Antisense inhibition of hyaluronan synthase-2 in human articular chondrocytes inhibits proteoglycan retention and matrix assembly.
In order to define the role of cell-associated hyaluronan in cartilage matrix retention, human articular chondrocytes as well as cartilage slices were treated with phosphorothioate oligonucleotides comprised of sequence antisense to the mRNA of human HA synthase-2 (HAS-2). As a prerequisite for these studies, it was necessary to determine which HA synthase (HAS), of three separate human genes capable of synthesizing HA, designated HAS-1, HAS-2, or HAS-3, is primarily responsible for HA synthesis in human articular chondrocytes. The copy number of each HAS mRNA expressed in cultured human articular chondrocytes was determined using quantitative (competitive) reverse transcription-polymerase chain reaction (RT-PCR). Only HAS-2 and HAS-3 mRNA expression was detected. The level of HAS-2 mRNA expression was 40-fold higher than that of HAS-3. Cultures of human articular chondrocytes and cartilage tissue slices were then transfected with HAS-2-specific antisense oligonucleotides. This treatment resulted in time-dependent inhibition of HAS-2 mRNA expression, as measured by quantitative RT-PCR, and a significant loss of cell-associated HA staining. Sense and reverse HAS-2 oligonucleotides showed no effect. The consequences of reduced HA levels (due to HAS-2 antisense inhibition) were a decrease in the diameter of the cell-associated matrix and a decreased capacity to retain newly synthesized proteoglycan. These results suggest that HA synthesized by HAS-2 plays a crucial role in matrix assembly and retention by human articular chondrocytes.
Department of Biochemistry, Rush Medical College, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois 60612, USA.
Y
Y
Nishida
C B
CB
Knudson
J J
JJ
Nietfeld
A
A
Margulis
W
W
Knudson
D001483:Base Sequence; D002358:Cartilage, Articular / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D005109:Extracellular Matrix / Q000187:drug effects* / Q000502:physiology / Q000648:ultrastructure; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D014453:Glucuronosyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics*; D006801:Humans; D000076002:Hyaluronan Synthases; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D011509:Proteoglycans / Q000187:drug effects / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013873:Thionucleotides; D014158:Transcription, Genetic / Q000187:drug effects*
D016376:Oligonucleotides, Antisense; D011509:Proteoglycans; D012333:RNA, Messenger; D013873:Thionucleotides; D014453:Glucuronosyltransferase; C101754:HAS2 protein, human; C105458:HAS3 protein, human; D000076002:Hyaluronan Synthases