1639 - Inflammatory Arthritis in Mice Is Dependent on the Mast Cell-Restricted Signaling Protein Ras Guanine Nucleotide Exchange Protein 4 (RasGRP4)

Monday, November 7, 2011: 3:30 PM
W470b (McCormick Place West)
Richard Stevens1, Roberto Adachi2, Peter A. Nigrovic3, Matthew J. Hamilton3 and Steven Krilis4, 1Brigham and Women's Hospital, Harvard Medical School, Boston, MA, 2University of Texas M. D. Anderson Cancer Center, Houston, TX, 3Brigham and Women's Hospital, Boston, MA, 4St George Hospital, Kogarah NSW, Australia
Presentation Number: 1639

Background/Purpose: Ras guanine nucleotide exchange protein-4 (RasGRP4) is a calcium-regulated, guanine nucleotide exchange factor and diacylglycerol/phorbol ester receptor that is expressed in human, rat, and mouse mast cells (MCs), and their circulating progenitors. Although the other three members of this family of intracellular signaling proteins participate in the development of T cells, B cells, and platelets, the importance of RasGRP4 in the development and function of MCs has not been elucidated. We created a transgenic RasGRP4-null C57BL/6 mouse to deduce the possible role(s) of this evolutionarily conserved signaling protein in MC-dependent inflammatory diseases.

Method: A targeting construct was prepared in which the Cre gene was placed at the translation-initiation site of the 17-exon, 19-kb mRasGRP4 gene. To ensure that no functional RasGRP4 protein could be expressed in the resulting transgenic mice, the first 8 exons of the gene were deleted, including those nucleotides that encode the protein’s guanine nucleotide exchange domain. Using homologous recombination methods and C57BL/6 mouse embryonic stem cells, a RasGRP4-null mouse was obtained which was then backcrossed 10 times with wild-type (WT) C57BL/6 mice to eliminate possible nonspecific mutations created during the homologous recombination step. The resulting transgenic mouse line was characterized and then subjected to inflammatory arthritis induced by K/BxN mouse serum.

Result: RasGRP4-null C57BL/6 mice were healthy and contained normal numbers of granulated MCs in their tissues that histochemically resembled those in WT C57BL/6 mice. Thus, RasGRP4 is not essential for the Kit-dependent development of tissue MCs. Immature MCs (mBMMCs) could be generated from RasGRP4-null mice by culturing their bone marrow cells for 4-6 weeks in IL-3 enriched conditioned media in the presence or absence of Kit ligand. Nevertheless, microarray analysis of IL-3-developed mBMMCs revealed altered expression of numerous transcripts compared with WT mBMMCs. Exposure of IL-3-developed RasGRP4-null mBMMCs to calcium ionophore resulted in the release of the cell’s preformed mediator β-hexosaminidase and increased expression of tumor necrosis factor-α and other cytokines and chemokines, as occurs in calcium ionophore-treated WT mBMMCs. Although the latter data revealed that signaling pathways downstream of RasGRP4 are intact and functional in MCs, arthritis was reduced >90% in RasGRP4-null mice that had received K/BxN mouse serum relative to similarly-treated WT mice. In support of these inflammatory arthritis data, MC-dependent colitis also was reduced in RasGRP4-null mice.

Conclusion: The exciting finding that RasGRP4 is essential in inflammatory arthritis in mice suggests that the pharmaceutical inactivation of this signaling protein might be of efficacy in the treatment of patients with inflammatory arthritis and other MC-dependent disorders.

Keywords: inflammation, inflammatory arthritis, mast cells and signal transduction

Disclosure: R. Stevens, None; R. Adachi, None; P. A. Nigrovic, None; M. J. Hamilton, None; S. Krilis, None.