Antisense Inhibition of Hyaluronan Synthase-2 in Human Articular Chondrocytes Inhibits Proteoglycan Retention and Matrix Assembly

Yoshihiro Nishida,Cheryl B Knudson,J Jaap Nietfeld,Arkady Margulis,Warren Knudson

doi:10.1074/jbc.274.31.21893

Abstract

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.

Highlights

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)
The consequences of reduced HA levels 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
HAS-1 mRNA expression was not detected in these chondrocytes even with the use of high cycle numbers (i.e. 35 cycles), whereas the same primer pairs successfully generated HAS-1, HAS-2, and HAS-3 products from RNA derived from human dermal fibroblasts (CCD-1093Sk, data not shown)

Summary

EXPERIMENTAL PROCEDURES

Materials—Dulbecco’s modified Eagle’s medium/Ham’s F12, Trizol reagent for RNA isolation, and LipofectAMINE reagent for transfection were obtained from Life Technologies, Inc. Following 1 day of culture for recovery, the tissue slices were washed and medium was replaced with fresh serum-free DMEM in the presence or absence of various phosphorothioate oligonucleotides, diluted as LipofectAMINE DNA-liposomes to a final concentration of 2 ␮M. Sections or cells were incubated with 2.0 ␮g/ml biotinylated HA-binding protein (HABP) probe for 2 h at room temperature followed by a streptavidin-peroxidase reagent (Vectastain kit) and diaminobenzidine-containing substrate solution (SIGMA FASTTM DAB). Following treatment of monolayer cultures of chondrocytes with or without incubation with various phosphorothioate oligonucleotides, and 24 h of recovery in oligonucleotide-free medium, the culture medium was removed and replaced with a 0.75-ml suspension of formalin-fixed erythrocytes (108 per ml) in PBS containing 0.1% bovine serum albumin. The sample and mimic cDNA products were co-amplified in the presence of the GAPDH-specific downstream primer together with 0.15 ␮M upstream primer (5Ј-ACT GCC ACC CAG AAG ACT GTG GAT GG-3Ј) using PCR conditions as described for HAS-2 amplification

RESULTS

Copy number

DISCUSSION

ADDITIONS AND CORRECTIONS