(A) Left: Representative flow cytometry analysis of membranal MT1-MMP and RECK expression on CD34⁺ cells enriched from the untreated (steady state) BM or PB, or PB of G-CSF–mobilized healthy human donors (MPB). Dotted lines indicate background labeling with secondary IgG. Numbers denote MFI values of MT1-MMP and RECK immunolabeling (mean ± SD of 3–6 independent experiments). Right: Representative flow cytometry analysis of MT1-MMP and RECK co-expression on human steady-state BM CD34⁺ progenitors. (B) G-CSF treatment ex vivo increases MT1-MMP expression on steady-state BM CD34⁺ cells. Flow cytometry analysis of MT1-MMP levels on CD34⁺ cells enriched from the BM of healthy donors (n = 6) and cultured for 48 hours in the presence of 100 ng/ml G-CSF, IL-6, SDF-1, or SCF or left untreated (–). Results are shown as fold change in MFI relative to untreated (mean ± SD; *P < 0.05). (C and D) Membranal MT1-MMP expression on MPB human CD34⁺ HPCs. Linear regression analysis (solid lines) and 95% confidence interval (dotted lines) are shown. (C) CD34⁺ cells were enriched from the PB of 21 consecutive G-CSF–treated healthy donors and immediately immunolabeled for MT1-MMP. (D) 29 consecutive patients were treated with chemotherapy and G-CSF, as described in Methods. PB cells were isolated on the first collection day and co-immunolabeled with anti–MT1-MMP, anti-CD34, and anti-CD45 Abs.

(A) MT1-MMP and RECK membranal levels on CD34⁺ progenitors detected in BM cells obtained from untreated chimeric mice following 48-hour treatment in vitro with G-CSF in the presence of the PI3K inhibitor LY294002 (PI3Ki) or DMSO vehicle (–). MFI was determined by flow cytometry and expressed as fold change compared with samples treated only with DMSO (mean ± SD of 3 independent experiments). *P < 0.05. (B) Representative immunohistochemical analysis of phospho-Akt levels (brown staining) in the BM sections of chimeric NOD/SCID mice treated (+) with G-CSF or untreated (–). Scale bar: 50 μm. (C) Relative changes in percentage of phospho-Akt–positive human hematopoietic cells repopulating the BM of chimeric mice treated with G-CSF or untreated, as determined by flow cytometry. Data are shown as fold change relative to PBS-treated (–) counterparts (mean ± SD of at least 3 independent experiments in duplicates). **P < 0.01. (D) Real-time PCR analysis of Mt1-mmp and Reck expression in the BM of Balb/c mice treated with G-CSF and with or without rapamycin (RAPA) or left untreated. Data are represented as fold change (mean ± SD for 10 and 5 independent experiments for G-CSF and G-CSF + RAPA, respectively, normalized to HPRT control). *P < 0.05, **P < 0.01. (E) Number of CFU cells (CFU-Cs; indicated as black circles) detected in 1 ml PB of Balb/c mice treated with G-CSF with or without RAPA or left untreated, as described in D. n = 6 mice for each treatment from 3 independent experiments. *P < 0.05 (left asterisk compares left and middle columns; right asterisk compares middle and right columns). Mean values for each group are indicated by horizontal lines.

(A and B) Enriched human MPB CD34⁺ cells were pre-incubated or not with anti–MT1-MMP or control anti-VLA6 Abs (A) or transfected with either control siRNA or MT1-MMP siRNA (B), as in Figure 4B, and transplanted into sub-lethally irradiated NOD/SCID mice. Results are shown as numbers of human cells per 10⁶ total cells detected in the BM of recipient mice. (A) Mean ± SD of 3–5 independent experiments, at least 3 mice per treatment. *P < 0.05. (B) Mean ± SD of 3 independent experiments, 2 mice per treatment, relative to control siRNA. *P < 0.05. Representative flow cytometry analysis of BM homing of human CD45⁺/CD34⁺ cells (numbers are indicated) is shown on the right. (C) Mouse BM cells were obtained from the WT (Mt1-mmp^+/+) and MT1-MMP KO (Mt1-mmp^–/–) littermates. Data are expressed as numbers of CFSE⁺ donor cells per 10⁶ total cells detected in the BM of transplanted NOD/SCID mice (mean ± SD of 3 independent experiments, 2 mice per treatment). **P = 0.02. Representative flow cytometry analysis is shown on the right, numbers indicate CFSE⁺ donor cells per 10⁶ total cells detected in the BM. (D) BM cells obtained from MT1-MMP KO or WT mice (CD45.2⁺) were transplanted at the indicated cell doses (5 × 10⁴ white circles and 2 × 10⁵ black circles) into sub-lethally irradiated B6.SJL (CD45.1⁺) recipients. Results are shown as percentage of donor-derived c-kit⁺CD45.2⁺ cells detected in the BM of recipients (CD45.1⁺ mice). Mean ± SD of 4 independent experiments. **P < 0.01. Average values for each group are indicated by horizontal lines.

(A) Representative immunohistochemical analysis (brown staining) of CD44 expression in the BM of highly engrafted NOD/SCID chimeric mice treated with G-CSF (+) or untreated (–). Scale bar: 100 μm. (B) Flow cytometry analysis of membranal CD44 expression on human CD34⁺ cells in the BM of chimeric mice treated as described in Figure 6A. Data are shown as fold change in MFI compared with sham-injected mice (mean ± SD of 4 independent experiments, n = 12 mice for G-CSF, n = 6 mice for anti–MT1-MMP). *P < 0.05; **P < 0.01. (C) Representative immunoblot of CD44 cleavage products in the BM supernatants of NOD/SCID chimeric mice treated with G-CSF with or without anti–MT1-MMP or anti-RECK as described in Figure 6. Mr, marker. (D) Representative flow cytometry analysis of MT1-MMP and CD44 expression in G-CSF–treated U937 cells transfected with MT1-MMP siRNA or control siRNA. (E and F) Adhesion to hyaluronan of human CD34⁺ cells from the BM of chimeric mice (E) treated with G-CSF with or without anti–MT1-MMP or left untreated (mean ± SD of 3 independent experiments, n = 4–6 mice per treatment; **P < 0.01) or (F) untreated following incubation with anti-CD44, anti-RECK, G-CSF, or G-CSF + anti–MT1-MMP (mean ± SD of 3 independent experiments performed multiple times; *P < 0.05; **P < 0.01). (G) CFU cells detected in 1 ml PB of CD44^+/+ and CD44^–/– mice. n = 8 mice from each genotype, 4 independent experiments performed in duplicate. *P < 0.05. (H) Representative flow cytometry analysis of the percentage of primitive lineage^–c-kit⁺Sca-1⁺ HPCs in the PB of CD44^+/+ and CD44^–/– mice.

(A) Real-time PCR analysis of human MT1-MMP and RECK expression in the BM cells of sham-injected (–) or G-CSF–treated (+) chimeric mice. mRNA levels upon G-CSF treatment are shown as fold change relative to sham-injected mice (mean ± SD of 4 independent experiments, as normalized to human GAPDH control). **P < 0.01. (B) Representative immunohistochemical analysis of MT1-MMP and RECK expression (brown staining) in BM section of highly engrafted G-CSF–treated or sham-injected control chimeric mice. Scale bar: 50 μm. (C) Relative MT1-MMP activity determined in crude plasma membrane extracts from the BM cells of sham-injected (control) or G-CSF–treated mice. Measurements of fluorogenic substrate cleavage (in arbitrary units) as a function of incubation time with MT1-MMP–containing extracts are shown as mean ± SD of 3 independent experiments with 2 mice per treatment. *P < 0.05 between the treatments. (D and E) MT1-MMP and RECK expression on CD45⁺, CD34⁺, and CD34⁺/CD38^-/low human cells detected in the BM and PB of sham-injected or G-CSF–treated chimeric mice. Flow cytometry analysis data are represented as fold change in MFI relative to human BM cells from sham-injected mice (mean ± SD of 6 independent experiments, 2 mice per treatment). **P < 0.01.

(A) Enriched human MPB CD34⁺ cells were treated with anti–MT1-MMP Abs, TIMP-2, or rabbit serum (control IgG). Enriched human BM CD34⁺ cells were treated with anti-RECK Abs (αRECK) or IgG1 (control IgG). Cells were allowed to migrate via Matrigel. Data are shown as percentage of migrating cells from the input cell number (mean ± SD of 3 independent experiments in triplicates). *P < 0.05, **P < 0.01. (B) Enriched human MPB CD34⁺ cells were transfected with control (siCTRL) or MT1-MMP (siMT1) siRNA. Data are shown as described in A (mean ± SD of 3 independent experiments in duplicates). *P < 0.05. Representative flow cytometry analysis of MT1-MMP expression in siCTRL and siMT1 transfected cells is shown. (C) BM mononuclear cells were obtained form G-CSF– or sham-injected chimeric mice (control) and treated with αMT1-MMP or TIMP-2. Following trans-Matrigel migration, numbers of human CD34⁺/CD38^–/low were determined by flow cytometry. Data are expressed as percentage change compared with migration of untreated cells from G-CSF–mobilized chimeric mice (set at 100%) (mean ± SD, 4 independent experiments performed in triplicate). *P < 0.05, **P < 0.01. (D) BM mononuclear cells were obtained from chimeric B6.SJL (CD45.1⁺) mice treated for 3 days with G-CSF and previously transplanted with CD45.2⁺ BM cells from MT1-MMP KO (Mt1-mmp^–/–) mice or WT littermates (Mt1-mmp^+/+) and allowed to migrate to SDF-1 via Matrigel-coated filters. Data are expressed as percentage of migrating CD45.2⁺c-kit⁺ cells from the input cell number as determined by flow cytometry (mean ± SD, 3 independent experiments performed in quadruplicate). **P < 0.01.

(A) NOD/SCID chimeric mice were treated with G-CSF for 5 consecutive days. Anti–MT1-MMP or anti-VLA6 Abs were injected i.p. 20 μg/mouse on days 3–5 of G-CSF administration. Numbers of human CD45⁺, CD34⁺, and CD34⁺/38^–/low cells in the PB were determined by flow cytometry and normalized as described in Methods. Data are presented as mean ± SD of 3–5 independent experiments, 3–4 mice per group. *P < 0.05. (B) Chimeric B6.SJL (CD45.1⁺) mice, previously transplanted with 2 × 10⁵ BM cells derived from MT1-MMP KO mice or WT littermates (CD45.2⁺) were treated with G-CSF for 3 consecutive days. Numbers of donor-derived c-kit⁺CD45.2⁺ in the PB were determined by flow cytometry and normalized as described in Methods. Data are represented as fold change compared with sham-injected mice. Mean ± SEM of 4 independent experiments, n = 2–3 mice per group. *P < 0.05. (C) NOD/SCID chimeric mice were injected i.p. with 20 μg/mouse of anti-RECK or IgG control Abs for 2 consecutive days. The number of CD45⁺ and CD34⁺ human cells per ml PB was determined 14–16 hours after last injection and expressed as mean ± SD of at least 3 independent experiments, 2 mice per treatment. *P < 0.05. No significant changes were detected in IgG-treated as compared with sham-injected mice. (D) Representative gelatin zymography and densitometry analysis (mean ± SD) of MMP-2 and MMP-9 levels in the BM supernatants from sham-injected (–) or anti-RECK–injected (+) mice are shown. **P < 0.01.

In the absence of mobilizing stimuli (e.g., G-CSF), proteolytic activities of MT1-MMP, MMP-2, and MMP-9 are relatively low due to inhibition by RECK. Functional membranal CD44 contributes to progenitor cell adhesion to the BM components (retention). G-CSF signaling induces PI3K-mediated Akt phosphorylation, increasing MT1-MMP and decreasing RECK expression. The opposed changes in MT1-MMP and RECK levels result in MT1-MMP–mediated CD44 proteolysis as well as MMP-2 and MMP-9 secretion and activation. Collectively, these changes reduce progenitor cell retention and facilitate their egress and mobilization.