Tissue inhibitor of metalloproteinase-3 (TIMP-3) regulates hematopoiesis and bone formation in vivo

Abstract

Background: Tissue inhibitor of metalloproteinases-3 (TIMP-3) inhibits matrix metalloproteinases and membrane-bound sheddases. TIMP-3 is associated with the extracellular matrix and is expressed in highly remodeling tissues. TIMP-3 function in the hematopoietic system is unknown.

Methodology/principal findings: We now report that TIMP-3 is highly expressed in the endosteal region of the bone marrow (BM), particularly by osteoblasts, endothelial and multipotent mesenchymal stromal cells which are all important cellular components of hematopoietic stem cell (HSC) niches, whereas its expression is very low in mature leukocytes and hematopoietic stem and progenitor cells. A possible role of TIMP-3 as an important niche component was further suggested by its down-regulation during granulocyte colony-stimulating factor-induced mobilization. To further investigate TIMP-3 function, mouse HSC were retrovirally transduced with human TIMP-3 and transplanted into lethally irradiated recipients. TIMP-3 overexpression resulted in decreased frequency of B and T lymphocytes and increased frequency of myeloid cells in blood and BM, increased Lineage-negative Sca-1(+)KIT(+) cell proliferation in vivo and in vitro and increased colony-forming cell trafficking to blood and spleen. Finally, over-expression of human TIMP-3 caused a late onset fatal osteosclerosis.

Conclusions/significance: Our results suggest that TIMP-3 regulates HSC proliferation, differentiation and trafficking in vivo, as well as bone and bone turn-over, and that TIMP-3 is expressed by stromal cells forming HSC niches within the BM. Thus, TIMP-3 may be an important HSC niche component regulating both hematopoiesis and bone remodeling.

Figure 1. Decreased TIMP-3 expression level during G-CSF induced mobilization.

(A) Detection by reverse zymography of TIMP-1 and TIMP-3 proteins from mouse BM fluids following mobilization with G-CSF. G-CSF was administered twice daily from day 0 to day 6 and the left to rest for 2 days (day 8 group). Each sample is a pool of BM fluids from 4 mice per time point. Controls were made with recombinant human TIMP-1 (0.1 and 0.3 ng) and TIMP-2 (1 ng). (B) Quantification of TIMP-3 and TIMP-1 protein concentrations during G-CSF induced mobilization from panel A. (C–D) TIMP-3 mRNA expression by RT-qPCR in cells from the endosteal region (C) and central BM (D). Data are normalized to B2M expression and are average ± SD of at least 4 mice per time-point. * indicates a p value of 0.03 compared to control mice. (E–F) TIMP-1 mRNA expression in endosteal (E) and central BM (F).

Figure 2. TIMP-3 expression pattern in the BM.

(A) Comparison of TIMP-3 mRNA expression between endosteal and central BM cells by RT-qPCR. Data are mean ± SD of at least 7 mice per group normalized to B2M. (B) TIMP-3 mRNA in cells sorted from collagenase-treated compact bones or central bones. Phenotypes used are described in Supplementary Materials and Methods. Data are mean ± SD of at least 3 mice per group normalized to B2M. (C) TIMP-3 immunohistofluorescence by confocal laser scanning microscopy on bone sections x40 magnification. Two left panels are with rabbit anti-TIMP-3 antibody, two right panel with control non-immune rabbit IgG. Green color shows anti-TIMP-3 fluorescence whereas the pink color shows nuclei stained by DAPI. Osteoblasts (OB), endothelial cells (endo) and megakayocytes (MK) are indicated. (D) Reverse zymography of mouse blood plasma, sera and platelet lysates. Glycosylated and unglycosylated TIMP-3, and TIMP-2 are indicated. M lane contains molecular weight markers, + lane contains 20 ng of rmuTIMP-3.

Figure 3. Retroviral vector to over-express huTIMP-3 in murine hematopoietic cells.

(A) Representation of the MXIE retroviral vector containing huTIMP-3, an IRES, and GFP thus allowing for the co-expression of huTIMP-3 and GFP. (B) PCR and (C) RT-PCR of genomic DNA and RNA respectively from retrovirally transduced mouse hematopoietic cell line. M is markers, Mock are non-transduced cells, MXIE are cells transduced with empty-MXIE vector and MXIE-huTIMP-3 are cells transduced with the MXIE-huTIMP3 vector. + lane is the original plasmid containing huTIMP-3 cDNA as positive control. (D) Reverse zymography of culture supernatants from different clones of FDCP1 cells transduced with empty MXIE or MXIE-huTIMP-3 vectors. M is molecular weight markers, and + is 25 ng rhuTIMP-3.

Figure 4. Flow cytometry analysis of leukocytes in mice over-expressing huTIMP-3 in hematopoietic cells

. Frequency of CD11b⁺ myeloid cells (A), B220⁺ B cells (B) and CD3⁺ T cells (C) among GFP⁺ transduced cells in the BM, blood and spleen of mice reconstituted with HSC transduced with empty MXIE vector (white columns) or MXIE-huTIMP3 vector (black column). Data are mean ± SD of 9 mice per group. (D) Proportion of CD11b⁺ myeloid cells, B220⁺ B cells and CD3⁺ T cells in the BM, spleen and blood of the same mice when GFP expression was not taken into consideration. Data are mean ± SD of 9 mice per group. (E) Reverse zymography of BM fluids from mice transplanted with HSC transduced with empty MXIE vector or MXIE-huTIMP3 (n = 5 per group). M is molecular weight marker, + is 25 ng of rhuTIMP-3. (F) Quantification of the integrated intensity of TIMP-3 bands from panel E.

Figure 5. Increased CFC trafficking in mice over-expressing huTIMP-3.

Colony-forming cell number in spleen (A), blood (B) and femoral BM (C) from mice transplanted with HSC transduced with empty MXIE vector (white columns) or MXIE-huTIMP3 vector (black columns). Data are mean ± SD of 9 mice per group.

Figure 6. huTIMP-3 increases LSK cell proliferation in vivo and in vitro.

(A) Proportion of transduced LSK cells that incorporated BrdU in vivo after 3 day BrdU administration. Data are mean ± SD of 5 mice per group. (B) In vitro proliferation of transduced LSK cells after 12 days of culture. LSK cells were seeded at 4,000/mL. Data are average ± SD of triplicates. (C) Day 7 effect of the addition of 200 ng/mL purified rhuTIMP-3 to liquid culture of LSK cells from non-manipulated mice. Data are mean ± SEM of 2 mice in quadruplicates.

Figure 7. Long-term over-expression of huTIMP-3 in hematopoietic cells leads to fatal osteosclerosis.

(A) Survival curve of mice transplanted with HSC transduced with empty MXIE vector or MXIE-huTIMP3 vector (n = 10 in each group). (B) von Kossa staining and toluidine blue staining (C) of tibial sections from mice transplanted with HSC transduced with empty MXIE vector or MXIE-huTIMP3 vector. In panel B, black staining shows calcified bone. In panel C, cuboidal osteoblasts covering the osteoid are indicated by empty arrow heads. Note that osteoblasts and osteoid are absent in mice transduced with MXIE-huTIMP3.