Seok Hee Park

Fibroblasts from chronic wounds show altered TGF-β-signaling and decreased TGF-β Type II Receptor expression†

Chronic wounds are characterized by failure to heal in a defined time frame. However, the pathogenic steps leading from the etiological factors to failure to heal are unknown. Recently, increasing evidence suggests that resident cells in chronic wounds display a number of critical abnormalities, including senescence and unresponsiveness to the stimulatory action of transforming growth factor‐β1 (TGF‐β1). In this study, we have determined some of the mechanisms that might be responsible for unresponsiveness to TGF‐β1. Using Northern analysis and affinity labeling, we show that venous ulcer fibroblasts have decreased TGF‐β Type II receptor expression. This finding is not the result of genetic mutation, as shown by experiments with Type II receptor satellite instability. Decreased Type II receptor expression was accompanied by failure of ulcer fibroblasts to phosphorylate Smad 2, Smad 3, and p42/44 mitogen activating protein kinase (MAPK), and was associated with a slower proliferative rate in response to TGF‐β1. We conclude that venous ulcer fibroblasts show decreased Type II receptor expression and display abnormalities in the downstream signaling pathway involving MAPK and the early Smad pathway. These findings suggest ways to address and treat the abnormal cellular phenotype of cells in chronic wounds.

Jab1/CSN5, a Component of the COP9 Signalosome, Regulates Transforming Growth Factor β Signaling by Binding to Smad7 and Promoting Its Degradation

ABSTRACT Smad7 inhibits responses mediated by transforming growth factor β (TGF-β) and acts in a negative-feedback loop to regulate the intensity or duration of the TGF-β signal. However, the aberrant expression and continued presence of Smad7 may cause TGF-β resistance. Here we report that Jab1/CSN5, which is a component of the COP9 signalosome complex, associates constitutively with Smad7 and that overexpression of Jab1/CSN5 causes the translocation of Smad7 from the nucleus to the cytoplasm, promoting its degradation. Overexpression of Jab1/CSN5 increases Smad2 phosphorylation and enhances TGF-β-induced transcriptional activity. The inhibition of endogenous Jab1/CSN5 expression by small interfering RNA (siRNA) induces Smad7 expression. This study thus defines Jab1/CSN5 as an adapter that targets Smad7 for degradation, thus releasing Smad7-mediated suppression of TGF-β signaling.

Transcriptional Regulation of the Transforming Growth Factor β Type II Receptor Gene by Histone Acetyltransferase and Deacetylase Is Mediated by NF-Y in Human Breast Cancer Cells

Transcriptional repression of the transforming growth factor-β (TGF-β) type II receptor (TβRII) gene is one of several mechanisms leading to TGF-β resistance. Previously, we have shown that MS-275, a synthetic inhibitor of histone deacetylase (HDAC), specifically induces the expression of theTβRII gene and restores the TGF-β signaling in human breast cancer cell lines. However, little is known about the mechanism by which inhibition of HDAC activates TβRII expression. MS-275 treatment of cells expressing a wild-type TβRIIpromoter/luciferase construct resulted in a 10-fold induction of the promoter activity. DNA transfection and an electrophoretic mobility shift assay showed that the induction of the TβRIIpromoter by MS-275 requires the inverted CCAAT box and its cognate binding protein, NF-Y. In addition, a DNA affinity pull-down assay indicated that the PCAF protein, a transcriptional coactivator with intrinsic histone acetyltransferase (HAT) activity, is specifically recruited to the NF-Y complex in the presence of either MS-275 or trichostatin A. Based on these results, we suggest that treatment with the HDAC inhibitor induces TβRII promoter activity by the recruitment of the PCAF protein to the NF-Y complex, interacting with the inverted CCAAT box in the TβRIIpromoter.

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.

Sequence-specific enhancer binding protein is responsible for the differential expression of ERT/ESX/ELF-3/ESE-1/jen gene in human gastric cancer cell lines : Implication for the loss of TGF-β type II receptor expression

Transcriptional repression of the TGF-β type II receptor (RII) is one of the mechanisms leading to TGF-β resistance. The newly identified epithelium-specific ets transcription factor ERT/ESX/ELF-3/ESE-1/jen binds to the TGF-β RII promoter and induces promoter activity. The human gastric cancer cell lines, which show undetectable level of TGF-β RII mRNA, do not express ERT mRNA. To study the molecular mechanisms of loss of ERT expression, we have cloned and characterized the human ERT promoter. DNA transfection experiments and electrophoretic mobility shift assays have revealed the existence of a distinct enhancer element (−186 to −177) which we named ESE (ERT promoter specific element). Deletion of the ESE markedly decreased expression of the target gene. ESE interacts with two distinct nuclear protein complexes, at least one of which appears to be inactivated in a cell line which does not express the ERT mRNA, compared to a cell line expressing the ERT mRNA. These results suggest the possibility that inactivation of the sequence-specific DNA binding protein to the region from −186 to −177 contributes to the loss of ERT expression, leading to the loss of TGF-β type II receptor mRNA in human gastric cancer cell lines.