Alternative messenger RNA splicing and enzyme forms of cathepsin B in human osteoarthritic cartilage and cultured chondrocytes.
OBJECTIVE
In previous studies, we suggested that cathepsin B, which is present at sites of cartilage remodeling in osteoarthritis (OA), may act as an antagonist of cartilage repair, an enhancer of the action of metalloproteinases, and a mediator of cartilage neovascularization and mineralization. Alternative splicing of cathepsin B pre-messenger RNA (pre-mRNA) and/or mRNA overexpression is a plausible regulatory mechanism. In the present study, we investigated the abundance of cathepsin B transcripts and the properties of cathepsin B protein in normal and OA cartilage, osteophytes, and cultured chondrocytes.


METHODS
Cathepsin B mRNA splice variants containing the full-length transcript (CB) and the variants lacking either exon 2 (CB[-2]) or lacking exons 2 and 3 (CB[-2,3]) were measured by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot assays and were localized by in situ RT-PCR. Cathepsin B protein was analyzed by electrophoretic, Western blot, and chromatographic methods.


RESULTS
The relative content of CB, CB(-2), and CB(-2,3) varied considerably in OA cartilage and osteophytes, with less variation in normal cartilage. The mean cathepsin B mRNA level was significantly higher in OA cartilage and osteophytes than in normal cartilage. Normal cultured chondrocytes attained cathepsin B mRNA levels similar to those in OA cartilage. Enzyme overexpression resulted in the secretion of procathepsin B, followed by activation to the proteolytically active form.


CONCLUSIONS
The high levels of CB and CB(-2) are consistent with an overproduction of secreted procathepsin B in OA. Up-regulation of chondrocyte cathepsin B, which takes place at both the transcriptional and the translational level, suggests a leading role of the enzyme in the progression of OA.
Berardi
S
S
University of Zurich, Switzerland.
Lang
A
A
Kostoulas
G
G
Hörler
D
D
Vilei
E M
EM
Baici
A
A
D000368:Aged; D017398:Alternative Splicing; D002356:Cartilage / Q000201:enzymology*; D002401:Cathepsin B / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D019902:Chondrocytes / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D017403:In Situ Hybridization; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D010003:Osteoarthritis / Q000201:enzymology* / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation
D007527:Isoenzymes; D012333:RNA, Messenger; D002401:Cathepsin B