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 matr ix retention, human articular chondrocytes as well as cartilage slices were treated with phosphorothioate oligonucleotides comprised of sequence antis ense to the mRNA of human HA synthase-2 (HAS-2), As a prerequisite for thes e studies, it was necessary to determine which HA synthase (HAS), of three separate human genes capable of synthesizing HA, designated HAS-1, HAS-2, o r HAS-3, is primarily responsible for HA synthesis in human articular chond rocytes, The copy number of each HAS mRNA expressed in cultured human artic ular chondrocytes was determined using quantitative (competitive) reverse t ranscription-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 hig her than that of HAS-3. Cultures of human articular chondrocytes and cartil age tissue slices were then transfected with HAS-2-specific antisense oligo nucleotides. 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 s howed no effect. The consequences of reduced HA levels (due to HAS-2 antise nse inhibition) were a decrease in the diameter of the cell-associated matr ix and a decreased capacity to retain newly synthesized proteoglycan, These results suggest that HA synthesized by HAS-2 plays a crucial role in matri x assembly and retention by human articular chondrocytes.