Calpains and calpastatin in SH‐SY5Y neuroblastoma cells during retinoic acid‐induced differentiation and neurite outgrowth: Comparison with the human brain calpain system

Abstract

Calpains have importance in human neurodegenerative disease pathogenesis, but these mechanisms are difficult to study in postmortem tissues. To establish a cellular model of the human calpain and calpastatin system, we characterized calpain I, calpain II, and calpastatin in SH-SY5Y human neuroblastoma cells in relation to their counterparts in human brain and investigated their expression and activity after inducing cellular differentiation with retinoic acid (RA), a physiological effector of normal brain development. Calpain I in both SH-SY5Y cells and human brain existed in the cytosolic and particulate fractions as three isoforms (80, 78, and 76 kDa) and exhibited atypical isoelectric focusing behavior. Calpain II in SH-SY5Y cells, as in human brain, migrated as a single predominantly cytosolic 76-kDa protein with an isoelectric point ranging from 5.9 to 6.3. Calpastatin from both sources was also 90% cytosolic. In the cells it was composed of four discrete bands, ranging in molecular weight from 110 to 127 kDa. Levels of activated (76 and 78 kDa) and precursor (80 kDa) calpain I isoforms rose 54% (P < 0.0001) in the particulate fraction and 26% (P < 0.0001) in the soluble fraction after 3 days of RA exposure. Because levels and activity of calpastatin remain unchanged during the first 7 days of RA exposure, the increased abundance of calpain I implies a net activation of the calpain system during differentiation. Calpain I activation may contribute to the remodeling of cell shape and neurite extension/retraction associated with neuronal differentiation.

Proteasome Inhibitor PS-341 (Bortezomib) Induces Calpain-dependent IκBα Degradation

“…In Western blotting, we detected multiple sizes (e.g. ϳ110, 70, and 40 kDa) of calpastatin, which are identical to published observations (27,28). siRNA-mediated silencing of calpastatin substantially reduced the levels of these proteins, indicating that they are all specific calpastatin proteins.…”

Mechanistic Role of Calpains in Postischemic Neurodegeneration

“…A similarly wide range of physiologic functions has been attributed to calpain within the brain and neuronal culture systems. Calpain has been implicated in the differentiation of neuronal cell lines (Grynspan et al, 1997), neurite outgrowth in hippocampal neuronal culture (Song et al, 1994), as well as synaptic remodeling and long-term potentiation (Tomimatsu et al, 2002).…”

A Brain-specific Grb2-associated Regulator of Extracellular Signal-regulated Kinase (Erk)/Mitogen-activated Protein Kinase (MAPK) (GAREM) Subtype, GAREM2, Contributes to Neurite Outgrowth of Neuroblastoma Cells by Regulating Erk Signaling

“…Therefore, GAREM2 regulates Erk activation after growth factor stimulation in SH-SY5Y cells. (23)(24)(25)(26)(27). Activation of Erk with IGF-I stimulation is necessary for this phenomenon (28).…”