Abstract
Human umbilical cord Wharton’s jelly derived mesenchymal stem cells (hUCMSCs), a source of cell therapy, have received a great deal of attention due to their homing or migrating ability in response to signals emanating from damaged sites. It has been found that IL-1β possesses the ability to induce the expression of matrix metalloproteinase-3 (MMP-3) in bone marrow MSCs. MMP-3 is involved in cell migration in various types of cells, including glioblastoma, vascular smooth muscle, and adult neural progenitor cells. In this study, we proposed that IL-1β influences hUCMSCs migration involving MMP-3. The expression level of MMP-3 in IL-1β-induced hUCMSCs was verified using cDNA microarray analysis, quantitative real-time PCR, ELISA and Western blot. Wound-healing and trans-well assay were used to investigate the cell migration and invasion ability of IL-1β-treated hUCMSCs. In addition, we pre-treated hUCMSCs with interleukin-1 receptor antagonist, MMP-3 inhibitors (ALX-260-165, UK 356618), or transfected with MMP-3 siRNA to confirm the role of MMP3 in IL-1β-induced cell migration. Our results showed that IL-1β induced MMP-3 expression is related to the migration of hUCMSCs. Moreover, extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) inhibitor U0126, p38 inhibitor SB205380, JNK inhibitor SP600125 and Akt inhibitor GSK 690693 decreased IL-1β-induced MMP-3 mRNA and protein expression. The migration and invasion ability analyses showed that these inhibitors attenuated the IL-1β-induced migration and invasion ability of hUCMSCs. In conclusion, we have found that IL-1β induces the expression of MMP-3 through ERK1/2, JNK, p38 MAPK and Akt signaling pathways to enhance the migration of hUCMSCs. These results provide further understanding of the mechanisms in IL-1β-induced hUCMSCs migration to injury sites.
Highlights
Human umbilical cord Wharton’s jelly derived mesenchymal stem cells are rapidly gaining attention for its therapeutic potential in regenerative medicine [1]
The cell viability assay data showed no significant difference between IL-1β-treated and control groups (Fig 1C), suggested that IL-1β stimulated stem cell migration was not affected by cell viability
To clarify that the matrix metalloproteinase-3 (MMP-3) expression was induced by IL-1β, cells were pretreated with IL-1β receptor antagonist IL-1 receptor antagonist (IL-1RA), the results showed that IL-1RA significantly suppressed IL-1β-induced matrix metalloproteinase (MMP)-3 expression
Summary
Cell cultureHuman umbilical cord derived mesenchymal stem cells (hUCMSCs) were purchased from Bioresource Collection and Research Center, Hsinchu, Taiwan. hUCMSCs were cultured in low serum defined medium consisting of 56% low-glucose Dulbecco’s Modified Eagle Medium (DMEM-LG; Invitrogen, CA, USA), 37% MCBD 201 (Sigma, MO, USA), 2% fetal bovine serum (Thermo, Logan, UT), 0.5 mg/ml of AlbuMAX1 I (Invitrogen, CA, USA), 1X insulin-transferrin-selenium-A (Invitrogen, CA, USA), 1X antibiotic antimycotic solution (Thermo, Logan, UT), 10 nM dexamethasone (Sigma, MO, USA), 50 nM L-ascorbic acid 2-phosphate (Sigma, MO, USA), 10 ng/ml of epidermal growth factor (PeproTech, NJ, USA), and 1 ng/ml of platelet-derived growth factor-BB (PeproTech, NJ, USA) at 37 ̊C and 5% CO2. Cytokines and inhibitors hUCMSCs were starved in serum-free DMEM-LG containing 0.1% bovine serum albumin (BSA) for 16 hours, treated with 2 μg/ml IL-1 receptor antagonist (IL-1RA) (Peprotech, NJ, USA), MMP-3 inhibitors UK356618 (20 nM) (Tocris, UK) and ALX260165 (20 μM) (Enzo life, UK), ERK1/2 inhibitor U0126 (10–30 μM) (Tocris, UK), p38 inhibitor SB205380 (50 nM) (Tocris, UK), Akt inhibitor GSK 690693 (20 μM) (Tocris, UK) and JNK inhibitor SP600125 (20 nM) (Tocris, UK) for 2 hours prior to human recombinant interleukin-1β (IL-1β) stimulation. Cell viability assay hUCMSCs were seeded in 96-well plates in serum-free DMEM-LG containing 0.1% BSA for 16 hours and stimulated with 100 ng/ml IL-1β for 36 hours. MTT assay reagent (3(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, SERVA Heidelberg German 20395) were added into the culture medium in 1 mg/mL and incubated with cells for 4 hours at 37 ̊C and 5% CO2. The MTT assay data were determined by Multimode microplate readers (Infinite 200, TECAN)
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