Aberrant splicing of the hRasGRP4 transcript and decreased levels of this signaling protein in the peripheral blood mononuclear cells in a subset of patients with rheumatoid arthritis

Introduction: An unidentified population of peripheral blood mononuclear cells (PBMCs) express Ras guanine nucleotide releasing protein 4 (RasGRP4). The aim of our study was to identify the cells in human blood that express hRasGRP4, and then to determine if hRasGRP4 was altered in any patient with rheumatoid arthritis (RA). Methods: Monocytes and T cells were purified from PBMCs of normal individuals, and were evaluated for their expression of RasGRP4 mRNA/protein. The levels of RasGRP4 transcripts were evaluated in the PBMCs from healthy volunteers and RA patients by real-time quantitative PCR. The nucleotide sequences of RasGRP4 cDNAs were also determined. RasGRP4 protein expression in PBMCs/monocytes was evaluated. Recombinant hRasGRP4 was expressed in mammalian cells. Results: Circulating CD14(+) cells in normal individuals were found to express hRasGRP4. The levels of the hRasGRP4 transcript were significantly higher in the PBMCs of our RA patients relative to healthy individuals. Sequence analysis of hRasGRP4 cDNAs from these PBMCs revealed 10 novel splice variants. Aberrantly spliced hRasGRP4 transcripts were more frequent in the RA patients than in normal individuals. The presence of one of these abnormal splice variants was linked to RA. The levels of hRasGRP4 protein in PBMCs tended to be lower. As expected, the defective transcripts led to altered and/or nonfunctional protein in terms of P44/42 mitogen-activated protein (MAP) kinase activation. Conclusions: The identification of defective isoforms of hRasGRP4 transcripts in the PBMCs of RA patients raises the possibility that dysregulated expression of hRasGRP4 in developing monocytes plays a pathogenic role in a subset of RA patients.