STAP-2 facilitates insulin signaling through binding to CAP/c-Cbl and regulates adipocyte differentiation.

Signal-transducing adaptor protein-2 (STAP-2) is an adaptor molecule involved in several cellular signaling cascades. Here, we attempted to identify novel STAP-2 interacting molecules, and identified c-Cbl associated protein (CAP) as a binding protein through the C-terminal proline-rich region of STAP-2. Expression of STAP-2 increased the interaction between CAP and c-Cbl, suggesting that STAP-2 bridges these proteins and enhances complex formation. CAP/c-Cbl complex is known to regulate GLUT4 translocation in insulin signaling. STAP-2 overexpressed human hepatocyte Hep3B cells showed enhanced GLUT4 translocation after insulin treatment. Elevated levels of Stap2 mRNA have been observed in 3T3-L1 cells and mouse embryonic fibroblasts (MEFs) during adipocyte differentiation. The differentiation of 3T3-L1 cells into adipocytes was highly promoted by retroviral overexpression of STAP-2. In contrast, STAP-2 knockout (KO) MEFs exhibited suppressed adipogenesis. The increase in body weight with high-fat diet feeding was significantly decreased in STAP-2 KO mice compared to WT animals. These data suggest that the expression of STAP-2 correlates with adipogenesis. Thus, STAP-2 is a novel regulatory molecule that controls insulin signal transduction by forming a c-Cbl/STAP-2/CAP ternary complex.

Tyrosinase regulates the motility of human melanoma cell line A375 through its hydroxylase enzymatic activity.

Melanoma, originating from melanocytes, is a highly aggressive tumor. Tyrosinase is involved in melanin production in melanocytes, and its overexpression is noted in malignant melanomas. However, the role of tyrosinase in melanomas remains unclear. Therefore, this study aimed to evaluate the potential functions of tyrosinase in the human melanoma cell line A375. The expression level of tyrosinase in A375 cells was undetectable. However, markedly increased expression level was observed in the mouse melanoma cell line B16F10 and the human melanoma cell line WM266-4. Subsequently, we investigated the effect of ectopic tyrosinase expression on A375 cell motility using wound-healing assay. The overexpression of tyrosinase resulted in enhanced cell migration in both stable and transient tyrosinase expression cells. The levels of filamentous actin were decreased in tyrosinase-expressing A375 cells, suggesting that tyrosinase regulates cell motility by modulating actin polymerization. Histidine residues in tyrosinase are important for its enzymatic activity for synthesizing melanin. Substitution of these histidine residues to alanine residues mitigated the promotion of tyrosinase-induced A375 cell metastasis. Furthermore, melanin treatment enhanced A375 cell metastasis and phosphorylation of Cofilin. Thus, our findings suggest that tyrosinase increases the migration of A375 cells by regulating actin polymerization through its enzymatic activity.

Scraping Assay as a Novel Strategy to Evaluate Axonal Regeneration Using Human-Induced Pluripotent Stem Cell-Derived Neurons.

Neuronal regrowth after traumatic injury is strongly inhibited in the central nervous system (CNS) of adult mammals. Cell-intrinsic and extrinsic factors limit the regulation of axonal growth and regrowth of fibers is minimal despite nearly all neurons surviving. Developing medical drugs to promote neurological recovery is crucial since neuronal injuries have few palliative cares and no pharmacological interventions. Herein, we developed a novel in vitro axonal regeneration assay system to screen the chemical reagents using human-induced pluripotent stem cell (hiPSC)-derived neurons. These neurons were cultured in a 96-well plate to form a monolayer and were scraped using a floating metal pin tool for axotomy. The cell number and plate coating conditions were optimized to score the regenerating axon. Treatment using the Rho-associated kinase (ROCK) inhibitor Y-27632 enhanced axonal regeneration in this regeneration assay system with hiPSC-derived neurons. Therefore, our novel screening method is suitable for drug screening to identify the chemical compounds that promote axonal regeneration after axotomy under in vitro conditions.