficiency of neurite extension is improved on some surfaces or diminished on others. These results indicate that the conformation of Fn(adsorbed) changes in specific ways on derivatized substrata. Change in Fn conformation was confirmed by FTIR/ATR spectroscopy experiments of Fn(adsorbed). Overall, these studies indicate changes in Fn conformations on chemically-derivatized self-assembled monolayers leading to up- or down-regulation of cell type-specific physiological responses from receptors via their signaling pathways. They also offer predictability for regulating responses of specific cell types when these cells interact with biomaterial implants in vivo.