Matrix metalloproteinase (MMP) 1, also known as interstitial collagenase, is an endopeptidase secreted as an inactive proenzyme that is then activated by proteolytic cleavage. MMP1 plays a significant role in tissue repair and remodeling by degrading structural components of the extracellular matrix (ECM), such as collagens, gelatin, and fibronectin. As such it is closely related to all matrix-dependent disorders, such as fibrotic disease, and some types of cancer that rely on cell adhesion, migration, and invasion.
HTRF assays offer many advantages over other technologies:
Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
Human MMP1 assay principle
The Human Matrix MetalloProteinase 1 (MMP1) assay measures MMP1 in cell supernatant. The assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. In presence of Human MMP1 in a sample, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor, and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the capsid present in the sample and therefore provides a means of assessing any changes caused by experimental variables, in a no-wash assay format.
Human MMP1 assay protocol
The Human MMP1 assay protocol, using a 384-well small volume white plate, is described on the right. 16 µL of sample or standard are dispensed directly into the detection plate for detection by HTRF® reagents. The antibodies labeled with HTRF fluorophores are added before an overnight incubation. The assay can be run in up to a 1536-well format by simply resizing each addition volume proportionally.
MMP1 upregulation induced by different treatments in human pulmonary fibroblast cell line MRC-5
The human lung fibroblast cell line MRC-5 was plated in a 96-well plate (12,500 cells/well). The day after, cells were treated overnight with 50 µL of increasing concentrations of PMA diluted in FBS-free medium supplemented with 1% BSA. Cell supernatants were collected for the quantitative measurement of secreted MMP1 levels.
16 µL of supernatants (diluted ¼) were then transferred into a white detection plate (384 low volume) and 4 µL of the HTRF Human MMP1 detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.
As expected, PMA treatment led to a dose-dependent increase in the secretion of human MMP1.
The human lung fibroblast cell line MRC-5 was plated in a 96-well plate (12,500 cells/well). The day after, cells were treated overnight with 50 µL of increasing concentrations of human TNF alpha or TGF-β1 diluted in FBS-free medium supplemented with 1% BSA. Cell supernatants were collected for the quantitative measurement of secreted MMP1 levels.
16 µL of supernatants (diluted ¼) were then transferred into a white detection plate (384 low volume) and 4 µL of the HTRF Human MMP1 detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.
As expected, cell treatment for 24h induced an upregulation of the secreted level of human MMP1, particularly after TNF alpha treatment.
MMP1 upregulation induced by PMA treatment in cell lysates and cell supernatant of human glioblastoma cancer cell line U87-MG
The human glioblastoma cancer cell line U87-MG was plated in a 96-well plate (100,000 cells/well). The day after, cells were treated overnight with 50 µL of PMA (50 ng/mL) diluted in FBS-free medium supplemented with 1% BSA.
Cell supernatants were collected, and cells were lysed with 50 µL of supplemented lysis buffer #3 (4X) for 30 min at room temperature under gentle shaking for the quantitative measurement of MMP1 levels.
16 µL of supernatants or lysates were then transferred into a white detection plate (384 low volume) and 4 µL of the HTRF Human MMP1 detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.
As expected, PMA treatment led to an increase in the intracellular and secreted levels of human MMP1 in this cancer cell line.
Versatility of Human MMP1 assay in human cell line supernatants
Different human cell lines were seeded in T175 flasks in 25 mL of complete culture medium at 37°C, 5% CO2 up to 80% confluence. Then the supernatant was collected. 16 µL of each cell line supernatant were transferred into a 384 well low volume white microplate and 4 µL of HTRF Human MMP1 detection antibody were added. The HTRF signal was recorded after an overnight incubation. The results reveal a differential expression pattern with a high concentration level in the human pulmonary fibroblast cell line and a lower level in the human glioblastoma cancer cell line (U87-MG) or Human foreskin BJ fibroblasts.
MMP1 signaling pathway
Matrix metalloproteinases (MMPs) are a family, 23 members of which have been identified, produced by diverse cell types, including immune cells, epithelial cells, and fibroblasts. MMP functions include degradation of components of the extracellular matrix (ECM) in normal physiological processes, such tissue remodeling, and in disease processes, such as cancer. Specifically, MMP-1 breaks down the interstitial collagens, types I, II, and III.
MMP1, or collagenase-1, plays critical roles in many physiologic processes by degrading these collagen types I, II, and III.
The regulation of MMP1 is complex and multifactorial. All the pathways involved seem to converge toward JNK and its resulting activation of transcription factors c-Jun and c-Fos. Various growth factors and cytoskeleton-related mechanical tension may be involved.
Cytokines are also involved in MMP1 regulation. Pro-inflammatory IL-1β in particular promotes them via the NF-kB pathway or via p38 MAP kinase, particularly in cancer.
MMP1 are secreted or expressed at the membrane as precursors and their activity is regulated by the TIMPs (tissue inhibitors of metalloproteinases) and by themselves, as they activate each other in a complex regulatory network.
