HSPC mobilization by Me6 is MMP9-dependent through activating the AKT, ERK, and p38 signal pathways. (A) Human Jurkat cells were stimulated with Me6 for 0, 5, 10, 20, and 40 minutes. The cell lysates were prepared and subjected to western blot analysis to detect the total protein level and phosphorylation level of AKT, ERK, and p38. (B) Human Jurkat cells were stimulated with Me6 for 40 minutes without pretreatment or following a 40-minute pretreatment with the selective PI3K/AKT inhibitor LY294002, ERK inhibitor PD98059, or p38 inhibitor SB203580. Total and phosphorylation level of AKT, ERK, and p38 were analyzed. (C) Jurkat cells (1 × 10⁶) were cultured with Me6 for 0, 12, 24, and 48 hours. Real-time quantitative polymerase chain reaction (qPCR) was performed to show that Me6 treatment significantly upregulated the expression of MMP-9 mRNA. (D) The expression level of MMP-2 mRNA was not affected by Me6. (E) Inhibition of PI3K/AKT, ERK, or p38 pathways significantly attenuated the expression of MMP-9 mRNA in the Me6-treated Jurkat cells. A total of 1 × 10⁶ Jurkat cells were cultured for 24 hours in medium alone, with Me6, or with Me6 and a selective signaling pathway inhibitor (LY294002, PD98059, or SB203580). The cells were collected for analysis of the MMP-9 mRNA levels by qPCR. (F) Me6 treatment upregulated the expression level of MMP-9 protein in Jurkat cells. (G) The upregulation of MMP-9 protein levels was significantly inhibited by LY294002, PD98059, or SB203580. (H-I) Analysis of (H) total CFU, and (I) CFU-G/GM/M, CFU-GEMM, and BFU-E in the PB of C57BL/6 mice single injected with PBS or 5 mg/kg Me6; or injected with both Me6 and LY294002, PD98059, SB203580, or neutralizing anti–MMP-9 antibody (clone 6-6B). LY294002, PD98059, SB203580, or anti–MMP-9 antibody was injected 1 hour prior to Me6 administration. n = 6 mice per group. **P < .01 vs the PBS group. ^#P < .05 and ^##P < .01 vs the Me6 group. (J) The expression of MMP-9 protein was increased in BM cells from mice treated with Me6. The secretion of MMP-9 protein was increased in BM fluids from Me6-treated mice compared with the control mice. The concentration of protein in the BM fluids was determined by using the bicinchoninic acid (BCA) protein assay system, and the samples with equivalent protein concentrations were loaded onto an sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. (K) The concentration of SDF-1α in BM fluids was detected by enzyme-linked immunosorbent assay. BM fluids were collected from mice treated with PBS or Me6 12 hours after injection. n = 3 mice per group. **P < .01 vs the control group. (L) Mobilization of WBCs by Me6 was significantly inhibited in MMP-9^−/− mice. (M) The percentages of Lin⁻Sca-1⁺ cells showed little change in Me6-treated MMP-9^−/− mice compared with PBS-treated MMP-9^−/− control mice. MMP-9^−/− and MMP-9^+/+ mice were treated with 5 mg/kg Me6 or PBS. After 12 hours, blood WBCs and HPCs (Lin⁻Sca-1⁺) were counted. n = 3 mice per group. *P < .05 vs the control MMP-9^+/+ group. (N-O) Me6 significantly induced mobilization of WBCs and HPCs (Lin⁻Sca-1⁺) in wild-type BM-MMP-9^−/− mice. BM cells from MMP-9^+/+ WT mice were transplanted into lethally irradiated MMP-9^−/− mice. Six weeks after the BM transplantation, these BM-reconstructed mice were subcutaneously injected with PBS or Me6; 12 hours later, blood WBCs and HPCs (Lin⁻Sca-1⁺) were counted. n = 4 mice per group. * P < .05 vs the PBS control group. L, LY294002; M, Me6; P, PD98059; S, SB203580.