Warren Knudson

Hyaluronan-binding proteins in development, tissue homeostasis, and disease.

The high molecular weight glycosaminoglycan hyaluronan plays an important role in tissue remodeling during development, normal tissue homeostasis, and disease. The interaction of hyaluronan with matrix hyaluronan‐binding proteins and cell‐surface hyaluronan receptors regulates many aspects of cell behavior such as cell migration, cell‐cell adhesion, and cell differentiation. Hyaluronan‐binding proteins have been grouped together as a family termed hyaladherins — further subdivided in matrix and cell‐surface hyaladherins (receptors). Specific hyaluronan‐hyaladherin interactions that affect cell behavior are the focus of this review. Both clearance and turnover of hyaluronan involve hyaluronan receptor‐mediated endocytosis. Pericellular matrix assembly and retention on many cells, especially chondrocytes, are mediated by hyaluronan receptors, in coordination with other matrix hyaladherins. Hyaluronan can also have an independent, direct effect on cell‐to‐cell adhesion as well as migration, again mediated by specific cell‐surface hyaluronan receptors. This is especially apparent in tumor cells, where metastatic potential is correlated with hyaluronan receptor expression. As migrating cells encounter new environments enriched in matrix hyaladherins, the capacity for matrix assembly may terminate cell migration. Thus, the temporal/spatial deposition of particular matrix hyaladherins also serves as signals or matrix cues to alter cell behavior.—Knudson, C. B., Knudson, W. Hyaluronan‐binding proteins in development, tissue homeostasis, and disease. FASEB J. 7: 1233‐1241; 1993.

CD44-mediated uptake and degradation of hyaluronan.

Hyaluronan turnover occurs systemically from the lymph and serum as well as locally by the same cells responsible for its synthesis. Local turnover involves receptor-mediated uptake and delivery to lysosomes. Of the many hyaluronan binding proteins/receptors known, the participation of CD44 in the internalization of hyaluronan has been best characterized. Some fraction of the hyaluronan bound to CD44 becomes internalized and delivered to lysosomes by a mechanism that is not dependent on clatherin, caveolae or pinocytosis. In cells such as chondrocytes, anabolic and catabolic cytokines can alter the activity of CD44 toward hyaluronan internalization. However, the mechanism of cellular regulation remains unclear. Regulation may involve the participation of alternatively spliced isoforms of CD44, changes in CD44 phosphorylation, changes in cytoskeletal binding proteins or, the activity or extracellular proteolytic activity.

Osteogenic protein 1 stimulates cell-associated matrix assembly by normal human articular chondrocytes: Up-regulation of hyaluronan synthase, CD44, and aggrecan

OBJECTIVE To determine the effects of osteogenic protein 1 (OP-1) on hyaluronan (HA), CD44, and aggrecan biosynthesis as well as the contribution of these molecules in promoting matrix assembly by human articular chondrocytes. METHODS Normal human chondrocytes were cultured with or without OP-1 treatment. Changes in the relative expression of messenger RNA (mRNA) for HA synthases 2 and 3 (HAS-2 and HAS-3), CD44, and aggrecan were determined by competitive quantitative reverse transcriptase-polymerase chain reaction. Accumulation of HA was characterized by indirect staining, CD44 by flow cytometry, and aggrecan biosynthesis by 35SO4 incorporation. RESULTS OP-1 stimulated the expression of HAS-2, CD44, and aggrecan mRNA in a time-dependent manner, resulting in increased expression of HA, CD44, and aggrecan. Prominent increases in HA-rich cell-associated matrices were also observed. CONCLUSION OP-1 stimulates not only the synthesis of matrix macromolecules such as aggrecan, but also the synthesis of other molecules required for matrix retention, namely, HA and CD44.

Hyaluronan oligosaccharides induce matrix metalloproteinase 13 via transcriptional activation of NFkappaB and p38 MAP kinase in articular chondrocytes.

Hyaluronan exerts a variety of biological effects on cells including changes in cell migration, proliferation, and matrix metabolism. However, the signaling pathways associated with the action of hyaluronan on cells have not been clearly defined. In some cells, signaling is induced by the loss of cell-hyaluronan interactions. The goal of this study was to use hyaluronan oligosaccharides as a molecular tool to explore the effects of changes in cell-hyaluronan interactions and determine the underlying molecular events that become activated. In this study, hyaluronan oligosaccharides induced the loss of extracellular matrix proteoglycan and collagen from cultured slices of normal adult human articular cartilage. This loss was coincident with an increased expression of matrix metalloproteinase (MMP)-13. MMP-13 expression was also induced in articular chondrocytes by hyaluronan (HA) hexasaccharides but not by HA tetrasaccharides nor high molecular weight hyaluronan. MMP-13 promoter-reporter constructs in CD44-null COS-7 cells revealed that both CD44-dependent and CD44-independent events mediate the induction of MMP-13 by hyaluronan oligosaccharides. Electromobility gel shift assays demonstrated the activation of chondrocyte NFκB by hyaluronan oligosaccharides. NFκB activation was also documented in C-28/I2 immortalized human chondrocytes by luciferase promoter assays and phosphorylation of IKK-α/β. The link between activation of NFκB and MMP-13 induction by HA oligosaccharides was further confirmed through the use of the NFκB inhibitor helenalin. Inhibition of MAP kinases also demonstrated the involvement of p38 MAP kinase in the hyaluronan oligosaccharide induction of MMP-13. Our findings suggest that hyaluronan-CD44 interactions affect matrix metabolism via activation of NFκB and p38 MAP kinase.

Acylation of CD44 and its association with lipid rafts are required for receptor and hyaluronan endocytosis

CD44 is a cell surface receptor for the extracellular matrix macromolecule hyaluronan. In addition, CD44 mediates the endocytosis of hyaluronan leading to its subsequent degradation within lysosomes. Using model systems of COS-7 and Flp-293 cells, we demonstrate that the association of CD44 with lipid rafts is essential for the endocytosis of hyaluronan but not the extracellular binding. Further, we demonstrate that palmitoylation of CD44 on two highly conserved cysteine residues is essential for the association with lipid rafts as determined by density gradient ultracentrifugation. Mutations of either cysteine residues or pretreatment of cells with the palmitic acid analog 2-bromopalmitate, reduced the [3H]palmitic acid incorporation into CD44 and prevented CD44-lipid rafts association. Preventing CD44 palmitoylation had no effect on the binding of hyaluronan but inhibited hyaluronan internalization. The turnover of the CD44 receptor itself was also affected by blocking its association with lipid rafts. Using cycloheximide to prevent de novo protein synthesis, palmitoylation-deficient cysteine mutants underwent slower turnover from cell surface compared with the palmitoylation-intact wild type, as determined by immunofluorescence and Western blotting. These results indicate that palmitoylation of CD44 is a critical driving determinant to CD44 association with lipid rafts and, concomitantly, the rates of hyaluronan endocytosis and CD44 turnover from cell surface.

The cell surface hyaluronate binding sites of invasive human bladder carcinoma cells

High-affinity, cell surface binding sites for hyaluronate were demonstrated on highly invasive human bladder carcinoma cells. These binding sites were shown to be specific for hyaluronate, saturable and exhibit a Km of 0.94 x 10(-9) M and a Bmax of 65 ng hyaluronate/10(6) cells. The binding of [3H]hyaluronate to a fixed cell-affinity column was competed with unlabeled hyaluronate and hyaluronate-hexasaccharide but not with hyaluronate-tetrasaccharide, chondroitin sulfate, heparin or non-sulfated dextran. Pre-treatment of cells with protease destroyed the binding activity whereas pretreatment with Streptomyces hyaluronidase to reveal occupied binding sites had no effect. No hyaluronate-binding activity was observed on normal human fibroblasts.

A Requirement for the CD44 Cytoplasmic Domain for Hyaluronan Binding, Pericellular Matrix Assembly, and Receptor-mediated Endocytosis in COS-7 Cells

CD44-negative COS-7 cells were transfected with expression constructs for CD44H (the predominant CD44 isoform), CD44E (epithelial isoform), or truncation mutant derivatives lacking the carboxyl-terminal 67 amino acids of the cytoplasmic domain, CD44HΔ67 and CD44EΔ67. The truncation mutant CD44HΔ67 is identical to a naturally occurring alternatively spliced “short tail” CD44 isoform (CD44st), which incorporates exon 19 in place of exon 20. CD44st lacks intracellular signaling motifs as well as protein domains necessary for interaction with cytoskeletal components. Transfection of COS-7 cells with each construct yielded equivalent levels of mRNA expression, whereas no CD44 expression was observed in parental, nontransfected COS-7 cells. Western analysis and immunostaining of COS-7 transfectants confirmed CD44 protein expression of the truncation mutant derivatives. COS-7 cells transfected with CD44H or CD44E gained the capacity to bind fluorescein-conjugated HA (fl-HA) and assemble HA-dependent pericellular matrices in the presence of exogenously added HA and proteoglycan. In addition, the CD44H- and CD44E-transfected cells were able to internalize surface-boundfl-HA. COS-7 cells transfected with the vector alone or with either of the mutant CD44 isoforms, CD44HΔ67 or CD44EΔ67, did not exhibit the capacity to assemble pericellular matrices or to bind and internalize the fl-HA. Cotransfection of CD44Δ67 mutants together with CD44H reduced the size of the HA-dependent pericellular matrices. Transfection of bovine articular chondrocytes with CD44Δ67 also inhibited pericellular matrix assembly. Collectively, these results indicate an obligatory requirement for the CD44 receptor cytoplasmic domain for ligand (HA) binding, formation and retention of the pericellular matrix, as well as CD44-mediated endocytosis of HA. In addition, the results suggest a potential regulatory role for the differentially expressed alternatively spliced short tail CD44 isoform.

CD44 modulates Smad1 activation in the BMP-7 signaling pathway

Bone morphogenetic protein 7 (BMP-7) regulates cellular metabolism in embryonic and adult tissues. Signal transduction occurs through the activation of intracellular Smad proteins. In this paper, using a yeast two-hybrid screen, Smad1 was found to interact with the cytoplasmic domain of CD44, a receptor for the extracellular matrix macromolecule hyaluronan. Coimmunoprecipitation experiments confirmed the interaction of Smad1 with full-length CD44—interactions that did not occur when CD44 receptors truncated within the cytoplasmic domain were tested. Chondrocytes overexpressing a truncated CD44 on a background of endogenous full-length CD44 no longer exhibited Smad1 nuclear translocation upon BMP-7 stimulation. Further, pretreatment of chondrocytes with Streptomyces hyaluronidase to disrupt extracellular hyaluronan–cell interactions inhibited BMP-7–mediated Smad1 phosphorylation, nuclear translocation of Smad1 or Smad4, and SBE4–luciferase reporter activation. These results support a functional link between the BMP signaling cascade and CD44. Thus, changes in hyaluronan–cell interactions may serve as a means to modulate cellular responsiveness to BMP.

Antisense inhibition of chondrocyte CD44 expression leading to cartilage chondrolysis.

OBJECTIVE To better define critical functions of the hyaluronan receptor CD44 in cartilage. METHODS Articular chondrocytes and cartilage tissue slices were treated with CD44 sequence-specific antisense phosphorothioate oligonucleotides. CD44 expression was probed by immunofluorescence microscopy, enzyme-linked immunosorbent assay, and Western blotting. RESULTS Antisense oligonucleotides demonstrated a dose- and time-dependent inhibition of CD44 protein expression; negative controls showed no effect. Similar to osteoarthritic cartilage, antisense-treated cartilage slices displayed a near-total loss of stainable proteoglycan-rich matrix. CONCLUSION CD44 expression is needed for maintenance of cartilage homeostasis.

Accumlation of Hyaluronate in Human Lung Carcinoma As Measured by A new Hyaluronate Elisa

We have developed a new ELISA to quantify hyaluronate. This ELISA takes advantage of an anti-keratan sulfate antibody to differentiate between the coated aggregating rat chondrosarcoma proteoglycan which captures the hyaluronate and the keratan sulfate-bearing aggregating proteoglycan added subsequently. Absorbance in this assay was shown to be linear to the logarithmic concentration of hyaluronate in the range of 15 to 1000 ng/ml. Pre-treatment of hyaluronate with papain or protease did not interfere with its quantification; in contrast, pre-treatment with 0.1N NaOH significantly interferes with the subsequent measurement of the hyaluronate molecules. The size of the hyaluronate molecule was found to be an important factor in quantification: only large size hyaluronate molecules could be quantified accurately. The ELISA was used to show that human lung carcinomas contain 2 to 500 times as much hyaluronate as normal lung tissue from the same patient.