Kruppel-Like-Factor 5 (Klf-5) Controls Hematopoietic Stem Cell/Progenitor Bone Marrow Homing and Lodging Through Rab5-Mediated Expression of Active β₁ Integrin

Abstract 113

The Kruppel-like factor 5 (Klf5) is a transcription factor that regulates cellular signaling and is involved in cell proliferation and oncogenesis. Recent studies have implicated Klf5 in the self-renewal and maintenance of pluripotent stem cells. In this project, we examined the role of Klf5 in hematopoietic stem/progenitor cells (HSC/P) activity. Using a murine model of inducible deficiency of Klf5 in hematopoietic tissue (Mx1-Cre/Klf5^flox/flox, Klf5-deficient), we observed a severe engraftment deficiency in competitive repopulation assays in primary and secondary recipients, although it did not cause any significant hematopoietic defects basally or after chemotherapy stress in primary mice. Engraftment failure of Klf5-deficient HSC was associated with a severe defect in homing (1.8±0.8% vs 6.0±2.0% for Klf5-deficient and WT HSC/P, respectively, p<0.001), endosteal lodging (9.09±12.58% vs 44.74±12.58% for Klf5-deficient and WT HSC/P, respectively, p<0.02) and adhesion to the fibronectin C-terminus (28.7±7.3% vs 44.4±3.2% of CFU-C adhesion for Klf5-deficient and WT HSC/P, respectively, p<0.001), without altering the adhesion to laminin or collagen, or CXCL12-directed chemotaxis. The homing defect associated with increased (3.5-fold) basal circulating of HSC/P and decreased HSC/P membrane β₁, but not of the β₁-integrin transcript or intracellular protein. In vivo gain-of-function of Klf5 in HSC in a stem cell leukemia (Scl) promoter-driven, tetracycline-inducible (ScltTA/TRE-Klf5) binary transgenic mouse model, resulted in increased adhesion, but not migration, of Lin⁻/c-kit⁺/Sca-1⁺ BM cells to fibronectin (32.1±7.8% vs 20.8±4.8% of CFU-C homing for WT HSC/P). The expression of Rab5 family members, mediators of β1-integrin recycling in the early endosome, was ∼50% decreased in Klf5-deficient HSC/P. Klf5 was shown to bind directly to the promoter of Rab5A and Rab5B, and lentiviral reintroduction of Rab5B rescued the expression of active membrane β₁-integrin, adhesion to fibronectin, and BM homing of Klf5-deficient HSC/P. Our data indicate that Klf5 expression is indispensable for adhesion, homing, lodging and retention of HSC/P in the BM through Rab5-dependent regulation of the expression of active β1-integrin in the cell membrane.