TIMP–1 Plays a Functional Role in CD34. ⁺ Hematopoietic Stem and Progenitor Cells.

Abstract 1487

Poster Board I-510

TIMP–1 protein (Tissue Inhibitor of Metalloproteinases) is a recently identified tetraspanin interacting cell surface protein in the immortalized human breast epithelial cell line MCF10A. Tetraspanins like CD63 are proteins that consist of four transmembrane domains and are known to interact with integrins. Integrins play a crucial role in hematopoietic stem cell homing and mobilization. We first screened gene array data sets of CD34⁺ human hematopoietic stem and progenitor cells (HSPCs) and found TIMP–1 mRNA expression. In this study we show that TIMP–1 co-localizes with the tetraspanin CD63 and Beta-1-Integrin. Furthermore, we found a functional interaction of TIMP-1 with its receptor complex on G-CSF mobilized HSPCs.

All experiments were carried out using highly enriched CD34⁺ cells. Co-immunoprecipitation shows that TIMP–1 binds to CD63. Using high resolution Stimulated Emission Depletion (STED) microscopy we could confirm co-localization of TIMP-1 and CD63 as well as Beta-1 Integrin and CD63. To further characterize the role of TIMP-1 in the Beta-1-Integrin signaling, we used an antibody specific to the active form of Beta-1- Integrin. Flow cytometric analysis revealed a significantly higher number of active Beta-1-Integrin in TIMP-1 stimulated cells suggesting TIMP-1 to activate the receptor complex on CD34⁺ cells. For functional analysis of the receptor complex formation, transwell migration assays were performed showing significantly increased migratory capacities of TIMP–1 treated cells. Additionally, TIMP–1 stimulation leads to a significantly increased adhesion rate of CD34⁺ cells to the fibronectin-coated dish. To assess a potential role of TIMP–1 in apoptosis, CD34⁺ HSPCs were co-incubated with thapsigargin and TIMP–1 or DMSO as a control. Subsequent flow cytometric analysis of cleaved Caspase-3 revealed a decrease of apoptotic cells in the TIMP-1 treated samples.

In summary, we can show that TIMP-1, CD63 and Beta-1-Integrin form a complex on CD34⁺ HSPCs. TIMP-1 activates the Beta-1-Integrin signaling in HSPCs and alters the adhesive as well as the migratory behavior of CD34⁺ HSPCs. Furthermore, TIMP-1 induces an antiapoptotic effect in CD34⁺ cells. The functional effects of TIMP-1 in HSPCs might be of relevance in clinical hematopoietic stem cell transplantation so that we are currently about to verify these effects in an in vivo model.