ß-Taxilin participates in differentiation of C2C12 myoblasts into myotubes.

Myogenesis is required for the development of skeletal muscle. Accumulating evidence indicates that the expression of several genes are upregulated during myogenesis and these genes play pivotal roles in myogenesis. However, the molecular mechanism underlying myogenesis is not fully understood. In this study, we found that ß-taxilin, which is specifically expressed in the skeletal muscle and heart tissues, was progressively expressed during differentiation of C2C12 myoblasts into myotubes, prompting us to investigate the role of ß-taxilin in myogenesis. In C2C12 cells, knockdown of ß-taxilin impaired the fusion of myoblasts into myotubes, and decreased the diameter of myotubes. We also found that ß-taxilin interacted with dysbindin, a coiled-coil-containing protein. Knockdown of dysbindin conversely promoted the fusion of myoblasts into myotubes and increased the diameter of myotubes in C2C12 cells. Furthermore, knockdown of dysbindin attenuated the inhibitory effect of ß-taxilin depletion on myotube formation of C2C12 cells. These results demonstrate that ß-taxilin participates in myogenesis through suppressing the function of dysbindin to inhibit the differentiation of C2C12 myoblasts into myotubes.

TSG101, a tumor susceptibility gene, bidirectionally modulates cell invasion through regulating MMP-9 mRNA expression.

BACKGROUND: Tumor susceptibility gene 101 (TSG101) was initially identified in fibroblasts as a tumor suppressor gene but subsequent studies show that TSG101 also functions as a tumor-enhancing gene in some epithelial tumor cells. Although previous studies have unraveled diverse biological functions of TSG101, the precise mechanism by which TSG101 is involved in carcinogenesis and tumor progression in a bidirectional and multifaceted manner remains unclear. METHODS: To reveal the mechanism underlying bidirectional modulation of cell invasion by TSG101, we used RNA interference to examine whether TSG101 depletion bidirectionally modulated matrix metalloproteinase (MMP)-9 expression in different cell types. RESULTS: TSG101 depletion promoted cell invasion of HT1080 cells but contrarily reduced cell invasion of HeLaS3 cells. In HT1080 cells, TSG101 depletion increased both baseline and phorbol 12-myristate 13-acetate (PMA)-induced MMP-9 secretion through enhancing MMP-9 mRNA expression, but did not affect the expression or activation of MMP-2. In contrast, TSG101 depletion decreased PMA-induced MMP-9 secretion through reducing MMP-9 mRNA expression in HeLaS3 cells. TSG101 depletion had little impact on the signaling pathways required for the activation of transcription of MMP-9 or MMP-9 mRNA stability in either cell line. CONCLUSION: TSG101 bidirectionally modulates cell invasion through regulating MMP-9 mRNA expression in different cell types. Our results provide a mechanistic context for the role of TSG101 in cell invasion as a multifaceted gene.

α-Taxilin interacts with sorting nexin 4 and participates in the recycling pathway of transferrin receptor.

Membrane traffic plays a crucial role in delivering proteins and lipids to their intracellular destinations. We previously identified α-taxilin as a binding partner of the syntaxin family, which is involved in intracellular vesicle traffic. α-Taxilin is overexpressed in tumor tissues and interacts with polymerized tubulin, but the precise function of α-taxilin remains unclear. Receptor proteins on the plasma membrane are internalized, delivered to early endosomes and then either sorted to the lysosome for degradation or recycled back to the plasma membrane. In this study, we found that knockdown of α-taxilin induced the lysosomal degradation of transferrin receptor (TfnR), a well-known receptor which is generally recycled back to the plasma membrane after internalization, and impeded the recycling of transferrin. α-Taxilin was immunoprecipitated with sorting nexin 4 (SNX4), which is involved in the recycling of TfnR. Furthermore, knockdown of α-taxilin decreased the number and length of SNX4-positive tubular structures. We report for the first time that α-taxilin interacts with SNX4 and plays a role in the recycling pathway of TfnR.

Expression of α-taxilin in the murine gastrointestinal tract: potential implication in cell proliferation.

α-Taxilin, a binding partner of the syntaxin family, is a candidate tumor marker. To gain insight into the physiological role of α-taxilin in normal tissues, we examined α-taxilin expression by Western blot and performed immunochemical analysis in the murine gastrointestinal tract where cell renewal vigorously occurs. α-Taxilin was expressed in the majority of the gastrointestinal tract and was prominently expressed in epithelial cells positive for Ki-67, a marker of actively proliferating cells. In the small intestine, α-taxilin was expressed in transient-amplifying cells and crypt base columnar cells intercalated among Paneth cells. In the corpus and antrum of the stomach, α-taxilin was expressed in cells localized in the lower pit and at the gland, respectively, but not in parietal or zymogenic cells. During development of the small intestine, α-taxilin was expressed in Ki-67-positive regions. Inhibition of cell proliferation by suppression of the Notch cascade using a γ-secretase inhibitor led to a decrease in α-taxilin- and Ki-67-positive cells in the stomach. These results suggest that expression of α-taxilin is regulated in parallel with cell proliferation in the murine gastrointestinal tract.

Interaction of α-taxilin localized on intracellular components with the microtubule cytoskeleton.

Intracellular vesicle traffic plays an essential role in the establishment and maintenance of organelle identity and biosynthetic transport. We have identified α-taxilin as a binding partner of the syntaxin family, which is involved in intracellular vesicle traffic. Recently, we have found that α-taxilin is over-expressed in malignant tissues including hepatocellular carcinoma and renal cell carcinoma. However, a precise role of α-taxilin in intracellular vesicle traffic and carcinogenesis remains unclear. Then, we first investigated here the intracellular distribution of α-taxilin in Hela cells. Immunofluorescence studies showed that α-taxilin distributes throughout the cytoplasm and exhibits a tubulo-vesicular pattern. Biochemical studies showed that α-taxilin is abundantly localized on intracellular components as a peripheral membrane protein. Moreover, we found that α-taxilin distributes in microtubule-dependent and syntaxin-independent manners, that α-taxilin directly binds to polymerized tubulin in vitro, and that N-ethylmaleimide but not brefeldin A affects the intracellular distribution of α-taxilin. These results indicate that α-taxilin is localized on intracellular components in a syntaxin-independent manner and that the α-taxilin-containing intracellular components are associated with the microtubule cytoskeleton and suggest that α-taxilin functions as a linker protein between the α-taxilin-containing intracellular components and the microtubule cytoskeleton.