Abstract

Mast cell activation plays a critical pathophysiologic role in asthma and allergy. A role for mast cell activation has also been described in multiple sclerosis, rheumatoid arthritis and coronary artery disease. In addition, these cells also play a prominent role in early phases of innate immunity to pathogenic bacteria. While several cytokines influence the growth, survival and maturation of mast cells; stem cell factor (SCF) and its interaction via the tyrosine kinase receptor, KIT is essential for normal mast cell development and function. However the intracellular signals that control mast cell growth, migration and maturation are not completely understood. In non-hematopoietic cells, Rho family GTPases are key regulators of many different biological processes including cell motility, growth, and differentiation. Cdc42, Rac and Rho are the most extensively studied members of this family. Although the role of Rac GTPases is becoming increasingly clear in mast cells and in hematopoietic cells in general, virtually nothing is known about the role of downstream effectors of Rho GTPases in these cells. RhoA and RhoC activate the serine/threonine protein kinases ROCKI and ROCKII. We show that both ROCKI and ROCKII are expressed in hematopoietic cells, including in bone marrow cells, splenocytes as well as in thymocytes. To determine the role of ROCK kinases in mast cells, we generated mice deficient in the expression of ROCKI. Here, we demonstrate that Rho-kinase ROCKI plays an essential role in regulating mast cell growth and maturation. We show that deficiency of ROCKI results in impaired maturation of bone marrow derived mast cells in response to IL-3 stimulation. Furthermore, the reduced maturation of ROCKI−/− mast cells is associated with reduced expression of KIT as well as reduced expression of the high affinity receptor for IgE at different stages of maturation (13% vs 7% at week1, 80% vs 52% at week2, and 93% vs 67% KIT/IgE receptor double positive cells at week3, n=3). KIT induced proliferation in response to SCF was also significantly reduced in ROCKI deficient mast cells, which was associated with reduced activation of MAPKinase Erk1 and Erk2. To test if the decreased growth in response to SCF was simply due to reduced KIT expression or due to cell intrinsic defects in ROCKI signaling, we isolated KIT positive WT and ROCKI−/− mast cells and measured growth in response to SCF and/or IL-3 stimulation by thymidine incorporation over a period of 24 and 48 hours. KIT positive ROCKI−/− mast cells showed reduced growth in response to SCF as well as in response to a combination of SCF and IL-3, suggesting a critical role of ROCKI in normal growth and maturation of mast cells. Since ROCK kinases also regulate migration in non-hematopoietic cells, we next examined the role of ROCKI in integrin (haptotactic) as well as in cytokine induced (chemotaxis) migration of mast cells. Mast cells deficient in ROCKI showed a 68% reduction in directional migration on fibronectin alone (64±7 [WT] vs 20±4, p<0.05) and a 31% reduction in the presence of SCF and fibronectin (181±16 [WT] vs 124±11 [ROCK1−/−], p<0.05), although no defects in SCF induced chemotaxis were observed. Taken together, our results identify ROCKI as a novel molecule that regulates growth, maturation and integrin-directed (haptotactic) migration of mast cells. Our results suggest that commercially available ROCK kinase inhibitors could prove to be useful small molecule inhibitors for treating diseases involving mast cells such as chronic inflammation and allergy.

Author notes

Disclosure:Research Funding: R01 HL075816 (PI: R. Kapur) 1/1/04-12/31/08, NIH $250,000, Mechanisms of adhesion and growth control in stem cells; R01 HL082981 -01 (Co-PI: R. Kapur) 9/15/05-8/31/09, NIH $250,000, Activating PTPN11 and c-kit Mutations in Myeloproliferative Disorder; R01 HL077177-01A2 (PI: R Kapur) 4/1/06-3/31/11, NIH $250,000, Novel Mechanisms of c-Kit regulation in mast cells.