TNF-alpha induces MMP2 gelatinase activity and MT1-MMP expression in an in vitro model of nucleus pulposus tissue degeneration.
METHODS
In vitro-formed bovine nucleus pulposus (NP) tissues were used as a model for tumor necrosis factor-alpha (TNF-alpha) induced NP degeneration.


OBJECTIVE
To elucidate the signal transduction mechanisms regulating TNF-alpha induced matrix metalloproteinase (MMP) activity.


BACKGROUND
TNF-alpha is thought to contribute to the pathophysiology of intervertebral disc (IVD) degeneration by up-regulating MMPs, such as MMP-2. MMP-2 has been implicated in influencing disease progression and in the induction of neovascularization.


METHODS
In vitro-formed bovine NP tissues were treated with TNF-alpha to examine its effect on MMP-2 gene and protein levels and activity. The effect of TNF-alpha on membrane type (MT)1-MMP, an activator of MMP-2, was also assessed. MT1-MMP functional activation by TNF-alpha was confirmed using promoter-reporter luciferase constructs. Immunoblots and electrophoretic mobility shift assays were used to examine the expression and DNA binding activity of transcription factors known to regulate transcriptional activation of MT1-MMP.


RESULTS
TNF-alpha treatment induced MMP-2 gelatinase activity, which occurred in the absence of any change in MMP-2 gene or protein expression, but did correlate with increased MT1-MMP mRNA and protein levels. Up-regulation of MMP-2 activity was dependent on the ERK-MAPK pathway. ERK-1/2 activation up-regulated early growth factor (Egr-1) expression and its DNA binding activity to the MT1-MMP promoter. There was no effect on Sp-1 binding activity. Reporter constructs demonstrated that TNF-alpha induced MT1-MMP transcriptional activation and that this response was dependant on ERK MAPK and Egr-1.


CONCLUSIONS
TNF-alpha induced MMP-2 gelatinase activity correlated with induction of MT1-MMP and not MMP-2 expression. MMP-2 activation was dependent on the ERK-MAPK pathway. As ERK also appeared to regulate MT1-MMP production, this suggests that TNF-alpha induction of MMP-2 gelatinase activity may be regulated by MT1-MMP. These findings elucidate the regulation of gelatinase activity and identify a mechanism whereby TNF-alpha may contribute to matrix degradation in NP tissue.
Séguin
Cheryle A
CA
BioEngineering of Skeletal Tissues Team, Mount Sinai Hospital, Toronto, Ontario, Canada.
Pilliar
Robert M
RM
Madri
Joseph A
JA
Kandel
Rita A
RA
D000818:Animals; D002417:Cattle; D024202:Electrophoretic Mobility Shift Assay; D048049:Extracellular Signal-Regulated MAP Kinases / Q000502:physiology; D015870:Gene Expression; D015151:Immunoblotting; D066298:In Vitro Techniques; D007403:Intervertebral Disc / Q000201:enzymology*; D008156:Luciferases / Q000235:genetics; D053511:Matrix Metalloproteinase 14 / Q000235:genetics / Q000378:metabolism*; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*; D015854:Up-Regulation
D014409:Tumor Necrosis Factor-alpha; D008156:Luciferases; D048049:Extracellular Signal-Regulated MAP Kinases; D020778:Matrix Metalloproteinase 2; D053511:Matrix Metalloproteinase 14