SOCS2 and DC Maturation Figure 3. SOCS2 siRNA inhibits TLR4 signaling of DC via inhibition of both MyD88-dependent (A, B) and -independent pathway (C). Enriched monocytes were cultured in the presence of GM-CSF and IL-4 for 6 days. iDCs were transfected with SOCS2 siRNA or control siRNA and incubated for 24 hours. (A). SOCS2 knock-down effect on phosphorylation of SAKP/JNK, P38, ERK and IkBa. Transfected cells were stimulated with LPS at the indicated time points. The level of phosphorylation of SAKP/JNK, P38, ERK and IkBa was determined by Western Blotting of whole-cell lysates using antibodies specific for the phosphorylated, activated forms of the proteins. The levels of total ERK was determined as loading controls. (B). SOCS2 knock-down effect on nuclear translocation of NF-kB. Transfected cells were stimulated with LPS at the indicated time points. Nuclear protein was extracted for Western Blotting measurement. The level of P65 NF-kB and Lamin A (as loading control) was detected. (C). SOCS2 knock- down effect on phosphorylation of IRF3. Transfected cells were stimulated with LPS at the indicated time points. The level of phosphorylated and total IRF3 was determined by Western Blotting of whole-cell lysates using antibodies specific for the forms of the proteins, respectively. The IRF3 bands were quantified, and the ratio of phosphorylated to total IRF3 were shown. doi:10.1371/journal.pone.0007178.g003 39%, for the HLA-DR marker, as compared to the levels in control cells (data no shown). Discussion Our data confirm and further extend the knowledge of SOCS involvement in DC maturation [25,26] and indicate that SOCS2 is an important SOCS family member involved in the maturation process. This was demonstrated by a clear increase of both SOCS2 mRNA and protein during DC maturation and the finding that SOCS2 silencing leads to an impaired maturation of DCs. LPS stimulation of iDCs where SOCS2 had been silenced failed to express CD83, a typical marker of DC maturation. In addition, such cells showed a lower expression of the co-stimulatory molecules CD40, CD86 and HLA-DR. We showed that SOCS2 silencing decreased both MyD88-dependent and -independent signaling in the TLR4 signaling pathway. This finding was corroborated by a corresponding decrease of LPS inducible genes encoding the effector cytokines TNF-a, IL-6, IL-1b and CCL-4. A SOCS2 dependency was also demonstrated for gene expression of INF-b, CXCL-9 and CXCL-10 i.e. genes related to the MyD88- independent signaling pathway. LPS stimulation under conditions of SOCS2 knock-down resulted in a decreased expression of all of these genes. Our results were substantiated at the protein level by Western Blots of protein extracts. Down-regulation of SOCS2 led to a decrease in NF-kB nuclear translocation in response to LPS stimulation [35] and also the phosphorylation of IRF3 [36] and the activation of MAP kinases [37] was reduced in LPS stimulated cells where SOCS2 had been silenced. Thus, SOCS2 regulates TLR signaling via the MyD88-dependent and -independent signaling pathway. SOCS2, as other members of this family is thought to function as a subunit of an E3 ubiquitin ligase complex [38]. The SOCS box, placed at the C-terminus serves to interact with Elongins C and B, cullin5 and Rbx2 forming the catalytic core of the complex [39,40]. The SH2 domain of SOCS2 serves to interact with phosphotyrosine residues the targeted substrate directing the specificity of the ubiquitin ligase reaction [39]. For other members of the family it has been demonstrated that SOCS targets are polyubiquitinated and subsequently degraded by the proteasome [40,41]. Consequently, the most common effect of SOCS proteins is to reduce signaling. In dendritic cells, this is the case with SOCS3 and SOCS1, which have been shown to inhibit TLR signaling targeting TRAF6 in the case of SOCS3 [42] and MAL in the case of SOCS1 [43]. In contrast, SOCS2 elimination reduce LPS induced signals suggesting that negative regulators of TLR4 signaling are themselves targeted for degradation by SOCS2. Such negative regulators should themselves be tyrosine phosphorylated in order to interact with SOCS2. Interestingly, SOCS2 can control levels of other SOCS proteins such as SOCS3 and SOCS1 [44], and such interactions may explain why LPS signaling is diminished in the absence of SOCS2. Other SOCS proteins are not the only possible targets and proteins such as DNAX- activating protein 12 [45] and the Tyro3/Axl/Mer family of receptor kinases [46] have been shown both to be tyrosine phosphorylated as well as to act as negative regulators of TLR signaling. Interestingly, both of these pathways target MAL an early signaling intermediaries in the TLR signaling cascade. Because both the MAL/MyD88 and the TRAM-TRIF pathways are both affected by SOCS2 knock-down, it seems plausible that SOCS2 targets act early on the signaling pathway, in line with possible actions of SOCS1 or the Tyro3/Axl/Mer receptor family. In a more complex scenario, it is possible that SOCS2 has separate targets for MyD88 dependent and independent pathways. In this case several negative regulators such as Src homology 1-containing tyrosine phosphatase 1 which targets IRAK1 [47], Src homology 2-containing tyrosine phosphatase which targets TANK binding kinase 1 [48] and mitogen-activated protein kinase phosphatase-1 PLoS ONE | www.plosone.org 4 September 2009 | Volume 4 | Issue 9 | e7178