The interaction of migrating newt epidermal cells with the extracellular matrix protein, fibronec... more The interaction of migrating newt epidermal cells with the extracellular matrix protein, fibronectin, was studied. Pieces of nitrocellulose coated with intact human plasma fibronectin or proteolytically derived fragments were implanted into wounded limbs so that the coated nitrocellulose served as wound bed for migrating epidermal cells as they attempted to form a wound epithelium. Epidermal cells migrated very poorly on nitrocellulose pieces coated with (a) a 27-kD amino-terminal heparin-binding fragment, (b) a 46-kD gelatin-binding fragment, (c) a combined 33-and 66-kD carboxy-terminal heparin-binding preparation representing peptide sequences in the A and B chains, respectively, or (d) a 31-kD carboxyterminal fragment from the A chain, containing a free sulfhydryl group. In contrast, epidermal cells readily migrated onto nitrocellulose coated with a mixture of fragments from the middle of the molecule (80-125kD) that bind neither heparin nor gelatin. Attempts to block migration on fibronectin-coated nitrocellulose using 1810, a monoclonal antibody that blocks Chinese hamster ovary cell attachment to fibronectin, were unsuccessful despite saturation of the epitope against which I810 is directed. In contrast, a polyclonal anti-fibronectin antibody did inhibit migration. These results show that the ability of fibronectin to support newt epidermal cell migration is not shared equally by all regions of the molecule, but is restricted to a domain in the middle third. They also suggest that the site supporting migration is separate and distinct from the site mediating Chinese hamster ovary cell attachment.
Cell adhesion and neurite outgrowth on fibronectin is a multistep process modulated by different ... more Cell adhesion and neurite outgrowth on fibronectin is a multistep process modulated by different extra- and intracellular signals. Fibronectin-mediated cell attachment and spreading can be affected in a negative way by tenascin-C, an extracellular matrix glycoprotein expressed in a temporally and spacially restricted manner during early morphogenesis. Tenascin-R (J1-160/180), consisting of two major isoforms of 160 kDa (tenascin-R 160) and 180 kDa (tenascin-R 180) in mammals, is an extracellular matrix glycoprotein of the central nervous system that shares high structural homologies with tenascin-C. Here we show that in relation to fibronectin-mediated adhesion, the two extracellular matrix molecules are also functionally closely related. When offered as mixed substrata with other extracellular matrix molecules, the two tenascin-R isoforms and tenascin-C derived from mouse brain selectively inhibit fibronectin-dependent cell adhesion and neurite outgrowth, and affect cell morphology...
This report outlines a method for rapid quantification of corneal epithelial cell movement in a b... more This report outlines a method for rapid quantification of corneal epithelial cell movement in a block organ culture system designed to model a penetrating or perforating wound of the cornea. Rabbit corneas were secured over a plastic conformer with a chalazion clamp. Two-millimeter, full-thickness corneal specimens were sampled from central and peripheral cornea and placed in tissue culture. The aim of the procedure was to produce full-thickness corneal specimens from well-defined geographic areas of the cornea of reproducible size and shape without undue trauma to the epithelium. The instruments used for measurements were simple, allowing for accumulation of a large number of observations which could be subjected to rigorous statistical evaluation. This method was used to determine if there was a detectable difference in the rate of epithelial cell movement over stromal cut surface of specimens from the periphery of the cornea, versus the rate over specimens from the central area o...
The glomerular filtration barrier consists of complex matrix constituents interposed between the ... more The glomerular filtration barrier consists of complex matrix constituents interposed between the glomerular endothelial and epithelial cells, which constitute the lining of the glomerular capillaries. The matrix components include collagen and noncollagenous molecules such as laminin and proteoglycans. This review describes herein the metabolic consequences of diabetes mellitus to which increased permeability of the filtration barrier may be attributed by altering matrix molecules.
A 70,000-mol-wt protein was isolated from A431 carcinoma cell extracellular matrix that promotes ... more A 70,000-mol-wt protein was isolated from A431 carcinoma cell extracellular matrix that promotes cell substratum adhesion of these epidermoid tumor cells. Extracellular matrix was isolated by a modification of a procedure described by Hedman et al. (Hedman, K ., M .
Integrin-mediated tumor cell adhesion to type IV collagen is believed to play a role in the invas... more Integrin-mediated tumor cell adhesion to type IV collagen is believed to play a role in the invasion of basement membrane proteins and the subsequent metastatic process. The cellular protein CAR (cell adhesion regulator) has been proposed to influence integrin-mediated binding to extracellular matrix proteins, including basement membrane (type IV) collagen. Three analogs of the CAR 138-142 have been tested for activity. The first contains the 138-142 sequence (CAR 138-142 , Val-Glu-Ile-Leu-Tyr-NH 2), the second contains the 138-142 sequence with a phosphorylated Tyr [pCAR 138-142 , Val-Glu-Ile-Leu-Tyr(PO 3 H 2)-NH 2 ], and the third contains the reversed 138-142 sequence (rCAR 138-142 , Tyr-Leu-Ile-Glu-Val-NH 2). When added extracellularly, none of the analogs had a significant affect on cell adhesion to type IV collagen. Using a novel reversible cell permeabilization method, we found that intracellular incorporation of both CAR 138-142 and pCAR 138-142 resulted in inhibition of cell adhesion in a dose-dependent fashion. The IC 50 values were ∼90 and ∼10 µM for CAR 138-142 and pCAR 138-142 , respectively. Intracellular incorporation of the rCAR 138-142 peptide had no affect on cell adhesion. Fluorescence microscopy of a fluorescein-labeled CAR 138-142 peptide revealed that the reversible permeabilization procedure resulted in the peptides crossing the cell membrane. Affinity chromatography of melanoma cell lysates with pCAR 138-142 or rCAR 138-142 attached to a solid support of magnetic beads suggested that one protein was bound uniquely by pCAR 138-142. Immunoprecipitation analysis identified vinculin, a protein associated with the actin cytoskeleton, as the protein specifically bound by pCAR 138-142. Immunoprecipitation with pp125 FAKor 1-integrin-derived mAbs gave negative results. Our study suggests that a possible therapeutic approach for inhibition of melanoma cell adhesion adhesion to extracellular matrix proteins is the use of CAR peptide analogs intracellularly.
Tumor cell adhesion to the triple-helical domain of basement membrane (type IV) collagen occurs a... more Tumor cell adhesion to the triple-helical domain of basement membrane (type IV) collagen occurs at several different regions. Cellular recognition of the sequence spanning ␣1(IV)531-543 has been proposed to be independent of triple-helical conformation (Miles, A. J., Skubitz, A. P. N., Furcht, L. T., and Fields, G. B. (1994) J. Biol. Chem. 269, 30939-30945). In the present study, integrin interactions with a peptide analog of the ␣1(IV)-531-543 sequence have been analyzed. Tumor cell adhesion (melanoma, ovarian carcinoma) to the ␣1(IV)531-543 chemically synthesized peptide was inhibited by a monoclonal antibody against the ␣ 3 integrin subunit, and to a lesser extent by monoclonal antibodies against the  1 and ␣ 2 integrin subunits. An anti-␣ 5 monoclonal antibody and normal mouse IgG were ineffective as inhibitors of tumor cell adhesion to the peptide. Two cell surface proteins of 120 and 150 kDa bound to an ␣1(IV)531-543 peptide affinity column and were eluted with 20 mM EDTA. When the eluted proteins were incubated with monoclonal antibodies against either the ␣ 3 or  1 integrin subunit, proteins corresponding in molecular weight to ␣ 3 and  1 integrin subunits were precipitated. No proteins were immunoprecipated with monoclonal antibodies against the ␣ 2 or ␣ 5 integrin subunits. Thus, the ␣ 3  1 integrin from two tumor cell types has been shown to bind directly to the ␣1(IV)531-543 peptide. The ␣1(IV)531-543 peptide is the first collagen-like sequence that has been shown to bind the ␣ 3  1 integrin.
Several regions within the triple-helical domain of type IV collagen function as cellular recogni... more Several regions within the triple-helical domain of type IV collagen function as cellular recognition sites. We have demonstrated previously that melanoma cell activities promoted by the alpha 1(IV)1263-1277 sequence are enhanced by triple helicity (Fields, C. G., Mickelson, D. J., Drake, S.L., McCarthy, J.B., and Fields, G.B. (1993) J. Biol. Chem. 268, 14153-14160), whereas Eble et al. reached similar conclusions for alpha 1 beta 1 integrin-mediated fibrosarcoma cell adhesion to [alpha 1(IV)]2 alpha 2(IV)434-472 (Eble, J. A., Golbik, R., Mann, K., and Kühn, K. (1993) EMBO J. 12, 4795-4802). In the present study, we have examined the cell adhesion activities of a third region in type IV collagen. A single-stranded peptide (SSP) incorporating the alpha 1(IV)531-543 sequence promoted the adhesion of melanoma, ovarian carcinoma, and Jurkat cells in a dose-dependent manner, with 40% cell adhesion observed at [SSP] = 1.8, 11.5, and 42.2 microM, respectively. Nearly identical results were obtained for cell adhesion to an all-D-enantiomer of the SSP, suggesting that the cell surface receptor(s) for this site do not discriminate based on chirality. The alpha 1(IV)531-543 sequence maintained its cell adhesion promoting activity when incorporated into a homotrimeric triple-helical polypeptide, although relative levels of adhesion were either slightly enhanced or slightly diminished compared with the SSP. Triple-helical conformation was thus not critical for cellular recognition of the alpha 1(IV)531-543 sequence. Single-site substitution experiments of the SSP showed no overall correlation between the biological effects of substitutions and SSP conformation. The SSP, D-SSP, triple-helical polypeptide, and SSP substitution results suggest that cell recognition of the alpha 1(IV)531-543 sequence is generally independent of substrate conformation. The present and prior studies indicate that "conformationally dependent" and "conformationally independent" cellular recognition sites exist within the triple-helical domain of type IV collagen.
Recent investigations have emphasized the role of activated granulocytes in mediating vascular en... more Recent investigations have emphasized the role of activated granulocytes in mediating vascular endothelial injury in the pathogenesis of shock lung. In vitro studies have indicated that tight adherence of the neutrophil to the endothelium is crucial for the development of cellular injury. Fibronectin is critical to cell-to- substratum and cell-to-cell interactions. Since fibronectin resides in plasma, on endothelial cell surfaces and is secreted into cell matrices, the adhesive properties of fibronectin must be modulated, lest universal cell agglomeration occur, yet be enhanced when cell attachment is appropriate. In these studies, treatment of fibronectin- coated surfaces with neutrophil release products increased the adhesion of activated neutrophils. Similarly, endothelial cells treated with neutrophil release products become a more adherent substrate for neutrophils. This enhanced adherence generated by treatment of fibronectin with neutrophil supernatants is inhibitable by heat...
The aim of this work was to identify the integrin subunits present on the cell surface of human c... more The aim of this work was to identify the integrin subunits present on the cell surface of human corneal epithelial cells. The authors determined to show whether type IV collagen, heparin-binding peptides of type IV collagen (Hep-I, Hep-II, and Hep-III), fibronectin, and GRGDSP promote cell adhesion of human corneal epithelial cells. Type IV collagen and heparin-binding peptides of type IV collagen may be important in corneal epithelial cell adhesion in normal and pathologic conditions and reepithelialization. The authors assess the role of cell surface integrins in mediating cell adhesion to these proteins and peptides. Fluorescence-activated cell sorter (FACS) analysis was used to determine the integrin subunits expressed at the cell surface of the cultured human corneal epithelial cells. Cell adhesion was assessed with type IV collagen, heparin-binding peptides of type IV collagen, fibronectin, and GRGDSP: Antibodies to the integrin subunits were used to determine the potential ro...
The aim of this work was to show epidermal growth factor (EGF)-dependent migration of human corne... more The aim of this work was to show epidermal growth factor (EGF)-dependent migration of human corneal epithelial cells to fibronectin and GRGDSP peptide. The authors assessed the role of cell surface integrin heterodimer alpha 5 beta 1 in mediating haptotactic cell migration to fibronectin by the use of specific function-blocking integrin antibodies. A haptotactic cell migration assay in a Boyden chamber was used to compare the relative migration of the cultured human corneal epithelial cells in the presence of fibronectin and GRGDSP peptide-coated filters. Epithelial cells were incubated in the presence of function-blocking integrin antibodies or anti-EGF-receptor antibodies to determine their role in haptotactic cell migration. Human corneal epithelial cells grown as primary cultures migrated in the presence of fibronectin or GRGDSP peptide, but only on stimulation with EGF. Antibodies to the EGF receptor blocked the EGF-mediated stimulation of haptotactic cell migration. Anti-beta ...
Herpes simplex virus (HSV) infection may be involved in various endothelial-injury syndromes, inc... more Herpes simplex virus (HSV) infection may be involved in various endothelial-injury syndromes, including vasculitis and atherosclerosis. In a previous study, it was reported that HSV-infected human umbilical endothelial cells are more vulnerable to detachment mediated by granulocyte-secreted proteases. To elucidate the molecular basis of this observation, the authors examined the interaction of infected endothelial cells with the purified basement membrane proteins, fibronectin, laminin, and type IV collagen. HSV-infected endothelial cells exhibited defects in their ability to adhere, spread, and migrate on all three matrix components. This defective adhesion could be partially overcome by increasing concentrations of fibronectin; in contrast, no abrogation of deficient binding occurs with increased levels of laminin or collagen type IV. This suggests that endothelial cells may use different surface constituents for binding to the three proteins and use multiple "receptors"...
The object of these studies was to examine the localization of type IV collagen (Coll‐IV) in the ... more The object of these studies was to examine the localization of type IV collagen (Coll‐IV) in the basement membranes (BM) of epithelial and stromal elements (smooth muscle, nerves, vessels) in normal, hyperplastic, and neoplastic (primary and metastatic) prostate. We also examined the relationship of Coll‐IV distribution to the degree of tumor differentiation (Gleason grading system). We compared immunoperoxidase (IP) and immunoalkaline phosphatase (AP) techniques in these studies and in selected samples we also evaluated immunofluorescence (IF) localization of Coll‐IV and the effects of tissue fixation and pepsin digestion. We found that IF localization of Coll‐IV was intense in unfixed sections. IP and AP reactions were absent in fixed, paraffin‐embedded sections but pepsin treatment yielded intense and uniform reaction products in these same preparations. Both the IP and AP techniques showed similar localization of Coll‐IV in the BM of normal, hyperplastic, and well‐differentiated...
Metastasis of prostate carcinoma requires invasion through the basement membrane, a thin extracel... more Metastasis of prostate carcinoma requires invasion through the basement membrane, a thin extracellular matrix that underlies the epithelial cells, which must be breached by tumor cells invading into surrounding tissue. The CXC-chemokines, which have been shown to promote the migration of neutrophils and carcinoma cells, are candidates to influence prostate carcinoma-cell invasion. CXC-chemokines were examined for the ability to stimulate prostate cell line PC3 invasion in vitro through a reconstituted basement membrane and long-term migration and short-term adhesion to laminin, a major component of the basement membrane. PC3 cells responded to IL-8 and GROalpha with a 1. 6-2-fold increase in invasion through reconstituted basement membrane. A corresponding 2-3-fold increase in chemotaxis toward IL-8 and GROa was seen on laminin. Anti-CXCR2 antibody inhibited IL-8-stimulated migration. Expression levels of the beta(1) integrins were not changed by IL-8, and alpha(6beta1) integrin was used for both stimulated and baseline migration. In addition to the increases in migration and invasion, 2-6-fold transient increases in adhesion on laminin were seen with both IL-8 and GROalpha. These results suggest that the CXC-chemokines stimulate migration and invasion in part by altering the activation state of the beta(1) integrins. The CXC-chemokines act on prostate carcinoma cells through the CXCR2 receptor to promote behavior important for metastasis, and as such may be important in prostate carcinoma progression and metastasis.
Recent studies using solid-phase-binding assays and electron microscopy suggested the presence of... more Recent studies using solid-phase-binding assays and electron microscopy suggested the presence of a heparin-binding domain between the inner globule of a lateral short arm and the cross region of laminin. Using the information from the amino acid sequence of the B1 chain of laminin, several peptides were synthesized from areas with a low hydropathy index and a high density of lysines and/or arginines. One of these, peptide F-9 (RYVVLPRPVCFEKGMNYTVR), which is derived from the inner globular domain of the lateral short arm, demonstrated specific binding to heparin. This was tested in direct solid-phase binding assays by coating the peptide either on nitrocellulose or on polystyrene and in indirect competition assays where the peptide was in solution and either laminin or heparin was immobilized on a solid support. The binding of [3H]heparin to peptide F-9 was dramatically reduced when heparin but not other glycosaminoglycans other than heparin (dextran sulfate, dermatan sulfate) were...
The interaction of migrating newt epidermal cells with the extracellular matrix protein, fibronec... more The interaction of migrating newt epidermal cells with the extracellular matrix protein, fibronectin, was studied. Pieces of nitrocellulose coated with intact human plasma fibronectin or proteolytically derived fragments were implanted into wounded limbs so that the coated nitrocellulose served as wound bed for migrating epidermal cells as they attempted to form a wound epithelium. Epidermal cells migrated very poorly on nitrocellulose pieces coated with (a) a 27-kD amino-terminal heparin-binding fragment, (b) a 46-kD gelatin-binding fragment, (c) a combined 33-and 66-kD carboxy-terminal heparin-binding preparation representing peptide sequences in the A and B chains, respectively, or (d) a 31-kD carboxyterminal fragment from the A chain, containing a free sulfhydryl group. In contrast, epidermal cells readily migrated onto nitrocellulose coated with a mixture of fragments from the middle of the molecule (80-125kD) that bind neither heparin nor gelatin. Attempts to block migration on fibronectin-coated nitrocellulose using 1810, a monoclonal antibody that blocks Chinese hamster ovary cell attachment to fibronectin, were unsuccessful despite saturation of the epitope against which I810 is directed. In contrast, a polyclonal anti-fibronectin antibody did inhibit migration. These results show that the ability of fibronectin to support newt epidermal cell migration is not shared equally by all regions of the molecule, but is restricted to a domain in the middle third. They also suggest that the site supporting migration is separate and distinct from the site mediating Chinese hamster ovary cell attachment.
Cell adhesion and neurite outgrowth on fibronectin is a multistep process modulated by different ... more Cell adhesion and neurite outgrowth on fibronectin is a multistep process modulated by different extra- and intracellular signals. Fibronectin-mediated cell attachment and spreading can be affected in a negative way by tenascin-C, an extracellular matrix glycoprotein expressed in a temporally and spacially restricted manner during early morphogenesis. Tenascin-R (J1-160/180), consisting of two major isoforms of 160 kDa (tenascin-R 160) and 180 kDa (tenascin-R 180) in mammals, is an extracellular matrix glycoprotein of the central nervous system that shares high structural homologies with tenascin-C. Here we show that in relation to fibronectin-mediated adhesion, the two extracellular matrix molecules are also functionally closely related. When offered as mixed substrata with other extracellular matrix molecules, the two tenascin-R isoforms and tenascin-C derived from mouse brain selectively inhibit fibronectin-dependent cell adhesion and neurite outgrowth, and affect cell morphology...
This report outlines a method for rapid quantification of corneal epithelial cell movement in a b... more This report outlines a method for rapid quantification of corneal epithelial cell movement in a block organ culture system designed to model a penetrating or perforating wound of the cornea. Rabbit corneas were secured over a plastic conformer with a chalazion clamp. Two-millimeter, full-thickness corneal specimens were sampled from central and peripheral cornea and placed in tissue culture. The aim of the procedure was to produce full-thickness corneal specimens from well-defined geographic areas of the cornea of reproducible size and shape without undue trauma to the epithelium. The instruments used for measurements were simple, allowing for accumulation of a large number of observations which could be subjected to rigorous statistical evaluation. This method was used to determine if there was a detectable difference in the rate of epithelial cell movement over stromal cut surface of specimens from the periphery of the cornea, versus the rate over specimens from the central area o...
The glomerular filtration barrier consists of complex matrix constituents interposed between the ... more The glomerular filtration barrier consists of complex matrix constituents interposed between the glomerular endothelial and epithelial cells, which constitute the lining of the glomerular capillaries. The matrix components include collagen and noncollagenous molecules such as laminin and proteoglycans. This review describes herein the metabolic consequences of diabetes mellitus to which increased permeability of the filtration barrier may be attributed by altering matrix molecules.
A 70,000-mol-wt protein was isolated from A431 carcinoma cell extracellular matrix that promotes ... more A 70,000-mol-wt protein was isolated from A431 carcinoma cell extracellular matrix that promotes cell substratum adhesion of these epidermoid tumor cells. Extracellular matrix was isolated by a modification of a procedure described by Hedman et al. (Hedman, K ., M .
Integrin-mediated tumor cell adhesion to type IV collagen is believed to play a role in the invas... more Integrin-mediated tumor cell adhesion to type IV collagen is believed to play a role in the invasion of basement membrane proteins and the subsequent metastatic process. The cellular protein CAR (cell adhesion regulator) has been proposed to influence integrin-mediated binding to extracellular matrix proteins, including basement membrane (type IV) collagen. Three analogs of the CAR 138-142 have been tested for activity. The first contains the 138-142 sequence (CAR 138-142 , Val-Glu-Ile-Leu-Tyr-NH 2), the second contains the 138-142 sequence with a phosphorylated Tyr [pCAR 138-142 , Val-Glu-Ile-Leu-Tyr(PO 3 H 2)-NH 2 ], and the third contains the reversed 138-142 sequence (rCAR 138-142 , Tyr-Leu-Ile-Glu-Val-NH 2). When added extracellularly, none of the analogs had a significant affect on cell adhesion to type IV collagen. Using a novel reversible cell permeabilization method, we found that intracellular incorporation of both CAR 138-142 and pCAR 138-142 resulted in inhibition of cell adhesion in a dose-dependent fashion. The IC 50 values were ∼90 and ∼10 µM for CAR 138-142 and pCAR 138-142 , respectively. Intracellular incorporation of the rCAR 138-142 peptide had no affect on cell adhesion. Fluorescence microscopy of a fluorescein-labeled CAR 138-142 peptide revealed that the reversible permeabilization procedure resulted in the peptides crossing the cell membrane. Affinity chromatography of melanoma cell lysates with pCAR 138-142 or rCAR 138-142 attached to a solid support of magnetic beads suggested that one protein was bound uniquely by pCAR 138-142. Immunoprecipitation analysis identified vinculin, a protein associated with the actin cytoskeleton, as the protein specifically bound by pCAR 138-142. Immunoprecipitation with pp125 FAKor 1-integrin-derived mAbs gave negative results. Our study suggests that a possible therapeutic approach for inhibition of melanoma cell adhesion adhesion to extracellular matrix proteins is the use of CAR peptide analogs intracellularly.
Tumor cell adhesion to the triple-helical domain of basement membrane (type IV) collagen occurs a... more Tumor cell adhesion to the triple-helical domain of basement membrane (type IV) collagen occurs at several different regions. Cellular recognition of the sequence spanning ␣1(IV)531-543 has been proposed to be independent of triple-helical conformation (Miles, A. J., Skubitz, A. P. N., Furcht, L. T., and Fields, G. B. (1994) J. Biol. Chem. 269, 30939-30945). In the present study, integrin interactions with a peptide analog of the ␣1(IV)-531-543 sequence have been analyzed. Tumor cell adhesion (melanoma, ovarian carcinoma) to the ␣1(IV)531-543 chemically synthesized peptide was inhibited by a monoclonal antibody against the ␣ 3 integrin subunit, and to a lesser extent by monoclonal antibodies against the  1 and ␣ 2 integrin subunits. An anti-␣ 5 monoclonal antibody and normal mouse IgG were ineffective as inhibitors of tumor cell adhesion to the peptide. Two cell surface proteins of 120 and 150 kDa bound to an ␣1(IV)531-543 peptide affinity column and were eluted with 20 mM EDTA. When the eluted proteins were incubated with monoclonal antibodies against either the ␣ 3 or  1 integrin subunit, proteins corresponding in molecular weight to ␣ 3 and  1 integrin subunits were precipitated. No proteins were immunoprecipated with monoclonal antibodies against the ␣ 2 or ␣ 5 integrin subunits. Thus, the ␣ 3  1 integrin from two tumor cell types has been shown to bind directly to the ␣1(IV)531-543 peptide. The ␣1(IV)531-543 peptide is the first collagen-like sequence that has been shown to bind the ␣ 3  1 integrin.
Several regions within the triple-helical domain of type IV collagen function as cellular recogni... more Several regions within the triple-helical domain of type IV collagen function as cellular recognition sites. We have demonstrated previously that melanoma cell activities promoted by the alpha 1(IV)1263-1277 sequence are enhanced by triple helicity (Fields, C. G., Mickelson, D. J., Drake, S.L., McCarthy, J.B., and Fields, G.B. (1993) J. Biol. Chem. 268, 14153-14160), whereas Eble et al. reached similar conclusions for alpha 1 beta 1 integrin-mediated fibrosarcoma cell adhesion to [alpha 1(IV)]2 alpha 2(IV)434-472 (Eble, J. A., Golbik, R., Mann, K., and Kühn, K. (1993) EMBO J. 12, 4795-4802). In the present study, we have examined the cell adhesion activities of a third region in type IV collagen. A single-stranded peptide (SSP) incorporating the alpha 1(IV)531-543 sequence promoted the adhesion of melanoma, ovarian carcinoma, and Jurkat cells in a dose-dependent manner, with 40% cell adhesion observed at [SSP] = 1.8, 11.5, and 42.2 microM, respectively. Nearly identical results were obtained for cell adhesion to an all-D-enantiomer of the SSP, suggesting that the cell surface receptor(s) for this site do not discriminate based on chirality. The alpha 1(IV)531-543 sequence maintained its cell adhesion promoting activity when incorporated into a homotrimeric triple-helical polypeptide, although relative levels of adhesion were either slightly enhanced or slightly diminished compared with the SSP. Triple-helical conformation was thus not critical for cellular recognition of the alpha 1(IV)531-543 sequence. Single-site substitution experiments of the SSP showed no overall correlation between the biological effects of substitutions and SSP conformation. The SSP, D-SSP, triple-helical polypeptide, and SSP substitution results suggest that cell recognition of the alpha 1(IV)531-543 sequence is generally independent of substrate conformation. The present and prior studies indicate that "conformationally dependent" and "conformationally independent" cellular recognition sites exist within the triple-helical domain of type IV collagen.
Recent investigations have emphasized the role of activated granulocytes in mediating vascular en... more Recent investigations have emphasized the role of activated granulocytes in mediating vascular endothelial injury in the pathogenesis of shock lung. In vitro studies have indicated that tight adherence of the neutrophil to the endothelium is crucial for the development of cellular injury. Fibronectin is critical to cell-to- substratum and cell-to-cell interactions. Since fibronectin resides in plasma, on endothelial cell surfaces and is secreted into cell matrices, the adhesive properties of fibronectin must be modulated, lest universal cell agglomeration occur, yet be enhanced when cell attachment is appropriate. In these studies, treatment of fibronectin- coated surfaces with neutrophil release products increased the adhesion of activated neutrophils. Similarly, endothelial cells treated with neutrophil release products become a more adherent substrate for neutrophils. This enhanced adherence generated by treatment of fibronectin with neutrophil supernatants is inhibitable by heat...
The aim of this work was to identify the integrin subunits present on the cell surface of human c... more The aim of this work was to identify the integrin subunits present on the cell surface of human corneal epithelial cells. The authors determined to show whether type IV collagen, heparin-binding peptides of type IV collagen (Hep-I, Hep-II, and Hep-III), fibronectin, and GRGDSP promote cell adhesion of human corneal epithelial cells. Type IV collagen and heparin-binding peptides of type IV collagen may be important in corneal epithelial cell adhesion in normal and pathologic conditions and reepithelialization. The authors assess the role of cell surface integrins in mediating cell adhesion to these proteins and peptides. Fluorescence-activated cell sorter (FACS) analysis was used to determine the integrin subunits expressed at the cell surface of the cultured human corneal epithelial cells. Cell adhesion was assessed with type IV collagen, heparin-binding peptides of type IV collagen, fibronectin, and GRGDSP: Antibodies to the integrin subunits were used to determine the potential ro...
The aim of this work was to show epidermal growth factor (EGF)-dependent migration of human corne... more The aim of this work was to show epidermal growth factor (EGF)-dependent migration of human corneal epithelial cells to fibronectin and GRGDSP peptide. The authors assessed the role of cell surface integrin heterodimer alpha 5 beta 1 in mediating haptotactic cell migration to fibronectin by the use of specific function-blocking integrin antibodies. A haptotactic cell migration assay in a Boyden chamber was used to compare the relative migration of the cultured human corneal epithelial cells in the presence of fibronectin and GRGDSP peptide-coated filters. Epithelial cells were incubated in the presence of function-blocking integrin antibodies or anti-EGF-receptor antibodies to determine their role in haptotactic cell migration. Human corneal epithelial cells grown as primary cultures migrated in the presence of fibronectin or GRGDSP peptide, but only on stimulation with EGF. Antibodies to the EGF receptor blocked the EGF-mediated stimulation of haptotactic cell migration. Anti-beta ...
Herpes simplex virus (HSV) infection may be involved in various endothelial-injury syndromes, inc... more Herpes simplex virus (HSV) infection may be involved in various endothelial-injury syndromes, including vasculitis and atherosclerosis. In a previous study, it was reported that HSV-infected human umbilical endothelial cells are more vulnerable to detachment mediated by granulocyte-secreted proteases. To elucidate the molecular basis of this observation, the authors examined the interaction of infected endothelial cells with the purified basement membrane proteins, fibronectin, laminin, and type IV collagen. HSV-infected endothelial cells exhibited defects in their ability to adhere, spread, and migrate on all three matrix components. This defective adhesion could be partially overcome by increasing concentrations of fibronectin; in contrast, no abrogation of deficient binding occurs with increased levels of laminin or collagen type IV. This suggests that endothelial cells may use different surface constituents for binding to the three proteins and use multiple "receptors"...
The object of these studies was to examine the localization of type IV collagen (Coll‐IV) in the ... more The object of these studies was to examine the localization of type IV collagen (Coll‐IV) in the basement membranes (BM) of epithelial and stromal elements (smooth muscle, nerves, vessels) in normal, hyperplastic, and neoplastic (primary and metastatic) prostate. We also examined the relationship of Coll‐IV distribution to the degree of tumor differentiation (Gleason grading system). We compared immunoperoxidase (IP) and immunoalkaline phosphatase (AP) techniques in these studies and in selected samples we also evaluated immunofluorescence (IF) localization of Coll‐IV and the effects of tissue fixation and pepsin digestion. We found that IF localization of Coll‐IV was intense in unfixed sections. IP and AP reactions were absent in fixed, paraffin‐embedded sections but pepsin treatment yielded intense and uniform reaction products in these same preparations. Both the IP and AP techniques showed similar localization of Coll‐IV in the BM of normal, hyperplastic, and well‐differentiated...
Metastasis of prostate carcinoma requires invasion through the basement membrane, a thin extracel... more Metastasis of prostate carcinoma requires invasion through the basement membrane, a thin extracellular matrix that underlies the epithelial cells, which must be breached by tumor cells invading into surrounding tissue. The CXC-chemokines, which have been shown to promote the migration of neutrophils and carcinoma cells, are candidates to influence prostate carcinoma-cell invasion. CXC-chemokines were examined for the ability to stimulate prostate cell line PC3 invasion in vitro through a reconstituted basement membrane and long-term migration and short-term adhesion to laminin, a major component of the basement membrane. PC3 cells responded to IL-8 and GROalpha with a 1. 6-2-fold increase in invasion through reconstituted basement membrane. A corresponding 2-3-fold increase in chemotaxis toward IL-8 and GROa was seen on laminin. Anti-CXCR2 antibody inhibited IL-8-stimulated migration. Expression levels of the beta(1) integrins were not changed by IL-8, and alpha(6beta1) integrin was used for both stimulated and baseline migration. In addition to the increases in migration and invasion, 2-6-fold transient increases in adhesion on laminin were seen with both IL-8 and GROalpha. These results suggest that the CXC-chemokines stimulate migration and invasion in part by altering the activation state of the beta(1) integrins. The CXC-chemokines act on prostate carcinoma cells through the CXCR2 receptor to promote behavior important for metastasis, and as such may be important in prostate carcinoma progression and metastasis.
Recent studies using solid-phase-binding assays and electron microscopy suggested the presence of... more Recent studies using solid-phase-binding assays and electron microscopy suggested the presence of a heparin-binding domain between the inner globule of a lateral short arm and the cross region of laminin. Using the information from the amino acid sequence of the B1 chain of laminin, several peptides were synthesized from areas with a low hydropathy index and a high density of lysines and/or arginines. One of these, peptide F-9 (RYVVLPRPVCFEKGMNYTVR), which is derived from the inner globular domain of the lateral short arm, demonstrated specific binding to heparin. This was tested in direct solid-phase binding assays by coating the peptide either on nitrocellulose or on polystyrene and in indirect competition assays where the peptide was in solution and either laminin or heparin was immobilized on a solid support. The binding of [3H]heparin to peptide F-9 was dramatically reduced when heparin but not other glycosaminoglycans other than heparin (dextran sulfate, dermatan sulfate) were...
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Papers by Leo Furcht