RAW cells were treated with 20 ng mL−1 of murine recombinant TNF-α and RCAN-1 levels were assessed 1.5, 4, and 8 h later. As shown in Fig. 6, RCAN1-4 was increased modestly, but these increases did not reach statistical significance and are therefore unlikely to contribute much, if at all, to the inductions observed MK-2206 nmr in Figs 1–5. The above data, as well as previous studies implicating RCAN1 in T-lymphocyte function (Rothermel et al., 2000; Narayan et al., 2005), suggest that RCAN1 plays an important overall role in immune function. In order to better determine the functional significance of RCAN1 in the macrophage and immune

response, we carried out in vivo infection analyses on RCAN1 KOs and WT controls. The animals used for these studies have been described previously (Ryeom et al.,

2003), and have a portion of these C-terminus coding region removed, leading to the total loss of expression of both major RCAN1 isoforms. KO and WT mice were nasally infected with 10 000 CFU of the gram-negative bacteria F. tularensis. After 7 days, the mice were sacrificed and the bacterial burden and proinflammatory cytokine levels were assessed in the lung (the main target of intranasally administered F. tularensis) and spleen. As shown in Fig. 7, no statistically significant change in bacterial burden was observed in the 7-day KO lung as compared with the WT when using a using a two-tailed Mann–Whitney test (note: significance was observed using a one-tailed Mann–Whitney test, but because the two-tailed test is a more stringent comparison, we have chosen to use these results). Spleen

bacterial MAPK inhibitor burden was also assessed, with much lower bacterial numbers observed and no differences found between KO and WT (data not shown). NFAT proteins are major transcription factors critical for the immune response, especially in the induction of cytokine genes such as IL-2, check details IL-4, IL-6, IFN-γ, and TNF-α (Rao et al., 1997; Crabtree, 1999; Rusnak & Mertz, 2000; Kiani et al., 2001; Peng et al., 2001; Crabtree & Olson, 2002; Ryeom et al., 2003). Because NFATs are tightly regulated by calcineurin and RCAN1 regulates calcineurin, it is reasonable to assume that RCAN1 may regulate calcineurin-dependent cytokine production. To assess this in vivo, KO and WT mice were nasally infected with 10 000 CFU of F. tularensis, and then evaluated for inflammatory cytokine levels in the lung and spleen 7 days after infection. As expected, a strong elevation in all of the proinflammatory cytokines examined, including MCP-1, IL-6, IFN-γ, and TNF-α, was observed in F. tularensis-infected vs. noninfected mice (N=6–7 for infected; N=2 for noninfected controls). Importantly, a statistically significant increase in all the tested F. tularensis-infected KO mice cytokine levels was observed in the lung as compared with F. tularensis-infected WT mice cytokines (Fig. 8).