Microarray studies on genes stimulated by RANKL in bone marrow macrophages in a cyclooxygenase-2-dependent manner. Mus musculus

Overall goal was to look for the differentially expressed genes between receptor activator of nuclear factor kappa-B ligand (RANKL)-stimulated wild type (WT) bone marrow macrophages (BMMs) and the BMMs in which the expression or the enzyme activity of cyclooxygenase-2 (Cox2), the major enzyme for prostaglandin E2 (PGE2) synthesis, is absent. For this purpose, two separate microarrays (experiment 1 and 2) were done. In both the experiments, Serum Amyloid A3 (Saa3) was one of the most highly differentially expressed gene. Overall design: For experiment 1, BMMs were isolated from the bone marrow of Cox2 knockout (KO) and WT mice. Out of the three sample groups, two were treated with 30 ng/ml each of macrophage-colony stimulating factor (M-CSF) and RANKL, while the third group was treated with M-CSF only, for 1 day. Two of the sample groups had n=3 biological replicates (i.e. samples), while the third one had n=4 biological replicates. The differential gene expression comparisons among the sample groups were done as (A) WT+MCSF+RANKL_d1 versus Cox2KO+MCSF+RANKL_d1 and (B) WT+MCSF+RANKL_d1 versus WT+MCSF_d1. For experiment 2 also, BMMs were obtained from Cox2 KO and WT mice. All the four sample groups were treated with M-CSF and RANKL for 3 days. All the four sample groups had n=4 biological replicates. One of the WT sample group was also treated with an inhibitor of Cox2 activity,NS398 (0.1 µm) and one of the KO sample group was also treated with the product of Cox2 enzyme, PGE2 (1 µm). The differential gene expression comparisons among the sample groups were done as (A) WT+MCSF+RANKL_d3 versus Cox2KO+MCSF+RANKL_d3; (B) WT+MCSF+RANKL_d3 versus WT+MCSF+RANKL+NS398_d3 and (C) Cox2KO+MCSF+RANKL+PGE2_d3 versus Cox2KO+MCSF+RANKL_d3.