Calpains and calpastatin in SH-SY5Y neuroblastoma cells during retinoic acid-induced differentiation and neurite outgrowth: comparison with the human brain calpain system.
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.
Grynspan
F
F
Laboratories of Molecular Neuroscience, Mailman Research Center, McLean Hospital, Belmont, MA 02178, USA.
Griffin
W B
WB
Mohan
P S
PS
Shea
T B
TB
Nixon
R A
RA
D001921:Brain / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002154:Calpain / Q000737:chemistry / Q000378:metabolism*; D002454:Cell Differentiation; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism*; D006801:Humans; D007536:Isomerism; D008970:Molecular Weight; D016501:Neurites / Q000502:physiology*; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D014212:Tretinoin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; D014212:Tretinoin; C033668:calpastatin; D002154:Calpain