Rab44 negatively regulates myoblast differentiation by controlling fusogenic protein transport and mTORC1 signaling.

Skeletal muscle is composed of multinucleated myotubes formed by the fusion of mononucleated myoblasts. Skeletal muscle differentiation, termed as myogenesis, have been investigated using the mouse skeletal myoblast cell line C2C12. It has been reported that several "small" Rab proteins, major membrane-trafficking regulators, possibly regulate membrane protein transport in C2C12 cells; however, the role of Rab proteins in myogenesis remains unexplored. Rab44, a member of "large" Rab GTPases, has recently been identified as a negative regulator of osteoclast differentiation. In this study, using C2C12 cells, we found that Rab44 expression was upregulated during myoblast differentiation into myotubes. Knockdown of Rab44 enhanced myoblast differentiation and myotube formation. Consistent with these results, Rab44 knockdown in myoblasts increased expression levels of several myogenic marker genes. Rab44 knockdown increased the surface accumulation of myomaker and myomixer, two fusogenic proteins required for multinucleation, implying enhanced cell fusion. Conversely, Rab44 overexpression inhibited myoblast differentiation and tube formation, accompanied by decreased expression of some myogenic markers. Furthermore, Rab44 was found to be predominantly localized in lysosomes, and Rab44 overexpression altered the number and size of lysosomes. Considering the underlying molecular mechanism, Rab44 overexpression impaired the signaling pathway of the mechanistic target of rapamycin complex1 (mTORC1) in C2C12 cells. Namely, phosphorylation levels of mTORC1 and downstream mTORC1 substrates, such as S6 and P70-S6K, were notably lower in Rab44 overexpressing cells than those in control cells. These results indicate that Rab44 negatively regulates myoblast differentiation into myotubes by controlling fusogenic protein transport and mTORC1 signaling.

Rab44 deficiency accelerates recovery from muscle damage by regulating mTORC1 signaling and transport of fusogenic regulators.

The skeletal muscle is a tissue that shows remarkable plasticity to adapt to various stimuli. The development and regeneration of skeletal muscles are regulated by numerous molecules. Among these, we focused on Rab44, a large Rab GTPase, that has been recently identified in immune cells and osteoclasts. Recently, bioinformatics data has revealed that Rab44 is upregulated during the myogenic differentiation of myoblasts into myotubes in C2C12 cells. Thus, Rab44 may be involved in myogenesis. Here, we have investigated the effects of Rab44 deficiency on the development and regeneration of skeletal muscle in Rab44 knockout (KO) mice. Although KO mice exhibited body and muscle weights similar to those of wild-type (WT) mice, the histochemical analysis showed that the myofiber cross-sectional area (CSA) of KO mice was significantly smaller than that of WT mice. Importantly, the results of muscle regeneration experiments using cardiotoxin revealed that the CSA of KO mice was significantly larger than that of WT mice, suggesting that Rab44 deficiency promotes muscle regeneration. Consistent with the in vivo results, in vitro experiments indicated that satellite cells derived from KO mice displayed enhanced proliferation and differentiation. Mechanistically, KO satellite cells exhibited an increased mechanistic target of rapamycin complex 1 (mTORC1) signaling compared to WT cells. Additionally, enhanced cell surface transport of myomaker and myomixer, which are essential membrane proteins for myoblast fusion, was observed in KO satellite cells compared to WT cells. Therefore, Rab44 deficiency enhances muscle regeneration by modulating the mTORC1 signaling pathway and transport of fusogenic regulators.

The potential effect of ethyl alcohol elution from liner type denture adhesives on breath alcohol concentration and determination of drunk driving.

Background/purpose: The application of liner type denture adhesives containing ethyl alcohol (EtOH) may result in the person being considered a drunk driver, which is a social problem. This study measured the amount of EtOH loss from the materials and its effect on breath alcohol concentration (BrAC). Materials and methods: The amount of EtOH loss of three liner type denture adhesives was measured using a gas chromatograph-mass spectrometer. Five specimens were measured for each material. The BrAC of ten participants who wore the palatal plate lined with the material having the highest amount of EtOH elution was also determined using an alcohol detector every 5 min for 60 min. The threshold for drunk driving was a BrAC of 0.15 mg/L or more. Results: Significant differences were found in the amount of EtOH elution among the three materials. For all materials, the elution amount from the start of immersion to 30 min was significantly larger than that of the following 30 min (P < 0.05). BrAC values of the participants reached their maxima 5 min after insertion of the materials, and 80% of participants surpassed the threshold for drunk driving. However, no participants reached the threshold for drunk driving after 50 min. Conclusion: The results suggest that a determination of drunk drinking will not be made when 1 h or more has passed after insertion of a denture lined with a liner type denture adhesive into the mouth, though a determination of drunk driving may exist due to EtOH from the materials.

Rab44 Deficiency Induces Impaired Immune Responses to Nickel Allergy.

Rab44 was recently identified as an atypical Rab GTPase that possesses EF-hand and coiled-coil domains at the N-terminus, and a Rab-GTPase domain at the C-terminus. Rab44 is highly expressed in immune-related cells such as mast cells, macrophages, osteoclasts, and granulocyte-lineage cells in the bone marrow. Therefore, it is speculated that Rab44 is involved in the inflammation and differentiation of immune cells. However, little is known about the role of Rab44 in inflammation. In this study, we showed that Rab44 was upregulated during the early phase of differentiation of M1- and M2-type macrophages. Rab44-deficient mice exhibited impaired tumor necrosis factor alpha and interleukin-10 production after lipopolysaccharide (LPS) stimulation. The number of granulocytes in Rab44-deficient mice was lower, but the lymphocyte count in Rab44-deficient mice was significantly higher than that in wild-type mice after LPS stimulation. Moreover, Rab44-deficient macrophages showed impaired nickel-induced toxicity, and Rab44-deficient mice showed impaired nickel-induced hypersensitivity. Upon nickel hypersensitivity induction, Rab44-deficient mice showed different frequencies of immune cells in the blood and ears. Thus, it is likely that Rab44 is implicated in immune cell differentiation and inflammation, and Rab44 deficiency induces impaired immune responses to nickel allergies.