title: Cysteine proteases in differentiation of embryonic stem cells into neural cells.
abstract: Glycosylated mouse cystatin C (mCysC), an endogenous inhibitor of cysteine cathepsin proteases (CP), has been suggested as a cofactor of β-FGF to induce the differentiation of mouse embryonic stem cells into neural progenitor cells (NPCs). To investigate the possible role of CP in neural differentiation, we treated embryoid bodies (EBs) with (i) E64, an inhibitor of papain-like CP and of calpains, (ii) an inhibitor of cathepsin L (iCatL), (iii) an inhibitor of calpains (iCalp), or (iv) cystatins, and their ability to differentiate into neural cells was assessed. We show that the inhibition of CP induces a significant increase in Pax6 expression in EBs, leading to an increase in the number of nestin-positive cells after 3 days. Fourteen days after E64 treatment, we observed increased numbers of β-III-tubulin-positive cells, showing greater percentage of immature neurons, and this feature persisted up to 24 days. At this point, we encountered higher numbers of neurons with inward Na(+) current compared with untreated EBs. Further, we show that mCysC and iCatL, but not unglycosylated egg white cystatin or iCalp, increased the numbers of NPCs. In contrast to E64 and iCatL, mCysC did not inhibit CP in EBs and its neural-inducing activity required β-FGF. We propose that the inhibition of CP induces the differentiation of mouse embryonic stem cells into NPCs and neurons through a mechanism that is distinct from CysC-induced neural differentiation.
authors.affiliation: Programa de Terapias Celulares/PROTECEL, Instituto de Biofísica Carlos Chagas Filho, Rio de Janeiro, Brazil. razevedo@biof.ufrj.br
authors.forename: Ricardo Luiz
authors.initials: RL
authors.lastname: de Azevedo-Pereira
authors.forename: Ana Paula C A
authors.initials: AP
authors.lastname: Lima
authors.forename: Deivid de Carvalho
authors.initials: Dde C
authors.lastname: Rodrigues
authors.forename: Edson
authors.initials: E
authors.lastname: Rondinelli
authors.forename: Emiliano Horácio
authors.initials: EH
authors.lastname: Medei
authors.forename: Regina Coeli
authors.initials: RC
authors.lastname: Goldenberg
authors.forename: Antonio Carlos Campos Campos
authors.initials: AC
authors.lastname: de Carvalho
authors.forename: Rosalia
authors.initials: R
authors.lastname: Mendez-Otero
mesh_terms: D000818:Animals; D000943:Antigens, Differentiation / Q000235:genetics / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors; D056668:Cathepsin L / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D022081:Cell Surface Extensions / Q000378:metabolism; D018920:Coculture Techniques; D055316:Cystatin C / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004268:DNA-Binding Proteins; D058732:Embryoid Bodies / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D053595:Embryonic Stem Cells / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D005136:Eye Proteins / Q000378:metabolism; D019922:GAP-43 Protein / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D007381:Intermediate Filament Proteins / Q000235:genetics / Q000378:metabolism; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008564:Membrane Potentials; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D064231:Nestin; D016900:Neurofilament Proteins / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000201:enzymology; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism
chemical_list: D000943:Antigens, Differentiation; C525031:Cst3 protein, mouse; D055316:Cystatin C; D015853:Cysteine Proteinase Inhibitors; D004268:DNA-Binding Proteins; D005136:Eye Proteins; D019922:GAP-43 Protein; D018398:Homeodomain Proteins; D007381:Intermediate Filament Proteins; C577484:NES protein, human; D009419:Nerve Tissue Proteins; C577485:Nes protein, mouse; D064231:Nestin; C492380:NeuN protein, mouse; D016900:Neurofilament Proteins; D009687:Nuclear Proteins; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D012097:Repressor Proteins; C051774:neurofilament protein H; D002154:Calpain; D056668:Cathepsin L; C534278:Ctsl protein, mouse; D007930:Leucine; C024974:E 64