Biomedical subjects

Cheryl B Knudson

Publications and source records attributed to Cheryl B Knudson.

11 recordsLinked to original sources

PubMed2005

Antisense inhibition of hyaluronan synthase-2 in human osteosarcoma cells inhibits hyaluronan retention and tumorigenicity.

Osteosarcoma is a common malignant bone tumor associated with childhood and adolescence. The results of numerous studies have suggested that hyaluronan plays an important role in regulating the aggressive behavior of various types of cancer cells. However, no studies have addressed hyaluronan with respect to osteosarcomas. In this investigation, the mRNA expression copy number of three mammalian hyaluronan synthases (HAS) was determined using competitive RT-PCR in the osteoblastic osteosarcoma cell line, MG-63. MG-63 are highly malignant osteosarcoma cells with an abundant hyaluronan-rich matrix. The results demonstrated that HAS-2 is the predominant HAS in MG-63. Accumulation of intracellular hyaluronan increased in association with the proliferative phase of these cells. The selective inhibition of HAS-2 mRNA in MG-63 cells by antisense phosphorothioate oligonucleotides resulted in reduced hyaluronan accumulation by these cells. As expected, the reduction in hyaluronan disrupted the assembly of cell-associated matrices. However, of most interest, coincident with the reduction in hyaluronan, there was a substantial decrease in cell proliferation, a decrease in cell motility and a decrease in cell invasiveness. These data suggest that hyaluronan synthesized by HAS-2 in MG-63 plays a crucial role in osteosarcoma cell proliferation, motility, and invasion.

Bone Neoplasms↗

PubMed2005

Hyaluronan oligosaccharide-induced activation of transcription factors in bovine articular chondrocytes.

OBJECTIVE: To document the activity profile of transcription factors following chondrocyte stimulation with hyaluronan (HA) hexasaccharides (HA(6)) and to determine the expression of genes whose transcriptional activation is tightly associated with the transcription factors. METHODS: Nuclear extracts from bovine articular chondrocytes treated with HA(6) were subjected to transcription factor protein-DNA array analysis. Electrophoretic mobility shift assay (EMSA) analyses were performed to confirm the results of protein-DNA array. The gene expressions of matrix metalloproteinase 3 (MMP-3), type II collagen, and cartilage oligomeric matrix protein (COMP) were examined by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), and protease activity was assessed by casein zymography. RESULTS: In the protein-DNA array analysis, 12 transcription factors were up-regulated and 2 transcription factors were down-regulated in the chondrocytes treated with HA(6). The transcription factors retinoic acid receptor (RAR), retinoid X receptor (RXR), and Sp-1 exhibited >2-fold increased activity by HA(6) treatment, as confirmed by EMSA. RT-PCR analysis showed that the expression levels of MMP-3, type II collagen, and COMP messenger RNA, which are tightly associated with the activation of RAR, RXR, or Sp-1, were up-regulated by treatment with HA(6). Addition of high molecular mass HA after HA(6) treatment resulted in abrogation of the MMP-3 induction. CONCLUSION: These results suggest that HA(6) increase the activity of multiple transcription factors in chondrocytes and signal the enhanced expression of key genes involved in cartilage-matrix remodeling and turnover. The data also demonstrate that high molecular mass HA has a potential to suppress the signaling activated by HA(6).

Adjuvants, Immunologic↗

PubMed2004

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.

Active Transport, Cell Nucleus↗

PubMed2004

Osteogenic Protein-1 inhibits matrix depletion in a hyaluronan hexasaccharide-induced model of osteoarthritis.

OBJECTIVE: To examine the capacity of recombinant osteogenic protein-1 (OP-1) to inhibit the cartilage extracellular matrix damage that follows treatment with hyaluronan hexasaccharides (HA6). DESIGN: The effects of OP-1 were examined on isolated human chondrocytes grown in alginate beads as well as articular cartilage slices treated with hyaluronan hexasaccharides. Changes in the relative expression of messenger RNA for hyaluronan synthase- 2, aggrecan and CD44 were determined by competitive quantitative reverse transcriptase-polymerase chain reaction. Cartilage proteoglycan biosynthesis was examined by a (35)S-sulfate incorporation assay. Cell-associated matrix of human chondrocytes was visualized by the use of particle exclusion assay, and alcian blue staining. Cartilage slices were examined for accumulation of proteoglycan by Safranin-O, and hyaluronan by a specific biotinylated probe. RESULTS: Combined OP-1 and HA6 treatment resulted in enhanced expression of mRNA for aggrecan and HAS-2, compared to the treatment with HA6 only. This increased expression of aggrecan mRNA was paralleled by an increased synthesis of cartilage proteoglycan especially retained in the cell-associated matrix. Co-treatment with OP-1 inhibited the HA6-induced depletion of cell-associated matrices as well as HA6-induced depletion of hyaluronan and proteoglycan within cartilage tissue slices. CONCLUSIONS: These results demonstrate that OP-1 can abrogate the catabolic events associated with a HA6-induced matrix depletion model of osteoarthritis. The mRNA levels of two major cartilage extracellular matrix components, aggrecan and hyaluronan synthase-2 are enhanced above values obtained by either OP-1 or HA6 treatments alone.

Activin Receptors, Type I↗

PubMed2004

Induction of CD44 and MMP expression by hyaluronidase treatment of articular chondrocytes.

In this study, the effects of fragmentation of the glycosoaminoglycans of the cell-associated matrix by hyaluronidase (HAase) on the expression of CD44 receptor and matrix metalloproteinase (MMP) mRNAs in cultured articular chondrocytes were examined. Chondrocytes, isolated from rabbit and bovine articular cartilage, were treated with bovine testicular HAase (0-200 units/ml) in the presence or absence of an antibody for CD44. The mRNA levels of CD44, CD44 variant (CD44v), MMPs (MMP-1, -3 and -9), and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) were determined by RT-PCR. The treatment of cultured chondrocytes with HAase resulted in the production of low molecular weight fragments of hyaluronan (HA). The expression of CD44, CD44v and MMP (MMP-1, -3 and -9) mRNAs, but not TIMP-1 or TIMP-2 mRNA, was up-regulated in the cultures treated with HAase, whereas this expression was not affected by treatment with purified HA of 1.0 x 10(5) Da. Furthermore, the induction of CD44 and MMPs on treatment with HAase was suppressed by an anti-CD44 antibody. The results suggest that the fragmentation of HA may lead to cartilage destruction in terms of the enhanced expression of MMPs as well as the upregulation of CD44.

Animals↗

PubMed2004

Hyaluronan and CD44: modulators of chondrocyte metabolism.

Stability of cell-matrix interactions promotes tissue homeostasis, and matrix receptors provide for the assembly and retention of matrix as well as the linkage to the signal transduction pathways activated in response to changes within the extracellular matrix. Therefore, changes in cell-matrix interactions may influence cell responses to cytokines and cell survival and may facilitate tissue remodeling. Matrix components sequester or present soluble morphogens to their signaling receptors. Another layer of complexity would be the establishment of new or uncoupling of cell-matrix interactions, leading to altered cellular response to morphogens. The hyaluronan receptor CD44 serves as the critical link for the retention of hyaluronan-proteoglycan aggregates to the chondrocyte cell surface. As we continue to define functional chondrocyte CD44, future studies will need to include analysis of the variant CD44 isoform expression, phosphorylation, cytoskeletal interactions, occupancy, and turnover. Disruption of chondrocyte CD44:hyaluronan interaction will induce a cascade of events resulting in the activation of both catabolic as well as anabolic gene products. Fragments of hyaluronan produced in free radical processes have the potential to augment the production of nitric oxide in a CD44-dependent mechanism. Data also support an emerging paradigm that CD44-mediated signaling affects both chondrocyte survival pathways as well as apoptotic pathways.

Animals↗

PubMed2003

Hyaluronan and CD44: strategic players for cell-matrix interactions during chondrogenesis and matrix assembly.

Embryonic induction, soluble and insoluble factors, receptors, and signal transduction are orchestrated for the morphogenesis of the cartilage elements. The interaction of cells with the extracellular matrix (ECM) may lead to altered cellular response to morphogens based on the formation of new adhesive contacts, or the uncoupling of cell-matrix interactions. Hyaluronan's influence on cell behavior, and its intimate association with cells are accomplished by a wide variety of specific binding proteins for hyaluronan. The temporal expression of the hyaluronan receptor CD44 (which is expressed as several alternatively spliced variants) may be strategic to many of these cell-matrix interactions during chondrogenesis. CD44 expression is temporally coincident with the reduction of intercellular spaces at the regions of future cartilage deposition. The spatial organization of CD44 at the cell surface may function to establish or regulate the structure of the pericellular matrix dependent on a hyaluronan scaffold. As the ECM is modified during embryogenesis, the cellular response to inductive signals may be altered. An uncoupling of chondrocyte-hyaluronan interaction leads to chondrocytic chondrolysis. Thus, consideration of cell-matrix interactions during chondrogenesis, in the light of our current understanding of the temporal and spatial expression of signaling morphogens, should become a promising focus of future research endeavors.

Animals↗

PubMed2002

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, CD44HDelta67 and CD44EDelta67. The truncation mutant CD44HDelta67 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-bound fl-HA. COS-7 cells transfected with the vector alone or with either of the mutant CD44 isoforms, CD44HDelta67 or CD44EDelta67, did not exhibit the capacity to assemble pericellular matrices or to bind and internalize the fl-HA. Cotransfection of CD44Delta67 mutants together with CD44H reduced the size of the HA-dependent pericellular matrices. Transfection of bovine articular chondrocytes with CD44Delta67 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.

3' Untranslated Regions↗

PubMed2002

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.

Animals↗

PubMed2002

Temporal expression of CD44 during embryonic chick limb development and modulation of its expression with retinoic acid.

Hyaluronan-cell interactions are initiated co-ordinately with mesenchymal condensation during chondrogenic differentiation in the limb bud. Hyaluronan is responsible for the retention and organization of proteoglycan within the cartilage matrix. Hyaluronan-CD44 binding also retains proteoglycan aggregates to the chondrocyte plasma membrane. A sequence for CD44 protein in chick has recently been reported, but never evaluated in chick chondrocytes. Total RNA was isolated from embryonic chick limb buds, stages 18, 19, 24, 25 and 30. Using semi-quantitative RT-PCR, expression of aggrecan, this chick CD44 orthologue and GAPDH mRNA was analyzed. Aggrecan expression was detected at all stages, but was increased at stage 30. CD44 mRNA was detected at extremely low levels at stage 18 to higher levels in the latter stages. Thus, the temporal expression of CD44 mRNA correlated with the onset of pre-cartilage condensation. The full-length chick chondrocyte CD44 cDNA was obtained following RT-PCR using RNA derived from tibial chondrocytes from stage 37 chick embryos. The nucleotide sequence was used to generate an amino acid sequence and analyses revealed homologies of 44.4% with mouse, 47.8% with bovine and 46.3% with human CD44. Tibial chondrocytes were cultured in the presence or absence of retinoic acid for 36 or 72 h. By RT-PCR, expression of aggrecan and the CD44 mRNA by chick chondrocytes was decreased after retinoic acid treatment, while GAPDH expression showed no change. As expected, control chondrocytes exhibited a round morphology while retinoic acid-treated chondrocytes were elongated. The retinoic acid-treated chondrocytes also exhibited reduced hyaluronan binding. This functional assay indicates a role for a CD44 receptor in matrix retention by chick chondrocytes.

Amino Acid Sequence↗

PubMed2002

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.

Animals↗