In this study, we investigated the effect of oxidative stress in hADMPCs on the induction of neuronal differentiation. Such mechanisms may explain how administration of hADMPCs to neurodegenerative lesions enhances endogenous repair mechanisms via neurogenesis of endogenous neural progenitor and stem cells. Damaged tissues, such as the brain tissue of patients who have suffered from ischemic stroke, are subject to inflammation and the generation of reactive oxygen species (ROS) [17
]. Our data demonstrated that hADMPCs, when exposed to oxidative stress, facilitate neuronal differentiation in rat pheochromocytoma cell line PC12 cells by upregulation of fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 2 (BMP2) secretion through p38 MAPK activation.
Our results show that BMP2 and FGF2 were upregulated in hADMPCs when exposed to buthionine sulfoximine (BSO), a glutathione-synthesis inhibitor that leads to oxidative stress. These findings may have therapeutic implications in neurodegenerative diseases. We concluded that BMP2 and FGF2 secreted from hADMPCs that had been exposed to oxidative stress were the main inducers of neurite outgrowth in PC12 cells. Erk1/2 and Smad1/5/8 were significantly activated in these cells (Figure
), while other growth factors known to induce neurite outgrowth in PC12 cells such as nerve growth factor (NGF) and vascular endothelial growth factor (VEGF) were not observed to be upregulated by BSO treatment (data not shown). We confirmed that BMP2 enhanced the effect that FGF2 had on the differentiation of PC12 cells (Figure
), supporting our idea that hADMPCs under oxidative stress conditions secrete BMP2 and FGF2 and that this contributes to neuronal differentiation. Consistent with our conclusions, it has been reported that BMP2, via activation of a Smad signaling pathway, facilitated FGF2-induced neuronal differentiation in PC12 cells [26
]. However, since hADMPCs have been reported to secrete many growth factor including NGF, VEGF, HGF, and IGF [11
], we cannot exclude the possibility that BMP2 and FGF2 are acting cooperatively with these growth factors to facilitate neurite outgrowth in PC12 cells. Thus, the precise molecular mechanisms of induction of PC12 differentiation and the precise expression profiles in BSO-treated hADMPCs need to be further investigated.
Recently, BMP signaling through Smad1/5/8 has been reported to contribute to neurite outgrowth in dorsal root ganglion neurons both in vitro and in vivo [34
]. Moreover, BMP2 has been shown to have neurotrophic effects on midbrain dopaminergic neurons [36
], ventral mesencephalic neurons [37
], mouse embryonic striatal neurons [38
], and nitrergic and catecholaminergic enteric neurons [39
]. Moreover, FGF2 is trophic for neurons, glias, and endothelial cells in the central nervous system. FGF2 also prevents downregulation of the anti-apoptotic protein Bcl-2 in ischemic brain tissue and limits excitotoxic damage to the brain through an activin-dependent mechanism [40
]. These findings are consistent with our hypothesis that hADMPCs secret BMP2 and FGF2 to induce neurogenesis in neurodegenerative lesions in response to oxidative stress.
As it has been shown that ROS activate ERKs, JNKs, and p38 MAPKs [28
], we examined the MAPK signaling pathway in hADMPCs exposed to oxidative stress and found that BSO treatment resulted in significant activation of ERK1/2 and p38 MAPK. Intriguingly, addition of SB203580, a specific inhibitor of p38 MAPK, but not the ERK inhibitor U0126, suppressed BMP2
expression in BSO-treated hADMPCs to control levels (Figure
), suggesting that p38 MAPK was contributing to upregulation of BMP2 and FGF2 in hADMPCs when exposed to oxidative stress. Moreover, lentiviral transduction of the constitutively active form of MKK6, a MAPKK that selectively activates p38 MAPK isoforms [30
], resulted in upregulation of BMP2 and FGF2 and this also demonstrated the crucial role of the p38 MAPK cascade in the regulation of BMP2 and FGF2. In primary human endothelial cells, p38-dependent regulation of BMP2 expression was reported previously. Viemann et al.
] investigated the genes that were induced by inflammatory stimulation with tumor necrosis factor α (TNF-α) and classified these genes into 2 categories based on whether they were regulated in an NF-κB-dependent or p38 MAPK-dependent manner. Consistent with our findings, they found that significant induction of BMP2 expression by TNF-α was markedly suppressed by SB202190, an inhibitor of p38 MAPK. These results support the hypothesis that activation of the p38 MAPK pathway in hADMPCs in response to inflammation surrounding neurodegenerative lesions leads to induction of BMP2 and FGF2, which in turn support regeneration of neuronal cells.
It has been known that NF-κB directly binds to the BMP2 promoter to induce its expression [31
], and MSK1, a downstream molecule of p38 MAPK, is involved in NF-κB transactivation [32
]. However, we did not observe an elevation of NF-κB transcriptional activity in hADMPCs when they were exposed to oxidative stress (Figure
). The mechanism of p38-dependent regulation of gene expression is not completely understood, and the precise mechanism by which p38 MAPK regulates the expression of BMP2 and FGF2 remains to be determined.
In this study, we also found that suppression of ERK1/2 MAPK by U0126 in BSO-treated hADMPCs resulted in slight activation of p38 MAPK (Figure
A). Consistent with this, the expression level of BMP2
mRNA was also upregulated when cells exposed to oxidative stress were pretreated with U0126 (Figure
C). Previously, “seesaw cross-talk” between ERK and p38 MAPK signaling has been reported; i.e., the MEK inhibitor caused a decrease in the phosphorylation level of ERK and an increase in that of p38, whereas the p38 inhibitor had the opposite effect [42
]. We did not investigate the phosphorylation of ERK1/2 in SB203580-treated hADMPCs, but it may be possible that seesaw cross-talk also occurs in our system.