TIMP-1 Deficiency Subverts Cell Cycle Dynamics In HSCs.

Abstract 1545

Although TIMP-1 was initially described as a mere inhibitor of Metalloproteinases (MMPs), recent findings have offered a different perspective on its biological role, contributing to unveil its multifaceted nature. In addition to inhibiting MMP activity, TIMP-1 has been proven to play MMP-independent, cytokine-like activities and to be involved in the regulation of numerous biological functions, including cell proliferation and survival. We therefore hypothesized that TIMP-1 might be involved in the homeostatic regulation of hematopoietic stem cells (HSCs), whose biological behavior is the synthesis of both microenvironmental and intrinsic cues. Bone marrow hematopoietic stem cells (HSCs) were isolated from TIMP-1^-/- mice based on the phenotype Side Population c-Kit⁺Lin^- Sca-1⁺ (SP^KLS). In vitro cultural assays as well as in vivo transplantation assays were employed to investigate how TIMP-1 obliteration affects murine hematopoiesis. Cell-cycle dynamics in KO SP^KLS HSCs were characterized by Pyronin Y/Hoechst staining, Ki-67 staining, as well as evaluation of RNA expression of cell cycle inhibitors, such as p53, p57, and p21.

We found that TIMP-1^-/- mice have decreased HSC numbers and, consistent with this finding, TIMP-1^-/- HSCs display reduced capability of long-term repopulation. Interestingly, the cell cycle distribution of TIMP-1^-/- LT-HSCs is profoundly distorted, with a consistent proportion of the stem cell pool arrested in the G₁ phase, suggesting that TIMP-1 is intrinsically involved in the regulation of the HSC proliferation dynamics. Of note, TIMP-1^-/- HSCs present decreased levels of CD44 glycoprotein, whose expression has been proven to be controlled by p53, the master regulator of the G₁/S transition. Interestingly, p53 RNA levels are indeed increased in TIMP-1^-/- SP^KLS HSCs compared to controls. Likewise, the expression level of other cell-cycle inhibitors, such as p57 and p21, were found to be higher in KO SP^KLS HSCs, indicating a disregulation of cell-cycle dynamics.Our study highlights a novel biological role of TIMP-1 in the regulation of the HSC compartment and suggest a novel mechanism presiding over stem cell quiescence in the framework of the BM milieu.