Youn Sook Lee

Smad6-specific recruitment of Smurf E3 ligases mediates TGF-β1-induced degradation of MyD88 in TLR4 signalling

Transforming growth factor-β (TGF-β) is a potent anti-inflammatory cytokine that regulates interleukin-1 receptor and Toll-like receptor (TLR) signalling. Here we show a novel mechanism where TGF-β1-induced K48-linked polyubiquitination and degradation of the adaptor MyD88 protein is dependent on the Smad6 protein, but not Smad7, and mediated by recruitment of the Smad ubiquitin regulator factor proteins, Smurf1 and Smurf2, which have E3-ubiquitin ligase activity. Smurf1 interaction with MyD88 appears to be mediated by Smad6, and Smurf2 interaction by Smurf1. Knockdown of endogenous Smurf1 or Smurf2 by RNA interference significantly suppresses the anti-inflammatory effects of TGF-β1 by preventing lipopolysaccharide-induced NF-κB nuclear translocation, resulting in de-suppression of pro-inflammatory gene expression. Similar effects are observed on the lipoteichoic-acid-induced TLR2 pathway, which is also MyD88-dependent, but not the MyD88-independent TLR3 pathway. Thus, our results suggest that MyD88 degradation driven by the Smad6-Smurf pathway is a novel mechanism for TGF-β1-mediated negative regulation of MyD88-dependent pro-inflammatory signalling.

Smad6 inhibits non-canonical TGF-β1 signalling by recruiting the deubiquitinase A20 to TRAF6

Transforming growth factor (TGF)-β, a pivotal cytokine involved in a variety of cellular functions, transmits signals through Smad-dependent canonical and Smad-independent noncanonical pathways. In contrast to the canonical TGF-β pathway, it is unknown how noncanonical TGF-β pathways are negatively regulated. Here we demonstrate that the inhibitory Smad Smad6, but not Smad7, negatively regulates TGF-β1-induced activation of the TRAF6-TAK1-p38 MAPK/JNK pathway, a noncanonical TGF-β pathway. TGF-β1-induced Smad6 abolishes K63-linked polyubiquitination of TRAF6 by recruiting the A20 deubiquitinating enzyme in AML-12 mouse liver cells and primary hepatocytes. In addition, the knockdown of Smad6 or A20 in an animal model or cell culture system maintains TAK1 and p38 MAPK/JNK phosphorylation and increases apoptosis, emphasizing the crucial role of the Smad6-A20 axis in negative regulation of the TGF-β1-TRAF6-TAK1-p38 MAPK/JNK pathway. Therefore, our findings provide insight into the molecular mechanisms underlying negative regulation of noncanonical TGF-β pathways.

Smad7 and Smad6 bind to discrete regions of Pellino-1 via their MH2 domains to mediate TGF-β1-induced negative regulation of IL-1R/TLR signaling

Transforming growth factor-beta1 (TGF-beta1) performs diverse cellular functions, including anti-inflammatory activity. The inhibitory Smad (I-Smad) Smad6 was previously shown to play an important role in TGF-beta1-induced negative regulation of Interleukin-1/Toll-like receptor (IL-1R/TLR) signaling through binding to Pellino-1, an adaptor protein of interleukin-1 receptor associated kinase 1(IRAK1). However, it is unknown whether Smad7, the other inhibitory Smad, also has a role in regulating IL-1R/TLR signaling. Here, we demonstrate that endogeneous Smad7 and Smad6 simultaneously bind to discrete regions of Pellino-1 upon TGF-beta1 treatment, via distinct regions of the Smad MH2 domains. In addition, the Smad7-Pellino-1 interaction abrogated NF-kappaB activity by blocking formation of the IRAK1-mediated IL-1R/TLR signaling complex, subsequently causing reduced expression of pro-inflammatory genes. Double knock-down of endogenous Smad6 and Smad7 genes by RNA interference further reduced the anti-inflammatory activity of TGF-beta1 than when compared with single knock-down of Smad7. These results provide evidence that the I-Smads, Smad6 and Smad7, act as critical mediators for effective TGF-beta1-mediated suppression of IL-1R/TLR signaling, by simultaneous binding to discrete regions of Pellino-1.

Inhibition of lethal inflammatory responses through the targeting of membrane‐associated Toll‐like receptor 4 signaling complexes with a Smad6‐derived peptide

We have previously reported that Smad6, one of the inhibitory Smads of transforming growth factor‐β (TGF‐β)/bone morphogenetic protein (BMP) signaling, inhibits Toll‐like receptor (TLR) 4 signaling by disrupting the Pellino‐1‐mediated TLR4 signaling complex. Here, we developed Smaducin‐6, a novel membrane‐tethered palmitic acid‐conjugated Smad6‐derived peptide composed of amino acids 422–441 of Smad6. Smaducin‐6 interacted with Pellino‐1, located in the inner membrane, thereby disrupting the formation of IRAK1‐, RIP1‐, IKKε‐mediated TLR4 signaling complexes. Systemic administration of Smaducin‐6 showed a significant therapeutic effect on mouse TLR4‐mediated inflammatory disease models, cecal‐ligation–puncture (CLP)‐induced sepsis, and lipopolysaccharide‐induced endotoxemia, by inhibiting pro‐inflammatory cytokine production and apoptosis while enhancing neutrophil migration and bacterial clearance. Our findings provide clues to develop new peptide‐based drugs to target Pellino‐1 protein in TLR4 signaling pathway for the treatment of sepsis.

Pellino-1, an Adaptor Protein of Interleukin-1 Receptor/Toll-like Receptor Signaling, Is Sumoylated by Ubc9

Covalent modifications of the Pellino-1 protein are essential for transmitting innate immune response signals downstream, as the phosphorylation and polyubiquitination of Pellino-1 mediated by the IRAK proteins appear to have roles in regulating Pellino-1 function. In this study, we demonstrate that the Pellino-1 protein is post-translationally modified by small-ubiquitin-related modifier-1 (SUMO-1). Sumoylation assays with Pellino-1 and SUMO-1 expression plasmids reveal that the Pellino-1 protein is sumoylated in vitro and in vivo. Treatment of SUMO-1 specific protease 1 (SENP1) inhibited the sumoylation of the Pellino-1 protein and a GST pull-down assay as well as a yeast two hybrid assay showed that Pellino-1 binds to the SUMO-conjugating enzyme, Ubc9. Furthermore, we identified the five lysine residues of the Pellino-1 protein where SUMO-1 covalently attaches. Some of the sumoylated sites overlap with previously identified ubiquitination sites, suggesting competition between sumoylation and ubiquitination, as well as suggesting that the sumoylated Pellino-1 protein may have a cellular function distinct from previously identified functions.

Decreased expression of glutaredoxin 1 is required for transforming growth factor-β1-mediated epithelial–mesenchymal transition of EpRas mammary epithelial cells

Transforming growth factor-beta (TGF-beta) is a cytokine important in inducing epithelial-mesenchymal transition (EMT), a crucial morphological event in a wide range of physiological and pathological cellular processes. In this study, we demonstrate that TGF-beta1 induces the EMT phenotype through decreasing the expression of the glutaredoxin 1 (Grx1) gene, an anti-oxidant enzyme, in H-Ras transformed EpH4 mammary epithelial cells (EpRas), but not in the parental EpH4 cells. TGF-beta1-induced reduction of Grx1 expression caused an increase of intracellular reactive oxygen species (ROS) in EpRas cells, and pre-treatment of the ROS scavenger N-acetylcysteine (NAC) inhibited TGF-beta1-induced EMT. Grx1-overexpressing EpRas cells showed a reduction in intracellular ROS generation and suppressed the expression of mesenchymal markers upon treatment of TGF-beta1. In addition, MEK/MAP kinase and phosphatidylinositol-3 kinase (PI3K) signaling were found to mediate the decrease in Grx1 expression upon TGF-beta1 treatment, depending on the presence of Ras protein. Thus our findings strongly suggest that TGF-beta1 promotes EMT by increasing intracellular ROS levels via down-regulation of the Grx1 gene in EpRas cells.

Therapeutic effects of mouse bone marrow-derived clonal mesenchymal stem cells in a mouse model of inflammatory bowel disease

Mouse bone marrow-derived clonal mesenchymal stem cells (mcMSCs), which were originated from a single cell by a subfractionation culturing method, are recognized as new paradigm for stem cell therapy featured with its homogenous cell population. Next to proven therapeutic effects against pancreatitis, in the current study we demonstrated that mcMSCs showed significant therapeutic effects in dextran sulfate sodium (DSS)-induced experimental colitis model supported with anti-inflammatory and restorative activities. mcMSCs significantly reduced the disease activity index (DAI) score, including weight loss, stool consistency, and intestinal bleeding and significantly increased survival rates. The pathological scores were also significantly improved with mcMSC. We have demonstrated that especial mucosal regeneration activity accompanied with significantly lowered level of apoptosis as beneficiary actions of mcMSCs in UC models. The levels of inflammatory cytokines including TNF-α, IFN-γ, IL-1β, IL-6, and IL-17 were all significantly concurrent with significantly repressed NF-κB activation compared to the control group and significantly decreased infiltrations of responsible macrophage and neutrophil. Conclusively, our findings provide the rationale that mcMSCs are applicable as a potential source of cell-based therapy in inflammatory bowel diseases, especially contributing either to prevent relapse or to accelerate healing as solution to unmet medical needs in IBD therapy.

14-3-3ε protein increases matrix metalloproteinase-2 gene expression via p38 MAPK signaling in NIH3T3 fibroblast cells

One of the 14-3-3 protein isoforms, 14-3-3ɛ, was previously shown to be increased during skin aging. We suggest here a possible role for the 14-3-3ɛ protein in skin aging by providing evidence that 14-3-3ɛ increases the expression of the matrix-metalloproteinase (MMP)-2 gene in NIH3T3 fibroblast cells. Expression of the 14-3-3ɛ gene in NIH3T3 cells primarily up-regulated the expression of the MMP-2 gene at the transcriptional level by inducing specific DNA binding proteins bound to an upstream region of the MMP-2 promoter from -1,629 to -1,612. Inhibition of endogenous 14-3-3ɛ gene expression by RNA interference also decreased endogenous MMP-2 gene expression. Furthermore, up-regulation of the MMP-2 gene by 14-3-3ɛ was suppressed by expression of a dominant-negative mutant of p38 MAP kinase. These findings strongly suggest that increased expression of 14-3-3ɛ contributes to remodeling of extracellular matrix in skin through increasing MMP-2 gene expression via p38 MAP kinase signaling.

A20 promotes metastasis of aggressive basal-like breast cancers through multi-monoubiquitylation of Snail1

Although the ubiquitin-editing enzyme A20 is a key player in inflammation and autoimmunity, its role in cancer metastasis remains unknown. Here we show that A20 monoubiquitylates Snail1 at three lysine residues and thereby promotes metastasis of aggressive basal-like breast cancers. A20 is significantly upregulated in human basal-like breast cancers and its expression level is inversely correlated with metastasis-free patient survival. A20 facilitates TGF-β1-induced epithelial–mesenchymal transition (EMT) of breast cancer cells through multi-monoubiquitylation of Snail1. Monoubiquitylated Snail1 has reduced affinity for glycogen synthase kinase 3β (GSK3β), and is thus stabilized in the nucleus through decreased phosphorylation. Knockdown of A20 or overexpression of Snail1 with mutation of the monoubiquitylated lysine residues into arginine abolishes lung metastasis in mouse xenograft and orthotopic breast cancer models, indicating that A20 and monoubiquitylated Snail1 are required for metastasis. Our findings uncover an essential role of the A20–Snail1 axis in TGF-β1-induced EMT and metastasis of basal-like breast cancers.

The deubiquitinating enzyme USP50 regulates inflammasome activation through targeting the ASC adaptor protein.

NOD‐like receptor family protein 3 (NLRP3)‐mediated inflammasome activation promotes caspase‐1‐dependent production of interleukin‐1β (IL‐1β) and requires the adaptor protein ASC. Compared with the priming and activation mechanisms of the inflammasome signaling pathway, post‐translational ubiquitination/deubiquitination mechanisms controlling inflammasome activation have not been clearly addressed. We here demonstrate that the deubiquitinating enzyme USP50 binds to the ASC protein and subsequently regulates the inflammasome signaling pathway by deubiquitinating the lysine 63‐linked polyubiquitination of ASC. USP50 knockdown in human THP‐1 cells and mouse bone marrow‐derived macrophages shows a significant decrease in procaspase‐1 cleavage, resulting in a reduced secretion of IL‐1β and interleukin‐18 (IL‐18) upon treatment with NLRP3 stimuli and a reduction in ASC speck formation and oligomerization. Thus, we elucidate a novel regulatory mechanism of the inflammasome signaling pathway mediated by the USP50 deubiquitinating enzyme.