Age-related changes in expression of tissue inhibitor of metalloproteinases-3 associated with transition from the notochordal nucleus pulposus to the fibrocartilaginous nucleus pulposus in rabbit intervertebral disc.
METHODS
Experimental study on age-related changes in expression of tissue inhibitor of metalloproteinases-3 (TIMP-3) associated with transition from notochordal nucleus pulposus (NP) to fibrocartilaginous NP in rabbit intervertebral disc (IVD).


OBJECTIVE
To identify roles of notochordal NP in extracellular matrix (ECM) metabolism of IVD.


BACKGROUND
One of most interesting properties of TIMP-3 is to inhibit aggrecanases in addition to matrix metalloproteinases. Balance of aggrecanase/TIMP-3 is critical to maintain homeostasis of ECM metabolism.


METHODS
Four-week-old and 160-week-old male Japanese white rabbits were used. Age-related changes in IVDs were evaluated histologically using previously established grading system. Immunohistochemistry of TIMP-3 and semiquantitative reverse transcriptase-polymerase reaction (RT-PCR) of TIMP-3, a disintegrin and metalloproteinases with thrombospondin motifs (ADAMTS) 4, 5, and transforming growth factor-beta1 (TGF-beta1), were conducted.


RESULTS
Semiquantitative assessment of histologic changes indicated that 4-week-old rabbit was equivalent to fetus to 2-year-old human and 160-week-old rabbit was equivalent to 11- to 30-year-old human, particularly 11- to 16-year-old, which corresponds to transition period from notochordal to fibrocartilaginous NP. Immunohistochemistry revealed that TIMP-3 was positive in 4-week-old rabbit only. Semiquantitative RT-PCR revealed that levels of expressions of TGF-beta1 and TIMP-3 mRNAs in 4-week-old were significantly higher than those in 160-week-old rabbits. There was no significant difference in expression of ADAMTS4 mRNA. ADAMTS5 mRNA was not detected or extremely low in both groups. Expression of TIMP-3 mRNA in NP was upregulated by TGF-beta1 but was not affected by IL-1beta. On the contrary, expression of ADAMTS4 mRNA was not upregulated by TGF-beta1 but was upregulated by IL-1beta.


CONCLUSIONS
Levels of expression of TIMP-3 in notochordal NP were significantly lower in 160-week-old rabbits than those in 4-week-old rabbits. Decrease in expression of TIMP-3, possibly mediated in part by TGF-beta1, may cause imbalance of ADAMTS4/TIMP-3 ratio at transition period from notochordal to fibrocartilaginous NP.
Tsuji
Takashi
T
Department of Orthopaedic Surgery, School of Medicine, Keio University, Tokyo, Japan.
Chiba
Kazuhiro
K
Imabayashi
Hideaki
H
Fujita
Yoshinari
Y
Hosogane
Naobumi
N
Okada
Yasunori
Y
Toyama
Yoshiaki
Y
D051722:ADAM Proteins / Q000235:genetics / Q000378:metabolism; D000071121:ADAMTS4 Protein; D000367:Age Factors; D000818:Animals; D005109:Extracellular Matrix / Q000201:enzymology; D051445:Fibrocartilage / Q000166:cytology / Q000201:enzymology* / Q000254:growth & development*; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D006706:Homeostasis / Q000502:physiology; D007403:Intervertebral Disc / Q000166:cytology / Q000201:enzymology* / Q000254:growth & development*; D008297:Male; D009672:Notochord / Q000166:cytology; D011348:Procollagen N-Endopeptidase / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000235:genetics* / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism
D012333:RNA, Messenger; D019717:Tissue Inhibitor of Metalloproteinase-3; D053773:Transforming Growth Factor beta1; D051722:ADAM Proteins; D011348:Procollagen N-Endopeptidase; D000071121:ADAMTS4 Protein; C000603918:ADAMTS4 protein, human