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. 2019 May 22:25:3815-3824.
doi: 10.12659/MSM.913623.

Klotho Protein Reduced the Expression of Matrix Metalloproteinase-1 (MMP-1) and Matrix Metalloproteinase-3 (MMP-3) in Fibroblasts from Patients with Pelvic Organ Prolapse (POP) by Down-Regulating the Phosphorylation of ERK1/2

Jun Qiu  1 , Menglu Qin  1 , Bozhen Fan  2 , Xinliang Chen  1
Affiliations

Klotho Protein Reduced the Expression of Matrix Metalloproteinase-1 (MMP-1) and Matrix Metalloproteinase-3 (MMP-3) in Fibroblasts from Patients with Pelvic Organ Prolapse (POP) by Down-Regulating the Phosphorylation of ERK1/2

Jun Qiu et al. Med Sci Monit. .

Abstract

BACKGROUND Pelvic organ prolapse (POP) is due to age-related atrophy and the weakening of the tissues of the pelvic floor, with degradation of collagen and extracellular matrix (ECM) by metalloproteinases (MMPs). This study aimed to investigates the role of the age-related enzyme klotho, encoded by the KL gene, in cultured fibroblasts obtained from patients with POP and the levels of reactive oxygen species (ROS), interleukin-6 (IL-6), and MMPs. MATERIAL AND METHODS Pelvic floor fibroblasts were obtained from connective tissue from three patients with POP and three normal subjects. Cell proliferation and ROS production were measured using a cell counting kit-8 (CCK-8) assay and flow cytometry. Levels of interleukin-6 (IL-6), klotho, metalloproteinase-1 (MMP-1), MMP-3, extracellular signal-regulated kinases 1/2 (ERK1/2), and p-ERK1/2 were measured by enzyme-linked immunoassay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. RESULTS In cultured pelvic floor fibroblasts from patients with POP, the expression of klotho protein and klotho mRNA were significantly down-regulated in fibroblasts from patients with POP compared with normal fibroblasts. Klotho supplementation in cultured fibroblasts for patients with POP included increased cell growth, reduced expression of ROS reduction, and reduced the secretion of IL-6. Using qRT-PCR and Western blot, klotho supplementation of fibroblasts from patients with POP increased cell growth and reduced the levels of IL-6 and ROS in a dose-dependent way. CONCLUSIONS Klotho protein reduced the expression of MMP-1 and MMP-3 in fibroblasts from patients with POP by down-regulating the phosphorylation of ERK1/2.

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Conflict of interest statement

Conflict of interest

None.

Figures

Figure 1
Figure 1
Reduced expression of klotho mRNA and protein in pelvic floor fibroblasts from patients with pelvic organ prolapse (POP) compared with fibroblasts from normal controls. (A) Quantitative real-time polymerase chain reaction (qRT-PCR) for klotho in fibroblasts from patients with pelvic organ prolapse (POP) compared with fibroblasts from normal controls. (B) Western blot analysis for klotho protein in fibroblasts from patients with pelvic organ prolapse (POP) compared with fibroblasts from normal controls. The expression of GAPDH served as an internal control. (C) Quantitative results of Western blot analysis of fibroblasts from patients with pelvic organ prolapse (POP) compared with fibroblasts from the normal control group. The asterisks indicate the significance of the differences (** p<0.01, *** p<0.001).
Figure 2
Figure 2
Flow cytometry for the measurement of reactive oxygen species (ROS) and interleukin-6 (IL-6) in pelvic floor fibroblasts from patients with pelvic organ prolapse (POP) compared with fibroblasts from normal controls. (A) Examination ROS levels in pelvic floor fibroblasts from patients with pelvic organ prolapse (POP) and normal control cells using flow cytometry using dichloro-dihydro-fluorescein diacetate (DCFH-DA). (B) IL-6 production increased in supernatants of cultured pelvic floor fibroblasts from patients with POP when compared with fibroblasts from the normal control group. The asterisks indicate the significance of differences (** p<0.01, *** p<0.001).
Figure 3
Figure 3
The effects of klotho supplementation on pelvic floor fibroblasts from patients with pelvic organ prolapse (POP) compared with fibroblasts from normal controls. (A) Supplementation with klotho protein increased fibroblast growth in a dose-dependent and time-dependent way for 72 hrs. (B) Treatment with klotho (50 ng/mL and 500 ng/mL) for 48 h significantly decreased the levels of reactive oxygen species (ROS). (C) Interleukin-6 (IL-6) production in supernatants was reduced following klotho administration (50 ng/ml and 500 ng/ml) for 24 h. The asterisks indicate the significance of the differences (* p<0.05, ** p<0.01, *** p<0.001).
Figure 4
Figure 4
Western blot for the measurement of matrix metalloproteinase- (MMP-1) and MMP-3 in pelvic floor fibroblasts from patients with pelvic organ prolapse (POP) compared with fibroblasts from normal controls. (A) The expression of MMP-1 and MMP-3 in pelvic floor fibroblasts in patients with or without pelvic organ prolapse (POP) measured by Western blot. (B) Quantitative results of the Western blot analysis in A. (C) Supplementation with klotho protein decreased the expression of MMP-1 and MMP-3 in pelvic floor fibroblasts in patients with POP. (D) Quantitative results of Western blot analysis in C. The asterisks indicate the significance of differences (** p<0.01, *** p<0.001).
Figure 5
Figure 5
Western blot for the measurement of phosphorylated ERK1/2 in pelvic floor fibroblasts from patients with pelvic organ prolapse (POP) compared with fibroblasts from normal controls. (A) The expression and phosphorylation of ERK1/2 in pelvic floor fibroblasts from patients with or without pelvic organ prolapse (POP) were measured using Western blot. (B) Quantitative results of the Western blot analysis from A. (C) Supplementation with klotho down-regulated the phosphorylation but not the expression of ERK1/2 in pelvic floor fibroblasts in fibroblasts from patients with POP. (D) Quantitative results of the Western blot analysis from C. The asterisks indicate the significance of differences (**p<0.01).
Figure 6
Figure 6
Western blot for the measurement of matrix metalloproteinase- (MMP-1) and MMP-3 in pelvic floor fibroblasts from patients with pelvic organ prolapse (POP) compared with fibroblasts from normal controls treated with klotho, U0126, and epidermal growth factor (EGF). (A) The effects of administration of klotho, U0126, and epidermal growth factor (EGF) on the expressions of MMP-1 and MMP-3 in pelvic floor fibroblasts in patients with pelvic organ prolapse (POP) were measured using Western blot. (B) Quantitative results of the Western blot analysis of A. The asterisks indicate the significance of differences (**p<0.01).
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