Nuclear corepressor SMRT acts as a strong regulator of both ß-oxidation and suppressor of fibrosis in the differentiation process of mouse skeletal muscle cells.

BACKGROUND: Silencing Mediator of Retinoid and Thyroid hormone receptors (SMRT; NCoR2) is a transcriptional corepressor (CoR) which has been recognized as an important player in the regulation of hepatic lipogenesis and in somatic development in mouse embryo. SMRT protein is also widely expressed in mouse connective tissues, for example adipocytes and muscle. We recently reported that mice with global deletion of SMRT develop significant obesity and muscle wasting which are independent from thyroid hormone (TH) signaling and thermogenesis. However, the tissue specific role of SMRT in skeletal muscle is still not clear. METHODS: To clarify role of SMRT in muscle differentiation, we made myogenic C2C12 clones which lack SMRT protein (C2C12-SKO) by using CRISPR-Cas9. Wild-type C2C12 (C2C12-WT) and C2C12-SKO cells were cultured in differentiation medium, and the resulting gene and protein profiles were compared between the two cell lines both before and after differentiation. We also analyzed muscle tissues which were dissected from whole body SMRT knockout (KO) mice and their controls. RESULTS: We found significant up-regulation of muscle specific ß-oxidation markers; Peroxisome proliferator-activated receptor δ (PPARδ) and PPARγ coactivator-1α (PGC-1α) in the C2C12-SKO cells, suggesting that the cells had a similar gene profile to what is found in exercised rodent skeletal muscle. On the other hand, confocal microscopic analysis showed the significant loss of myotubes in C2C12-SKO cells similar to the morphology found in immature myoblasts. Proteomics analysis also confirmed that the C2C12-SKO cells had higher expression of markers of fibrosis (ex. Collagen1A1; COL1A1 and Fibroblast growth factor-2; FGF-2), indicating the up-regulation of Transforming growth factor-ß (TGF-ß) receptor signaling. Consistent with this, treatment with a specific TGF-ß receptor inhibitor ameliorated both the defects in myotube differentiation and fibrosis. CONCLUSION: Taken together, we demonstrate that SMRT functions as a pivotal transcriptional mediator for both ß-oxidation and the prevention for the fibrosis via TGF-ß receptor signaling in the differentiation of C2C12 myoblasts. In contrast to the results from C2C12 cells, SMRT does not appear to play a role in adult skeletal muscle of whole body SMRT KO mice. Thus, SMRT plays a significant role in the differentiation of myoblasts.

A novel human organic anion transporter NPT4 mediates the transport of ochratoxin A.

In the present study, we investigated the transport of nephrotoxic mycotoxin ochratoxin A (OTxA) by a novel human organic anion transporter hNPT4 using the Xenopus oocyte expression system. hNPT4 mediated time- and concentration-dependent uptake of OTxA (K(m): 802.8 µM) in a pH- and voltage-sensitive manner. Cis-inhibition experiments suggest that the substrate selectivity of hNPT4 is similar to that of hOAT4. The fact that the K(m) of OTxA for the efflux transporter hNPT4 was much higher than those for the uptake transporters hOAT1 and hOAT3 may favor the accumulation of OTxA in the tubular cell and lead to nephrotoxicity.

The antiallergic drug oxatomide promotes human eosinophil apoptosis and suppresses IL-5-induced eosinophil survival.

BACKGROUND: Eosinophils accumulated in sites of allergic inflammation are thought to play a crucial role in the pathogenesis of allergic disorders including asthma, allergic rhinitis, and atopic dermatitis, and tissue eosinophilia is attributable to increased eosinophil survival or decreased eosinophil apoptosis. OBJECTIVE: Effects of the antiallergic, histamine H1 blocker oxatomide on viability and apoptosis of eosinophils isolated from the peripheral blood of atopic subjects were studied. METHODS: Eosinophil viability and apoptosis were evaluated by using a colorimetric assay and annexin V-labeling, caspase-3 activity, and DNA fragmentation assay. RESULTS: The viability of eosinophils increased in the presence of IL-5 (10 ng/mL), confirming that IL-5 prolongs eosinophil survival in vitro. Application of oxatomide at concentrations over 20 micromol/L for 24 hours decreased the IL-5-induced enhancement of eosinophil viability. Double staining of the cells with annexin V and propidium iodide showed that deprivation of IL-5 promoted spontaneous eosinophil apoptosis and that oxatomide facilitated apoptosis and suppressed the prolongation of eosinophil survival stimulated by IL-5. In the absence of IL-5, approximately 71% and 96% of eosinophils after 24 and 48 hours, respectively, underwent spontaneous apoptosis. IL-5 decreased the rate of eosinophil apoptosis to 38% and 52% after 24 and 48 hours, respectively. Oxatomide increased eosinophil apoptosis in a concentration-dependent manner in the presence of IL-5. Furthermore, oxatomide increased caspase-3 activity and DNA fragmentation. CONCLUSION: We demonstrated that oxatomide possesses a novel therapeutic effect of apoptosis promotion on eosinophils and prevents the antiapoptotic effects of IL-5, suggesting that oxatomide may contribute to resolution of tissue eosinophilia in allergic inflammation.