Shuang Li

Manganese-containing superoxide dismutase (MnSOD) is an essential primary antioxidant enzyme that converts superoxide radical to hydrogen peroxide and molecular oxygen within the mitochondrial matrix. Cytosolic glutathione peroxidase (GPX) converts hydrogen peroxide into water. MnSOD is reduced in a variety of tumor types and has been proposed to be a new kind of tumor suppressor gene, but the mechanism(s) by which MnSOD suppresses malignancy is unclear. According to the enzymatic reactions catalyzed by MnSOD and cytosolic GPX, change in the cellular redox status, especially change attributable to accumulation of hydrogen peroxide or other hydroperoxides, is a possible reason to explain the suppression of tumor growth observed in MnSOD-overexpressing cells. To test this possible mechanism, we transfected human cytosolic GPX cDNA into human glioma cells overexpressing MnSOD. The results showed that GPX overexpression not only reversed the tumor cell growth inhibition caused by MnSOD overexpression but also altered the cellular contents of total glutathione, reduced glutathione, oxidized glutathione, and intracellular reactive oxygen species. Overexpression of GPX also inhibited degradation of the inhibitory subunit alpha of nuclear factor-KB. These results suggest that hydrogen peroxide or other hydroperoxides appear to be key reactants in the tumor suppression by MnSOD overexpression, and growth inhibition correlates with the intracellular redox status. This work suggests that manipulations that inhibit peroxide removal should enhance the tumor suppressive effect of MnSOD overexpression.

Infection and inflammation can result in the rapid loss of muscle mass and myofibrillar proteins (muscle atrophy). In addition, aspartate (Asp) is necessary for protein synthesis in mammalian cells. We hypothesized that Asp could attenuate LPS-induced muscle atrophy in a piglet model. Twenty-four weaning piglets were allotted to four treatments, including non-challenged control, LPS challenged control, LPS+0.5% Asp and LPS+1.0% Asp. On d 21, the piglets were injected with i.p. injection of LPS (100 ug/kg BM) or saline. At 4 h post-injection, blood, gastrocnemius and longissimus dorsi muscles samples were collected for biochemical and molecular analyses. Asp decreased the concentrations of cortisol and glucagon in plasma. In addition, Asp increased protein and RNA contents in muscles, and decreased mRNA expression of muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1). Moreover, Asp decreased phosphorylation of AMPKα but increased phosphorylation of Akt and Forkhead Box O (FOXO) 1 in the muscles. Our results indicate that Asp suppresses LPS-induced MAFbx and MuRF1 expression via activation of Akt signaling, and inhibition of AMPKα and FOXO1 signaling.

Podocan, a small leucine-rich repeat protein, is a negative regulator of cell proliferation. In this study, we demonstrated that podocan is involved in the differentiation of C2C12 murine myoblasts. Podocan expression increased with the progression of C2C12 differentiation. As expect, siRNA-mediated podocan knockdown inhibited C2C12 differentiation, as indicated by inhibition of MYOG, MYH2, and desmin expression, as well as reductions in the differentiation and fusion indices. Overexpression of podocan using dCas9 technology promoted C2C12 cell differentiation. In addition, supplementation of culture medium with podocan influenced C2C12 differentiation. Podocan knockdown reduced Wnt/β-catenin signaling, characterized by a reduction in the nuclear translocation of β-catenin, whereas podocan overexpression had the opposite effect. Furthermore, treatment with XAV939, an inhibitor of Wnt/β-catenin, reduced the podocan-mediated promotion of C2C12 differentiation. Induction of muscle injury in mice by bupivacaine administration suggested that podocan may play a role in muscle regeneration. In summary, our results suggest that podocan is required for normal C2C12 differentiation and that its role in myogenesis is mediated by the Wnt/β-catenin pathway.

BACKGROUND: Small leucine-rich repeat proteins (SLRPs) are highly effective and selective modulators of cell proliferation and differentiation. Podocan is a newly discovered member of the SLRP family. Its potential roles in the differentiation of bovine muscle-derived satellite cells (MDSCs) and its underlying functional mechanism remain unclear. Our study aimed to characterize the function of the podocan gene in the differentiation of bovine MDSCs and to clarify the molecular mechanism by which podocan functions in order to contribute to a better understanding of the molecular mechanism by which extracellular matrix promotes bovine MDSC differentiation and provide a theoretical basis for the improvement of beef quality. METHODS: Bovine MDSCs were transfected with vectors to overexpress or inhibit podocan, and podocan protein was added to differentiation culture medium. qRT-PCR, western blotting, and immunofluorescence were performed to investigate the effects of podocan on MDSC differentiation. Confocal microscopy and western blotting were used to assess the nuclear translocation and expression of β-catenin. An inhibitor and activator of β-catenin were used to assess the effects of the Wnt/β-catenin signaling pathway on MDSC differentiation. We inhibited β-catenin while overexpressing podocan in MDSCs. Then, we performed mass spectrometry to identify which proteins interact with podocan to regulate the Wnt/β-catenin signaling pathway. Finally, we confirmed the relationship between podocan and Wnt4 by co-immunoprecipitation and western blotting. RESULTS: Podocan protein expression increased significantly during bovine MDSC differentiation. Differentiation of bovine MDSC was promoted and suppressed by podocan overexpression or inhibition, respectively. Podocan was also shown to modulate the Wnt/β-catenin signaling pathway. Treatment of bovine MDSCs with β-catenin inhibitor and activator showed that the Wnt/β-catenin pathway is involved in bovine MDSC differentiation. Furthermore, the effect of podocan on bovine MDSC differentiation was suppressed when this pathway was inhibited. We also found that podocan interacts with Wnt4. When Wnt4 was inhibited, podocan-induced promotion of bovine MDSC differentiation was attenuated through Wnt/β-catenin signaling. CONCLUSION: Podocan regulates Wnt/β-catenin through Wnt4 to promote bovine MDSC differentiation.

Muscle satellite cells are usually at rest, and when externally stimulated or regulated, they can be further differentiated by cell fusion to form new myotubes and muscle fibers. WD repeat domain 13 (WDR13) is highly conserved in vertebrates. Studies have shown that mice lacking the Wdr13 gene develop mild obesity, hyperinsulinemia, and increased islet β cell proliferation. However, the role of WDR13 in bovine cells is unclear. Here, we investigated the effect of WDR13 on bovine skeletal muscle-derived satellite cells (MDSCs). We found that WDR13 was upregulated in bovine MDSCs using western blotting and immunofluorescence experiments. Moreover, activation and inhibition of WDR13 expression increased and decreased cell differentiation, respectively, suggesting that WDR13 promotes bovine MDSC differentiation. To further understand the mechanism of action of WDR13, we examined changes in the PI3K/AKT signaling pathway following WDR13 activation or inhibition. In addition, cells were treated with a phosphoinositide kinase 3 (PI3K) inhibitor, LY294004, to observe cell differentiation. The results showed that activation of WDR13 inhibited the PI3K/AKT signaling pathway and enhanced cell differentiation. These data suggest that WDR13 can promote the differentiation of bovine MDSCs by affecting the PI3K/AKT signaling pathway.