Fig 1: Relationship between MMP-7 and collagen expression. (A) Col1a2 and Col3a1 expression changes in MMP-7 overexpressing cell as determined by real-time PCR. Casc3 was used as the reference gene. The upregulation determined by Illumina sequencing was 3.9- and 2.1-fold for Col1a2 and Col3a1 in WT cells, and 5.0 and 1.4 in active mutant MMP-7 cells (A1) compared to vector control. (B) Fibrotic changes are visualized by sirius red staining of collagen deposition. Caloric-restricted (CR) 24-month-old rats are comparable to young, 4-month control animals (top panels). Confirmation of increased collagen levels in older animals as determined by the hydroxyproline assay (bottom graph). *P < 0.05 relative to 4 AL, #relative to 24 AL. (C) Col1a2 and Col3a1 expression (left y-axis) correlates with MMP-7 expression (right y-axis) in individual F344 rats and increases with age as determined by real-time PCR. Casc3 was used as the reference gene.
Fig 2: Age-dependent changes in MMP/TIMP expression in the kidney. (A) Relative expression of MMPs and TIMPs in young (4 AL), old (24 AL), and calorie-restricted animals (24 CR) as determined by real-time PCR. ß-actin was used as the reference gene. Expression of MMP-2, -3, -7, -9, -12, -13, -14, -16, -17, -19, -20, -23, and -25, as well as TIMP-1 changed significantly as a function of age. Of these, the increased expression of MMP-2, -7, -9, -12, -13, -14, -16, -20, -23, and -25 was attenuated by caloric restriction, as was TIMP-1, with P < 0.05. (B) MMP-7 expression in aging rat kidneys is significantly increased as early as 16 months. *P < 0.05. (C) MMP-7 protein expression is increased in the 24-month-old rat kidney, but not CR controls. Each lane represents a lysate from an individual animal.
Fig 3: MMP-7 induced up-regulation of Col1a2 and Col3a1 is regulated by distinct pathways as visible by differential responses to selected pathway inhibitors, specifically the PI3K inhibitor LY294002 and the src inhibitor PP2.
Fig 4: Generation of MMP-7 overexpressing cell lines. Normal rat kidney cells (NRK-52E) were stably transfected with full-length human MMP-7 (WT), a catalytically active mutant and an inactive mutant form. Immunofluorescence staining with anti-MMP-7 antibody in vector and MMP-7 WT overexpressing cells, DAPI counterstain (bottom panels). Concentrated conditioned medium immunoblotted with anti-MMP-7 antibody shows bands for proform ∼30 kDa and active form ∼18 kDa (insert).
Fig 5: : MMP-7 activates src, PKA, and ERK1/2. (A) Col1a2 is upregulated in NRK-52E vector control cells after 24-h treatment with exogenous human MMP-7 and conditioned medium (CM) from WT MMP-7 overexpressing cells. *P < 0.05. (B) Col1a2 upregulation in NRK-52E MMP-7 overexpressing cells is attenuated by inhibition of PI3K (LY294002, 25 μmol\L), src (PP2, 1 μmol\L), p38 (SB203580, 10 μmol\L), ERK1/2 (FR180204, 5 μmol\L), PKA/PKC (Staurosporine, 100 nmol\L), and PKA (KT5720, 1 μmol\L) at 24-h exposure. A second p38 inhibitor (2-(4-Chlorophenyl)-4-(4-fluorophenyl)-5-pyridin-4-yl-1,2-dihydropyrazol-3-one) failed to reproduce the inhibition of SB203508. *P < 0.05. (C) Phosphorylation of ERK, src, and PKA increased in WT MMP-7 overexpressing NRK-52E cells compared to vector control cells as determined by immunofluorescent staining (top panels) and in-cell Western blot (bottom graph). *P < 0.05. (D) Transient (2 h) MMP-7 treatment activates ERK, src, and PKA in vector control NRK-52E cells as determined by immunofluorescent staining for phosphospecific antibodies.
Supplier Page from MilliporeSigma for Anti-MMP-7 antibody produced in rabbit