Human Keratinocytes Adhere to Two Distinct Heparin-Binding Synthetic Peptides Derived from Fibronectin

Fibronectin is present at the dermal-epidermal junction in normal skin and is increased in skin tissues in inflammatory diseases, skin cancers and wound repair. The present studies focused on further characterizing the interaction between fibronectin and keratinocytes, specifically addressing whether human keratinocytes utilize multiple adhesion promoting sequences within fibronectin. Initially, direct cell-binding assays were utilized in which keratinocyte adhesion to plastic substrata coated with fibronectin or proteolytic fragments of fibronectin was quantified. Intact fibronectin, a 75-kD proteolytic fragment containing the RGD sequence, and 33/66-kD cell adhesion/heparin binding fragments lacking the RGD sequence derived from the A and B chains of fibronectin, all promoted keratinocyte adhesion in a concentration-dependent manner. To further define putative cell-binding domains within the 33/66kD fibronectin fragments, we studied three chemically synthesized peptides derived from the amino acid sequence of the 33-kD fragment of the fibronectin A chain: FN-C/H-I (YEKPGSPPREVVPRPRPGV), FN-C/H-II (KNNQKSEPLIGRKKT) and CS1 (DELPQLVTLPHPNLHGPEILDVPST). Substrata coated with either FN-C/H-I or FN-C/H-II promoted keratinocyte adhesion in a concentration-dependent and saturable manner, whereas peptide CS1 promoted no significant keratinocyte adhesion. In solution, both exogenous FN-C/H-I and FN-C/H-II partially inhibited keratinocyte adhesion to the 33/66-kD fibronectin fragments. Furthermore, antibodies prepared against these peptides also inhibited keratinocyte adhesion to the 33/66-kD fibronectin fragments. These data indicate that keratinocyte adhesion to fibronectin is mediated by multiple distinct amino acid sequences, at least two of which are localized to the carboxy-terminal heparin binding domain of fibronectin

Hyaluronan Synthase Elevation in Metastatic Prostate Carcinoma Cells Correlates with Hyaluronan Surface Retention, a Prerequisite for Rapid Adhesion to Bone Marrow Endothelial Cells

Bone marrow is the primary site of metastasis in patients with advanced stage prostate cancer. Prostate carcinoma cells metastasizing to bone must initially adhere to endothelial cells in the bone marrow sinusoids. In this report, we have modeled that interaction in vitro using two bone marrow endothelial cell (BMEC) lines and four prostate adenocarcinoma cell lines to investigate the adhesion mechanism. Highly metastatic PC3 and PC3M-LN4 cells were found to adhere rapidly and specifically (70-90%) to BMEC-1 and trHBMEC bone marrow endothelial cells, but not to human umbilical vein endothelial cells (15-25%). Specific adhesion to BMEC-1 and trHBMEC was dependent upon the presence of a hyaluronan (HA) pericellular matrix assembled on the prostate carcinoma cells. DU145 and LNCaP cells were only weakly adherent and retained no cell surface HA. Maximal BMEC adhesion and RA encapsulation were associated with high levels of HA synthesis by the prostate carcinoma cells. Up-regulation of HA synthase isoforms Has2 and Has3 relative to levels expressed by normal prostate corresponded to elevated HA synthesis and avid BMEC adhesion. These results support a model in which tumor cells with up-regulated HA synthase expression assemble a cell surface hyaluronan matrix that promotes adhesion to bone marrow endothelial cells. This interaction could contribute to preferential bone metastasis by prostate carcinoma cells

Epidermal Growth Factor Stimulates Integrin-Mediated Cell Migration of Cultured Human Corneal Epithelial Cells on Fibronectin and Arginine-Glycine-Aspartic Acid Peptide

urpose. The aim of this work was lo show epidermal growth (actor (KGF)-dependent migration of human corneal epithelial cells to fibronectin and GRGDSP peptide. The authors assessed the role of cell surface integrin heterodimer ab(5\ in mediating haptotactic cell migration to fibronectin by the use of specific function-blocking integrin antibodies. Methods. 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. Results. 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-/?1 and anti-a5 antibodies each inhibited haptotactic cell migration to fibronectin and GRGDSP peptide. Conclusions. Epidermal growth factor provides an important stimulus of haptotactic cell migration of human corneal epithelial cells. Stimulation of cell migration by EGF was maximal in the range of 5 to 10 ng/ml; this response was completely blocked by incubation with an anti-EGF receptor antibody. Function-blocking integrin antibodies, specifically anti-/?1 and antiab, inhibited integrin-niediated cell migration to fibronectin and GRGDSP peptide. These data suggest that EGF represents an essential initial stimulus for haptotactic cell migration of human corneal epithelial cells; furthermore, integrins are important in mediating cell migration to fibronectin and GRGDSP. Invest Ophthalmol Vis Sci. 1995;36:2120-2120 L he corneal epithelium imparts not only a layer of protection to the underlying cornea, it also plays an important role in the maintenance of the basement membrane and the optical transparency of the underlying stroma. 1 The migration of corneal epithelial cells across the basement membrane is an early event in the rcconstitution of a functional corneal epithelium; 2 Fiow the * Department oj hilmmloiy Medmue and I'atholofry, tlw[litoinedi(al F.iiffnrmiifr ('.ente

Human Keratinocytes Adhere to a Unique Heparin-Binding Peptide Sequence Within the Triple Helical Region of Type IV Collagen

The present studies were aimed at further characterizing the interaction between basement membrane molecules and normal cultured human keratinocytes because of the intimate association between basal keratinocytes and the basement membrane. The studies show that keratinocytes adhere to type IV collagen-coated substrata to a greater degree than substrata coated with similar concentrations of fibronectin and laminin. To further define cell-binding regions within type IV collagen, studies were performed using purified pepsin-generated triple helical fragments of type IV collagen and show that keratinocytes bind to sites within the triple-helical region of type IV collagen. To delineate specific cell adhesion promoting sequences, we studied a series of chemically synthesized peptides derived from the triple-helical region of type IV collagen. One peptide, designated Hep III, which is thirteen amino acids in length and binds heparin, was active in directly promoting keratinocyte adhesion. Furthermore, in competition assays, this peptide in solution was shown to inhibit keratinocyte adhesion to substrata coated with Hep Ill or intact type IV collagen. These studies show that keratinocytes hind directly to type IV collagen and chemically define a major cell-adhesion -promoting site within the triple-helical region