BACKGROUND
mesenchymal stem cells  are pluripotent stem cells that can differentiate into various types of cells  <cit> .
these cells have been isolated from bone marrow  <cit> , umbilical cord blood  <cit> , and adipose tissue  <cit>  and can be easily obtained and expanded ex vivo under appropriate culture conditions.
thus, mscs are an attractive material for cell therapy and tissue engineering.
human adipose tissue-derived mesenchymal stem cells, also referred to as human adipose tissue-derived multilineage progenitor cells , are especially advantageous because they can be easily and safely obtained from lipoaspirates, and the ethical issues surrounding other sources of stem cells can be avoided  <cit> .
moreover, hadmpcs have more pluripotent properties for regenerative medical applications than other stem cells, since these cells have been reported to have the ability to migrate to the injured area and differentiate into hepatocytes  <cit> , cardiomyoblasts  <cit> , pancreatic cells  <cit> , and neuronal cells  <cit> .
in addition, it is known that hadmpcs secrete a wide variety of cytokines and growth factors necessary for tissue regeneration including nerve growth factor , brain-derived neurotrophic factor , fibroblast growth factors , vascular endothelial growth factor  and hepatocyte growth factor   <cit> .
recently, several groups have reported that hadmpcs facilitate neurological recovery in experimental models of stroke  <cit>  and parkinson’s disease  <cit> .
despite the superiority of hadmpcs over other stem cells, the potential use of hadmpcs for the treatment of these neurodegenerative disorders has not been fully investigated.
it has been reported that administration of hadmpcs in animal models of acute ischemic stroke markedly decreased brain infarct size, improved neurological function by enhancing angiogenesis and neurogenesis, and showed anti-inflammatory and anti-apoptotic effects  <cit> .
these effects were due in part to increased secretion levels of vegf, hgf and bfgf under hypoxic conditions  <cit> , indicating the role of hadmpcs in reducing the severity of hypoxia-ischemic lesions.
in addition to hypoxic stress, ischemic lesions are generally subject to inflammation, which leads to the generation of reactive oxygen species   <cit> .
ros are generated as a natural byproduct of normal aerobic metabolism, and mitochondrial respiration, together with oxidative enzymes such as plasma membrane oxidase, is considered to be the major intracellular source of ros production  <cit> .
although appropriate levels of ros play an important role in several physiological processes, oxidative damage initiated by excessive ros causes many pathological conditions including inflammation, atherosclerosis, aging, and cancer.
neuronal cells are especially vulnerable to oxidative stress, and numerous studies have examined the crucial roles of oxidative stress in neurodegenerative disorders such as stroke  <cit> , alzheimer’s disease  <cit> , and parkinson’s disease  <cit> .
in these diseases, microglia, the macrophages of the central nervous system , are activated in response to a local inflammation  <cit>  and generate large amounts of reactive oxygen and nitrogen species, thereby exposing nearby neurons to stress  <cit> .
thus, the influence of oxidative stress generated by neurodegenerative lesion on hadmpcs needs to be further studied.
in this study, we examined the role of oxidative stress on hadmpcs in neurite outgrowth in cells of the rat pheochromocytoma cell line .
upon treatment with buthionine sulfoximine , an inhibitor of the rate-limiting enzyme in the synthesis of glutathione, hadmpcs accumulated ros, which resulted in the upregulation of expression levels of the neurotrophic factors bmp <dig> and fgf <dig>  our present data thus provide new insights into understanding the mechanism of how hadmpcs exposed to oxidative stress contribute to neurogenesis, and this may explain the effects of stem cell transplantation therapy with hadmpcs in treating ischemic stroke.
RESULTS
hadmpcs exposed to oxidative stress stimulate neurite outgrowth in pc <dig> cells
hadmpcs were treated with 1 mm bso for 24 h; a group of hadmpcs that were not given any treatment was used as the control group.
as shown in figure 1a and b, bso treatment resulted in significant reduction of intracellular reduced glutathione levels, followed by accumulation of intracellular reactive oxygen species  in hadmpcs.
to investigate whether accumulation of ros affects secretion of cytokines from hadmpcs, conditioned medium from bso-treated ) or bso-untreated ) hadmpcs was added to pc <dig> cells.
as expected, addition of ngf significantly induced neurite outgrowth in the pc <dig> cells .
hadmpcs, like other mesenchymal stem cells derived from bone marrow or adipose tissue, may secrete many cytokines including ngf, bdnf and fgf <dig>  and this may account for the slight induction of neurite outgrowth seen in the cm-bso  treated cells .
in contrast, the number and length of neurite outgrowth of pc <dig> cells in cm-bso   was markedly enhanced compared with those in cm-bso  .
conditioned medium from bso-treated hadmpcs activates erk1/ <dig> mapk and smad signaling in pc <dig> cells
to investigate which intracellular signaling pathways were involved in the neurite outgrowth of pc <dig> cells in cm-bso , we used western blotting to determine the phosphorylation levels of erk1/ <dig> mapk, p <dig> mapk, smad1/5/ <dig> and akt in pc <dig> cells in various culture conditions.
ngf significantly activated erk1/ <dig> mapk and akt signaling pathway .
in contrast, erk1/ <dig> mapk was not activated in pc <dig> cells exposed to cm-bso , while an increase in phosphorylated smad1/5/ <dig> was observed.
interestingly, cm-bso  treatment led to both a significant increase in smad1/5/ <dig> phosphorylation levels as well as activation of the erk1/ <dig> mapk signaling pathway in pc <dig> cells .
akt was 2-fold activated in both cm-bso  and cm-bso  treated pc <dig> cells, but no significant difference between the  <dig> groups was observed.
fgf <dig> and bmp <dig> are upregulated through p <dig> mapk signaling in hadmpcs exposed to oxidative stress
we next examined which growth factors or cytokines from bso-treated hadmpcs were involved in stimulation of neurite outgrowth.
we found that both mrna  and protein  levels for bmp <dig> and fgf <dig> were markedly increased in hadmpcs treated with bso.
to determine if this upregulation was caused by ros, all cells were exposed to the antioxidant n-acetylcysteine .
as we expected, addition of nac to bso-treated hadmpcs reduced the expression levels of bmp <dig> and fgf <dig> to control levels .
as bmp <dig> together with fgf <dig> has previously been shown to induce neurite outgrowth in pc <dig> cells  <cit> , we examined the effect of bmp <dig> and fgf <dig> on neurite outgrowth.
we confirmed that pc <dig> cells did not differentiate effectively by bmp <dig> treatment alone, but bmp <dig> significantly augmented fgf2-induced neurite outgrowth in pc <dig> cells , as previously reported.
moreover, in order to confirm the effect of bmp <dig> on neurite outgrowth in pc <dig> cells, 200 ng/ml of noggin, an antagonist of bmp signaling, was added to cm-bso.
addition of noggin significantly suppressed the cm-bso -evoked phosphorylation of smad1/5/ <dig>  and shortened the length of neurite outgrowth in pc <dig> cells .
to address the question of which intracellular signaling pathways are affected by oxidative stress in hadmpcs, we focused on mapk signaling since previous studies had suggested that accumulation of ros in cells led to the activation of erk1/ <dig>  p <dig>  and jnk mapk  <cit> .
western blotting revealed that bso treatment markedly activated the p <dig> mapk pathway; sb <dig> could inhibit the activation, and u <dig> treatment stimulated the activation .
erk1/ <dig> mapk was significantly phosphorylated by bso treatment, and erk1/ <dig> activation was reduced to the control level by treatment with u <dig> .
in contrast, jnk activation was not observed in bso-treated hadmpcs .
therefore, we further investigated the relationship between increases in bmp <dig> and fgf <dig> expression and activation of the p <dig> and erk1/ <dig> mapk signaling pathways by oxidative stress.
treatment with the p <dig> mapk inhibitor sb <dig> dramatically downregulated the expression levels of bmp <dig> and fgf <dig> to control levels .
in contrast, the erk1/ <dig> mapk inhibitor u <dig> had no effect on fgf <dig> expression levels and led to a slight increase in bmp <dig> expression .
mkk6-mediated activation of p <dig> mapk increases bmp <dig> and fgf <dig> expression in hadmpcs
to further confirm the involvement of p <dig> mapk in the regulation of bmp <dig> and fgf <dig>  hadmpcs were transduced with a lentiviral vector expressing constitutively active mkk <dig> )  <cit>  from an ef1α promoter.
as shown in figure 5a, lentiviral transduction of mkk <dig>  led to expression of flag-tagged mkk <dig>  in hadmpcs.
moreover, the expression of mkk <dig>  resulted in activation of p <dig> mapk as expected  <cit>  , and upregulation of bmp <dig> and fgf <dig> expression .
nf-κb is not activated in hadmpcs exposed to oxidative stress
it has been reported that nf-κb directly binds to the bmp <dig> promoter to induce its expression  <cit> , and msk <dig>  a downstream molecule of p <dig> mapk, is involved in nf-κb transactivation  <cit> .
therefore, we hypothesized that p <dig> mapk-mediated activation of nf-κb might contribute to elevated expression of bmp <dig> mrna.
to confirm this hypothesis, transcriptional activation of nf-κb was examined by measuring luciferase activity driven by the synthetic nf-κb response element.
we found that transcriptional activity of nf-κb was not stimulated by bso treatment , and immunocytochemical analysis also revealed that nf-κb was not activated  in bso-treated hadmpcs .
these results suggested that elevated expression of bmp <dig> mrna is not mediated by nf-κb signaling.
our current data thus demonstrate the crucial role of ros, via activation of the p <dig> mapk signaling pathway, in regulating expression levels of the neurotrophic factors bmp <dig> and fgf <dig> in hadmpcs.
the overall model that we propose, based upon our findings, is shown in figure  <dig>
discussion
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   <cit> .
our data demonstrated that hadmpcs, when exposed to oxidative stress, facilitate neuronal differentiation in rat pheochromocytoma cell line pc <dig> cells by upregulation of fibroblast growth factor  <dig>  and bone morphogenetic protein  <dig>  secretion through p <dig> mapk activation.
our results show that bmp <dig> and fgf <dig> were upregulated in hadmpcs when exposed to buthionine sulfoximine , a glutathione-synthesis inhibitor that leads to oxidative stress.
these findings may have therapeutic implications in neurodegenerative diseases.
we concluded that bmp <dig> and fgf <dig> secreted from hadmpcs that had been exposed to oxidative stress were the main inducers of neurite outgrowth in pc <dig> cells.
erk1/ <dig> and smad1/5/ <dig> were significantly activated in these cells , while other growth factors known to induce neurite outgrowth in pc <dig> cells such as nerve growth factor  and vascular endothelial growth factor  were not observed to be upregulated by bso treatment .
we confirmed that bmp <dig> enhanced the effect that fgf <dig> had on the differentiation of pc <dig> cells , supporting our idea that hadmpcs under oxidative stress conditions secrete bmp <dig> and fgf <dig> and that this contributes to neuronal differentiation.
consistent with our conclusions, it has been reported that bmp <dig>  via activation of a smad signaling pathway, facilitated fgf2-induced neuronal differentiation in pc <dig> cells  <cit> .
however, since hadmpcs have been reported to secrete many growth factor including ngf, vegf, hgf, and igf  <cit> , we cannot exclude the possibility that bmp <dig> and fgf <dig> are acting cooperatively with these growth factors to facilitate neurite outgrowth in pc <dig> cells.
thus, the precise molecular mechanisms of induction of pc <dig> differentiation and the precise expression profiles in bso-treated hadmpcs need to be further investigated.
recently, bmp signaling through smad1/5/ <dig> has been reported to contribute to neurite outgrowth in dorsal root ganglion neurons both in vitro and in vivo  <cit> .
moreover, bmp <dig> has been shown to have neurotrophic effects on midbrain dopaminergic neurons  <cit> , ventral mesencephalic neurons  <cit> , mouse embryonic striatal neurons  <cit> , and nitrergic and catecholaminergic enteric neurons  <cit> .
moreover, fgf <dig> is trophic for neurons, glias, and endothelial cells in the central nervous system.
fgf <dig> also prevents downregulation of the anti-apoptotic protein bcl- <dig> in ischemic brain tissue and limits excitotoxic damage to the brain through an activin-dependent mechanism  <cit> .
these findings are consistent with our hypothesis that hadmpcs secret bmp <dig> and fgf <dig> to induce neurogenesis in neurodegenerative lesions in response to oxidative stress.
as it has been shown that ros activate erks, jnks, and p <dig> mapks  <cit> , we examined the mapk signaling pathway in hadmpcs exposed to oxidative stress and found that bso treatment resulted in significant activation of erk1/ <dig> and p <dig> mapk.
intriguingly, addition of sb <dig>  a specific inhibitor of p <dig> mapk, but not the erk inhibitor u <dig>  suppressed bmp <dig> and fgf <dig> expression in bso-treated hadmpcs to control levels , suggesting that p <dig> mapk was contributing to upregulation of bmp <dig> and fgf <dig> in hadmpcs when exposed to oxidative stress.
moreover, lentiviral transduction of the constitutively active form of mkk <dig>  a mapkk that selectively activates p <dig> mapk isoforms  <cit> , resulted in upregulation of bmp <dig> and fgf <dig> and this also demonstrated the crucial role of the p <dig> mapk cascade in the regulation of bmp <dig> and fgf <dig>  in primary human endothelial cells, p38-dependent regulation of bmp <dig> expression was reported previously.
viemann et al.
<cit>  investigated the genes that were induced by inflammatory stimulation with tumor necrosis factor α  and classified these genes into  <dig> categories based on whether they were regulated in an nf-κb-dependent or p <dig> mapk-dependent manner.
consistent with our findings, they found that significant induction of bmp <dig> expression by tnf-α was markedly suppressed by sb <dig>  an inhibitor of p <dig> mapk.
these results support the hypothesis that activation of the p <dig> mapk pathway in hadmpcs in response to inflammation surrounding neurodegenerative lesions leads to induction of bmp <dig> and fgf <dig>  which in turn support regeneration of neuronal cells.
it has been known that nf-κb directly binds to the bmp <dig> promoter to induce its expression  <cit> , and msk <dig>  a downstream molecule of p <dig> mapk, is involved in nf-κb transactivation  <cit> .
however, we did not observe an elevation of nf-κb transcriptional activity in hadmpcs when they were exposed to oxidative stress .
the mechanism of p38-dependent regulation of gene expression is not completely understood, and the precise mechanism by which p <dig> mapk regulates the expression of bmp <dig> and fgf <dig> remains to be determined.
in this study, we also found that suppression of erk1/ <dig> mapk by u <dig> in bso-treated hadmpcs resulted in slight activation of p <dig> mapk .
consistent with this, the expression level of bmp <dig> mrna was also upregulated when cells exposed to oxidative stress were pretreated with u <dig> .
previously, “seesaw cross-talk” between erk and p <dig> 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 p <dig>  whereas the p <dig> inhibitor had the opposite effect  <cit> .
we did not investigate the phosphorylation of erk1/ <dig> in sb203580-treated hadmpcs, but it may be possible that seesaw cross-talk also occurs in our system.
CONCLUSIONS
in summary, the results obtained in this study have demonstrated the potential use of hadmpcs for the treatment of neurodegenerative diseases such as ischemic stroke, parkinson’s disease, alzheimer’s disease, and spinal cord injury, in which the transplanted hadmpcs might be exposed to oxidative stress.
moreover, the p38-dependent modulation of bmp <dig> and fgf <dig> expression observed in this study is expected to be a new therapeutic target for neurodegenerative disorders.
materials and methods
adipose tissue samples
subcutaneous adipose tissue samples  were resected during plastic surgery in  <dig> females  as excess discards.
the study protocol was approved by the review board for human research of kobe university graduate school of medicine, foundation for biomedical research and innovation and kinki university pharmaceutical research and technology institute .
each subject provided a signed informed consent.
cell culture
pc <dig> cells were obtained from the health science research resources bank  and maintained in rpmi <dig> media supplemented with 10% heat-inactivated horse serum and 5% fetal bovine serum.
for differentiation, the cells were plated in 6-well culture plates coated with collagen type i  and the medium was replaced with differentiation medium  or conditioned medium from hadmpcs.
ngf , bmp <dig>  or fgf <dig>  were added to the differentiation medium.
recombinant murine noggin  was added to conditioned medium from bso-treated hadmpcs.
hadmpcs were isolated as previously reported  <cit>  and maintained in a medium containing 60% dmem-low glucose, 40% mcdb- <dig> medium , 1× insulin-transferrin-selenium ,  <dig> nm dexamethasone , 100 mm ascorbic acid 2-phosphate , 10 ng/ml epidermal growth factor , and 5% fetal bovine serum.
the cells were plated to a density of 5 ×  <dig> cells/cm <dig> on fibronectin-coated dishes, and the medium was replaced every 2 days.
preparation of conditioned medium from hadmpcs
two days after plating, hadmpcs were treated with bso  for 16 h. the medium was replaced with fresh culture medium for 2 days followed by replacement with pc <dig> cell differentiation medium.
after  <dig> more days, the medium was removed for use as conditioned medium.
for preparation of the conditioned medium from hadmpcs in which one of the three, p <dig>  erk1/ <dig>  or jnk mapk, was inhibited, hadmpcs were pretreated with 10 μm sb <dig> , 10 μm u <dig> , or 10 μm jnk inhibitor ii , respectively, for 2 h and subsequently treated with 1 mm bso.
measurement of gsh/gssg ratio
ratios of reduced glutathione  to oxidized glutathione  were measured using the gsh/gssg-glo assay kit  following the manufacturer’s protocol.
measurement of reactive oxygen species production
cells were harvested and incubated with 10 μm 5--chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester .
the amount of intracellular ros production was proportional to green fluorescence, as analyzed with a guava easycyte 8ht flow cytometer  using an argon laser at 488 nm and a 525/30 nm band pass filter, and dead cells were excluded with the live/dead fixable far red dead cell stain kit .
western blot analysis
cells were washed with ice-cold phosphate-buffered saline and lysed with m-per mammalian protein extraction reagent  following the manufacturer’s instructions.
equal amounts of proteins were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis , transferred to polyvinylidene fluoride  membranes , and probed with antibodies against phospho-erk1/ <dig> , erk1/ <dig> , phospho- <dig> , p <dig> , phospho-smad1/5/ <dig> , phospho-akt , akt , phospho-jnk , jnk   and actin .
horseradish peroxidase -conjugated anti-rabbit and anti-mouse secondary antibodies  were used as probes and immunoreactive bands were visualized with the immobilon western chemiluminescent hrp substrate .
the band intensity was measured using imagej software.
rna extraction, cdna generation, and quantitative polymerase chain reaction
total rna was extracted using the rneasy mini kit  following the manufacturer’s instructions.
cdna was generated from 1 μg of total rna using the verso cdna synthesis kit  and purified with the minelute pcr purification kit .
q-pcr analysis was carried out using the ssofast evagreen supermix  according to the manufacturer’s protocols.
the relative expression value of each gene was calculated using a ΔΔct method and the most reliable internal control gene was determined using the genorm software .
details of the primers used in these experiments are available on request.
enzyme-linked immunosorbent assay
enzyme-linked immunosorbent assay  was performed using the quantikine bmp- <dig> immunoassay system and quantikine fgf- <dig> immunoassay system  following the manufacturer’s protocols.
plasmid construction and lentivirus production
flag-tagged mkk <dig>   <cit>  was provided by addgene ; addgene plasmid 13518).
flag-tagged mkk <dig>  was cloned into a pentr <dig> vector .
an iresgfp fragment was subsequently cloned into the plasmid to produce the entry vector pentr11-mkk <dig> -iresgfp.
the entry vector and csii-ef-rfa  were incubated with lr clonase ii enzyme mix  to generate csii-ef-mkk <dig> -iresgfp.
the resultant plasmid was mixed with packaging plasmids  and transfected into 293 t cells.
the supernatant medium, which contained lentiviral vectors, was collected 2 days after transduction and concentrated by centrifugation .
luciferase assay
hadmpcs were transfected with pgl <dig>   and either ptal-luc or pnf-κb-luc by transit- <dig> .
the cells were then treated with 10 μm of sb <dig> or  <dig> % dmso for 2 h followed by addition of 1 mm of bso.
after 24 h, the medium was changed to fresh medium and cells were cultured for an additional 2 days.
the activity of nf-κb was measured using the dual luciferases assay system  according to the manufacturer’s protocol.
immunocytochemistry
hadmpcs were fixed with 4% paraformaldehyde in pbs for 10 min at 4°c and then washed  <dig> times in pbs.
blocking was performed with pbsmt  for 1 h at room temperature.
the cells were then incubated with rabbit monoclonal antibody against nf-κb p <dig>  overnight at 4°c.
after washing with pbs, cells were incubated with alexa  <dig> conjugated anti-rabbit igg  for 1 h. the cells were counterstained with 4′-6-diamidino-2-phenylindole   to identify cellular nuclei.
competing interests
none of the authors have any competing interests related to the manuscript.
authors’ contributions
mm carried out the facs analysis, qpcr analysis, elisa, immunofluorescent staining, and cell culture, participated in the study design, and drafted the manuscript.
hm participated in the study design, carried out the western blot analysis, luciferase assay, and cell culture, and drafted the manuscript.
au carried out western blot analysis, constructed the plasmids, and generated the lentiviral vectors.
yn carried out qpcr analysis and performed the statistical analysis.
ai resected subcutaneous adipose tissue samples during plastic surgery.
ho and am isolated hadmpcs from human adipose tissues.
th conceived the study, participated in its design and coordination, and helped to draft the manuscript.
all authors read and approved the final manuscript.

 mesenchymal stem cells are pluripotent stem cells that can differentiate into various types of cells. these cells have been isolated from bone marrow, umbilical cord blood, and adipose tissue and can be easily obtained and expanded ex vivo under appropriate culture conditions. thus, mscs are an attractive material for cell therapy and tissue engineering. human adipose tissue-derived mesenchymal stem cells, also referred to as human adipose tissue-derived multilineage progenitor cells, are especially advantageous because they can be easily and safely obtained from lipoaspirates, and the ethical issues surrounding other sources of stem cells can be avoided. moreover, hadmpcs have more pluripotent properties for regenerative medical applications than other stem cells, since these cells have been reported to have the ability to migrate to the injured area and differentiate into hepatocytes, cardiomyoblasts, pancreatic cells, and neuronal cells. in addition, it is known that hadmpcs secrete a wide variety of cytokines and growth factors necessary for tissue regeneration including nerve growth factor, brain-derived neurotrophic factor, fibroblast growth factors, vascular endothelial growth factor and hepatocyte growth factor. recently, several groups have reported that hadmpcs facilitate neurological recovery in experimental models of stroke and parkinson’s disease. despite the superiority of hadmpcs over other stem cells, the potential use of hadmpcs for the treatment of these neurodegenerative disorders has not been fully investigated. it has been reported that administration of hadmpcs in animal models of acute ischemic stroke markedly decreased brain infarct size, improved neurological function by enhancing angiogenesis and neurogenesis, and showed anti-inflammatory and anti-apoptotic effects. these effects were due in part to increased secretion levels of vegf, hgf and bfgf under hypoxic conditions, indicating the role of hadmpcs in reducing the severity of hypoxia-ischemic lesions. in addition to hypoxic stress, ischemic lesions are generally subject to inflammation, which leads to the generation of reactive oxygen species. ros are generated as a natural byproduct of normal aerobic metabolism, and mitochondrial respiration, together with oxidative enzymes such as plasma membrane oxidase, is considered to be the major intracellular source of ros production. although appropriate levels of ros play an important role in several physiological processes, oxidative damage initiated by excessive ros causes many pathological conditions including inflammation, atherosclerosis, aging, and cancer. neuronal cells are especially vulnerable to oxidative stress, and numerous studies have examined the crucial roles of oxidative stress in neurodegenerative disorders such as stroke, alzheimer’s disease, and parkinson’s disease. in these diseases, microglia, the macrophages of the central nervous system, are activated in response to a local inflammation and generate large amounts of reactive oxygen and nitrogen species, thereby exposing nearby neurons to stress. thus, the influence of oxidative stress generated by neurodegenerative lesion on hadmpcs needs to be further studied. in this study, we examined the role of oxidative stress on hadmpcs in neurite outgrowth in cells of the rat pheochromocytoma cell line. upon treatment with buthionine sulfoximine, an inhibitor of the rate-limiting enzyme in the synthesis of glutathione, hadmpcs accumulated ros, which resulted in the upregulation of expression levels of the neurotrophic factors bmp and fgf our present data thus provide new insights into understanding the mechanism of how hadmpcs exposed to oxidative stress contribute to neurogenesis, and this may explain the effects of stem cell transplantation therapy with hadmpcs in treating ischemic stroke.  hadmpcs exposed to oxidative stress stimulate neurite outgrowth in pc cells hadmpcs were treated with 1 mm bso for 24 h; a group of hadmpcs that were not given any treatment was used as the control group. as shown in figure 1a and b, bso treatment resulted in significant reduction of intracellular reduced glutathione levels, followed by accumulation of intracellular reactive oxygen species in hadmpcs. to investigate whether accumulation of ros affects secretion of cytokines from hadmpcs, conditioned medium from bso-treated ) or bso-untreated ) hadmpcs was added to pc cells. as expected, addition of ngf significantly induced neurite outgrowth in the pc cells. hadmpcs, like other mesenchymal stem cells derived from bone marrow or adipose tissue, may secrete many cytokines including ngf, bdnf and fgf and this may account for the slight induction of neurite outgrowth seen in the cm-bso treated cells. in contrast, the number and length of neurite outgrowth of pc cells in cm-bso was markedly enhanced compared with those in cm-bso. conditioned medium from bso-treated hadmpcs activates erk1/ mapk and smad signaling in pc cells to investigate which intracellular signaling pathways were involved in the neurite outgrowth of pc cells in cm-bso, we used western blotting to determine the phosphorylation levels of erk1/ mapk, p mapk, smad1/5/ and akt in pc cells in various culture conditions. ngf significantly activated erk1/ mapk and akt signaling pathway. in contrast, erk1/ mapk was not activated in pc cells exposed to cm-bso, while an increase in phosphorylated smad1/5/ was observed. interestingly, cm-bso treatment led to both a significant increase in smad1/5/ phosphorylation levels as well as activation of the erk1/ mapk signaling pathway in pc cells. akt was 2-fold activated in both cm-bso and cm-bso treated pc cells, but no significant difference between the groups was observed. fgf and bmp are upregulated through p mapk signaling in hadmpcs exposed to oxidative stress we next examined which growth factors or cytokines from bso-treated hadmpcs were involved in stimulation of neurite outgrowth. we found that both mrna and protein levels for bmp and fgf were markedly increased in hadmpcs treated with bso. to determine if this upregulation was caused by ros, all cells were exposed to the antioxidant n-acetylcysteine. as we expected, addition of nac to bso-treated hadmpcs reduced the expression levels of bmp and fgf to control levels. as bmp together with fgf has previously been shown to induce neurite outgrowth in pc cells, we examined the effect of bmp and fgf on neurite outgrowth. we confirmed that pc cells did not differentiate effectively by bmp treatment alone, but bmp significantly augmented fgf2-induced neurite outgrowth in pc cells, as previously reported. moreover, in order to confirm the effect of bmp on neurite outgrowth in pc cells, 200 ng/ml of noggin, an antagonist of bmp signaling, was added to cm-bso. addition of noggin significantly suppressed the cm-bso -evoked phosphorylation of smad1/5/ and shortened the length of neurite outgrowth in pc cells. to address the question of which intracellular signaling pathways are affected by oxidative stress in hadmpcs, we focused on mapk signaling since previous studies had suggested that accumulation of ros in cells led to the activation of erk1/ p and jnk mapk. western blotting revealed that bso treatment markedly activated the p mapk pathway; sb could inhibit the activation, and u treatment stimulated the activation. erk1/ mapk was significantly phosphorylated by bso treatment, and erk1/ activation was reduced to the control level by treatment with u. in contrast, jnk activation was not observed in bso-treated hadmpcs. therefore, we further investigated the relationship between increases in bmp and fgf expression and activation of the p and erk1/ mapk signaling pathways by oxidative stress. treatment with the p mapk inhibitor sb dramatically downregulated the expression levels of bmp and fgf to control levels. in contrast, the erk1/ mapk inhibitor u had no effect on fgf expression levels and led to a slight increase in bmp expression. mkk6-mediated activation of p mapk increases bmp and fgf expression in hadmpcs to further confirm the involvement of p mapk in the regulation of bmp and fgf hadmpcs were transduced with a lentiviral vector expressing constitutively active mkk ) from an ef1α promoter. as shown in figure 5a, lentiviral transduction of mkk led to expression of flag-tagged mkk in hadmpcs. moreover, the expression of mkk resulted in activation of p mapk as expected, and upregulation of bmp and fgf expression. nf-κb is not activated in hadmpcs exposed to oxidative stress it has been reported that nf-κb directly binds to the bmp promoter to induce its expression, and msk a downstream molecule of p mapk, is involved in nf-κb transactivation. therefore, we hypothesized that p mapk-mediated activation of nf-κb might contribute to elevated expression of bmp mrna. to confirm this hypothesis, transcriptional activation of nf-κb was examined by measuring luciferase activity driven by the synthetic nf-κb response element. we found that transcriptional activity of nf-κb was not stimulated by bso treatment, and immunocytochemical analysis also revealed that nf-κb was not activated in bso-treated hadmpcs. these results suggested that elevated expression of bmp mrna is not mediated by nf-κb signaling. our current data thus demonstrate the crucial role of ros, via activation of the p mapk signaling pathway, in regulating expression levels of the neurotrophic factors bmp and fgf in hadmpcs. the overall model that we propose, based upon our findings, is shown in figure  discussion 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. our data demonstrated that hadmpcs, when exposed to oxidative stress, facilitate neuronal differentiation in rat pheochromocytoma cell line pc cells by upregulation of fibroblast growth factor and bone morphogenetic protein secretion through p mapk activation. our results show that bmp and fgf were upregulated in hadmpcs when exposed to buthionine sulfoximine, a glutathione-synthesis inhibitor that leads to oxidative stress. these findings may have therapeutic implications in neurodegenerative diseases. we concluded that bmp and fgf secreted from hadmpcs that had been exposed to oxidative stress were the main inducers of neurite outgrowth in pc cells. erk1/ and smad1/5/ were significantly activated in these cells, while other growth factors known to induce neurite outgrowth in pc cells such as nerve growth factor and vascular endothelial growth factor were not observed to be upregulated by bso treatment. we confirmed that bmp enhanced the effect that fgf had on the differentiation of pc cells, supporting our idea that hadmpcs under oxidative stress conditions secrete bmp and fgf and that this contributes to neuronal differentiation. consistent with our conclusions, it has been reported that bmp via activation of a smad signaling pathway, facilitated fgf2-induced neuronal differentiation in pc cells. however, since hadmpcs have been reported to secrete many growth factor including ngf, vegf, hgf, and igf, we cannot exclude the possibility that bmp and fgf are acting cooperatively with these growth factors to facilitate neurite outgrowth in pc cells. thus, the precise molecular mechanisms of induction of pc differentiation and the precise expression profiles in bso-treated hadmpcs need to be further investigated. recently, bmp signaling through smad1/5/ has been reported to contribute to neurite outgrowth in dorsal root ganglion neurons both in vitro and in vivo. moreover, bmp has been shown to have neurotrophic effects on midbrain dopaminergic neurons, ventral mesencephalic neurons, mouse embryonic striatal neurons, and nitrergic and catecholaminergic enteric neurons. moreover, fgf is trophic for neurons, glias, and endothelial cells in the central nervous system. fgf also prevents downregulation of the anti-apoptotic protein bcl- in ischemic brain tissue and limits excitotoxic damage to the brain through an activin-dependent mechanism. these findings are consistent with our hypothesis that hadmpcs secret bmp and fgf to induce neurogenesis in neurodegenerative lesions in response to oxidative stress. as it has been shown that ros activate erks, jnks, and p mapks, we examined the mapk signaling pathway in hadmpcs exposed to oxidative stress and found that bso treatment resulted in significant activation of erk1/ and p mapk. intriguingly, addition of sb a specific inhibitor of p mapk, but not the erk inhibitor u suppressed bmp and fgf expression in bso-treated hadmpcs to control levels, suggesting that p mapk was contributing to upregulation of bmp and fgf in hadmpcs when exposed to oxidative stress. moreover, lentiviral transduction of the constitutively active form of mkk a mapkk that selectively activates p mapk isoforms, resulted in upregulation of bmp and fgf and this also demonstrated the crucial role of the p mapk cascade in the regulation of bmp and fgf in primary human endothelial cells, p38-dependent regulation of bmp expression was reported previously. viemann et al. investigated the genes that were induced by inflammatory stimulation with tumor necrosis factor α and classified these genes into categories based on whether they were regulated in an nf-κb-dependent or p mapk-dependent manner. consistent with our findings, they found that significant induction of bmp expression by tnf-α was markedly suppressed by sb an inhibitor of p mapk. these results support the hypothesis that activation of the p mapk pathway in hadmpcs in response to inflammation surrounding neurodegenerative lesions leads to induction of bmp and fgf which in turn support regeneration of neuronal cells. it has been known that nf-κb directly binds to the bmp promoter to induce its expression, and msk a downstream molecule of p mapk, is involved in nf-κb transactivation. however, we did not observe an elevation of nf-κb transcriptional activity in hadmpcs when they were exposed to oxidative stress. the mechanism of p38-dependent regulation of gene expression is not completely understood, and the precise mechanism by which p mapk regulates the expression of bmp and fgf remains to be determined. in this study, we also found that suppression of erk1/ mapk by u in bso-treated hadmpcs resulted in slight activation of p mapk. consistent with this, the expression level of bmp mrna was also upregulated when cells exposed to oxidative stress were pretreated with u. previously, “seesaw cross-talk” between erk and p 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 p whereas the p inhibitor had the opposite effect. we did not investigate the phosphorylation of erk1/ in sb203580-treated hadmpcs, but it may be possible that seesaw cross-talk also occurs in our system.  in summary, the results obtained in this study have demonstrated the potential use of hadmpcs for the treatment of neurodegenerative diseases such as ischemic stroke, parkinson’s disease, alzheimer’s disease, and spinal cord injury, in which the transplanted hadmpcs might be exposed to oxidative stress. moreover, the p38-dependent modulation of bmp and fgf expression observed in this study is expected to be a new therapeutic target for neurodegenerative disorders. materials and methods adipose tissue samples subcutaneous adipose tissue samples were resected during plastic surgery in females as excess discards. the study protocol was approved by the review board for human research of kobe university graduate school of medicine, foundation for biomedical research and innovation and kinki university pharmaceutical research and technology institute. each subject provided a signed informed consent. cell culture pc cells were obtained from the health science research resources bank and maintained in rpmi media supplemented with 10% heat-inactivated horse serum and 5% fetal bovine serum. for differentiation, the cells were plated in 6-well culture plates coated with collagen type i and the medium was replaced with differentiation medium or conditioned medium from hadmpcs. ngf, bmp or fgf were added to the differentiation medium. recombinant murine noggin was added to conditioned medium from bso-treated hadmpcs. hadmpcs were isolated as previously reported and maintained in a medium containing 60% dmem-low glucose, 40% mcdb- medium, 1× insulin-transferrin-selenium, nm dexamethasone, 100 mm ascorbic acid 2-phosphate, 10 ng/ml epidermal growth factor, and 5% fetal bovine serum. the cells were plated to a density of 5 ×  cells/cm on fibronectin-coated dishes, and the medium was replaced every 2 days. preparation of conditioned medium from hadmpcs two days after plating, hadmpcs were treated with bso for 16 h. the medium was replaced with fresh culture medium for 2 days followed by replacement with pc cell differentiation medium. after more days, the medium was removed for use as conditioned medium. for preparation of the conditioned medium from hadmpcs in which one of the three, p erk1/ or jnk mapk, was inhibited, hadmpcs were pretreated with 10 μm sb, 10 μm u, or 10 μm jnk inhibitor ii, respectively, for 2 h and subsequently treated with 1 mm bso. measurement of gsh/gssg ratio ratios of reduced glutathione to oxidized glutathione were measured using the gsh/gssg-glo assay kit following the manufacturer’s protocol. measurement of reactive oxygen species production cells were harvested and incubated with 10 μm 5--chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester. the amount of intracellular ros production was proportional to green fluorescence, as analyzed with a guava easycyte 8ht flow cytometer using an argon laser at 488 nm and a 525/30 nm band pass filter, and dead cells were excluded with the live/dead fixable far red dead cell stain kit. western blot analysis cells were washed with ice-cold phosphate-buffered saline and lysed with m-per mammalian protein extraction reagent following the manufacturer’s instructions. equal amounts of proteins were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, and probed with antibodies against phospho-erk1/, erk1/, phospho-, p, phospho-smad1/5/, phospho-akt, akt, phospho-jnk, jnk and actin. horseradish peroxidase -conjugated anti-rabbit and anti-mouse secondary antibodies were used as probes and immunoreactive bands were visualized with the immobilon western chemiluminescent hrp substrate. the band intensity was measured using imagej software. rna extraction, cdna generation, and quantitative polymerase chain reaction total rna was extracted using the rneasy mini kit following the manufacturer’s instructions. cdna was generated from 1 μg of total rna using the verso cdna synthesis kit and purified with the minelute pcr purification kit. q-pcr analysis was carried out using the ssofast evagreen supermix according to the manufacturer’s protocols. the relative expression value of each gene was calculated using a ΔΔct method and the most reliable internal control gene was determined using the genorm software. details of the primers used in these experiments are available on request. enzyme-linked immunosorbent assay enzyme-linked immunosorbent assay was performed using the quantikine bmp- immunoassay system and quantikine fgf- immunoassay system following the manufacturer’s protocols. plasmid construction and lentivirus production flag-tagged mkk was provided by addgene; addgene plasmid 13518). flag-tagged mkk was cloned into a pentr vector. an iresgfp fragment was subsequently cloned into the plasmid to produce the entry vector pentr11-mkk -iresgfp. the entry vector and csii-ef-rfa were incubated with lr clonase ii enzyme mix to generate csii-ef-mkk -iresgfp. the resultant plasmid was mixed with packaging plasmids and transfected into 293 t cells. the supernatant medium, which contained lentiviral vectors, was collected 2 days after transduction and concentrated by centrifugation. luciferase assay hadmpcs were transfected with pgl and either ptal-luc or pnf-κb-luc by transit-. the cells were then treated with 10 μm of sb or % dmso for 2 h followed by addition of 1 mm of bso. after 24 h, the medium was changed to fresh medium and cells were cultured for an additional 2 days. the activity of nf-κb was measured using the dual luciferases assay system according to the manufacturer’s protocol. immunocytochemistry hadmpcs were fixed with 4% paraformaldehyde in pbs for 10 min at 4°c and then washed times in pbs. blocking was performed with pbsmt for 1 h at room temperature. the cells were then incubated with rabbit monoclonal antibody against nf-κb p overnight at 4°c. after washing with pbs, cells were incubated with alexa conjugated anti-rabbit igg for 1 h. the cells were counterstained with 4′-6-diamidino-2-phenylindole to identify cellular nuclei. competing interests none of the authors have any competing interests related to the manuscript. authors’ contributions mm carried out the facs analysis, qpcr analysis, elisa, immunofluorescent staining, and cell culture, participated in the study design, and drafted the manuscript. hm participated in the study design, carried out the western blot analysis, luciferase assay, and cell culture, and drafted the manuscript. au carried out western blot analysis, constructed the plasmids, and generated the lentiviral vectors. yn carried out qpcr analysis and performed the statistical analysis. ai resected subcutaneous adipose tissue samples during plastic surgery. ho and am isolated hadmpcs from human adipose tissues. th conceived the study, participated in its design and coordination, and helped to draft the manuscript. all authors read and approved the final manuscript. 
we found that glutathione depletion in hadmpcs, caused by treatment with buthionine sulfoximine, resulted in the promotion of neurite outgrowth in pc cells through upregulation of bone morphogenetic protein and fibroblast growth factor transcription in, and secretion from, hadmpcs. addition of n-acetylcysteine, a precursor of the intracellular antioxidant glutathione, suppressed the bso-mediated upregulation of bmp and fgf moreover, bso treatment caused phosphorylation of p mapk in hadmpcs. adipose tissues contain populations of pluripotent mesenchymal stem cells that also secrete various cytokines and growth factors to support repair of damaged tissues. in this study, we examined the role of oxidative stress on human adipose-derived multilineage progenitor cells in neurite outgrowth in cells of the rat pheochromocytoma cell line. our results clearly suggest that glutathione depletion, followed by accumulation of reactive oxygen species, stimulates the activation of p mapk and subsequent expression of bmp and fgf in hadmpcs. inhibition of p mapk was sufficient to suppress bmp and fgf expression, while this expression was significantly upregulated by overexpression of a constitutively active form of mkk which is an upstream molecule from p mapk. human adipose-derived multilineage progenitor cellsadult stem cellsreactive oxygen speciesp mapkneurite outgrowthbmp2fgf2neurodegenerative disorders thus, transplantation of hadmpcs into neurodegenerative lesions such as stroke and parkinson’s disease, in which the transplanted hadmpcs are exposed to oxidative stress, can be the basis for simple and safe therapies.
 adipose tissues contain populations of pluripotent mesenchymal stem cells that also secrete various cytokines and growth factors to support repair of damaged tissues. in this study, we examined the role of oxidative stress on human adipose-derived multilineage progenitor cells in neurite outgrowth in cells of the rat pheochromocytoma cell line.  we found that glutathione depletion in hadmpcs, caused by treatment with buthionine sulfoximine, resulted in the promotion of neurite outgrowth in pc cells through upregulation of bone morphogenetic protein and fibroblast growth factor transcription in, and secretion from, hadmpcs. addition of n-acetylcysteine, a precursor of the intracellular antioxidant glutathione, suppressed the bso-mediated upregulation of bmp and fgf moreover, bso treatment caused phosphorylation of p mapk in hadmpcs. inhibition of p mapk was sufficient to suppress bmp and fgf expression, while this expression was significantly upregulated by overexpression of a constitutively active form of mkk which is an upstream molecule from p mapk.  our results clearly suggest that glutathione depletion, followed by accumulation of reactive oxygen species, stimulates the activation of p mapk and subsequent expression of bmp and fgf in hadmpcs. thus, transplantation of hadmpcs into neurodegenerative lesions such as stroke and parkinson’s disease, in which the transplanted hadmpcs are exposed to oxidative stress, can be the basis for simple and safe therapies. human adipose-derived multilineage progenitor cellsadult stem cellsreactive oxygen speciesp mapkneurite outgrowthbmp2fgf2neurodegenerative disorders 