Pericellular Coat Research Articles

Synthesis and Assembly of the Hyaluronan-Containing Coats around Normal Human Mesothelial Cells

In this study we examined the capacity of normal human mesothelial (NHM) cells and human malignant mesothelioma cells to form hyaluronan-containing pericellular matrices or "coats." The assembly of the pericellular coats was visualized by a particle exclusion assay. We found that large hyaluronan-containing coats were formed around NHM cells whereas their transformed counterparts had no or very limited coats. The coats were removed by treatment with Streptomyces hyaluronidase, which specifically degrades hyaluronan. NHM cells exhibited hyaluronan-containing pericellular matrix within 5 h after seeding. The formation of the coats was stimulated by platelet-derived growth factor and epidermal growth factor. Interestingly, the assembly of the hyaluronan-dependent pericellular matrices was inhibited by the addition of hyaluronan dodecasaccharides. The inhibitory effect on the formation of the coats was due to a destabilization of pericellular matrix and not due to an inhibitory effect of hyaluronan dodecasaccharides on hyaluronan synthesis. In contrast, hyaluronan hexasaccharides, an inhibitor of the interaction between polymeric hyaluronan and its cell surface receptors, had no effect on the size of the coat. Thus, our results are compatible with the possibility that the pericellular matrix surrounding NHM cells consists of newly synthesized hyaluronan which is extruded from the cell and independent of hyaluronan receptors on the cell surface. The coat seems to be stabilized by interactions (hyaluronan-hyaluronan or hyaluronan-protein bridges) which can be prevented by hyaluronan dodecasaccharides.

Hyaluronan-dependent pericellular coats of chick embryo limb mesodermal cells: Induction by basic fibroblast growth factor

Basic fibroblast growth factor (FGF) has been shown previously to be present in the chick embryonic limb during early stages of its development, at which time the limb mesodermal cells are proliferating within a hyaluronan-rich extracellular matrix. In this study, basic FGF was found to stimulate hyaluronan synthesis and production of hyaluronan-dependent pericellular coats by mesodermal cells from the chick embryo limb; acidic FGF, platelet-derived growth factor, epidermal growth factor, and retinoic acid either had a much smaller effect than basic FGF or an inhibitory effect. Transforming growth factor-β stimulated hyaluronan synthesis and coat formation but, unlike basic FGF, this factor also stimulated chondroitin sulfate production by the mesodermal cells.

Membrane‐associated hyaluronate‐binding activity of chondrosarcoma chondrocytes

The association of hyaluronate with the surface of chondrocytes was examined by several approaches using primary cultures of chondrocytes derived from the Swarm rat chondrosarcoma. In culture, chondrosarcoma chondrocytes produced large pericellular coats, which can be visualized by particle exclusion, and which can be removed by Streptomyces hyaluronidase. Exposure of chondrocytes, which had been metabolically labelled with 3H-acetate, to exogenous hyaluronate or to Streptomyces hyaluronidase resulted in the release of 36-38% of the endogenous, labelled chondroitin sulfate from the cell layer into the incubation solution. These results imply that at least 37% of the cell layer chondroitin sulfate proteoglycan is retained there by an interaction with hyaluronate. Thus membranes were prepared from cultured chondrocytes and examined for sites which bind 3H-hyaluronate. Binding was observed and found to be saturable, specific for hyaluronate, of high affinity (Kd = approximately 10(-10) M), and destroyed by treating the membranes with trypsin. The 3H-hyaluronate-binding activity was inhibited competitively by hyaluronate decasaccharides but not by hexasaccharides or octasaccharides, indicating that the binding sites recognize a sequence of hyaluronate composed of five disaccharide repeats. The binding activity was partially purified from a detergent extract of chondrocyte membranes by ion exchange chromatography on DEAE-cellulose, followed by affinity chromatography on wheat germ agglutinin-agarose. Analysis of the partially purified binding activity by SDS-PAGE revealed five protein bands of 48,000-66,000 daltons in silver-stained gels. SDS-PAGE followed by Western blotting and exposure to monoclonal antibodies which recognize epitopes present in link protein and in the hyaluronate-binding region of cartilage proteoglycan revealed no immunoreactive protein bands in the partially purified material. We conclude that one mechanism by which hyaluronate associates with the chondrocyte surface may be via interaction with a membrane-bound hyaluronate-binding protein which is distinct from link protein and proteoglycan.

Connective tissue components in synovial fibroblast cultures exposed to interleukin 1 and prostaglandin E2.

Long-term synovial fibroblast cultures were exposed to interleukin 1 (IL-1) or prostaglandin E2 (PGE2). The normally spindle-shaped fibroblasts changed to stellate-shaped cells, resembling the HLA-DR-positive, collagenase-producing cells which are normally seen only in primary cultures from enzyme-digested rheumatoid synovial tissue. However, the IL-1- or PGE2-induced fibroblasts were not HLA-DR-positive. This suggests that these cell populations represent originally different cell lines or that the expression of HLA-DR antigens is not induced by the agents used. For further characterization of these stellate cells, the location of fibronectin and type I collagen was studied by specific antibodies and the pericellular coat around fibroblasts was visualized by the erythrocyte exclusion method. Both IL-1 and PGE2 treatments destroyed the intercellular fibronectin network. Type I collagen was detected as intracellular granules. The stellate fibroblasts were usually full of these granules in contrast to intact fibroblasts in which the number of collagen fluorescence granules varied greatly. The pericellular coat known to be formed mainly by hyaluronic acid was similar around spindle and stellate-shaped fibroblasts. Rheumatoid arthritis-derived fibroblasts did not differ from their non-rheumatoid counterparts in any of the experiments. The effect of IL-1 and PGE2 on fibroblasts simulates the interaction between mononuclear cells and fibroblasts in synovial stroma and also potentially the interactions between different cell types in synovial lining.

Changes in the pericellular matrix during differentiation of limb bud mesoderm

Mesodermal cells in the developing chick embryo limb bud appear morphologically homogeneous until stage 21. At stage 22 the prechondrogenic and premyogenic areas begin to condense, culminating in the appearance of cartilage and muscle by stage 25–26. We have examined changes in the hyaluronate-dependent pericellular matrices elaborated by mesodermal cells of the limb bud from different developmental stages and the corresponding changes in production of cell surface-associated and secreted glycosaminoglycans. When placed in culture, most early mesodermal cells (stage 17 lateral plate and stage 19 limb bud) exhibited pericellular coats as visualized by the exclusion of particles. These coats were removed by treatment of the cultures with Streptomyces hyaluronidase. Cells from stage 20–21 limb buds (precondensation) had smaller coats, whereas cells derived from stage 22, 24, and 26 limb buds (condensed chondrogenic and myogenic regions) lacked coats. However, coats were reformed during subsequent cytodifferentiation of chondrocytes; chondrocytes from stage 28 and 30 limb buds, and more mature chondrocytes from stage 38 tibiae, had pericellular coats. Thus, cytodifferentiation of cartilage is accompanied by extensive intercellular matrix accumulation in vivo and reacquisition of pericellular coats in vitro. Although their structure was still dependent on hyaluronate, chondrocyte coats were associated with increased proteoglycan content compared to the coats of early mesodermal cells. The amount of incorporation of [ 3H]acetate into cell surface hyaluronate remained relatively constant from stages 17 to 38, whereas in the medium compartment, incorporation into hyaluronate was more than 4-fold greater by stage 17 and 19 mesodermal cells than by cells from stages between 20 and 38. However, there was a progressive increase in incorporation into cell surface and medium chondroitin sulfate throughout these developmental stages. Thus, at the time of cellular condensation in the limb bud in vivo, we have observed a reduction in size of hyaluronate-dependent pericellular coats and a dramatic change in the relative proportion of hyaluronate and chondroitin sulfate produced by the mesodermal cells in vitro.

Glycoproteins on the surface of smooth muscle cells involved in their interaction with type V collagen.

Type V collagen is a major component of the pericellular coat of smooth cells (SMC). The purpose of the present study was to assess biochemically the nature of an in vitro interaction between bovine aortic SMC and type V collagen from the same source. This interaction was originally shown to be mediated by a cell-surface glycoconjugate. Data obtained in the present study suggests that the binding system consists of integral membrane glycoproteins which act alone or in combination with a surface glycolipid in type V attachment. The nature of this system was indicated by the finding of 80 000 and 50 000 components in the plasma membrane fractions which were specifically retained by type V collagen--Sepharose columns and incorporated both methionine and mannose label. Moreover, inhibition of protein synthesis lowered SMC attachment by 25%. The mannose label associated with these components was probably in the form of a simple oligosaccharide at the attachment site since it bound to concanavalin A (ConA) and was sensitive to endoglycosidase H. Iodinated ConA labelling indicated elevated levels of these components were associated with SMC--type V collagen interaction. The attachment region on the type V molecule was localized within the cyanogen bromide peptide 6 of the alpha 2 (V) chain.

Loss of hyaluronate-dependent coat during myoblast fusion

Cultured myoblasts were found to exhibit extensive, Streptomyces hyaluronidase-sensitive pericellular coats as revealed by exclusion of particles (fixed red blood cells). These coats are not discernible subsequent to fusion of the myoblasts to form myotubes. The myoblasts contained 2.5 times more hyaluronate attached to their cell surface than myotubes when the data was expressed per unit of protein, but no change in hyaluronate was evident on a per DNA basis. Hyaluronidase activities in the cultures were equivalent when expressed per unit of protein. We conclude that, although the myotubes accumulate larger amounts of protein than myoblasts, there is no compensatory increase in hyaluronate.

Pericellular coat of chick embryo chondrocytes: structural role of hyaluronate.

Chondrocytes produce large pericellular coats in vitro that can be visualized by the exclusion of particles, e.g., fixed erythrocytes, and that are removed by treatment with Streptomyces hyaluronidase, which is specific for hyaluronate. In this study, we examined the kinetics of formation of these coats and the relationship of hyaluronate and proteoglycan to coat structure. Chondrocytes were isolated from chick tibia cartilage by collagenase-trypsin digestion and were characterized by their morphology and by their synthesis of both type II collagen and high molecular weight proteoglycans. The degree of spreading of the chondrocytes and the size of the coats were quantitated at various times subsequent to seeding by tracing phase-contrast photomicrographs of the cultures. After seeding, the chondrocytes attached themselves to the tissue culture dish and exhibited coats within 4 h. The coats reached a maximum size after 3-4 d and subsequently decreased over the next 2-3 d. Subcultured chondrocytes produced a large coat only if passaged before 4 d. Both primary and first passage cells, with or without coats, produced type II collagen but not type I collagen as determined by enzyme-linked immunosorbent assay. Treatment with Streptomyces hyaluronidase (1.0 mU/ml, 15 min), which completely removed the coat, released 58% of the chondroitin sulfate but only 9% of the proteins associated with the cell surface. The proteins released by hyaluronidase were not digestible by bacterial collagenase. Monensin and cycloheximide (0.01-10 microM, 48 h) caused a dose-dependent decrease in coat size that was linearly correlated to synthesis of cell surface hyaluronate (r = 0.98) but not chondroitin sulfate (r = 0.2). We conclude that the coat surrounding chondrocytes is dependent on hyaluronate for its structure and that hyaluronate retains a large proportion of the proteoglycan in the coat.

Endogenous hyaluronate-cell surface interactions in 3T3 and simian virus-transformed 3T3 cells.

3T3 cells have a large, pericellular coat which contains 30 times more hyaluronate than the amount of cell surface hyaluronate associated with simian virus 40-transformed 3T3 (SV-3T3) cells. On the other hand, SV-3T3 cells have high affinity binding sites for exogenously added hyaluronate, whereas 3T3 cells have much lower affinity sites. Removal of cell surface hyaluronate from SV-3T3 cells by treatment with hyaluronidase caused a reproducible increase in their maximum binding capacity for exogenous hyaluronate but no significant change in binding affinity or specificity. For 3T3 cells, however, the maximum amount of binding decreased and the affinity of binding increased after hyaluronidase treatment. When endogenous cell surface hyaluronate was labeled metabolically and then the cells incubated in the presence of exogenous unlabeled hyaluronate, the labeled cell surface hyaluronate was quantitatively displaced from the SV-3T3 cells but was not displaced from the 3T3 cells. Chondroitin sulfate and heparin did not displace cell surface hyaluronate from either cell type. Membranes isolated from SV-3T3 cells bound hyaluronate specifically and with high affinity, whereas membranes from 3T3 cells did not consistently bind a significant amount of hyaluronate. We conclude from these studies that the retention of endogenous hyaluronate on the surface of SV-3T3 cells is mediated by binding sites similar to those detected by the addition of exogenous hyaluronate, and the mechanism of retention of endogenous hyaluronate on the surface of 3T3 cells differs from SV-3T3 cells.

Hyaluronate coat formation and cell spreading in rat fibrosarcoma cells

Hyaluronate-containing pericellular coats have been demonstrated around rat fibrosarcoma cells by exclusion of particles (fixed red blood cells). The cell coats normally form during spreading of the rat fibrosarcoma cells subsequent to subculturing. Monensin, a drug which disrupts the Golgi and which also inhibits hyaluronate synthesis in these cells, inhibits the regeneration of these coats after hyaluronidase or trypsin treatment but does not inhibit cell spreading. Cycloheximide, a drug which inhibits protein but not hyaluronate synthesis, does not prevent coat regeneration but partially inhibits cell spreading. Thus by exploiting the opposing effects of cycloheximide and monensin on coat regeneration and cell spreading, we have been able to dissociate these two phenomena.

Monensin inhibition of hyaluronate synthesis in rat fibrosarcoma cells.

Evidence is presented that monensin-sensitive membranes, presumably the Golgi apparatus, are involved in the synthesis of hyaluronate in rat fibrosarcoma cells. Monensin caused the inhibition of incorporation of metabolic precursors into hyaluronate produced by rat fibrosarcoma cells in a concentration- and time-dependent manner. Maximum inhibition (70-80%) was obtained on treatment with 10(-7) M monensin for 24 h. Incorporation of label into secreted hyaluronate and into that associated with the cell surface was inhibited, but incorporation into intracellular hyaluronate was not inhibited. In 3T3 cells, treatment for 24 h with 10(-7) M monensin inhibited incorporation of label only into secreted hyaluronate. The hyaluronate-rich pericellular coat, revealed by exclusion of fixed red blood cells, was depleted on treatment with monensin under the same conditions which caused inhibition of hyaluronate synthesis in the fibrosarcoma cells. Cell proliferation, as measured by DNA content/culture and [3H]thymidine incorporation, was also inhibited in a dose-dependent manner by monensin (10(-8)-10(-6) M). Protein synthesis was not inhibited at these doses, nor was monensin cytotoxic as judged by a 51Cr release assay. The inhibition of hyaluronate synthesis was independent of the antiproliferative effect of monensin because it was obtained during log phase growth or confluency and in the presence or in the absence of serum.

Hyaluronate degradation in 3T3 and simian virus-transformed 3T3 cells.

The cellular control of hyaluronate levels was examined in cultures of simian virus 40-transformed 3T3 (SV3T3) and 3T3 cells which are known to differ in their metabolism of hyaluronate. When [3H]hyaluronate was added to cultures of the two cell lines, four times more ligand was bound per mg of protein by the SV3T3 cells than by the 3T3 cells. Of the bound [3H] hyaluronate, 40% was degraded by the SV3T3 cells to oligosaccharides characteristic of the breakdown of hyaluronate, but only 2% was degraded by 3T3 cells. Hyaluronidase activity was found in the cell layer and medium of the SV3T3 cultures, but was not detectable in 3T3 cells. The SV3T3 enzyme was active only at acidic pH, but at neutral pH the secreted SV3T3 hyaluronidase was thermally more stable then the cell-associated enzyme. In contrast, both cell lines were found to contain similar amounts of beta-glucuronidase and beta-N-acetylglucosaminidase activity. We conclude that the elevated capacity of SV3T3 cells to degrade hyaluronate may be partially responsible for their lack of the hyaluronate-containing pericellular coat which is prominent around 3T3 cells.

Explant Culture of Human and Rabbit Articular Chondrocytes

Copious outgrowth of chondrocytes was obtained by explantation from each of three rabbit and one surgically-resected human articular cartilages pretreated briefly with trypsin. In lapine explants, ascorbate (40 micrograms/ml) increased DNA three-fold over control values and resulted in deposition of a chondroid matrix. It doubled radiosulfate incorporation by the outgrowths. Up to 56% of the sulfated glycosaminoglycan synthesized was located in the trypsin-digestible pericellular coat compared with about 15% in previous monolayer cultures. The collagens synthesized were characterized partially. In rabbit cell cultures, the alpha 1:alpha 2 ratio varied from 2.9 to 3.8. In human cultures, an unusual post-alpha 2 peak was observed. The findings suggest an uncoupling of the phenotypic expression of the major cartilaginous macromolecules in the cultures. There were no distinctive differences between chondrocytes derived from normal and fibrillated human cartilage of the same individual.