Identification and differential expression of human collagenase-3 mRNA species derived from internal deletion, alternative splicing, and different polyadenylation and transcription initiation sites.
OBJECTIVE
Collagenase-3 is a metalloprotease that plays a role in tissue remodeling and pathological processes including arthritis. The human gene is transcribed into major (3.0 and 2.5 kb) and minor (2.2/2.0 kb) transcripts, as seen in Northern blot assays. We investigated the possibility that other transcripts, not detectable by Northern blot, were synthesized as either coding or regulatory RNAs that would modulate collagenase-3 expression and function/activity.


METHODS
We screened a cDNA library and total RNA from human chondrocytes by plaque hybridization and RT-PCR, and expressed the transcripts in a cellular environment. The levels of expression of each transcript in normal and osteoarthritic joint cells and cartilage were monitored by RT-PCR.


RESULTS
We identified five different collagenase-3 RNA species derived from alternative polyadenylation sites (COL3-APS), internal deletion (COL3-DEL), alternative splicing (COL3-9B/COL3-9B-2), and different transcription initiation site (COL3-ATS and COL3-ATS-INT). Each transcript could be translated in a cellular environment. Interestingly, the proteins translated from the COL3-DEL and COL3-9B-2 transcripts had a modified hemopexin-like domain, suggesting altered collagenolytic activities. The transcript types COL3-APS, COL3-9B-2, and COL3-ATS were up-regulated in the osteoarthritic samples and expressed in the chondrosarcoma cell line SW1353.


CONCLUSIONS
Our data show that the human collagenase-3 gene is subjected to different levels of regulation and constitutes a more complex system than was originally thought.
Tardif
G
G
Osteoarthritis Research Unit, Hôpital Notre-Dame, Centre Hospitalier de l'Université de Montreal, 1560 Sherbrooke Street East, Quebec, H2L 4M1, Montreal, Canada.
Dupuis
M
M
Reboul
P
P
Geng
C S
CS
Pelletier
J-P
JP
Ranger
P
P
Martel-Pelletier
J
J
D017398:Alternative Splicing / Q000502:physiology; D000595:Amino Acid Sequence; D002358:Cartilage, Articular / Q000201:enzymology*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000201:enzymology; D017364:Collagenases / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods; D007719:Knee Joint / Q000201:enzymology; D053509:Matrix Metalloproteinase 13; D008875:Middle Aged; D008969:Molecular Sequence Data; D020370:Osteoarthritis, Knee / Q000201:enzymology*; D026723:Polyadenylation / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D017385:Sequence Homology; D013583:Synovial Membrane / Q000201:enzymology; D024363:Transcription Initiation Site / Q000502:physiology; D014158:Transcription, Genetic
D018076:DNA, Complementary; D012333:RNA, Messenger; D017364:Collagenases; C507079:MMP13 protein, human; D053509:Matrix Metalloproteinase 13