Related Experiment Videos
Impairment of NF-kappaB activation and modulation of gene expression by calpastatin
F Chen1, L M Demers, V Vallyathan
1Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia 26505, USA.
Abstract:
To address the involvement of the calpain system in both basal and silica-induced nuclear factor (NF)-kappaB activation, several human bronchial epithelial cell lines were established in which an intracellular inhibitor of calpain, calpastatin, was stably expressed. Reduced basal and silica-induced inhibitor (IkappaBalpha) degradation and NF-kappaB activation were observed in cells stably overexpressing calpastatin. In addition, the cells in which calpain was constitutively inhibited by the overexpression of calpastatin exhibited a notable morphological change. Whereas empty vector-transfected cells displayed a morphology indistinguishable from that of parental cells, cells overexpressing calpastatin exhibited a mosaic morphological change with reduced formation of lamella 30 min after the cells were seeded. Genefilter microarray experiments, in which 3,965 human genes can be evaluated for their expression at the same time, showed that calpastatin downregulated genes encoding several membrane-associated proteins or nuclear proteins and upregulated genes of collagen alpha2, DAZ, and mitochondrial capsule selenoprotein. These results suggest that, in addition to their proteolytic activities on cytoskeletal proteins and other cellular regulatory proteins, calpain-calpastatin systems can also affect the expression levels of genes encoding structural or regulatory proteins.
Insights
The calpain-calpastatin system influences nuclear factor-kappaB (NF-κB) activation and cell morphology. Overexpressing calpastatin, an inhibitor, reduced NF-κB signaling and altered cell structure, impacting gene expression.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The calpain system plays a role in cellular processes.
- Nuclear factor-kappaB (NF-κB) activation is crucial in cellular responses.
- Silica exposure can induce inflammatory responses.
Purpose of the Study:
- To investigate the role of the calpain system in basal and silica-induced NF-κB activation.
- To determine the effects of inhibiting calpain on bronchial epithelial cells.
Main Methods:
- Established human bronchial epithelial cell lines stably expressing calpastatin, an intracellular inhibitor of calpain.
- Assessed inhibitor of kappaBalpha (IκBα) degradation and NF-κB activation.
- Analyzed cell morphology using microscopy.
- Performed Genefilter microarray experiments to evaluate gene expression.
Main Results:
- Overexpression of calpastatin reduced basal and silica-induced IκBα degradation and NF-κB activation.
- Cells overexpressing calpastatin showed altered morphology with reduced lamella formation.
- Microarray analysis revealed calpastatin downregulated genes for membrane-associated and nuclear proteins, while upregulating collagen alpha2, DAZ, and mitochondrial capsule selenoprotein.
Conclusions:
- The calpain-calpastatin system is involved in regulating NF-κB activation in bronchial epithelial cells.
- Calpain inhibition affects cell morphology and gene expression, including structural and regulatory proteins.
- These findings suggest a broader role for calpains beyond proteolytic activity.