Motogenic activity of IGD-containing synthetic peptides

Although the IGD amino acid motif (iso-gly-asp) is a highly conserved feature of the fibronectin type I module, no biological activity has as yet been ascribed to it. We have previously reported that the gelatin-binding domain of fibronectin stimulates the migration of human skin fibroblasts into native, but not denatured, type I collagen substrata. Two IGD-containing type I modules are present within the gelatin-binding domain. The object of this study was to ascertain whether soluble synthetic peptides containing the IGD motif stimulate fibroblast migration. We found that IGD peptides stimulated fibroblast migration in the following order of activity: IGDS (as present in the ninth type I module) > IGDQ (as present in the seventh type I module) > IGD. The scrambled SDGI peptide and the well-characterised RGDS peptide were devoid of motogenic activity. The migratory response of fibroblasts to IGD-containing peptides consisted of two distinct phases: an initial period of peptide-mediated cell activation and a subsequent period of enhanced migration manifest in the absence of further IGD peptide. Cell activation was substratum-independent (occurring equally well on both native and denatured type I collagen substrata), whilst the manifestation of enhanced migration was persistent and substratum-dependent (being evident only by cells adherent to a native collagen substratum). Our data further indicated that cell activation (1) is elicited by a signal transduction cascade occurring within minutes of cell exposure to IGD-containing peptides, (2) is dependent upon integrin alphavbeta3 functionality, (3) involves the tyrosine phosphorylation of focal adhesion kinase (ppFAK125) and (4) is inhibited by signalling mediated through integrin alpha5beta1. The expression of migration stimulating activity by soluble IGD-containing peptides clearly distinguishes them from their RGD counterparts. This is the first identified biological activity of the highly conserved IGD motif and provides a rational platform for the development of a novel family of therapeutic compounds designed to stimulate cell migration in relevant clinical situations, such as impaired wound healing.

Substratum-dependent stimulation of fibroblast migration by the gelatin-binding domain of fibronectin

Nanomolar concentrations of native fibronectin and its RGDS-containing cell-binding domain have previously been reported to stimulate fibroblast migration in the transmembrane (or ‘Boyden chamber’) assay; in contrast, the gelatin-binding domain (GBD) of fibronectin has consistently been reported to be devoid of migration-stimulating activity in this assay. We have examined the effects of fibronectin and several of its purified functional domains on the migration of human skin fibroblasts in what is presumably a more physiologically relevant assay involving the movement of cells into a 3-D matrix of native type I collagen fibrils. We report that: (a) femtomolar concentrations of GBD stimulate fibroblast migration into such collagen matrices; and (b) fibronectin, as well as peptides containing all other of its functional domains, do not exhibit migration-stimulating activity when tested in the femtomolar to nanomolar concentration range (i.e. 0.1 pg/ml to 1 microgram/ml). The correct assignment of migration-stimulating activity to GBD, rather than to a contaminant, was confirmed by: (a) the use of several fibronectin and GBD purification protocols; (b) the neutralization of GBD migration-stimulating activity by monoclonal antibodies directed against epitopes present in this domain; (c) the time-dependent generation of migration-stimulating activity by the proteolytic degradation of native fibronectin; and (d) obtaining an identical dose-response curve with a genetically engineered GBD peptide. The cryptic migration-stimulating activity of GBD was not affected by the presence of serum or native fibronectin, but was inhibited by TGF-beta 1. Parallel experiments using the transmembrane assay confirmed that GBD was devoid of migration-stimulating activity in this assay when membranes coated with gelatin were used, but revealed that significant stimulation of migration was achieved with membranes coated with native type I collagen. Cells preincubated with GBD for 24 hours whilst growing on plastic tissue culture dishes and then plated onto native collagen matrices in the absence of further GBD also displayed an elevated migration compared to controls. Taken together, these observations suggest that: (a) the interaction of GBD with a putative cell surface receptor (and not the collagen substratum) initiates a persistent alteration in cell phenotype which is manifest by an increase in migratory activity when these cells are cultured on a native collagen substratum; and (b) GBD may play a hitherto unrecognised role in the control of cell migration in response to the local release of proteases during pathological processes, such as tumour invasion and wound repair.

Human osteoblasts in culture synthesize collagenase and other matrix metalloproteinases in response to osteotropic hormones and cytokines

Collagenase production by rodent osteoblasts in response to calciotropic hormones has led to the hypothesis that bone cells play a major role in bone resorption by degrading the surface osteoid layer, thereby exposing the underlying mineralized matrix to osteoclastic action. Many studies suggest, however, that this model might not apply to bone resorption in the human. Human osteoblasts have been shown to produce gelatinase-A (72 kDa) and TIMP-1 (tissue inhibitor of metalloproteinases), but previous investigators have been unable to demonstrate the synthesis of collagenase by human osteoblasts either constitutively or in response to bone resorptive agents. In the present study the ability of human osteoblasts to produce the matrix metalloproteinases (MMPs) collagenase, gelatinase and stromelysin, and their specific inhibitors TIMPs-1 and 2, was examined using highly sensitive and specific antisera and by zymography. Semi-quantitative histomorphometric data showed that cells cultured on either glass or a type I collagen substratum constitutively synthesized gelatinase-A and TIMP-1. On type I collagen, however, a small proportion of unstimulated cells produce both collagenase (7%) and gelatinase-B (95 kDa; 3%). Treatment of cells with either parathyroid hormone (PTH), 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3), or partially purified mononuclear cell conditioned medium (MCM), stimulated the synthesis of collagenase, gelatinase-B and stromelysin; MCM was 2- to 3-fold more potent than either PTH or 1,25(OH)2D3. Zymography using SDS/PAGE on conditioned media from cells cultured on type I collagen films revealed the presence of active gelatinase-A and that MCM stimulated progelatinase-B synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of beta-xylosides on proteoglycan biosynthesis and morphology of PC12 pheochromocytoma cells and primary cultures of rat cerebellum

We have examined the effects of beta-xylosides, which act as exogenous acceptors for glycosaminoglycan chain initiation, on the morphology and proteoglycan biosynthesis of PC12 pheochromocytoma cells, and on monolayer, aggregate and explant cultures of early postnatal rat cerebellum. PC12 cells cultured for 13 days in the presence of nerve growth factor (NGF) and beta-xyloside, and labeled during days 11–13 with sodium [35S]sulfate, showed an 8- to 11-fold increase in [35S]sulfate-labeled macromolecules released into the culture medium. Most of the increase was accounted for by chondroitin sulfate, which was in the form of free glycosaminoglycan chains, which were not acid-precipitable. The presence of beta-xyloside also led to a 65–115% increase in [35S]sulfate incorporation into cell-associated glycosaminoglycans and glycoproteins of untreated and NGF-treated PC12 cells, respectively. beta-Xyloside treatment reduced the size of the chondroitin sulfate chains in both the cells and medium from approximately 34,000 to 10,000 Mr, but had much less effect on heparan sulfate, which decreased in size from 16,000 to 13,000-14,500 Mr (in the medium and cells, respectively). beta-Xyloside inhibition of proteoglycan biosynthesis was accompanied by significant morphological effects in NGF-treated PC12 cells, consisting of an increase in length and decrease in the branching, diameter and adhesion to the collagen substratum of the PC12 cell processes. p-Nitrophenyl- and 4-methylumbelliferyl-beta-D-xylosides produced similar effects, which were not seen with p-nitrophenyl-beta-D-galactoside. beta-Xylosides also produced distinct alterations in the adhesion and morphology of monolayer, aggregate, and explant cultures of early postnatal rat cerebellum, which occurred together with inhibition of chondroitin sulfate proteoglycan biosynthesis and a decrease in glycosaminoglycan chain size. These studies indicate that chondroitin sulfate (and probably also heparan sulfate) proteoglycans play a significant role in modulating cell-cell and cell-matrix interactions in nervous tissue development and differentiation.

Pericytes derived from the retinal microvasculature undergo calcification in vitro

Pericytes isolated from the bovine retinal microvasculature retain characteristic features of their in vivo counterparts, such as the presence of glycogen deposits, long filamentous processes, prominent microfilament bundles and the ability to display two distinct and reversible phenotypes. Time-lapse video-microscopy demonstrated that pericytes tend to overlap and aggregate, even in sparse cultures. After reaching confluence, they form multilayered areas that retract away from each other, resulting in the formation of multicellular nodules. These nodules increase in size and cellularity by going through repeated 5- to 6-h cycles of anchoring, spreading, cell proliferation and retraction. Alkaline phosphatase was not detected in pericytes at subconfluent or confluent densities, but this enzyme was expressed in areas of high cell density, such as multilayers and nodules. Pericytes synthesise and deposit an extracellular matrix at all stages of their in vitro development, including nodule formation. The matrix within the nodules contains cross-striated collagen fibres and matrix vesicles. Needle-like crystals of hydroxyapatite appear to be deposited within the matrix, thus leading to massive calcification of the nodule. Calcification, as assessed by electron microscopy, histochemical staining and X-ray microprobe analysis, occurred on plastic and collagen substrate in the absence of disodium-beta-glycerophosphate. The addition of this compound at 5 or 10 mM or the use of a collagen substratum (rather than plastic), brought forward the process of nodule formation and calcification by 3–6 days. Our results suggest that retinal pericytes may differentiate in vitro along the osteogenic pathway.

Substratum-induced stress fiber assembly in vascular endothelial cells during spreading in vitro

We tested whether aortic endothelial cell (EC)-synthesized substrata, which modulate smooth muscle cell proliferation and EC motility following injury, could influence EC actin cytoskeleton and spreading in vitro. A partial characterization of the substrata indicates that the substratum prepared by deoxycholic acid extraction (DOC-derived substratum) is enriched with fibronectin and type IV collagen. Substratum prepared by removal of the intact monolayer with 20 mM EGTA in PBS (EGTA-derived substratum) contains fibronectin and heparan sulfate proteoglycan, but no type IV collagen. Morphometric analyses were performed on fixed and cytoskeletal antibody treated EC in order to quantitate the extent of spreading and stress fiber (SF) assembly. Compared to plastic, the DOC-derived substratum, a collagenase-treated DOC-derived substratum (CT-DOC-derived substratum) and the EGTA-derived substratum promote EC spreading 2.3-, 2.9- and 1.7-fold, respectively. In addition, there are 4.2-, 4.1- and 2.0-fold more SF on DOC-, CT-DOC- and EGTA-derived substrata, respectively, when compared to plastic. Subcellular fractionation and immunoprecipitation of cytoskeletal proteins from metabolically labeled EC were performed prior to electrophoresis and fluorography. The DOC-derived substratum increases immunoprecipitable actin and myosin 3- to 4.5-fold in both fractions compared to the EGTA-derived substratum and plastic. Collagenase treatment of the DOC-derived substratum partially inhibits this increase. Cycloheximide treatment prevents the rise in soluble actin and myosin as well as causing a reduction in SF number by 1/2 on the DOC-derived substratum and 2/3 on CT-DOC-derived substratum. We propose that fibronectin-collagen interactions are, in part, responsible for inducing endothelial synthesis of cytoskeletal proteins required for SF assembly. This substratum-induced actin-cytoskeletal reorganization facilitates EC spreading in vitro.