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Co-expression of tissue inhibitor and matrix metalloproteinase in myocardium
S C Tyagi1, S G Kumar, J Banks
1Department of Internal Medicine, Dalton Cardiovascular Research Center, University of Missouri-Health Sciences Center, Columbia 65212, USA.
Abstract:
Matrix metalloproteinases (MMP) are present in the latent form in normal myocardium. To examine the stringent balance between MMP and tissue inhibitor of metalloproteinase (TIMP) and to determine whether MMP are secreted simultaneously and in co-ordination with their inhibitors, we analysed MMP and TIMP by immunological, isolation by gel-permeation and affinity chromatography, and enzymatic assays in tissues and extracts. Using antibodies to MMP-1 and TIMP-1, we found strong in situ staining of MMP-1 and TIMP-1 in tissues. The staining was uniform in the endo- and subendomyocardium as well as in the interstitial space. TIMP-1 was present wherever MMP-1 was localized. From the tissue extract, proteins were separated on a gel-filtration column (Sephacryl S-200) and analysed for MMP and TIMP activity by zymography as well as by using succinyl-Gly-Pro-Leu-Gly-Pro-4-amido-7-methyl coumarin (Suc-GPLGP-AMC) as a selective fluorogenic substrate for collagenase. TIMP and MMP were further purified on collagen-Sepharose affinity column. The results indicated that MMP activity was co-eluted with TIMP activity. MMP-1, MMP-2 and TIMP-1 were further analysed by Northern blot for mRNA levels in the heart, skin, lung, liver and kidney. Results suggested co-expression of MMP-1 and TIMP-1 at the transcription level in all tissues. The level of MMP-2 mRNA was specifically higher in the heart tissue, which suggests a role of MMP-2 in the integrity of cardiovascular structure. The study indicated that myocardium as well as other tissue have an endogenous inhibitory system, suggesting that the MMPs activity is co-ordinated by their inhibitors at both the gene and protein levels. Furthermore, MMP and TIMP were co-expressed and were tightly regulated in maintaining the architecture of the interstitial tissue.
Insights
Matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) are tightly regulated together at gene and protein levels. This coordinated expression maintains interstitial tissue architecture in myocardium and other tissues.
Area of Science:
- Biochemistry
- Molecular Biology
- Cardiovascular Research
Background:
- Matrix metalloproteinases (MMP) are crucial enzymes involved in tissue remodeling.
- A balance between MMPs and their inhibitors, tissue inhibitors of metalloproteinases (TIMP), is essential for tissue homeostasis.
- The coordinated expression and regulation of MMPs and TIMPs in normal myocardium are not fully understood.
Purpose of the Study:
- To investigate the stringent balance between MMPs and TIMPs in normal myocardium.
- To determine if MMPs are secreted simultaneously and in coordination with their inhibitors.
- To analyze the co-expression and regulation of MMPs and TIMPs at both protein and gene levels.
Main Methods:
- Immunohistochemical staining for MMP-1 and TIMP-1.
- Gel-permeation chromatography and affinity chromatography for protein isolation.
- Enzymatic assays, zymography, and Northern blot analysis for MMP and TIMP activity and mRNA levels.
- Use of fluorogenic substrate (Suc-GPLGP-AMC) for collagenase activity.
Main Results:
- MMP-1 and TIMP-1 were co-localized in myocardial tissues.
- MMP activity consistently co-eluted with TIMP activity.
- Co-expression of MMP-1 and TIMP-1 mRNA was observed in multiple tissues, with higher MMP-2 mRNA levels in the heart.
- Evidence of an endogenous inhibitory system coordinating MMP activity at gene and protein levels.
Conclusions:
- MMPs and TIMPs are tightly regulated and co-expressed at both transcriptional and protein levels.
- This coordinated regulation is crucial for maintaining the architecture of interstitial tissue.
- MMP-2 may play a specific role in cardiovascular structure integrity.