RESUMO
Human perivascular stem/stromal cells (hPSC) are a multipotent mesenchymogenic stromal cell population defined by their perivascular locale. Recent studies have demonstrated the high potential for clinical translation of this fluorescence-activated cell sorting (FACS)-derived cell population for autologous bone tissue engineering. However, the mechanisms underlying the osteogenic differentiation of PSC are incompletely understood. The current study investigates the roles of canonical and noncanonical Wnt signaling in the osteogenic and adipogenic differentiation of PSC. Results showed that both canonical and noncanonical Wnt signaling activity transiently increased during PSC osteogenic differentiation in vitro. Sustained WNT3A treatment significantly decreased PSC osteogenic differentiation. Conversely, sustained treatment with Wnt family member 16 (WNT16), a mixed canonical and noncanonical ligand, increased osteogenic differentiation in a c-Jun N-terminal kinase (JNK) pathway-dependent manner. Conversely, WNT16 knockdown significantly diminished PSC osteogenic differentiation. Finally, WNT16 but not WNT3A increased the adipogenic differentiation of PSC. These results indicate the importance of regulation of canonical and noncanonical Wnt signaling for PSC fate and differentiation. Moreover, these data suggest that WNT16 plays a functional and necessary role in PSC osteogenesis.
Assuntos
Adipogenia/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Proteínas Wnt/farmacologia , Proteína Wnt3A/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Engenharia Tecidual/métodos , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Neural epidermal growth factor-like (NEL)-like protein 1 (NELL-1) has been identified as an osteoinductive differentiation factor that promotes mesenchymal stem cell (MSC) osteogenic differentiation. In addition to full-length NELL-1, there are several NELL-1-related transcripts reported. We used rapid amplification of cDNA ends to recover potential cDNA of NELL-1 isoforms. A NELL-1 isoform with the N-terminal 240 amino acid (aa) residues truncated was identified. While full-length NELL-1 that contains 810 aa residues (NELL-1810 ) plays an important role in embryologic skeletal development, the N-terminal-truncated NELL-1 isoform (NELL-1570 ) was expressed postnatally. Similar to NELL-1810 , NELL-1570 induced MSC osteogenic differentiation. In addition, NELL-1570 significantly stimulated MSC proliferation in multiple MSC-like populations such as murine C3H10T1/2 MSC cell line, mouse primary MSCs, and perivascular stem cells, which is a type of stem cells proposed as the perivascular origin of MSCs. In contrast, NELL-1810 demonstrated only limited stimulation of MSC proliferation. Similar to NELL-1810 , NELL-1570 was found to be secreted from host cells. Both NELL-1570 expression lentiviral vector and column-purified recombinant protein NELL-1570 demonstrated almost identical effects in MSC proliferation and osteogenic differentiation, suggesting that NELL-1570 may function as a pro-osteogenic growth factor. In vivo, NELL-1570 induced significant calvarial defect regeneration accompanied by increased cell proliferation. Thus, NELL-1570 has the potential to be used for cell-based or hormone-based therapy of bone regeneration.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Glicoproteínas/genética , Células-Tronco Mesenquimais/citologia , Proteínas do Tecido Nervoso/genética , Osteogênese/fisiologia , Animais , Proteínas de Ligação ao Cálcio/biossíntese , Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular , Proliferação de Células/fisiologia , Glicoproteínas/biossíntese , Glicoproteínas/metabolismo , Células HEK293 , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/metabolismo , Isoformas de Proteínas , Ratos , Ratos Sprague-DawleyRESUMO
all-trans retinoic acid (atRA), the oxidative metabolite of vitamin A, is essential for normal embryonic development. Also, high levels of atRA are teratogenic in many species and can effectively induce cleft palate in the mouse. Most cleft palate resulted from the failed fusion of secondary palate shelves, and maintenance of the normal cell proliferation is important in this process of shelf growth. To clarify the mechanism by which atRA causes cleft palate, we investigated the effect of atRA on proliferation activity and cell cycle distribution in mouse embryonic palatal mesenchymal (MEPM) cells. atRA inhibited the growth of MEPM cells by inducing apoptosis in a dose-dependent manner. atRA also caused a G1 block in the cell cycle with an increase in the proportion of cells in G0/G1 and a decrease in the proportion of cells in S phase, as determined by flow cytometry. We next investigated the effects of atRA on molecules that regulate the G1 to S phase transition. These studies demonstrated that atRA inhibited expression of cyclins D and E at the protein level. Furthermore, atRA treatment reduced phosphorylated Rb and decreased cdk2 and cdk4 kinase activity. These data suggest that atRA had antiproliferative activity by modulating G1/S cell cycle regulators and by inhibition of Rb phosphorylation in MEPM cells, which might account for the pathogenesis of cleft palate induced by retinoic acid.