Calpastatin upregulation in Mycoplasma hyorhinis-infected cells is promoted by the mycoplasma lipoproteins via the NF-κB pathway.

Mycoplasma hyorhinis frequently contaminates cultured cells, with effects on synthetic and metabolic pathways. We demonstrated for the first time that contamination of cells by a strain of M. hyorhinis (NDMh) results in increased levels of calpastatin (the endogenous inhibitor of the ubiquitous Ca(2+) -dependent protease calpain). We now show that the calpastatin upregulation by NDMh in neuroblastoma SH-SY5Y cells resides in the NDMh lipoprotein fraction (LPP), via the NF-κB transcription pathway. NF-κB activation requires dissociation of the cytoplasmic NF-κB/IκB complex followed by NF-κB translocation to the nucleus. NDMh-LPP induced translocation of the NF-κB RelA subunit to the nucleus and upregulated calpastatin. RelA translocation and calpastatin elevation were prevented when dissociation of the NF-κB/IκB complex was inhibited either by transfection with the non-phosphorylatable IκB mutant ΔNIκBα, or by using PS1145, an inhibitor of the IκB kinase (IKK complex). Increased calpastatin levels attenuate calpain-related amyloid-ß-peptide and Ca(2+) -toxicity (these are central to the pathogenesis of Alzheimer's Disease). LPP-induced elevation of calpastatin provides an example of effects on non-inflammatory intracellular proteins, the outcome being significant alterations in host cell functions. Since calpastatin level is important in the control of calpain activity, mycoplasmal LPP may be of interest in treating some pathological processes involving excessive calpain activation.

Neuroprotective effects of Mycoplasma hyorhinis against amyloid-ß-peptide toxicity in SH-SY5Y human neuroblastoma cells are mediated by calpastatin upregulation in the mycoplasma-infected cells.

Mycoplasmas are frequent contaminants of cell cultures. Contamination leads to altered synthetic and metabolic pathways. We have found that contamination of neuroblastoma SH-SY5Y cells by a strain of Mycoplasma hyorhinis derived from SH-SY5Y cell culture (NDMh) leads to increased levels of calpastatin (the endogenous inhibitor of the Ca(2+)-dependent protease, calpain) in NDMh-infected cells. We have now examined effects of amyloid-ß-peptide (Aß) (central to the pathogenesis of Alzheimer's disease) on uncontaminated (clean) and NDMh-infected SH-SY5Y cells. Aß was toxic to clean cells, resulting in necrotic cell damage. Aß treatment led to activation of calpain and enhanced proteolysis, cell swelling, cell membrane permeability to propidium iodide (PI) (without nuclear apoptotic changes), and diminished mitochondrial enzyme activity (XTT reduction). Aß-toxicity was attenuated in the high calpastatin-containing NDMh-infected cells, as shown by inhibition of calpain activation and activity, no membrane permeability, normal cell morphology, and maintenance of mitochondrial enzyme activity (similar to attenuation of Aß-toxicity in non-infected cells overexpressing calpastatin following calpastatin-plasmid introduction into the cells). By contrast, staurosporine affected both clean and infected cells, causing apoptotic damage (cell shrinkage, nuclear apoptotic alterations, caspase-3 activation and caspase-promoted proteolysis, without PI permeability, and without effect on XTT reduction). The results indicate that mycoplasma protects the cells against certain types of insults involving calpain. The ratio of calpastatin to calpain is an important factor in the control of calpain activity. Exogenous pharmacological means, including calpastatin-based inhibitors, have been considered for therapy of various diseases in which calpain is implicated. Mycoplasmas provide the first naturally occurring biological system that upregulates the endogenous calpain inhibitor, and thus may be of interest in devising treatments for some disorders, such as neurodegenerative diseases.

Mycoplasma hyorhinis upregulates calpastatin and inhibits calpain-dependent proteolysis in SH-SY5Y neuroblastoma cells.

Mycoplasmas often contaminate cultured cells, leading to alterations in cellular gene expression, protein synthesis, signal transduction and metabolic pathways. Mycoplasmal contamination is often unnoticed, so that mycoplasma-induced alterations in cell functions may not be appreciated, unless specifically studied. Here, we show for the first time that contamination of SH-SY5Y cells by Mycoplasma hyorhinis leads to increased levels of calpastatin (the endogenous inhibitor of the Ca(2+)-dependent protease calpain), resulting in inhibition of Ca(2+)-induced calpain activation and inhibition of calpain-promoted proteolysis in the mycoplasmal-infected cells. Calpain activity is recovered upon calpastatin removal from extracts of contaminated cells. The calpain-calpastatin system has been implicated in a variety of physiological and pathological processes (signal transduction, motility, cell cycle, cell differentiation, membrane damage and apoptosis). Because the ratio of calpastatin to calpain is an important factor in the control of calpain activity within the cell, the elevated calpastatin may protect the mycoplasma-infected cells against certain types of damage (e.g. caused by high Ca(2+)). Thus, our results are important for studies on the modulation of host cells by mycoplasmas, and relevant to the pathobiology of processes involving mycoplasmal infections. The mycoplasma-infected cells provide a system for identifying factors that participate in the regulation of cellular calpastatin.

Amyloid beta peptide toxicity in differentiated PC12 cells: calpain-calpastatin, caspase, and membrane damage.

Amyloid beta peptide (Abeta) is implicated in the pathogenesis of Alzheimer's disease (AD). The peptide is toxic to neurons, possibly by causing initial synaptic dysfunction and neuronal membrane dystrophy, promoted by increased cellular Ca(2+). Calpain (Ca(2+)-dependent protease) and caspase have also been implicated in AD. There is little information on communication between the two proteases or on the involvement of calpastatin (the specific calpain inhibitor) in Abeta toxicity. We studied the effects of Abeta25-35 (sAbeta) on calpain, calpastatin, and caspase in neuronal-like differentiated PC12 cells. sAbeta-treated cells exhibited primarily cell membrane damage (varicosities along neurites, enhanced membrane permeability to propidium iodide, without apparent nuclear changes of apoptosis, and little poly (ADP-ribose) polymerase [PARP] degradation). The sAbeta-induced membrane damage is in contrast with staurosporine-induced damage (nuclear apoptotic changes, PARP degradation, without membrane propidium iodide permeability). sAbeta led to activation of caspase-8 and calpain, promotion of calpastatin degradation (by caspase-8 and by calpain), and enhanced degradation of fodrin (mainly by calpain). The results support the idea that Abeta causes primarily neuronal membrane dysfunction, and point to cross-talk between calpain and caspase (protease activation and degradation of calpastatin) in Abeta toxicity. Increased expression of calpastatin and/or decrease in calpain and caspase-8 may serve as means for ameliorating early symptoms of AD.