Inhibition of hyaluronan degradation by dextran sulphate facilitates characterisation of hyaluronan synthesis: an in vitro and in vivo study.
The concentration and molecular weight of hyaluronan often dictates its physiological function. Consequently full characterisation of the anabolic products and turnover rates of HA could facilitate understanding of the role that HA metabolism plays in disease processes. In order to achieve this it is necessary to interrupt the dynamic balance between concurrent HA synthesis and degradation, achievable through the inhibition of the hyaluronidases, a group of enzymes which degrade HA. The sulphated polysaccharide, dextran sulphate has been demonstrated to competitively inhibit testicular hyaluronidase in a non-biological system, but its application to in vitro biological systems had yet to be developed and evaluated. This study determined the inhibitory concentrations of dextran sulphate against both testicular and Streptomyces hyaluronidase in a cell-free and breast cancer model followed by characterisation of the effect that hyaluronidase inhibition exerted on HA synthesis and degradation. The IC(100) of dextran sulphate for both hyaluronidases in a cell-free and biological system was determined to be >or=400 microg/ml. At concentrations up to 10 mg/ml the dextran sulphate did not effect breast cancer cell proliferation or morphology, while at 400 microg/ml HA degradation was totally inhibited, enabling an accurate quantitation of HA production as well as characterisation of the cell-associated and liberated HA. FACS quantitation of the HA receptor CD44, HA synthase and the hyaluronidases HYAL 1 and HYAL 2 demonstrated that dextran sulphate down-regulated CD44 and HA synthase while upregulating the hyaluronidases. These results suggest dynamic feedback signalling and complex mechanisms occur in the net deposition of HA in vivo.
Laboratory for Hyaluronan Research, Department of Biochemistry and Molecular Biology, Monash University, Wellington Road, Clayton, Victoria, 3800 Australia.
Lishanthi
L
Udabage
Gary R
GR
Brownlee
Robert
R
Stern
Tracey J
TJ
Brown
D000818:Animals; D002417:Cattle; D015815:Cell Adhesion Molecules / Q000378:metabolism; D045744:Cell Line, Tumor; D002474:Cell-Free System / Q000187:drug effects; D016264:Dextran Sulfate / Q000494:pharmacology*; D015536:Down-Regulation; D058851:GPI-Linked Proteins; D014453:Glucuronosyltransferase; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D000076002:Hyaluronan Synthases; D006820:Hyaluronic Acid / Q000096:biosynthesis / Q000378:metabolism*; D006821:Hyaluronoglucosaminidase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology; D014166:Transferases / Q000378:metabolism; D015854:Up-Regulation
D015815:Cell Adhesion Molecules; D058851:GPI-Linked Proteins; D018960:Hyaluronan Receptors; D006820:Hyaluronic Acid; D016264:Dextran Sulfate; D014166:Transferases; D014453:Glucuronosyltransferase; D000076002:Hyaluronan Synthases; C473096:Hyal2 protein, human; D006821:Hyaluronoglucosaminidase