Matrix metalloproteinase expression levels suggest distinct enzyme roles during lumbar disc herniation and degeneration

Abstract

The disruption of the extracellular disc matrix is a major hallmark of disc degeneration. This has previously been shown to be associated with an up-regulation of major matrix metalloproteinase (MMP) expression and activity. However, until now hardly any data are available for MMP/TIMP regulation and thereby no concept exists as to which MMP/TIMP plays a major role in disc degeneration. The objective of this study was, therefore, to identify and quantify the putative up-regulation of MMPs/TIMPs on the mRNA and protein level and their activity in disc material in relation to clinical data and histological evidence for disc degeneration. A quantitative molecular analysis of the mRNA expression levels for the MMPs (MMPs-1, -2, -3, -7, -8, -9, -13) and the MMP inhibitors (TIMPs-1 and -2) was performed on 37 disc specimens obtained from symptomatic disc herniation or degeneration. In addition, disc specimens from patients without disc degeneration/herniation (=controls) were analyzed. Expression of MMPs-1, -2, -3, -7, -8, -9, -13 and TIMPs-1, -2 was analyzed using quantitative RT-PCR, normalized to the expression level of a house keeping gene (GAPDH). Gene expression patterns were correlated with MMP activity (in situ zymography), protein expression patterns (immunohistochemistry), degeneration score (routine histology) and clinical data. MMP-3 mRNA levels were consistently and substantially up-regulated in samples with histological evidence for disc degeneration. A similar but less pronounced up-regulation was observed for MMP-8. This up-regulation was paralleled by the expression of TIMP-1 and to a lesser extent TIMP-2. In general, these findings could be confirmed with regard to protein expression and enzyme activity. This study provides data on the gene and protein level, which highlights the key role of MMP-3 in the degenerative cascade leading to symptomatic disc degeneration and herniation. Control of the proteolytic activity of MMP-3 may, therefore, come into the focus when aiming to develop new treatment options for early disc degeneration.

Fig. 1

Expression profile of MMP-3 and MMP-7. Pie charts representing the amount of cases with an increase of RNA levels for MMP-3 (a) and MMP-7 (b) compared to the housekeeping gene. A 50-fold increase was termed minor, up to 500-fold moderate, up to 5,000-fold strong and more than 5,000-fold very strong

Fig. 2

Correlation of MMP-3 with histo-morphologic alterations. Histo-morphologic age-related alterations of the disc indicated by cell proliferation (a), tears and clefts formation (b), and mucoid degeneration (c) exhibited a significant correlation to the increase in mRNA expression for MMP-3. This was also reflected in the overall histologic degeneration score (d). Error bars represent standard error of the mean

Fig. 3

Expression profile of collagenases MMP-1, MMP-8 and MMP-13. Pie charts representing the amount of cases with an increase of RNA level for MMP-1 (a), MMP-1 (b) and MMP-13 (c) compared to the housekeeping gene. A 50-fold increase was termed minor, up to 500-fold moderate, up to 5,000-fold strong and more than 5,000-fold very strong

Fig. 4

Correlation of MMP 8 with pain duration and histo-morphologic alterations. Pain duration (a) and histo-morphologic age-related alterations (HDS) (b) exhibited a significant correlation to the increase in mRNA expression for MMP-8. Error bars represent standard error of the mean

Fig. 5

Expression profile of gelatinases MMP-2 and MMP-9. Pie charts representing the amount of case with an increase of RNA level for MMP-2 (a) and MMP-9 (b) compared to the housekeeping gene. A 50-fold increase was termed minor, up to 500-fold moderate, up to 5,000-fold strong and more than 5,000-fold very strong

Fig. 6

Expression profile of TIMP-1 and TIMP-2. Pie representing the amount of cases with an increase of RNA level for TIMP-1 (a) and TIMP-2 (b) compared to the housekeeping gene. A 50-fold increase was termed minor, up to 500-fold moderate, up to 5,000-fold strong and more than 5,000-fold very strong

Fig. 7

Immunohistochemical detection of MMP-1 and MMP-3. a Immunostaining for MMP-3 at a cleft margin with numerous positively labeled cells (red staining, case #28). b Several nuclear cells are also positive for MMP-1 (red staining, case #5)

Fig. 8

In situ zymography of intervertebral disc samples. a Section through a disc sample with minimal gelatinolytic activity with mostly preserved gelatin matrix (red staining, Score 1, case #22). b Extensive gelatinolytic activity (HDS 10, case #5) with loss of the red-stained gelatin matrix substrate (asterisk). Note focal clefts along the lightly stained activity areas (arrows)