An intracellular fragment of osteoactivin formed by ectodomain shedding translocated to the nucleoplasm and bound to RNA binding proteins.

Osteoactivin is a type I transmembrane protein upregulated by unloading stresses, including denervation, prolonged bed rest, and space flight, but the regulatory mechanisms of its expression and activation under these conditions remain undefined. Here we report that osteoactivin protein exists in two forms: an intact transmembrane form and a secreted form. The secreted form, the extracellular fragment of osteoactivin, was produced by ectodomain shedding and was released into a culture medium. Amino acid sequence analysis of the carboxy-terminal fragment of osteoactivin (OA-CTF) revealed that cleavage of osteoactivin by proteases occurred both at the cell surface and within the cell membrane. Localization analysis demonstrated translocalization of OA-CTF to the nucleus and the endoplasmic reticulum. Moreover, RNA binding proteins, which regulate pre-mRNA splicing, were identified as OA-CTF binding proteins. These results suggest that OA-CTF formed by ectodomain shedding is involved in the regulation of pre-mRNA splicing.

[Space flight/bedrest immobilization and bone. Development of inhibitors for atrophy caused by unloading stress].

Muscle atrophy caused by unloading stress is a serious problem in bed rest patients or astronauts. In our previous studies, we revealed that induction and activation of ubiquitin ligase Cbl-b played an important role in skeletal muscle atrophy caused by unloading stress. Under muscle atrophy conditions, Cbl-b interacted with and degraded IRS-1 (insulin receptor substrate 1) that is a central molecule in the IGF-1 signaling pathway. In addition, we developed a Cbl-b inhibitor (Cblin) that a pentapeptide mimetic of tyrosin608-phosphorylated IRS-1, DGpYMP. This Cblin peptide inhibited Cbl-b mediated IRS-1 ubiquitination and strongly decreased the Cbl-b-mediated induction of MAFbx/atrogin-1. We are further developing Cbl-b inhibitors that are more effective than an original Cblin peptide.

Cbl-b is a critical regulator of macrophage activation associated with obesity-induced insulin resistance in mice.

We previously reported the potential involvement of casitas B-cell lymphoma-b (Cbl-b) in aging-related murine insulin resistance. Because obesity also induces macrophage recruitment into adipose tissue, we elucidated here the role of Cbl-b in obesity-related insulin resistance. Cbl-b(+/+) and Cbl-b(-/-) mice were fed a high-fat diet (HFD) and then examined for obesity-related changes in insulin signaling. The HFD caused recruitment of macrophages into adipose tissue and increased inflammatory reaction in Cbl-b(-/-) compared with Cbl-b(+/+) mice. Peritoneal macrophages from Cbl-b(-/-) mice and Cbl-b-overexpressing RAW264.7 macrophages were used to examine the direct effect of saturated fatty acids (FAs) on macrophage activation. In macrophages, Cbl-b suppressed saturated FA-induced Toll-like receptor 4 (TLR4) signaling by ubiquitination and degradation of TLR4. The physiological role of Cbl-b in vivo was also examined by bone marrow transplantation and Eritoran, a TLR4 antagonist. Hematopoietic cell-specific depletion of the Cbl-b gene induced disturbed responses on insulin and glucose tolerance tests. Blockade of TLR4 signaling by Eritoran reduced fasting blood glucose and serum interleukin-6 levels in obese Cbl-b(-/-) mice. These results suggest that Cbl-b deficiency could exaggerate HFD-induced insulin resistance through saturated FA-mediated macrophage activation. Therefore, inhibition of TLR4 signaling is an attractive therapeutic strategy for treatment of obesity-related insulin resistance.