Limits...
Mincle-mediated translational regulation is required for strong nitric oxide production and inflammation resolution.

Lee WB, Kang JS, Choi WY, Zhang Q, Kim CH, Choi UY, Kim-Ha J, Kim YJ - Nat Commun (2016)

Bottom Line: Here we show that Mincle, the inducible receptor for mycobacterial cord factor, is the key switch for the transition of macrophages from cytokine expression to high nitric oxide production.In addition to its stimulatory role on TLR-mediated transcription, Mincle enhanced the translation of key genes required for nitric oxide synthesis through p38 and eIF5A hypusination, leading to granuloma resolution.Thus, Mincle has dual functions in the promotion and subsequent resolution of inflammation during anti-mycobacterial defence using both transcriptional and translational controls.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Republic of Korea.

ABSTRACT
In response to persistent mycobacteria infection, the host induces a granuloma, which often fails to eradicate bacteria and results in tissue damage. Diverse host receptors are required to control the formation and resolution of granuloma, but little is known concerning their regulatory interactions. Here we show that Mincle, the inducible receptor for mycobacterial cord factor, is the key switch for the transition of macrophages from cytokine expression to high nitric oxide production. In addition to its stimulatory role on TLR-mediated transcription, Mincle enhanced the translation of key genes required for nitric oxide synthesis through p38 and eIF5A hypusination, leading to granuloma resolution. Thus, Mincle has dual functions in the promotion and subsequent resolution of inflammation during anti-mycobacterial defence using both transcriptional and translational controls.

No MeSH data available.


Related in: MedlinePlus

eIF5A hypusination is required for Mincle-mediated iNOS translation.(a,b) WT BMDMs were stimulated with TDM, Pam3 or co-stimulated with Pam3 and TDM in the presence of GC7 for 12 h. (a) Nitric oxide release in culture supernatants. (b) qRT-PCR (left) and immunoblot (right) analysis of iNOS mRNA or protein expression. (c) Fluorographic analysis of hypusinated eIF5A from WT BMDMs treated with Pam3, TDM or co-treated with Pam3 and TDM in the presence of control (con) or GC7. (d) Fluorographic analysis of hypusinated eIF5A from WT and Mincle−/− (KO) BMDMs treated with Pam3, TDM or co-treated with Pam3 and TDM. (e) RIP analysis of iNOS and Actb mRNAs from NIH-3T3 cells co-transfected with expression vector for Flag alone (Flag-Mock) or Flag-tagged eFI5A WT or K50A mutant, plus an iNOS mRNA expression vector. Agarose gel electrophoresis (top) and qRT-PCR analysis (bottom) for the indicated genes. Data are expressed as per cent recovery relative to input RNA. *P<0.05, ***P<0.001 (Student's t-test). Data are representative of three (a,b,e) or two (c,d) independent experiments (a,b,e: mean and s.d.).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4837483&req=5

f5: eIF5A hypusination is required for Mincle-mediated iNOS translation.(a,b) WT BMDMs were stimulated with TDM, Pam3 or co-stimulated with Pam3 and TDM in the presence of GC7 for 12 h. (a) Nitric oxide release in culture supernatants. (b) qRT-PCR (left) and immunoblot (right) analysis of iNOS mRNA or protein expression. (c) Fluorographic analysis of hypusinated eIF5A from WT BMDMs treated with Pam3, TDM or co-treated with Pam3 and TDM in the presence of control (con) or GC7. (d) Fluorographic analysis of hypusinated eIF5A from WT and Mincle−/− (KO) BMDMs treated with Pam3, TDM or co-treated with Pam3 and TDM. (e) RIP analysis of iNOS and Actb mRNAs from NIH-3T3 cells co-transfected with expression vector for Flag alone (Flag-Mock) or Flag-tagged eFI5A WT or K50A mutant, plus an iNOS mRNA expression vector. Agarose gel electrophoresis (top) and qRT-PCR analysis (bottom) for the indicated genes. Data are expressed as per cent recovery relative to input RNA. *P<0.05, ***P<0.001 (Student's t-test). Data are representative of three (a,b,e) or two (c,d) independent experiments (a,b,e: mean and s.d.).

Mentions: Previously, iNOS translation was shown to be regulated by p38-dependent eIF5A hypusination in islet β-cells2627. This led us to ask whether the apparent translational effect of the Mincle-p38 axis might be related to eIF5A function. The hypusine modification of eIF5A is processed sequentially by two enzymes, deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH)28. First, we examined the effect of DHS inhibition by GC7 (N′-guanyl-1,7-diaminoheptane, a DHS inhibitor) on Mincle-dependent NO synthesis. GC7 completely inhibited NO release (Fig. 5a and Supplementary Fig. 14a) and iNOS protein expression, but not mRNA expression, when induced by co-stimulation of TDM and Pam3 (Fig. 5b) or co-stimulation of TDM and LPS (Supplementary Fig. 14b). These results indicate the involvement of eIF5A hypusination in iNOS expression in activated macrophages. To confirm Mincle-dependent eIF5A hypusination, we added 3H-spermidine to the bone marrow-derived macrophage (BMDM) culture and examined eIF5A hypusination (eIF5Ahyp) under various stimulation conditions. Basal levels of eIF5Ahyp in BMDMs were highly stimulated by TDM in a Mincle-dependent manner, but completely inhibited by the addition of GC7 or p38 inhibitor (Fig. 5c,d and Supplementary Fig. 14c,d). We obtained a similar result when eIF5A hypusination was inhibited by ciclopirox (CPX), a DOHH inhibitor2930. CPX inhibited NO release and iNOS protein expression from the macrophages stimulated with Pam3 and TDM, without causing a defect in iNOS mRNA expression (Supplementary Fig. 15a,b). Therefore, these results demonstrate that Mincle-p38-mediated eIF5A hypusination is required for iNOS translation in activated macrophages.


Mincle-mediated translational regulation is required for strong nitric oxide production and inflammation resolution.

Lee WB, Kang JS, Choi WY, Zhang Q, Kim CH, Choi UY, Kim-Ha J, Kim YJ - Nat Commun (2016)

eIF5A hypusination is required for Mincle-mediated iNOS translation.(a,b) WT BMDMs were stimulated with TDM, Pam3 or co-stimulated with Pam3 and TDM in the presence of GC7 for 12 h. (a) Nitric oxide release in culture supernatants. (b) qRT-PCR (left) and immunoblot (right) analysis of iNOS mRNA or protein expression. (c) Fluorographic analysis of hypusinated eIF5A from WT BMDMs treated with Pam3, TDM or co-treated with Pam3 and TDM in the presence of control (con) or GC7. (d) Fluorographic analysis of hypusinated eIF5A from WT and Mincle−/− (KO) BMDMs treated with Pam3, TDM or co-treated with Pam3 and TDM. (e) RIP analysis of iNOS and Actb mRNAs from NIH-3T3 cells co-transfected with expression vector for Flag alone (Flag-Mock) or Flag-tagged eFI5A WT or K50A mutant, plus an iNOS mRNA expression vector. Agarose gel electrophoresis (top) and qRT-PCR analysis (bottom) for the indicated genes. Data are expressed as per cent recovery relative to input RNA. *P<0.05, ***P<0.001 (Student's t-test). Data are representative of three (a,b,e) or two (c,d) independent experiments (a,b,e: mean and s.d.).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837483&req=5

f5: eIF5A hypusination is required for Mincle-mediated iNOS translation.(a,b) WT BMDMs were stimulated with TDM, Pam3 or co-stimulated with Pam3 and TDM in the presence of GC7 for 12 h. (a) Nitric oxide release in culture supernatants. (b) qRT-PCR (left) and immunoblot (right) analysis of iNOS mRNA or protein expression. (c) Fluorographic analysis of hypusinated eIF5A from WT BMDMs treated with Pam3, TDM or co-treated with Pam3 and TDM in the presence of control (con) or GC7. (d) Fluorographic analysis of hypusinated eIF5A from WT and Mincle−/− (KO) BMDMs treated with Pam3, TDM or co-treated with Pam3 and TDM. (e) RIP analysis of iNOS and Actb mRNAs from NIH-3T3 cells co-transfected with expression vector for Flag alone (Flag-Mock) or Flag-tagged eFI5A WT or K50A mutant, plus an iNOS mRNA expression vector. Agarose gel electrophoresis (top) and qRT-PCR analysis (bottom) for the indicated genes. Data are expressed as per cent recovery relative to input RNA. *P<0.05, ***P<0.001 (Student's t-test). Data are representative of three (a,b,e) or two (c,d) independent experiments (a,b,e: mean and s.d.).
Mentions: Previously, iNOS translation was shown to be regulated by p38-dependent eIF5A hypusination in islet β-cells2627. This led us to ask whether the apparent translational effect of the Mincle-p38 axis might be related to eIF5A function. The hypusine modification of eIF5A is processed sequentially by two enzymes, deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH)28. First, we examined the effect of DHS inhibition by GC7 (N′-guanyl-1,7-diaminoheptane, a DHS inhibitor) on Mincle-dependent NO synthesis. GC7 completely inhibited NO release (Fig. 5a and Supplementary Fig. 14a) and iNOS protein expression, but not mRNA expression, when induced by co-stimulation of TDM and Pam3 (Fig. 5b) or co-stimulation of TDM and LPS (Supplementary Fig. 14b). These results indicate the involvement of eIF5A hypusination in iNOS expression in activated macrophages. To confirm Mincle-dependent eIF5A hypusination, we added 3H-spermidine to the bone marrow-derived macrophage (BMDM) culture and examined eIF5A hypusination (eIF5Ahyp) under various stimulation conditions. Basal levels of eIF5Ahyp in BMDMs were highly stimulated by TDM in a Mincle-dependent manner, but completely inhibited by the addition of GC7 or p38 inhibitor (Fig. 5c,d and Supplementary Fig. 14c,d). We obtained a similar result when eIF5A hypusination was inhibited by ciclopirox (CPX), a DOHH inhibitor2930. CPX inhibited NO release and iNOS protein expression from the macrophages stimulated with Pam3 and TDM, without causing a defect in iNOS mRNA expression (Supplementary Fig. 15a,b). Therefore, these results demonstrate that Mincle-p38-mediated eIF5A hypusination is required for iNOS translation in activated macrophages.

Bottom Line: Here we show that Mincle, the inducible receptor for mycobacterial cord factor, is the key switch for the transition of macrophages from cytokine expression to high nitric oxide production.In addition to its stimulatory role on TLR-mediated transcription, Mincle enhanced the translation of key genes required for nitric oxide synthesis through p38 and eIF5A hypusination, leading to granuloma resolution.Thus, Mincle has dual functions in the promotion and subsequent resolution of inflammation during anti-mycobacterial defence using both transcriptional and translational controls.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Republic of Korea.

ABSTRACT
In response to persistent mycobacteria infection, the host induces a granuloma, which often fails to eradicate bacteria and results in tissue damage. Diverse host receptors are required to control the formation and resolution of granuloma, but little is known concerning their regulatory interactions. Here we show that Mincle, the inducible receptor for mycobacterial cord factor, is the key switch for the transition of macrophages from cytokine expression to high nitric oxide production. In addition to its stimulatory role on TLR-mediated transcription, Mincle enhanced the translation of key genes required for nitric oxide synthesis through p38 and eIF5A hypusination, leading to granuloma resolution. Thus, Mincle has dual functions in the promotion and subsequent resolution of inflammation during anti-mycobacterial defence using both transcriptional and translational controls.

No MeSH data available.


Related in: MedlinePlus