Hyaluronan receptor-directed assembly of chondrocyte pericellular matrix

Initial assembly of extracellular matrix occurs within a zone immediately adjacent to the chondrocyte cell surface termed the cell- associated or pericellular matrix. Assembly within the pericellular matrix compartment requires specific cell-matrix interactions to occur, that are mediated via membrane receptors. The focus of this study is to elucidate the mechanisms of assembly and retention of the cartilage pericellular matrix proteoglycan aggregates important for matrix organization. Assembly of newly synthesized chondrocyte pericellular matrices was inhibited by the addition to hyaluronan hexasaccharides, competitive inhibitors of the binding of hyaluronan to its cell surface receptor. Fully assembled chondrocyte pericellular matrices were displaced using hyaluronan hexasaccharides as well. When exogenous hyaluronan was added to matrix-free chondrocytes in combination with aggrecan, a pericellular matrix equivalent in size to an endogenous matrix formed within 30 min of incubation. Addition of hyaluronan and aggrecan to glutaraldehyde-fixed chondrocytes resulted in matrix assembly comparable to live chondrocytes. These matrices could be inhibited from assembling by the addition of excess hyaluronan hexasaccharides or displaced once assembled by subsequent incubation with hyaluronan hexasaccharides. The results indicate that the aggrecanrich chondrocyte pericellular matrix is not only on a scaffolding of hyaluronan, but actually anchored to the cell surface via the interaction between hyaluronan and hyaluronan receptors.     

Hyaluronan promotes the malignant phenotype

Hyaluronan is a high-molecular-weight, negatively charged polysaccharide with unusual physical and interactive properties. Hyaluronan is localized in the extracellular matrix, at the cell surface, and inside cells. Its tissue distribution is ubiquitous, but it is particularly concentrated in pericellular matrices surrounding proliferating and migrating cells. Hyaluronan contributes to cell behavior in at least three ways. Its unique physical properties influence the biomechanical properties of extracellular and pericellular matrices; it is a template for assembly of other pericellular macromolecules; and it interacts directly with cell surface receptors that transduce intracellular signals. Experimental studies in animal models have documented a crucial role for hyaluronan in tumor growth and metastasis. Cellular manipulations have shown that hyaluronan promotes anchorage-independent growth and invasiveness, hallmarks of the malignant phenotype.     

Hyaluronan Controls the Deposition of Fibronectin and Collagen and Modulates TGF-β1 Induction of Lung Myofibroblasts

The contribution of hyaluronan-dependent pericellular matrix to TGF-β1-driven induction and maintenance of myofibroblasts is not understood. Hyaluronan is an extracellular matrix (ECM) glycosaminoglycan important in cell adhesion, proliferation and migration, and is implicated in myofibroblast formation and maintenance. Reduced turnover of hyaluronan has been linked to differentiation of myofibroblasts and potentiation of lung fibrosis. Fibronectin is a fibril forming adhesive glycoprotein that is also upregulated following induction with TGF-β1. Although they are known to bind each other, the interplay between hyaluronan and fibronectin in the pericellular matrix during myofibroblast induction and matrix assembly is not clear. This study addresses the role of hyaluronan and its interaction with fibrillar matrix components during myofibroblast formation. Hyaluronan and fibronectin were increased and co-localized in the ECM following myofibroblast induction by TGF-β1. Inhibition of hyaluronan synthesis in TGF-β1-induced lung myofibroblasts over a 4 day period with 4-methyl umbelliferone (4-MU) further enhanced myofibroblast morphology, caused increased deposition of fibronectin and type I collagen in the ECM, and increased expression of alpha-smooth muscle actin and hyaluronan synthase 2 (HAS2) mRNA. Hyaluronan oligosaccharides or hyaluronidase treatment, which more effectively disrupted the pericellular matrix, had similar effects. CD44 and β1 integrins co-localized in the cell membrane and along some stress fibers. However, CD44 and hyaluronan were specifically excluded from focal adhesions, and associated primarily with cortical actin. Time-lapse imaging of the immediate effects of hyaluronidase digestion showed that hyaluronan matrix primarily mediates attachment of membrane and cortical actin between focal contacts, suggesting that surface adhesion through hyaluronan and CD44 is distinct from focal adhesion through β1 integrins and fibronectin. Fluorescein-labeled hyaluronan bound regularly along fibronectin fibers and co-localized more with β1 integrin and less with CD44. Therefore, the hyaluronan matrix can interfere with the assembly of fibrillar ECM components, and this interplay regulates the degree of myofibroblast formation. These data also suggest that adhesion through hyaluronan matrix impacts cytoskeletal organization, and is potentially part of a clutch mechanism that regulates stick and slip of myofibroblasts by affecting the adhesion to and organization of fibronectin and collagen.     

Proteoglycans: many forms and many functions.

Proteoglycans are produced by most eukaryotic cells and are versatile components of pericellular and extracellular matrices. They belong to many different protein families. Their functions vary from the physical effects of the proteoglycan aggrecan, which binds with link protein to hyaluronan to form multimolecular aggregates in cartilage; to the intercalated membrane protein CD44 that has a proteoglycan form and is a receptor and a cell-binding site for hyaluronan; to heparan sulfate proteoglycans of the syndecan and other families that provide matrix binding sites and cell-surface receptors for growth factors such as fibroblast growth factor (FGF). One feature that recurs in proteoglycan biology is that their structure is open to extensive modulation during cellular expression. Examples of protein changes are known, but a major source of structural variation is in the glycosaminoglycan chains. The number of chains and their length can vary, as well as their pattern of sulfation. This may result in the switching of different chain types with different properties, e.g., chondroitin sulfate and heparan sulfate, and it may also result in the selective expression of sulfated chain sequences that have specific functions. The control of glycosaminoglycan structure is not well understood, but it does appear to be used to change the properties of proteoglycans to suit different biological needs. Proteoglycan forms of proteins are thus important modifiers of the organization of the pericellular and extracellular matrices and modulators of the processes that occur there.     

Hyaluronan and morphogenesis

In the past decade, there has been an explosion of interest in hyaluronan, an often misunderstood, biochemically simple, yet functionally complex carbohydrate polymer that is a resident of many extracellular matrices. Previously thought of as a passive, space-filling component of the extracellular matrix, the so-called "goo" concept, hyaluronan has risen to a much higher regard in recent years, even being called "magic glue" in a recent perspective. Hyaluronan is likely to be the common thread in many morphogenetic processes, including condensation events and epithelial-to-mesenchymal transformation. Hyaluronan is comparatively unique as a component of the extracellular matrix as it is solely composed of carbohydrate. In order to truly understand this biopolymer, one must first understand its biosynthesis, then understand its uptake and turnover, then identify its binding proteins and receptors. Major advances have been made in all of these arenas within the past decade. Hyaluronan synthases, hyaluronidases, and the hyaladherins have been molecularly identified and cloned. Furthermore, many have now been inactivated, employing gene targeting strategies, to create mice deficient in the respective gene product function. Collectively, huge strides have been made in our understanding of the diverse biological functions for this fascinating molecule. Hyaluronan appeared in metazoans immediately prior to the arrival of the vertebrates, and may be required for the differentiation, development, and/or function of most cell lineages, structures, and tissues that we associate with vertebrates, such as the neural crest, the skeleton, including the teeth, skin, and hair, and the chambered heart. In this review, we will update the reader on the advances of the past decade and provide insight into those morphogenetic processes through which hyaluronan regulates vertebrate development.     

CD44-Anchored Hyaluronan-Rich Pericellular Matrices: An Ultrastructural and Biochemical Analysis

The chondrocyte pericellular matrix is an essential zone for cartilage matrix assembly and turnover. Electron micrographs of native endogenous and composition-defined exogenous pericellular matrices, both preserved via ruthenium hexaminetrichloride fixation procedures, depict strikingly similar networks of hyaluronan and proteoglycan extending out from the cell surface. Biochemical and morphological analyses of matrix regrowth show that monoclonal antibodies directed against the hyaluronan receptor CD44 blocked chondrocyte pericellular matrix assembly. Immunoperoxidase electron microscopy was used to display regular repeating spacing patterns of hyaluronan/proteoglycan assembly at the cell surface. These patterns compared well with the ultrastructural immunolocalization of CD44 at the cell surface. All of these data suggest that the hyaluronan receptor CD44 retains and participates in the assembly of the chondrocyte pericellular matrix.     

Depth-dependent analysis of the role of collagen fibrils, fixed charges and fluid in the pericellular matrix of articular cartilage on chondrocyte mechanics

The pericellular matrix of articular cartilage has been shown to regulate the mechanical environment of chondrocytes. However, little is known about the mechanical role of collagen fibrils in the pericellular matrix, and how fibrils might help modulate strains acting on chondrocytes when cartilage is loaded. The primary objective was to clarify the effect of pericellular collagen fibrils on cell volume changes and strains during cartilage loading. Secondary objectives were to investigate the effects of pericellular fixed charges and fluid on cell responses. A microstructural model of articular cartilage, in which chondrocytes and pericellular matrices were represented with depth-dependent structural and morphological properties, was created. The extracellular matrix and pericellular matrices were modeled as fibril-reinforced, biphasic materials with swelling capabilities, while chondrocytes were assumed to be isotropic and biphasic with swelling properties. Collagen fibrils in the extracellular matrix were represented with an arcade-like architecture, whereas pericellular fibrils were assumed to run tangential to the cell surface. In the early stages of a stress-relaxation test, pericellular fibrils were found to sensitively affect cell volume changes, even producing a reversal from increasing to decreasing cell volume with increasing fibril stiffness in the superficial zone. Consequently, steady-state volume of the superficial zone cell decreased with increasing pericellular fibril stiffness. Volume changes in the middle and deep zone chondrocytes were smaller and opposite to those observed in the superficial zone chondrocyte. An increase in the pericellular fixed charge density reduced cell volumes substantially in every zone. The sensitivity of cell volume changes to pericellular fibril stiffness suggests that pericellular fibrils play an important, and as of yet largely neglected, role in regulating the mechanical environment of chondrocytes, possibly affecting matrix synthesis during cartilage development and degeneration, and affecting biosynthetic responses associated with articular cartilage loading.     

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.     

The dynamic structure of the pericellular matrix on living cells

Although up to several microns thick, the pericellular matrix is an elusive structure due to its invisibility with phase contrast or DIC microscopy. This matrix, which is readily visualized by the exclusion of large particles such as fixed red blood cells is important in embryonic development and in maintenance of cartilage. While it is known that the pericellular matrix which surrounds chondrocytes and a variety of other cells consists primarily of proteoglycans and hyaluronan with the latter binding to cell surface receptors, the macromolecular organization is still speculative. The macromolecular organization previously could not be determined because of the collapse of the cell coat with conventional fixation and dehydration techniques. Until now, there has been no way to study the dynamic arrangement of hyaluronan with its aggregated proteoglycans on living cells. In this study, the arrangement and mobility of hyaluronan-aggrecan complexes were directly observed in the pericellular matrix of living cells isolated from bovine articular cartilage. The complexes were labeled with 30- to 40-nm colloidal gold conjugated to 5-D-4, an antibody to keratan sulfate, and visualized with video-enhanced light microscopy. From our observations of the motion of pericellular matrix macromolecules, we report that the chondrocyte pericellular matrix is a dynamic structure consisting of individual tethered molecular complexes which project outward from the cell surface. These complexes undergo restricted rotation or wobbling. When the cells were cultured with ascorbic acid, which promotes production of matrix components, the size of the cell coat and the position of the gold probes relative to the plasma membrane were not changed. However, the rapidity and extent of the tethered motion were reduced. Treatment with Streptomyces hyaluronidase removed the molecules that displayed the tethered motion. Addition of hyaluronan and aggrecan to hyaluronidase-treated cells yielded the same labeling pattern and tethered motion observed with native cell coats. To determine if aggrecan was responsible for the extended configuration of the complexes, only hyaluronan was added to the hyaluronidase-treated cells. The position and mobility of the hyaluronan was detected using biotinylated hyaluronan binding region (b- HABR) and gold streptavidin. The gold-labeled b-HABR was found only near the cell surface. Based on these observations, the hyaluronan- aggrecan complexes composing the cell coat are proposed to be extended in a brush-like configuration in an analogous manner to that previously described for high density, grafted polymers in good solvents.     

Platelet-derived growth factor stimulates the formation of versican-hyaluronan aggregates and pericellular matrix expansion in arterial smooth muscle cells

Hyaluronan and versican-rich pericellular matrices form around arterial smooth muscle cells (ASMC) preferentially during the detachment phase of proliferation and migration. PDGF is a potent mitogen and chemotactic agent for ASMC and also stimulates the production of extracellular matrix molecules which may regulate the proliferative and migratory capacity of the cells. We have examined the effect of PDGF on the formation of hyaluronan-dependent pericellular matrices, and on the synthesis and interaction of several major pericellular coat constituents. As demonstrated using a particle exclusion assay, PDGF stimulated the formation of pericellular matrices and was seen both in an increased proportion of cells with a coat and a greater coat size. This increase was accompanied by a transient increase in hyaluronan synthase 2 (HAS2) expression and an increase in hyaluronan synthesis and polymer length. PDGF also increased the synthesis of versican and link protein as measured at the mRNA and protein levels. The amount of native versican-hyaluronan aggregates and link-stabilized aggregate was also increased following PDGF treatment. Time lapse imaging showed that pericellular matrix formation occurred around trailing cell processes prior to their detachment. These data suggest that PDGF modulates the synthesis and organization of ASMC pericellular coat-forming molecules such as versican, hyaluronan, and link protein, which leads to extracellular matrix expansion and alterations in ASMC phenotype.     

Hyaluronan.

Hyaluronan (hyaluronic acid) is a high-molecular-mass polysaccharide found in the extracellular matrix, especially of soft connective tissues. It is synthesized in the plasma membrane of fibroblasts and other cells by addition of sugars to the reducing end of the polymer, whereas the nonreducing end protrudes into the pericellular space. The polysaccharide is catabolized locally or carried by lymph to lymph nodes or the general circulation, from where it is cleared by the endothelial cells of the liver sinusoids. The overall turnover rate is surprisingly rapid for a connective tissue matrix component (t1/2 0.5 to a few days). Hyaluronan has been assigned various physiological functions in the intercellular matrix, e.g., in water and plasma protein homeostasis. Hyaluronan production increases in proliferating cells and the polymer may play a role in mitosis. Extensive hyaluronidase-sensitive coats have been identified around mesenchymal cells. They are either anchored firmly in the plasma membrane or bound via hyaluronan-specific binding proteins (receptors). Such receptors have now been identified on many different cells, e.g., the lymphocyte homing receptor CD 44. Interaction between a hyaluronan receptor and extracellular polysaccharide has been connected with locomotion and cell migration. Hyaluronan seems to play an important role during development and differentiation and has other cell regulatory activities. Hyaluronan has also been recognized in clinical medicine. A concentrated solution of hyaluronan (10 mg/ml) has, through its tissue protective and rheological properties, become a device in ophthalmic surgery. Analysis of serum hyaluronan is promising in the diagnosis of liver disease and various inflammatory conditions, e.g., rheumatoid arthritis. Interstitial edema caused by accumulation of hyaluronan may cause dysfunction in various organs.     

Organization and adhesive properties of the hyaluronan pericellular coat of chondrocytes and epithelial cells

Hyaluronan is a megadalton glycosaminoglycan composed of repeating units of D-N-acetylglucosamine-beta-D-Glucuronic acid. It is known to form a highly hydrated pericellular coat around chondrocytes, fibrosarcoma, and smooth muscle cells. Using environmental scanning electron microscopy we detected fully hydrated hyaluronan pericellular coats around rat chondrocytes (RCJ-P) and epithelial cells (A6). Hyaluronan mediates early adhesion of both chondrocytes and A6 cells to glass surfaces. We show that chondrocytes in suspension establish early "soft contacts" with the substrate through a thick, hyaluronidase-sensitive coat (4.4 +/- 0.7 microm). Freshly-attached cells drift under shear stress, leaving hyaluronan "footprints" on the surface. This suggests that chondrocytes are surrounded by a multilayer of entangled hyaluronan molecules. In contrast, A6 cells have a 2.2 +/- 0.4- microm-thick hyaluronidase-sensitive coat, do not drift under shear stress, and remain firmly anchored to the surface. We consider the possibility that in A6 cells single hyaluronan molecules, spanning the whole thickness of the pericellular coat, mediate these tight contacts.     

Internalization of the Hyaluronan Receptor CD44 by Chondrocytes

Chondrocytes express CD44 as a primary receptor for the matrix macromolecule hyaluronan. Hyaluronan is responsible for the retention and organization of proteoglycan within cartilage, and hyaluronan-chondrocyte interactions are important for the assembly and maintenance of the cartilage matrix. Bovine articular chondrocytes were used to study the endocytosis and turnover of CD44 and the effects of receptor occupancy on this turnover. Matrix-intact chondrocytes exhibit approximately a 6% internalization of cell surface CD44 by 4 h. Treatment with Streptomyces hyaluronidase to remove endogenous pericellular matrix increased internalization to approximately 20% of cell surface CD44 at 4 h. This turnover could be partially inhibited by the addition of exogenous hyaluronan to these matrix-depleted chondrocytes. Cell surface biotin-labeled CD44 was internalized by chondrocytes and this internalization was decreased in the presence of hyaluronan. Colocalization of internalized CD44 and fluorescein-labeled hyaluronan in intracellular vesicles correlates with the previous results of receptor-mediated endocytosis pathway for the degradation of hyaluronan by acid hydrolases. Taken together, our results indicate that CD44 is internalized by chondrocytes and that CD44 turnover is modulated by occupancy with hyaluronan.     

Hyaluronan: Biosynthesis and signaling

Background

Hyaluronan is a critical component of extracellular matrix with several different roles. Besides the contribution to the tissue hydration, mechanical properties and correct architecture, hyaluronan plays important biological functions interacting with different molecules and receptors.

Scope of review

The review addresses the control of hyaluronan synthesis highlighting the critical role of hyaluronan synthase 2 in this context as well as discussing the recent findings related to covalent modifications which influence the enzyme activity. Moreover, the interactions with specific receptors and hyaluronan are described focusing on the importance of polymer size in the modulation of hyaluronan signaling.

Major conclusions

Due to its biological effects on cells recently described, it is evident how hyaluronan is to be considered not only a passive component of extracellular matrix but also an actor involved in several scenarios of cell behavior.

General significance

The effects of metabolism on the control of hyaluronan synthesis both in healthy and pathologic conditions are critical and still not completely understood. The hyaluronan capacity to bind several receptors triggering specific pathways may represent a valid target for new approach in several therapeutic strategies. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Intracellular localization of hyaluronan in proliferating cells.

Hyaluronan is a high molecular weight glycosaminoglycan found in the extracellular matrix of many tissues, where it is believed to promote cell migration and proliferation. It was recently shown that hyaluronan-dependent pericellular matrix formation is a rapid process that occurs as cells detach during mitosis. Growing evidence for intracellular hyaluronan in tissues in vivo, together with evidence of intracellular hyaluronan binding molecules, prompted us to examine hyaluronan distribution and uptake as well as hyaluronan binding sites in cells and their relationship to cell proliferation in vitro, using a biotinylated hyaluronan binding protein and fluorescein-labeled hyaluronan. In permeabilized smooth muscle cells and fibroblasts, hyaluronan staining was seen in the cytoplasm in a diffuse, network-like pattern and in vesicles. Nuclear hyaluronan staining was observed and confirmed by confocal microscopy and was often associated with nucleoli and nuclear clefts. After serum stimulation of 3T3 cells, there was a dramatic increase in cytoplasmic hyaluronan staining, especially during late prophase/early prometaphase of mitosis. In contrast, unstimulated cells were negative. There was a pronounced alteration in the amount and distribution of hyaluronan binding sites, from a mostly nucleolar distribution in unstimulated cells to one throughout the cytoplasm and nucleus after stimulation. Exogenous fluorescein-labeled hyaluronan was taken up avidly into vesicles in growing cells but was localized distinctly compared to endogenous hyaluronan, suggesting that hyaluronan in cells may be derived from an intracellular source. These data indicate that intracellular hyaluronan may be involved in nucleolar function, chromosomal rearrangement, or other events in proliferating cells. (J Histochem Cytochem 47:1331-1341, 1999)     

Latrunculin and cytochalasin decrease chondrocyte matrix retention.

The proteoglycan-rich extracellular matrix (ECM) directly associated with the cells of articular cartilage is anchored to the chondrocyte plasma membrane via interaction with the hyaluronan receptor CD44. The cytoplasmic tail of CD44 interacts with the cortical cytoskeleton. The objective of this study was to determine the role of the actin cytoskeleton in CD44-mediated matrix assembly by chondrocytes and cartilage matrix retention and homeostasis. Adult bovine articular cartilage tissue slices and isolated chondrocytes were treated with latrunculin or cytochalasin. Tissues were processed for histology and chondrocytes were examined for CD44 expression and pericellular matrix assembly. Treatments that disrupt the actin cytoskeleton reduced chondrocyte pericellular matrix assembly and the retention of proteoglycan within cartilage explants. There was enhanced detection of a neoepitope resulting from proteolysis of aggrecan. Cytoskeletal disruption did not reduce CD44 expression, as monitored by flow cytometry, but detergent extraction of CD44 was enhanced and hyaluronan binding was decreased. Thus, disruption of the cytoskeleton reduces the anchorage of CD44 in the chondrocyte membrane and the capacity of CD44 to bind its ligand. The results suggest that cytoskeletal disruption within cartilage uncouples chondrocytes from the matrix, resulting in altered metabolism and deleterious changes in matrix structure.     

Single-Molecule Unbinding Forces between the Polysaccharide Hyaluronan and Its Binding Proteins

The extracellular polysaccharide hyaluronan (HA) is ubiquitous in all vertebrate tissues, where its various functions are encoded in the supramolecular complexes and matrices that it forms with HA-binding proteins (hyaladherins). In tissues, these supramolecular architectures are frequently subjected to mechanical stress, yet how this affects the intermolecular bonding is largely unknown. Here, we used a recently developed single-molecule force spectroscopy platform to analyze and compare the mechanical strength of bonds between HA and a panel of hyaladherins from the Link module superfamily, namely the complex of the proteoglycan aggrecan and cartilage link protein, the proteoglycan versican, the inflammation-associated protein TSG-6, the HA receptor for endocytosis (stabilin-2/HARE), and the HA receptor CD44. We find that the resistance to tensile stress for these hyaladherins correlates with the size of the HA-binding domain. The lowest mean rupture forces are observed for members of the type A subgroup (i.e., with the shortest HA-binding domains; TSG-6 and HARE). In contrast, the mechanical stability of the bond formed by aggrecan in complex with cartilage link protein (two members of the type C subgroup, i.e., with the longest HA-binding domains) and HA is equal or even superior to the high affinity streptavidin?biotin bond. Implications for the molecular mechanism of unbinding of HA?hyaladherin bonds under force are discussed, which underpin the mechanical properties of HA?hyaladherin complexes and HA-rich extracellular matrices.     

Role of CD44 in the organization of keratinocyte pericellular hyaluronan

CD44 is a ubiquitous cell surface glycoprotein, involved in important cellular functions including cell adhesion, migration, and modulation of signals from cell surface receptors. While most of these CD44 functions are supposed to involve hyaluronan, relatively little is known about the contribution of CD44 to hyaluronan maintenance and organization on cell surface, and the role of CD44 in hyaluronan synthesis and catabolism. Blocking hyaluronan binding either by CD44 antibodies, CD44-siRNA or hyaluronan decasaccharides (but not hexasaccharides) removed most of the hyaluronan from the surfaces of both human (HaCaT) and mouse keratinocytes, resembling results on cells from CD44−/− animals. In vitro, compromising CD44 function led to reduced and increased amounts, respectively, of intracellular and culture medium hyaluronan, and specific accumulation below the cells. In vivo, CD44-deficiency caused no marked differences in hyaluronan staining intensity or localization in the fetal skin or in adult ear skin, while tail epidermis showed a slight reduction in epidermal hyaluronan staining intensity. However, CD44-deficient tail skin challenged with retinoic acid or tape stripping revealed diffuse accumulation of hyaluronan in the superficial epidermal layers, normally negative for hyaluronan. Our data indicate that CD44 retains hyaluronan in the keratinocyte pericellular matrix, a fact that has not been shown unambiguously before, and that hyaluronan abundance in the absence of CD44 can result in hyaluronan trapping in abnormal locations possibly interfering there with normal differentiation and epidermal barrier function.     

Collagen V localizes to pericellular sites during tendon collagen fibrillogenesis

During tendon development collagen fibrillogenesis occurs in extracellular micro-domains defined by the tenocytes. This permits cellular regulation of the extracellular steps involved in the tissue-specific matrix assembly required for function. The hypothesis tested here is that collagen V associates with the tenocyte surface where it functions in regulation of collagen assembly and cell-directed fibril deposition. The in vitro and in vivo data demonstrate that collagen V is a quantitatively minor component of the tendon. It is preferentially localized on the tenocyte surface as distinct foci in tendons and in cell culture. In vitro data indicate that this interaction with the tenocyte is not HSPG GAG-dependent. Collagen V is present as the mature, processed form, is absent from the media, and is a significant part of the detergent-insoluble cell layer, presumably as part of a membrane-associated complex. In contrast, procollagen I is not efficiently processed and is found predominantly in the culture media. Our data suggest that the regulatory role of collagen V requires collagen V to occupy a different cellular niche from the structural collagen I. In monolayer cultures, the conversion to the tissue form of collagen V and its deposition with the cell layer suggest efficient engagement of procollagen V with pericellular receptors and processing enzymes. The secretion of collagen I into the media and inefficient processing of procollagen I suggest reduced accessibility to these pericellular molecules due to disengagement from the cell surface. This all points to differential spatial localization of collagen V as a mechanism to optimize its regulatory roles during the cell-surface directed steps in tendon collagen fibril assembly.     

A Role for Versican in the Development of Leiomyosarcoma

Leiomyosarcoma (LMS) is a mesenchymal cancer that occurs throughout the body. Although LMS is easily recognized histopathologically, the cause of the disease remains unknown. Versican, an extracellular matrix proteoglycan, increases in LMS. Microarray analyses of 80 LMSs and 24 leiomyomas showed a significant elevated expression of versican in human LMS versus benign leiomyomas. To explore the importance of versican in this smooth muscle cell tumor, we used versican-directed siRNA to knock down versican expression in a LMS human cell line, SK-LMS-1. Decreased versican expression was accompanied by slower rates of LMS cell proliferation and migration, increased adhesion, and decreased accumulation of the extracellular matrix macromolecule hyaluronan. Addition of purified versican to cells expressing versican siRNA restored cell proliferation to the level of LMS controls, increased the pericellular coat and the retention of hyaluronan, and decreased cell adhesion in a dose-dependent manner. The presence of versican was not only synergistic with hyaluronan in increasing cell proliferation, but the depletion of versican decreased hyaluronan synthase expression and decreased the retention of hyaluronan. When LMS cells stably expressing versican siRNA were injected into nude mice, the resulting tumors displayed significantly less versican and hyaluronan staining, had lower volumes, and had reduced levels of mitosis as compared with controls. Collectively, these results suggest a role for using versican as a point of control in the management and treatment of LMS.