Fig 1.
IL-1β stimulates mesenchymal stem cell migration.
(A) Cell wound healing assay for IL-1β stimulated mesenchymal stem cells in the presence or absence of 2 μg/ml IL-1RA (IL-1β inhibitor) at 12 and 24 hours. Scale bars = 300 μm. (B) The wound area of hUCMSCs were indicated by MetaMorph and the data were normalized with control and shown as the mean ± SD (n = 3, *P<0.05, **P<0.01 versus control cells, #p<0.05, ##p<0.01 versus IL-1β treated cells). (C) Cell viability assay for IL-1β stimulation. Data were quantified by multimode micro-plate readers. Data are shown as the mean ± SD (n = 3) (N.S.: nonsignificance).
Fig 2.
Effects of IL-1β in hUCMSCs on MMP-3 RNA and protein levels.
(A) Quantitation of changes in gene expression of MMP-3 detected by real-time PCR after IL-1β inhibitor IL-1RA (2 μg/ml) pre-treatment and stimulation with IL-1β for 12–48 hours. (B) Example of Western blot results of the MMP-3 (54 kDa) from the lysates of cells treated with IL-1β and IL-1RA. The full-length Western blots was showed in S2 Fig. (C) Quantitative graphs of the Western blot results of MMP-3 protein expression of (B). (D) MMP-3 protein expression was measured using ELISA, pre-treatment with IL-1RA at concentration of 2 μg/ml and stimulated with IL-1β for 36 hours. Data are shown as the mean ± SD (n = 3, **P<0.01, ***P<0.005 versus control cells, #p<0.05, ##p<0.01 versus IL-1β treated cells).
Table 1.
Total RNA microarray based screening for the expression of MMPs with IL-1β stimulation for 24 hours in mesenchymal stem cells.
Fig 3.
Effects of MMP-3 inhibitor in hUCMSCs on MMP-3 secretion and MMP3 activity, cell migration and invasion.
(A) MMP-3 protein expression measured by ELISA. hUCMSCs treated with ALX 260165 and UK 356618 at concentration of 20 μM. (B) MMP-3 activity was measured by Fluorogenic peptide Assays. hUCMSCs treated with ALX 260165 and UK 356618 at concentration of 20 μM. (C) Cell wound healing assay. Cultures were treated with MMP-3 inhibitors ALX 260165, UK 356618 at concentrations of 20 μM, 20 nM, respectively as indicated. Scale bars = 300 μm. (D) Quantitative graph showing the migration ability of stem cells into the wound area at 24 hours. (E) Cell invasion assay, cultures were treated with MMP-3 inhibitors ALX 260165, UK 356618 at concentrations of 20 μM, 20 nM, respectively as indicated. Scale bars = 1 mm. (F) Graph indicates the invasion ability of stem cells. Data are shown as the mean ± SD (n = 3, **P<0.01, ***P<0.005, versus control cells, #p<0.05, ##p<0.01, ###p<0.005 versus IL-1β treated cells).
Fig 4.
Effect of knockdown MMP-3 on IL-1β treated hUCMSCs.
(A) Quantitation of changes in gene expression of MMP-3 detected by real-time PCR. hUCMSCs were transfected with MMP-3 siRNA and stimulated with IL-1β for 24 hours. (B) MMP-3 protein expression was measured using ELISA, transfected with MMP-3 siRNA and stimulated with IL-1β for 36 hours. (C) Cell wound healing assay for IL-1β stimulated mesenchymal stem cells after MMP-3 siRNA transfection. Scale bars = 300 μm. (D) The wound area of hUCMSCs were indicated by MetaMorph. (E) Cell invasion assay for IL-1β stimulated mesenchymal stem cells after MMP-3 siRNA transfection. Scale bars = 1 mm. (F) Graph indicates the invasion ability of stem cells. Data are shown as the mean ± SD (n = 3, **P<0.01, ***P<0.005 versus control cells, #p<0.05, ##p<0.01, ###p<0.005, versus IL-1β treated cells).
Fig 5.
Effects of ERK1/2 pathway on IL-1β-mediated MMP-3 expression, cell migration and cell invasion.
(A) Quantitation of changes in gene expression of MMP-3 detected by real-time PCR after ERK1/2 inhibitor U0126 (10–30 μM) treatment and stimulation with IL-1β for 24 hours. (B) Example of Western blot results of the MMP-3 (54 kDa) from the lysates of cells treated with IL-1β and ERK1/2 inhibitor U0126 (20 μM). The full-length Western blots was showed in S2 Fig. (C) Quantitative graphs of the Western blot results of MMP-3 protein expression of (B). (D) MMP-3 protein expression was measured using ELISA, treated with U0126 (20 μM) and stimulated with IL-1β for 36 hours. (E) MMP-3 activity was measured by Fluorogenic peptide Assays. hUCMSCs treated with U0126 at concentration of 20 μM. (F) Cell wound healing assay for IL-1β stimulated mesenchymal stem cells in the presence of ERK1/2 inhibitor U0126 (20 μM) at 24 hours. Scale bars = 300 μm. (G) The wound area of hUCMSCs were indicated by MetaMorph and the data were normalized with control and shown as the mean ± SD (n = 3, ***P<0.01 versus control cells, ##p<0.01 versus IL-1β treated cells). (H) Cell wound healing assay for IL-1β stimulated mesenchymal stem cells in the presence of ERK1/2 inhibitor U0126 (20 μM) at 24 hours. Scale bars = 1 mm. (I) Graph indicates the invasion ability of stem cells. Data are shown as the mean ± SD (n = 3, *P<0.05, *** P <0.005 versus control cells, #p<0.05, ##p<0.01, ###p<0.005 versus IL-1β treated cells).
Fig 6.
Effects of JNK, p38, and Akt pathways on IL-1β induced MMP-3 expression on hUCMSCs.
(A) Quantitation of changes in gene expression of MMP-3 detected by real-time PCR after p38 inhibitor (50 nM SB205380), Akt inhibitor (20 μM GSK690693), and JNK inhibitor (20 nM SP600125) treatment and stimulation with IL-1β for 24 hours. (B) Cell wound healing assay for IL-1β stimulated mesenchymal stem cells in the presence 50 nM SB205380, 20 μM GSK690693, and 20 nM SP600125 at 24 hours. Scale bars = 300 μm. (C) Graph indicates the migration ability of stem cells into the wound area. Data are shown as the mean ± SD (n = 3, *P<0.05, ***P<0.005 versus control cells, ###p<0.005 versus IL-1β treated cells).
Fig 7.
Schematic diagram of IL-1β signaling pathway in hUCMSCs migration.
A schematic diagram depicts the proposed role of IL-1β signaling pathway in hUCMSCs migration. The process of cell migration is initiated by IL-1β through ERK1/2 induced expression of MMP-3 in hUCMSCs.