Role of Focal Adhesion Kinase in Myelotoxicity, Inflammatory Stress Response and Myeloid Cell Functions.

Abstract 3615

Poster Board III-551

Focal adhesion kinase (FAK) is a key signaling molecule in focal adhesion signaling and a potential integrator of integrin and growth factor receptor mediated signals. FAK has been implicated in various cellular functions such as growth, survival, migration, adhesion and cytoskeletal reorganization in fibroblasts but its role in hematopoietic stem and progenitors is unknown. To demonstrate the role of FAK in normal and stress-induced hematopoiesis, we generated FAK deficient mice by using a Cre/loxP method from here on termed FAK^flox/flox (WT) mice. FAK deletion was induced by injecting poly (I)-poly(C) to FAK ^flox/flox mice containing the Mx.Cre transgene for one month (FAK-/-). PCR and western blot analysis revealed that after one month of poly (I)-poly(C) induction, hematopoietic cells failed to express detectable levels of FAK in bone marrow (BM), spleen and thymus. To determine the effect of FAK deletion on the development of hematopoietic cells a thorough analysis of the hematopoietic compartment in FAK-/- mice was performed. Total and differential cell counts of peripheral blood revealed significantly high red blood cell distribution width {RDW (%)} and mean cell volume (MCV) in FAK-/- mice compared to WT (n=13, WT; 18.6, 47.2 vs. FAK-/-; 20.06, 48.7, *p<0.05), respectively. In addition, differential basophil counts were significantly less in FAK-/- mice compared to WT (n=13, WT; 0.68 vs. FAK-/-; 0.3 *p<0.04) but all leukocyte populations were present at normal frequencies. Furthermore, platelet counts were significantly higher in FAK-/- peripheral blood compared to WT controls (n=13, WT; 759 vs FAK-/-; 978, *p<0.01). Under basal steady-state conditions, granulopoiesis appeared to be significantly altered in FAK deficient bone marrow (BM), as frequency of granulocytes, but not of other myeloid cells was reduced (n=10, WT; 44.14% vs. FAK-/-; 34.4%, *p<0.0001). Interestingly the frequency of Lin^-, c-Kit⁺, Sca-1⁺ was also impaired in FAK deficient BM compared to controls (n=9, *p<0.05). FAK deficient BM progenitors displayed significantly lower frequency of colony-forming units compared to WT controls in response to various cytokine combinations (n=6, *p<0.01), which was associated with higher apoptosis in vitro (n=9, *p<0.006). Under conditions of stress, recovery of BM myeloid compartment and Lin⁻,c-Kit⁺, Sca-1⁺ cells following 5-Fluorouracil myeloablation was much slower in FAK-/- mice compared to WT controls (n=3, *p<0.05). Furthermore, the response of myeloid cells to acute inflammatory stress inflicted by intraperitoneal injection of thioglycollate was impaired in FAK-/- mice compared to WT mice (Macrophages: WT; 7.47 × 10⁶ vs. FAK−/−; 3.1 × 10⁶, n=8, *p <0.01. Neutrophils: WT; 5.47 × 10⁶ vs. FAK−/−; 2.1 × 10⁶, n=3, *p <0.05). These results led us to more closely examine the myeloid compartment in these mice. In vitro, FAK-/- macrophage progenitors show reduced growth in response to M-CSF stimulation (n=4, *p <0.01). In addition, deficiency of FAK in macrophages resulted in significant reduction in haptotactic migration in response to M-CSF on extracellular matrix proteins such as fibronectin, laminin and collagen (n=4, *p <0.01). Consistently, a significant reduction in the migration of FAK-/- macrophages was also observed in a wound healing assay which was associated with reduced activation of Rho GTPases including Rac. The reduction in migration of FAK-/- macrophages was associated with a significant decrease in adhesion on fibronectin, laminin and collagen. The impaired migration and adhesion of FAK-/- macrophages was observed in spite of comparable levels of F4/80 as well as integrin (α4β1 & α5β1) expression. Consistent with enhanced neutrophil apoptosis and reduced frequency under basal conditions, FAK deficient BM derived neutrophil progenitors (BMNs) show reduced growth and cycling in response to G-CSF stimulation (n=4, *p <0.01). Deletion of FAK in BMNs led to increased apoptosis upon cytokine withdrawal, which was associated with reduced activation of AKT and increased caspase-3 cleavage compared to controls. Taken together, our findings indicate that FAK plays a vital role in modulating physiological stress response to myeloablation, inflammation as well as in regulating several functions in macrophages and neutrophils.