The phosphorylated, activated cytoplasmic domains of the transforming growth factor- (TGF) receptors were used as probes to screen an expression library that was prepared from a highly TGF-responsive intestinal epithelial cell line. One of the TGF receptor-interacting proteins isolated was identified to be the mammalian homologue of the LC7 family (mLC7) of dynein light chains (DLCs). This 11-kDa cytoplasmic protein interacts with the TGF receptor complex intracellularly and is phosphorylated on serine residues after ligand-receptor engagement. Forced expression of mLC7-1 induces specific TGF responses, including an activation of Jun N-terminal kinase (JNK), a phosphorylation of c-Jun, and an inhibition of cell growth. Furthermore, TGF induces the recruitment of mLC7-1 to the intermediate chain of dynein. A kinase-deficient form of TGF RII prevents both mLC7-1 phosphorylation and interaction with the dynein intermediate chain (DIC). This is the first demonstration of a link between cytoplasmic dynein and a natural growth inhibitory cytokine. Furthermore, our results suggest that TGF pathway components may use a motor protein light chain as a receptor for the recruitment and transport of specific cargo along microtublules. INTRODUCTIONTransforming growth factor- (TGF) is the prototype for the TGF superfamily of highly conserved growth regulatory polypeptides that also includes the activins, inhibins, bone morphogenetic proteins, decapentaplegic (Dpp), nodal, Lefty, and others (Roberts, 1998;Sporn and Vilcek, 2000;Yue and Mulder, 2001). Alterations in the TGF signaling components and pathways have been implicated in a vast array of human pathologies, including cancer (Massague et al., 2000;Sporn and Vilcek, 2000;Derynck et al., 2001).TGF binds to two types of transmembrane serine/threonine kinase receptors (RI and RII) in a heterotetrameric complex, to activate downstream components (Roberts, 1998; Massague et al., 2000;Sporn and Vilcek, 2000;Yue and Mulder, 2001). The Smad family of signaling intermediates plays an important role in mediating TGF responses (Attisano and Wrana, 2000;ten Dijke et al., 2000;Yue and Mulder, 2001). Moreover, TGF has been shown to regulate Ras (Mulder and Morris, 1992;Hartsough et al., 1996;Yue et al., 1998) and several components of the mitogen-activated protein kinase (Mapk) pathways (Hartsough and Mulder, 1995;Frey and Mulder, 1997;Mulder, 2000;Sporn and Vilcek, 2000;Yue and Mulder, 2001). In addition to the Ras/Mapk and Smad pathways, several proteins have been identified based upon their interaction with the TGF receptors (Yue and Mulder, 2001). Furthermore, various Smad-interacting proteins have also been identified, including SARA and Dab2, which interact with both Smads and the TGF receptors (Tsukazaki et al., 1998;Hocevar et al., 2001;Yue and Mulder, 2001).Despite advances in our understanding of the mechanisms by which the Smad and Ras/Mapk cascades mediate some TGF effects, these pathways seem to regulate primarily transcriptional events (Hocevar et al., 1...
We have identified km23-1 as a novel transforming growth factor- (TGF) receptor (TR)-interacting protein that is also a light chain of the motor protein dynein (dynein light chain). Herein, we demonstrate by sucrose gradient analyses that, in the presence of TGF but not in the absence, km23-1 was present in early endosomes with the TRs. Further, confocal microscopy studies indicate that endogenous km23-1 was co-localized with endogenous Smad2 at early times after TGF treatment, prior to Smad2 translocation to the nucleus. In addition, immunoprecipitation/blot analyses showed that TGF regulated the interaction between endogenous km23-1 and endogenous Smad2 in vivo. Blockade of km23-1 using a small interfering RNA approach resulted in a reduction in both total intracellular Smad2 levels and in nuclear levels of phosphorylated Smad2 after TGF treatment. This decrease was reversed by lactacystin, a specific inhibitor of the 26 S proteasome, suggesting that knockdown of km23-1 causes proteasomal degradation of phosphorylated (i.e. activated) Smad2. Blockade of km23-1 also resulted in a reduction in TGF/Smad2-dependent ARE-Lux transcriptional activity, which was rescued by a km23-1 small interfering RNA-resistant construct. In contrast, a reduction in TGF/Smad3-dependent SBE2-Luc transcriptional activity did not occur under similar conditions. Furthermore, overexpression of the dynactin subunit dynamitin, which is known to disrupt dynein-mediated intracellular transport, blocked TGF-stimulated nuclear translocation of Smad2. Collectively, our findings indicate for the first time that a dynein light chain is required for a Smad2-dependent TGF signaling pathway.
BackgroundNonalcoholic fatty liver disease (NAFLD) is a prevalent complication of extreme obesity. Loading of the liver with fat can progress to inflammation and fibrosis including cirrhosis. The molecular factors involved in the progression from simple steatosis to fibrosis remain poorly understood.MethodsGene expression profiling using microarray, PCR array, and RNA sequencing was performed on RNA from liver biopsy tissue from patients with extreme obesity. Patients were grouped based on histological findings including normal liver histology with no steatosis, lobular inflammation, or fibrosis, and grades 1, 2, 3, and 4 fibrosis with coexistent steatosis and lobular inflammation. Validation of expression was conducted using quantitative PCR. Serum analysis was performed using ELISA. Expression analysis of hepatocytes and hepatic stellate cells in response to lipid loading were conducted in vitro using quantitative PCR and ELISA.ResultsThree orthogonal methods to profile human liver biopsy RNA each identified the chemokine CCL20 (CC chemokine ligand 20 or MIP-3 alpha) gene as one of the most up-regulated transcripts in NAFLD fibrosis relative to normal histology, validated in a replication group. CCL20 protein levels in serum measured in 224 NAFLD patients were increased in severe fibrosis (p < 0.001), with moderate correlation of hepatic transcript levels and serum levels. Expression of CCL20, but not its cognate receptor CC chemokine receptor 6, was significantly (p < 0.001) increased in response to fatty acid loading in LX-2 hepatic stellate cells, with relative increases greater than those in HepG2 hepatocyte cells.ConclusionsThese results suggest that expression of CCL20, an important inflammatory mediator, is increased in NAFLD fibrosis. CCL20 serves as a chemoattractant molecule for immature dendritic cells, which have been shown to produce many of the inflammatory molecules that mediate liver fibrosis. These data also point to hepatic stellate cells as a key cell type that may respond to lipid loading of the liver.Electronic supplementary materialThe online version of this article (10.1186/s12967-018-1490-y) contains supplementary material, which is available to authorized users.
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