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Respiratory viruses, such as influenza, decrease airway cilia function and expression, which leads to reduced mucociliary clearance and inhibited overall immune defense. Ubiquitination is a posttranslational modification using E3 ligases, which plays a role in the assembly and disassembly of cilia. We examined the role of membrane-associated RING-CH (MARCH) family of E3 ligases during influenza infection and determined that MARCH10, specifically expressed in ciliated epithelial cells, is significantly decreased during influenza infection in mice, human lung epithelial cells, and human lung tissue. Cellular depletion of MARCH10 in differentiated human bronchial epithelial cells (HBECs) using CRISPR/Cas9 showed a decrease in ciliary beat frequency. Furthermore, MARCH10 cellular knockdown in combination with influenza infection selectively decreased immunoreactive levels of the ciliary component, dynein axonemal intermediate chain 1. Cellular overexpression of MARCH10 significantly decreased influenza hemagglutinin protein levels in the differentiated HBECs and knockdown of MARCH10 increased IL-1b cytokine expression, whereas overexpression had the reciprocal effect. These findings suggest that MARCH10 may have a protective role in airway pulmonary host defense and innate immunity during influenza infection. [ABSTRACT FROM AUTHOR]

FBXL2 is an important ubiquitin E3 ligase component that modulates inflammatory signaling and cell cycle progression, but its molecular regulation is largely unknown. Here we show that TNFα, a critical cytokine linked to the inflammatory response during skeletal muscle regeneration, suppressed Fbxl2 mRNA expression in C2C12 myoblasts and triggered significant alterations in cell cycle, metabolic, and protein translation processes. Gene silencing of Fbxl2 in skeletal myoblasts resulted in increased proliferative responses characterized by activation of mitogen activated protein (MAP) kinases and nuclear factor kappa B and decreased myogenic differentiation as reflected by reduced expression of myogenin and impaired myotube formation. TNFa did not destabilize the Fbxl2 transcript (t 10 h), but inhibited SP1 transactivation of its core promoter, localized to 160/+42 base pairs within the proximal 5' flanking region of the Fbxl2 gene. Chromatin immunoprecipitation and gel shift studies indicated that SP1 interacted with the Fbxl2 promoter during cellular differentiation, an effect less pronounced during proliferation or after TNFα exposure. TNFα, via activation of JNK, mediated phosphorylation of SP1 that impaired its binding to the Fbxl2 promoter resulting in reduced transcriptional activity. The results suggest that SP1 transcriptional activation of Fbxl2 is required for skeletal muscle differentiation, a process that is interrupted by a key pro-inflammatory myopathic cytokine. [ABSTRACT FROM AUTHOR]