Oxidative stress contributes to acceleration of muscle atrophy. However, it is still not completely understood what triggers the production of reactive oxygen species (ROS) during muscle atrophy. The objective of this study was to investigate redox balance during muscle atrophy. ROS generators and antioxidants were analyzed in atrophied soleus muscles after 2 weeks of hindlimb suspension (HLS) in mice. The HLS group showed an increase in lipid peroxidation, upregulated NOX1 and NOXO1, and downregulated mitochondrial complex I subunits NDUFS5 and NDUFV2. Additionally, HLS mice demonstrated a decrease in Prdx5 and MnSOD, but an increase in GPX2 and GPX3 in both mRNA and protein levels. As expected, MnSOD activity declined in the HLS group, while GPX activity was enhanced. These results suggest that redox imbalance occurs during muscle atrophy through NOX1 activation, mitochondrial complex I deficiency, and disturbance of antioxidants. Antioxidants altered by HLS may represent potential therapeutic targets for the protection against muscle atrophy.

Periodontal diseases are infectious polymicrobial inflammatory diseases that lead to destruction of the periodontal ligament, gingiva, and alveolar bone. Sequential colonization of a broad range of bacteria, including Fusobacterium nucleatum and Porphyromonas gingivalis , is an important phenomenon in this disease model. F. nucleatum is a facultative anaerobic species thought to be a key mediator of dental plaque maturation due to its extensive coaggregation with other oral bacteria, while P. gingivalis is an obligate anaerobic species that induces gingival inflammation by secreting various virulence factors. The formation of a bacterial complex by these two species is central to the pathogenesis of periodontal disease. Reactive oxygen species (ROS) are produced during bacterial infections and are involved in intracellular signaling. However, the impact of oral bacteria-induced ROS on the ecology of F. nucleatum and P. gingivalis has yet to be clarified. In the present study, we investigated ROS production induced in primary human oral cells by F. nucleatum and P. gingivalis and its effect on the formation of their bacterial complexes and further host cell apoptosis. We found that in primary human gingival fibroblasts (GFs), two NADPH oxidase isoforms, NOX1 and NOX2, were activated in response to F. nucleatum infection but not P. gingivalis infection. Accordingly, increased NADPH oxidase activity and production of superoxide anion were observed in GFs after F. nucleatum infection, but not after P. gingivalis infection. Interestingly, in NOX1, NOX2, or NOX1/NOX2 knockdown cells, the number of P. gingivalis decreased when the cells were coinfected with F. nucleatum . A similar pattern of host cell apoptosis was observed. This implies that F. nucleatum contributes to attachment of P. gingivalis by triggering activation of NADPH oxidase in host cells, which may provide an environment more favorable to strict anaerobic bacteria and have a subsequent effect on apoptosis of host cells.

It is well established that aging is associated with increased susceptibility to infectious diseases. Fusobacterium nucleatum is a well-known bacterial species that plays a central bridging role between early and late colonizers in the human oral cavity. Further, the ability of F. nucleatum to invade gingival fibroblasts (GFs) is critical to the development of periodontal diseases. However, the mechanisms underlying the age-related infection of GFs by F. nucleatum remain unknown. We used young (fourth passage) and senescent (22nd passage) GFs to investigate the mechanisms of F. nucleatum infection in aged GFs and first observed increased invasion of F. nucleatum in senescent GFs. We also found that the co-localization of caveolin-1 (Cav-1), a protein marker of aging, with F. nucleatum and the knockdown of Cav-1 in GFs reduced F. nucleatum invasion. Additionally, F. nucleatum infection triggered the production of reactive oxygen species (ROS) through activation of NADPH oxidase in GFs, but senescent GFs exhibited significantly lower levels of NADPH oxidase activity and ROS production compared with young GFs in both the uninfected and infected conditions. Also, senescent GFs exhibited a decline in proinflammatory cytokine production and extracellular signal regulated kinase (ERK) phosphorylation following F. nucleatum infection. Interestingly, the knockdown of Cav-1 in senescent GFs increased NADPH oxidase activity and caused the upregulation of interleukin-6 and interleukin-8 and the phosphorylation of ERK. Collectively, the increased expression of Cav-1 might play a critical role in F. nucleatum invasion and could hinder the host response in senescent GFs.

The periodontium undergoes age-related cellular and clinical changes, but the involved genes are not yet known. Here, we investigated age-related genetic changes in gingiva at the transcriptomic level. Genes that were differentially expressed between young and old human gingiva were identified by RNA sequencing and verified by real-time PCR. A total of 1939 mRNA transcripts showed significantly differential expression between young and old gingival tissues. Matrix metalloprotease (MMP) regulation was the top pathway involved in gingival aging. MMP3, MMP9, MMP12, and MMP13 were upregulated in old gingival tissues, concomitantly with interleukin-1 beta (IL1B) expression. In vitro experiments using human gingival fibroblasts (hGFs) showed that MMP12 was upregulated in old hGFs compared to young hGFs. Moreover, the MMP3, MMP9 and IL1B levels were more highly stimulated by infection with the oral bacterium, Fusobacterium nucleatum, in old hGFs compared to young hGFs. Collectively, these findings suggest that, in gingiva, the upregulation of MMP12 may be a molecular hallmark of natural aging, while the upregulations of MMP3, MMM9, and IL1B may indicate externally (e.g., infection)-induced aging. These findings contribute to our understanding of the molecular targets involved in gingival aging.

ABSTRACT Objectives: In tissue samples from patients with colorectal cancer (CRC), oxidation of C420 and C457 of plakoglobin (Pg) within tumor tissue was identified by proteomic analysis. The aim of this study was to identify the roles of Pg C420 and C457. Methods: Human CRC tissues, CRC and breast cancer cells, and normal mouse colon were prepared to validate Pg oxidation. MC38 cells were co-transfected with E-cadherin plus wild type (WT) or mutant (C420S or C457S) Pg to evaluate protein interactions and cellular localization, proliferation, and migration. Results: Pg was more oxidized in stage III CRC tumor tissue than in non-tumor tissue. Similar oxidation of Pg was elicited by H2O2 treatment in normal colon and cancer cells. C457S Pg exhibited diminished binding to E-cadherin and α-catenin, and reduced the assembly of E-cadherin–α-/β-catenin complexes. Correspondingly, immunofluorescent analysis of Pg cellular localization suggested impaired binding of C457S Pg to membranes. Cell migration and proliferation were also suppressed in C457S-expressing cells. Discussion: Pg appears to be redox-sensitive in cancer, and the C457 modification may impair cell migration and proliferation by affecting its interaction with the E-cadherin/catenin axis. Our findings suggest that redox-sensitive cysteines of Pg may be the targets for CRC therapy.

BACKGROUND:Peroxiredoxin V (Prdx V) plays a major role in preventing oxidative damage as an effective antioxidant protein within a variety of cells through peroxidase activity. However, the function of Prdx V is not limited to peroxidase enzymatic activity per se. It appears to have unique function in regulating cellular response to external stimuli by directing interaction with signaling protein. In this study, we identified Prdx V interacting partners in mouse kidney under hypoxic stress using immunoprecipitation and shotgun proteomic analysis (LC-MS/MS).RESULTS:Immunoprecipitation coupled with nano-UPLC-MS(E) shotgun proteomics was employed to identify putative interacting partners of Prdx V in mouse kidney in the setting of hypoxia. A total of 17 proteins were identified as potential interacting partners of Prdx V by a comparative interactomics analysis in kidney under normoxia versus hypoxia. Dihydrolipoamide branched chain transacylase E2 (DBT) appeared to be a prominent candidate protein displaying enhanced interaction with Prdx V under hypoxic stress. Moreover, hypoxic kidney exhibited altered DBT enzymatic activity compared to normoxia. An enhanced colocalization of these two proteins under hypoxic stress was successfully observed in vitro. Furthermore, peroxidatic cysteine residue (Cys48) of Prdx V is likely to be responsible for interacting with DBT.CONCLUSIONS:We identified several proteins interacting with Prdx V under hypoxic condition known to induce renal oxidative stress. In hypoxic condition, we observed an enhanced interaction of Prdx V and DBT protein as well as increased DBT enzymatic activity. The results from this study will contribute to enhance our understanding of Prdx V's role in hypoxic stress and may suggest new directions for future research.