Regulation of sterile α- and armadillo motif (SARM) containing protein expression in Pam2CSK4- and Pam3CSK4-activated mouse macrophage cell line (RAW264.7) requires TLR9
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We aimed to investigate the involvement of surface TLRs and endosomal TLRs in the regulation of SARM expression by TLR2 ligands (Pam2CSK4 and Pam3CSK4).
Materials and methods
Mouse macrophage cell line (RAW264.7) was treated with either Pam2CSK4 or Pam3CSK4 (TLR2 ligands) at a concentration of 100 ng/ml. At indicated time points, the treated cells were lysed. The gene and protein expression of SARM were determined by RT-PCR and immunoblotting, respectively. For silencing of TLR9 function, the cells were transfected with TLR9 siRNAs before stimulation by these two TLR2 ligands
The SARM expression was upregulated at both transcriptional and translational levels in time-dependent manner during activation of Pam2CSK4 and Pam3CSK4 in mouse macrophages. Blocking of ligand internalization by cytochalasin D showed interference effect with SARM expression. Moreover, our results also demonstrated that endosomal acidification and TLR9 were required for SARM expression suggesting the essential role of endosomal compartment acidification and TLR9 in regulating SARM expression.
Our findings suggested the collaboration of TLR2–TLR9 at least in the regulation of SARM expression. However, the underlying mechanism that participated in these two TLRs cooperation is underinvestigated.
KeywordsPam2CSK4 Pam3CSK4 RAW264.7 SARM TLR9
Matsayapan Pudla acknowledges support from the Talent Management Program of Mahidol University. Panthong Kulsantiwong was supported by the Udon Thani Rajabhat University scholarship for development of educational personnel. This research was supported by a research grant from Thailand Research Fund (Grant number BRG5980004 and MRG5980057) and Mahidol University.
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