PMC:3320587 / 40933-46128 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3320587","sourcedb":"PMC","sourceid":"3320587","source_url":"http://www.ncbi.nlm.nih.gov/pmc/3320587","text":"MTb induces NFAT5 gene expression via the MyD88-dependent signaling cascade\nTo begin to decipher the cellular mechanisms underlying NFAT5-dependent, MTb-induced HIV-1 replication, we next investigated the requirement for specific signaling molecules in the TLR pathway that are required for MTb stimulation of NFAT5 gene expression. We began by examining the requirement of TLR2 ligation in enhancing NFAT5 mRNA levels. When THP-1 cells were treated with the TLR2-specific agonist Pam3Cys for 16 hours, NFAT5 mRNA levels were significantly increased in Pam3Cys-stimulated cells as compared to mock-stimulated control cells (Figure 7A). As a positive control for this experiment we also infected THP-1 cells with CDC1551 (Figure 7A). Notably, MTb infection enhanced NFAT5 mRNA levels to a greater extent than Pam3Cys stimulation, suggesting that additional PRRs may be triggered during actual MTb infection, augmenting its stimulatory effect on NFAT5 gene expression (Figure 7A).\n10.1371/journal.ppat.1002620.g007 Figure 7 The MyD88-mediated signaling cascade is important for MTb induction of NFAT5 expression in human monocytes.\n(A) Engagement of TLR2 significantly enhances NFAT5 mRNA synthesis in monocytes. THP-1 cells were incubated with the TLR2-specific ligand Pam3Cys (5 µg/ml), infected with MTb strain CDC1551 (1∶1 cells∶bacilli), or left untreated. After 16 hours, NFAT5 mRNA levels were measured. NFAT5 mRNA expression was significantly increased when the cells were incubated with Pam3Cys (*, p\u003c0.05) or infected with MTb (**, p\u003c0.01). (B) MyD88 is important for MTb induction of NFAT5 gene expression in human monocytes. THP-1 cells that constitutively express lentivirally-delivered shRNA targeting MyD88 or control shRNA targeting GFP were constructed as described in Materials and Methods. Cells were infected with MTb strain CDC1551 (1∶1 cells∶bacilli) or left uninfected for 16 hours at 37°C, and RNA was analyzed. NFAT5 mRNA levels were significantly inhibited in the cells transduced with MyD88-specific shRNA (black bars) compared with the cells transduced with control shRNA (open bars) in both, the absence or presence of MTb infection. TNF and CD86 mRNAs, the expression of which are MyD88-dependent and -independent, respectively, during MTb infection, were measured as controls. (C, D) IRAK1 and TRAF6 are required for MTb induction of NFAT5 gene expression in human monocytes. THP-1 cells that constitutively express lentivirally-delivered IRAK1- or TRAF6-specific shRNA were constructed as described in Materials and Methods. THP-1 cells expressing control shRNA were used as described in 7B. Cells were infected with MTb strain CDC1551 or left uninfected for 16 hours at 37°C, and RNA was analyzed. NFAT5 expression was significantly inhibited in the MTb-infected cells expressing either IRAK1 (C) or TRAF6 (D) shRNA (black bars) compared to cells expressing control shRNA (open bars). TNF and CD86 mRNAs, both of which require IRAK1 and TRAF6 to be induced by PRR activation, were measured as controls. (*, p\u003c0.05; **, p\u003c0.01). The adaptor molecule MyD88 transmits signals upon TLR2 ligation (reviewed in [53]). To confirm that this protein and, therefore, that this TLR-dependent pathway is important for MTb induction of NFAT5 gene expression, we constructed a THP-1 cell line that constitutively expresses a lentivirally-delivered short hairpin (sh)RNA targeting MyD88. Real-time PCR analysis demonstrated that MyD88 mRNA levels in these cells were constitutively and significantly reduced in comparison to control THP-1 cells transduced with a lentivirus expressing control GFP shRNA (data not shown). These MyD88 knockdown THP-1 cells were then infected with MTb (CDC1551) and NFAT5 mRNA levels were examined. Since TNF is a MyD88-dependent gene [21], [54], [55], as a positive control we measured TNF mRNA levels. For a negative control, we measured mRNA levels of the MyD88-independent gene CD86 [55], [56]. As shown in Figure 7B, knockdown of MyD88 specifically inhibited MTb-induced NFAT5 and TNF mRNA levels and had no impact on CD86 mRNA levels. Thus, MTb regulation of NFAT5 is dependent upon MyD88.\nTo futher dissect the pathway mediating MTb upregulation of NFAT5 transcription, we also constructed THP-1 cells transduced with lentivirally-delivered shRNAs targeting IRAK1 or TRAF6, two signaling molecules downstream of MyD88. As shown in Figures 7C and 7D, knockdown of both IRAK1 and TRAF6 significantly inhibited MTb induced NFAT5 mRNA levels. As expected, MTb-induced TNF gene expression was also significantly reduced (Figures 7C–7D), consistent with a role for IRAK1 and TRAF6 in TNF gene expression secondary to MTb infection as previously reported. Although CD86 gene expression did not depend on MyD88 (Figure 7B), abrogation of IRAK1 and TRAF6 gene expression significantly reduced CD86 mRNA levels, indicating that CD86 expression is IRAK1- and TRAF6-dependent, but MyD88-independent in MTb-infected monocytes (Figures 7C–7D). Taken together, these results clearly demonstrate that MTb upregulation of NFAT5 gene expression in human monocytic cells requires activation of MyD88 and the MyD88-associated adaptor molecules IRAK1 and TRAF6.","divisions":[{"label":"title","span":{"begin":0,"end":75}},{"label":"p","span":{"begin":76,"end":978}},{"label":"figure 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