Inflammatory Chemokines MIP-1δ and MIP-3α Are Involved in the Migration of Multipotent Mesenchymal Stromal Cells Induced by Hepatoma Cells

Abstract

In vivo, bone marrow-derived multipotent mesenchymal stromal cells (MSC) have been identified at sites of tumors, suggesting that specific signals mobilize and activate MSC to migrate to areas surrounding tumors. The signals and migratory mechanisms that guide MSC are not well understood. Here, we investigated the migration of human MSC induced by conditioned medium of Huh-7 hepatoma cells (Huh-7 CM). Using a transwell migration system, we showed that human MSC migration was increased in the presence of Huh-7 CM. Using a human cytokine antibody array, we detected increased levels of MIP-1δ and MIP-3α in Huh-7 CM. Recombinant chemokines MIP-1δ and MIP-3α induced MSC migration. Anti-MIP-1δ and anti-MIP-3α antibodies added to Huh-7 CM decreased MSC migration, further suggesting that MIP-1δ and MIP-3α were implicated in the Huh-7 CM-induced MSC migration. By real-time polymerase chain reaction, we observed an absence of chemokine receptors CCR2 and CXCR2 and low expression of CCR1, CCR5, and CCR6 in MSC. Expression of these chemokine receptors was not regulated by Huh-7 CM. Furthermore, matrix metalloproteinase 1 (MMP-1) expression was strongly increased in MSC after incubation with Huh-7 CM, suggesting that MSC migration depends on MMP-1 activity. The signaling pathway MAPK/ERK was activated by Huh-7 CM but its inhibition by PD98059 did not impair Huh-7 CM-induced MSC migration. Further, long-term incubation of MSC with MIP-1δ increased α-smooth muscle actin expression, suggesting its implication in the Huh-7 CM-induced evolvement of MSC into myofibroblasts. In conclusion, we report that two inflammatory cytokines, MIP-1δ and MIP-3α, are able to increase MSC migration in vitro. These cytokines might be responsible for migration and evolvement of MSC into myofibroblasts around tumors.

FIG. 1.

Huh-7 CM and PDGF-BB induces MSC migration. (a) 6×10³ human MSC per condition were seeded in transwell with 8 μm pores. After 24 h of incubation, cells were rinsed, fixed by methanol, and stained by Hoechst. Nucleus were counted after whipping of nonmigrating cells on the top of the transwell. Incubation with PDGF-BB (20 ng/mL) and Huh-7 CM reached, respectively, 3.9±0.9 and 3.5±1.3-fold increased migration compared with control condition. HepG2 CM as well as conditioned medium from primary human hepatocytes did not induce MSC migration. The data are presented as mean values±SD from four independent experiments. (b) Combining Huh-7 CM and PDGF-BB did not potentiate the number of migrating cells. (c) Huh-7 CM were preincubated for 30 min with antibodies (0.5 μg/mL) against human PDGF-BB and then added to the migration assay for 24 h. For (b) and (c), mean values±SD of three independent experiments were performed as duplicated experiments. Migration of MSC under Huh-7 CM and Huh-7 CM with the anti-PDGF-BB antibody is not significantly different (*P=0.148). (**P≤0.01); (***P≤0.001); n.s., no significance. MSC, mesenchymal stromal cells; PDGF-BB, platelet derived growth factor BB.

FIG. 2.

Huh-7 cells produce increased levels of angiogenin, IGFBP-2, MIP-1δ, and MIP-3α. (a) Membranes of the human inflammatory cytokine antibody array were incubated for 2 h with control medium (DMEM 5% FCS) and two different preparations of Huh-7 CM. Several chemokines with increased levels such as MIP-1δ, MIP-3α, and angiogenin were detected in Huh-7. (b) Cytokines and their position on the membranes are shown in the table. (c) The graph shows the intensity of signals that was evaluated by analyzing the optical density of the spots. The experiment was performed twice with two different preparations of Huh-7 CM. DMEM, Dulbecco's modified Eagle medium.

FIG. 3.

Recombinant chemokines increased transwell migration of MSC in three out of five experiments. (a) Huh-7 CM were pre-incubated for 30 min with antibodies (10 μg/mL) against human MIP-3α and MIP-1δ. MSC were then treated with control medium PDGF-BB, Huh-7 CM, or Huh-7 CM containing the antibody against MIP-3α or MIP-1δ or both antibodies and migration was performed. Data are presented as mean±SD of three independent experiments each performed as duplicate (*P<0.05). (b) Transwell migration of MSC in control medium containing either recombinant MIP-3α, MIP-1δ, angiogenin, and IGFBP-2 or PDGF-BB compared with Huh-7 CM. Data are presented as mean±SD of three independent experiments each performed as duplicate (*P<0.05). n.s., no significance.

FIG. 4.

MSC express low levels of chemokine receptors and high levels of proteins involved in EM degradation. MSC isolated from at least six different donors and expanded for three to five passages have been used to perform real-time PCR on total RNA extractions. (a) Expression level of all five chemokine receptors CCR1, CCR2, CCR5, CCR6, and CXCR2. Expression level of CCR6 was expressed at higher levels in three of seven donors. (b) Expression level of the matrix metallo proteinase 1 (MMP-1) and 2 (MMP-2) as well as TIMP-1. PCR, polymerase chain reaction.

FIG. 5.

Expression of chemokine receptors and of proteins involved in EM degradation after incubation with Huh-7 CM. MSC isolated from four different donors have been treated with control condition (CTRL), PDGF-BB, and Huh-7 CM for 24 h. (a) Total RNA were extracted, and real-time PCR was performed on CCR1, CCR6, and CXCR2. (b) Real-time PCR was performed for MMP-1, MMP-2, and TIMP-1. All experimental data are mean±SD from at least three independent experiments (**P<0.02). (c) MSC were seeded at a density of 30,000 cells per well in a 24-well plate. Cells were incubated with control medium (CTRL), control medium containing MIP-1δ and MIP-3α, as well as Huh-7 CM. Western blotting were performed using an anti-MMP1 antibody. GAPDH was used as loading control. This figure shows one representative experiment out of three. (d) The graph presents intensity of signals that was evaluated by analyzing the optical density of the spots. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. n.s., no significance.

FIG. 6.

Huh-7 CM, MIP-1δ, and MIP-3α mediate activation of ERK pathway in MSC. (a) 30×10³ MSC per well were seeded into 24-well plates. Two days later, MSC are incubated in control medium (CTRL) containing PDGF-BB (20 ng/mL), Huh-7 CM, MIP-1δ, or MIP-3α with or without MAP kinase inhibitor PD98059 (50 μM). Cells were rinsed with PBS containing orthovanadate, and proteins were loaded on 12% SDS/polyacrylamide gel. Phospho-ERK and ERK total were detected using polyclonal antibodies. GAPDH is shown as a control for loading. (b) The p-ERK signal was quantified by densitometry and normalized to GAPDH. Error bars represent the SD from duplicates of one representative experiment out of three independent experiments. (c, d) Migration assay was performed by seeding 6×10³ MSC per well at the top of membrane inserts with 8 μm pores. PD98059 was added or not during 24 h of migration. Cells that have migrated on the lower side of the membrane were counted and expressed as fold increase to the control condition. (b, c) Represent mean values±SD from three independent experiments. (*, P≤0.05); n.s., no significance. PBS, phosphate-buffered saline.

FIG. 7.

MIP-1δ induces α-SMA expression in MSC after 5 days of exposure. MSC were seeded at a density of 30,000 cells per well in a 24-well plate. Cells were incubated with control medium (CTRL), control medium containing PDGF-BB (25 ng/mL), MIP-1δ (25 ng/mL), or TGF-β (50 ng/mL) as well as with Huh-7 CM. (a) Western blotting was performed using an anti-αSMA antibody. GAPDH was used as loading control. This figure shows one representative experiment out of three. (b) The graph shows the intensity of signals of the western blot spots that was evaluated by analyzing the optical density of the spots.