Effects of TGF-β1 on alternative splicing of Superficial Zone Protein in articular cartilage cultures

Abstract

Superficial Zone Protein (SZP) is expressed by the superficial zone chondrocytes and is involved in boundary lubrication of the articular cartilage surface. SZP protein expression is dependent on anatomical location and is regulated by the transforming growth factor-β (TGF-β) pathway. The hypothesis of this study was that between load-bearing, and non-load-bearing locations, of the femoral medial condyle alternative splice isoforms of SZP are different, and regulated by TGF-β1. Using reverse transcription-polymerase chain reaction (RT-PCR) we identified differentially expressed SZP alternative splicing. Using recombinant proteins of the N-terminal region produced from these isoforms, we identified differences in binding to heparin and the extracellular matrix. We identified a novel splice form of SZP (isoform E), lacking exons 2-5. Differences in alternative splicing were observed between anterior load-bearing locations of the femoral medial condyle (M1) compared to the posterior non-load-bearing location (M4). TGF-β1 increased splicing out of exons 4 and 5 encoding a heparin binding domain. The minimal induction time for changes in splicing by TGF-β1 at the M1 location was 1h, although this did change total SZP mRNA levels. Inhibition of Smad3 phosphorylation inhibited TGF-β1 induced splicing, and SZP protein expression. Recombinant proteins corresponding to isoforms upregulated by TGF-β1 had reduced binding. The SZP dimerization domain is located within exon 3. In conclusion, alternative splicing of SZP is regulated by TGF-β1 signaling and may regulate SZP interaction with heparin/heparan sulfate or other components in the extracellular matrix of articular cartilage by splicing out of the heparin binding domain.