RESUMO
BACKGROUND: The ability of human bone marrow mesenchymal stem cells (BM-MSCs) to migrate and localize specifically to injured tissues is central in developing therapeutic strategies for tissue repair and regeneration. Fibroblast activation protein (FAP) is a cell surface serine protease expressed at sites of tissue remodeling during embryonic development. It is also expressed in BM-MSCs, but not in normal tissues or cells. The function of FAP in BM-MSCs is not known. PRINCIPAL FINDINGS: We found that depletion of FAP proteins significantly inhibited the migration of BM-MSCs in a transwell chemotaxis assay. Such impaired migration ability of BM-MSCs could be rescued by re-expressing FAP in these cells. We then demonstrated that depletion of FAP activated intracellular RhoA GTPase. Consistently, inhibition of RhoA activity using a RhoA inhibitor rescued its migration ability. Inhibition of FAP activity with an FAP-specific inhibitor did not affect the activation of RhoA or the migration of BM-MSCs. Furthermore, the inflammatory cytokines interleukin-1beta (IL-1ß) and transforming growth factor-beta (TGF-ß) upregulated FAP expression, which coincided with better BM-MSC migration. CONCLUSIONS: Our results indicate FAP plays an important role in the migration of BM-MSCs through modulation of RhoA GTPase activity. The peptidase activity of FAP is not essential for such migration. Cytokines IL-1ß and TGF-ß upregulate the expression level of FAP and thus enhance BM-MSC migration.
Assuntos
Gelatinases/genética , Proteínas de Membrana/genética , Células-Tronco Mesenquimais/metabolismo , Serina Endopeptidases/genética , Proteína rhoA de Ligação ao GTP/genética , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Linhagem Celular , Quimiotaxia , Endopeptidases , Inibidores Enzimáticos/farmacologia , Gelatinases/antagonistas & inibidores , Gelatinases/deficiência , Regulação da Expressão Gênica , Teste de Complementação Genética , Humanos , Interleucina-1beta/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/deficiência , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Cultura Primária de Células , Serina Endopeptidases/deficiência , Transdução de Sinais , Fator de Crescimento Transformador beta/farmacologia , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Fibroblast activation protein (FAP) is a prolyl-cleaving endopeptidase proposed as an anti-cancer drug target. It is necessary to define its cleavage-site specificity to facilitate the identification of its in vivo substrates and to understand its biological functions. We found that the previously identified substrate of FAP, α(2)-anti-plasmin, is not a robust substrate in vitro. Instead, an intracellular protein, SPRY2, is cleavable by FAP and more suitable for investigation of its substrate specificity in the context of the full-length globular protein. FAP prefers uncharged residues, including small or bulky hydrophobic amino acids, but not charged amino acids, especially acidic residue at P1', P3 and P4 sites. Molecular modelling analysis shows that the substrate-binding site of FAP is surrounded by multiple tyrosine residues and some negatively charged residues, which may exert least preference for substrates with acidic residues. This provides an explanation why FAP cannot cleave interleukins, which have a glutamate at either P4 or P2', despite their P3-P2-P1 sites being identical to SPRY2 or α-AP. Our study provided new information on FAP cleavage-site specificity, which differs from the data obtained by profiling with a peptide library or with the denatured protein, gelatin, as the substrate. Furthermore, our study suggests that negatively charged residues should be avoided when designing FAP inhibitors.