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Negative regulation of adipogenesis from human mesenchymal stem cells by Jun N-terminal kinase
BBRC Biochemical and Biophysical Research Communications 326 (2005) 499–504 www.elsevier.com/locate/ybbrc
Negative regulation of adipogenesis from human mesenchymal stem cells by Jun N-terminal kinase Sachiko Tominagaa, Tomohiro Yamaguchia, Shin-Ichiro Takahashib, Fumiko Hirosea, Takashi Osumia,* a
Graduate School of Life Science, Himeji Institute of Technology, University of Hyogo, 3-2-1 Koto, Kamigori, Hyogo 678-1297, Japan b Department of Animal Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Received 1 November 2004 Available online 20 November 2004
Abstract Human mesenchymal stem cells (hMSCs) are capable of differentiating into several cell types including adipocytes, osteoblasts, and chondrocytes, under appropriate culture conditions. We found that SP600125, an inhibitor of Jun N-terminal kinase (JNK), promoted adipogenesis whereas it repressed osteogenesis from hMSCs. SP600125 increased the expression of adipogenic transcription factors, CCAAT/enhancer-binding proteins a and b as well as peroxisome proliferator-activated receptor c2, which suggested that the chemical acted on the early steps of transcriptional regulatory cascade in adipogenesis. A gene reporter assay showed that SP600125 and a dominant negative JNK promoted a transcriptional activity dependent on the cAMP-response element (CRE). Thus, JNK represses adipogenesis from hMSCs probably by, at least in part, inhibiting the transactivating function of CRE-binding protein. Another action of JNK, phosphorylation at Ser307 of insulin receptor substrate-1, was also predicted to contribute to the repression of adipogenesis. 2004 Elsevier Inc. All rights reserved. Keywords: Human mesenchymal stem cells; Adipogenesis; Osteogenesis; Jun N-terminal kinase; cAMP-response element-binding protein; Insulin receptor substrate-1; Inhibitor; SP600125
Adipocytes are derived from mesenchymal stem cells that are also potent to differentiate into myocytes, osteoblasts, and chondrocytes. Adipogenesis is a complex process including commitment of the multipotent cells to the adipogenic lineage, proliferation of the preadipocytes, and terminal differentiation. Although the mechanism of terminal differentiation into adipocytes has been intensively studied using the established murine preadipocyte lines such as 3T3-L1 and F442A, key information is lacking on the commitment of multipotent precursor cells into individual cell lineages including preadipocytes. This was in part because of the difficulty in obtaining appropriate multipotent stem cells. In this *
Corresponding author. Fax: +81 791 58 0193. E-mail address: [email protected] (T. Osumi).
0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.11.056
regard, human mesenchymal stem cells (hMSCs) seem to be promising tools, because they have multipotency to differentiate into several cell types such as adipocytes, osteoblasts, and chondrocytes, under appropriate culture conditions [1]. In fact, successful adipogenesis from hMSCs was reported [2]. MAP kinases are a group of serine/threonine kinases conserved from yeast to mammals, being involved in versatile cellular processes, such as cell growth, apoptosis, and differentiation [3]. The classic MAP kinase, extracellular signal-regulated protein kinase (ERK), is a ubiquitous component of signal transduction pathways and activated by diverse extracellular stimuli. In animals two other major members of the MAP kinase family, Jun N-terminal kinase (JNK), also known as stress-activated protein kinase, and p38/reactivating
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kinase (p38) have been described. They are activated by a variety of cytokines, environmental stress, as well as ultraviolet and ionizing radiation. Using hMSCs, it was shown that inhibition of the ERK signaling pathway blocked osteogenic conversion, resulting instead in adipocyte formation [4]. This study suggested that hMSCs would be suitable for characterizing the early processes of adipogenesis and osteogenesis. In this study, we investigated the contribution of MAP kinases to adipogenesis and osteogenesis from hMSCs, using specific inhibitors. JNK signaling seemed to regulate osteogenesis positively, whereas adipogenesis negatively by inhibiting the function of an adipogenic transcription factor, cAMP-response element (CRE)binding protein (CREB).
Materials and methods Materials. PD98059 was purchased from Sigma, SP600125 from ALEXIS, and SB203580 from BIOMOL, respectively. Polyclonal antiC/EBPb (sc-150) and anti-C/EBPa (sc-9314) antibodies were obtained from Santa Cruz and phospho-specific anti-IRS-1 (Ser307) antibody was from Upstate. Polyclonal anti-insulin receptor substrate (IRS)-1 antibody was as described [5]. Cell culture. hMSCs were purchased from BioWhittaker. The cells were maintained in DulbeccoÕs modified EagleÕs medium (DMEM)high glucose (4.5 g glucose/L) containing 10% fetal bovine serum (FBS) (maintenance medium), in 5% CO2 at 37 C. They were passaged every 3 days at 70% confluence. For adipogenesis experiments, 2.1 · 104 hMSCs in 0.2–0.3 ml of maintenance medium per cm2 were seeded in 12-well plates or 35 mm dishes. After confluence, the cells were treated for 3 days with a hormone cocktail containing 1 lM dexamethasone (Dex), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.2 mM indomethacin, and 10 lg/ml insulin [6]. The cocktail was then removed, and the cells were cultured in maintenance medium containing 10 lg/ml insulin for further 6 days. For osteogenesis experiments, 3.1 · 103 cells in 0.2–0.3 ml of maintenance medium per cm2 were used. Cells were kept for 4–24 h in maintenance medium at 37 C in 5% CO2 to allow adhering to the bottom of plates. The medium was then replaced with maintenance medium supplemented with 0.1 lM Dex, 0.05 mM ascorbic acid, and 10 mM glycerol-2-phosphate (osteogenic medium) [6]. The cells were cultured for 8 days by changing the medium every 3 days. For simultaneous induction of adipocytes and osteoblasts, we optimized the culture conditions by preliminary experiments with regard to the cell density as well as the composition of supplements. In this protocol, the cells were plated as in the adipogenesis experiment. After confluence, the cells were treated for 3 days with the maintenance medium containing 0.1 lM Dex, 0.5 mM IBMX, 0.2 mM indomethacin, 10 lg/ml insulin, 0.05 mM ascorbic acid, and 10 mM glycerol-2phosphate. The cocktail was then removed, and the cells were cultured in the osteogenic medium for 6 days. In the experiments using MAP kinase inhibitors, PD98059 was used at 20 lM, whereas SB203580 and SP600125, 10 lM. The inhibitors were dissolved in dimethyl sulfoxide (DMSO), and the same amount of vehicle was added to the control samples. Estimation of differentiation. Glycerol-3-phosphate dehydrogenase (GPDH) activity and triglyceride content were measured as described previously [7]. Alkaline phosphatase (ALP) was assayed using an Alkaline Phospha K-test Wako kit (Wako Pure Chemicals) according to the manufacturerÕs protocol. Extracellularly deposited calcium was stained by the von Kossa method [8]. Assays were performed in trip-
licate, and the averages ± SD are given. Statistical significance was analyzed by StudentÕs t test and presented as: *P < 0.05; **P < 0.01; ***P < 0.001 and NS, not significant. Determination of the adipocyte marker gene expression. Total RNA was prepared with a RNeasy Mini Kit (Qiagen). RT-PCR was performed as described previously [7], using a ribosomal protein gene, 36B4, as a standard for the amount of RNA. The sequences of primers were: PPARc, forward (5 0 -gaggaattcatgggtgaaactctgggagattc-3 0 ) and reverse (5 0 -ccactggatctgttcttgtg-3 0 ); aP2, forward (5 0 -tactgggccaggaatttgac-3 0 ) and reverse (5 0 -taaactcttgtggaagtcacg-3 0 ); LPL, forward (5 0 cctgtacaagagagaacctgac-3 0 ) and reverse (5 0 -gcttctccaatgttgcatcctg-3 0 ); and 36B4, forward (5 0 -ttcgtgttcaccaaggaggac-3 0 ) and reverse (5 0 -atgatcagcccgaaggagaag-3 0 ). Western blot analysis was carried out as described previously [9].
Results To clarify the key signaling pathways in adipogenesis and osteogenesis from hMSCs, we investigated the contributions of MAP kinases, using specific inhibitors. When the cells were treated under the adipogenic conditions with PD98059, an ERK kinase (MEK1) inhibitor, the specific activity of GPDH and triglyceride content were increased as compared with the control cells (Fig. 1A). A JNK inhibitor, SP600125 [10], increased both parameters even more significantly, whereas a p38 inhibitor, SB203580, severely repressed them. These results suggest that all three MAP kinases are involved in adipogenesis from hMSCs, ERK and JNK acting negatively, whereas p38 positively. Next, we examined the effect of each inhibitor on osteogenic conversion of hMSCs. Under the osteogenic conditions, PD98059 did not affect the expression of ALP, an osteoblast marker (Fig. 1B). On the other hand, both SB203580 and SP600125 significantly decreased the ALP specific activity. Under these culture conditions, a small number of cells exhibited a round-shaped adipocyte-like morphology for the samples treated with SP600125, but not with PD98059. These results suggest that p38 and JNK participate in osteogenesis and adipogenesis from hMSCs, p38 positively regulating both processes, whereas JNK regulating positively the former and negatively the latter. We were particularly interested in JNK, because it seemed to affect the balance of forming adipogenic and osteogenic lineages from hMSCs. We asked whether the inhibitors exerted similar effects under the conditions that allowed simultaneous osteogenesis and adipogenesis. We established a protocol by which both types of cells differentiated, as confirmed by the increase in triglyceride content and ALP specific activity (see below). The lipid-containing cells were negative for the histochemical staining of ALP, and vice versa (data not shown), indicating that the differentiated adipocytes and osteoblasts expressed their own marker genes mutually exclusively. Under these conditions, PD98059 did not affect the ALP specific activity significantly, but SB203580 and SP600125
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Fig. 1. Effects of MAP kinase inhibitors on the adipogenesis and osteogenesis from hMSCs. (A) Specific activity of GPDH (top) and triglyceride content (bottom) of the cells after induction of adipogenesis. Cells were treated and cultured under the adipogenic conditions for 9 days in total, in the presence or absence of MAP kinase inhibitors. (B) Specific activity of ALP of the cells induced for osteogenesis. Cells were cultured for 8 days under the osteogenic conditions in the presence or absence of inhibitors. (C) Specific activity of ALP (top) and triglyceride content (bottom) of the cells induced for adipogenesis and osteogenesis simultaneously (see Materials and methods). (D) Staining of calcium deposition. Cells were cultured under the conditions of (C) and stained by the von Kossa method. Cells were cultured in the absence (left panels) and presence (right panels) of SP600125. Upper panels show the staining of whole culture dishes and lower panels the phase-contrast micrograms.
decreased it (Fig. 1C). On the other hand, PD98059 slightly increased the triglyceride accumulation, whereas SP600125 increased it 2.5-fold as compared with the control. In contrast, SB203580 severely repressed the triglyceride accumulation. As another measure of osteogenesis, we examined calcium deposition by von Kossa staining. On day 8 of post-confluence under the conditions for inducing both adipogenesis and osteogenesis, the SP600125-treated cells exhibited a reduced level of calcium staining as compared with the control (Fig. 1D, upper panels). Increase in the number of adipocytes by SP600125 was in parallel with the decrease in calcium deposition, which was mainly observed in the areas devoid of adipocytes microscopically (dark deposits in Fig. 1D, lower
panels). These results supported the notion that JNK might regulate adipogenesis negatively and osteogenesis positively, in the early stages of differentiation. We next investigated by RT-PCR the effect of SP600125 on the expression of adipocyte markers during the differentiation of hMSCs into adipocytes (Fig. 2A). In the presence of SP600125, the expression of lipoprotein lipase (LPL) and peroxisome proliferated activated receptor (PPAR) c, early to middle adipocytemarkers [11], was increased. The expression of aP2 was not affected significantly by SP600125. We failed to detect the expression of osteopontin, an osteoblast marker [12], in any sample (data not shown). We also studied the expression of CCAAT/enhancer-binding protein (C/EBP) a and b by Western blotting (Fig. 2B). In the
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Fig. 2. Effect of SP600125 on the expression of adipocyte markers. Total RNA and cell lysates were prepared on the days indicated from the cells induced for adipogenesis in the presence of SP600125 or the vehicle. (A) RT-PCR of mRNA. Duplicate samples were analyzed. (B) Immunoblot analysis of C/EBPb and C/EBPa. Arrowheads indicate the isoforms of C/EBPb and C/EBPa, together with the sizes in kiloDalton. Lactate dehydrogenase (LDH) was taken as a control that was not affected by differentiation. The primary antibodies were used at 1:1000 dilutions.
absence of SP600125, expression of C/EBPb increased at day 1 and then decreased. In the presence of SP600125, on the other hand, the expression of C/EBPb also once decreased after the initial increase at day 1, though to a smaller extent than the control, and again increased at day 7. On the other hand, expression of the two C/EBPa isoforms peaked at days 3–5, which was strongly promoted by SP600125. In a transcriptional cascade of adipocyte differentiation, C/EBPb has been suggested to play a key role, by inducing PPAEc and C/EBPa [13]. Thus, SP600125 seems to promote adipogenesis from hMSCs by acting on the early steps of the differentiation cascade. A transcription factor, CREB, was reported to be involved in the expression of key transcription factors including C/EBPb during the induction of adipogenesis [14]. Accordingly, the effect of JNK inhibition on the transactivating function of CREB was examined by a gene reporter assay, in the presence or absence of IBMX, an agent expected to increase the cellular cAMP level. With a reporter plasmid containing four copies of CRE (pCRE-Luc), an increase in the reporter expression was observed with SP600125, in both the presence and absence of IBMX (Fig. 3A). To confirm that the activating effect of SP600125 was due to the inhibition of JNK, we employed a dominant negative
Fig. 3. Effect of JNK inhibition on the CRE-mediated transcriptional activation. A CRE-containing vector, pCRE-Luc (Stratagene) (filled bar), or its CRE-deleted version (gray bar) was used as a reporter. (A) Effect of SP600125. Trypsinized hMSCs (1 · 106 cells) were suspended in 100 ll phosphate-buffered saline and mixed with 10 lg of a reporter plasmid and 1 lg of a Renila luciferase expression vector, pRL-tk (Promega). Electroporation was performed in a cuvette with electrodes 2 mm apart, at the settings of 100 V, 1000 lF, and 72 X, with an electroporator, Model BT-600 (BTX). Transfected cells were plated in 24-well plates, cultured for 12 h, and then treated with the adipogenic hormone cocktail, with or without IBMX and SP600125. Luciferase activity was measured after additional 20 h. (B) Effect of dominant negative (dn) JNK. Trypsinized hMSCs (6 · 105 cells) were suspended in 100 ll of the Human MSC Nucleofector solution (Amaxa). The cell suspension was mixed with 0.4 lg of a reporter plasmid, 1.2 lg of a wild-type (WT) or dn JNK expression plasmid (gift from E. Nishida), 0.2 lg of a GFP-expression vector, phGFP(105)-C1 [23], and 0.2 lg of a b-galactosidase expression vector, pCMVb. Electroporation was performed in a Nucleofector (Amaxa) as recommended by the manufacturer, using a program C-17. Luciferase activity was normalized with Renila luciferase or b-galactosidase activity for transfection efficiency. Activities are shown as relative values, taking as 1 the activity of CRE-deleted reporter in the absence of both IBMX and a JNK expression vector. Inset, Western blot examining for the expression of WT and dn JNK.
JNK in the reporter assay, and observed an increase in the reporter activity (Fig. 3B), despite a lower expression level as compared with the wild-type JNK (Fig. 3B, inset). In these experiments, the effect of IBMX was slight, possibly due to the elevated level
S. Tominaga et al. / Biochemical and Biophysical Research Communications 326 (2005) 499–504
Fig. 4. Effect of SP600125 on the level of phosphorylation at Ser307 of IRS-1. Cells were induced for adipogenesis in the presence or absence of SP600125 and harvested at the periods as indicated. Samples were immunoblotted with an anti-phospho Ser307 IRS-1 antibody at a dilution of 1:1500 and an anti-IRS-1 antibody at a dilution of 1:200.
of basal CREB activation. Consistent with the increase in the CRE-dependent transcriptional activity by JNK inhibition in the absence of IBMX, SP600125 also promoted adipogenesis in the absence of IBMX, as assessed by GPDH specific activity and expression of adipocyte markers including aP2 (data not shown). These results suggest that SP600125 increases CREB activity through the inhibition of JNK, acting apparently in a similar manner to IBMX. It was reported [15] that phosphorylation of IRS-1 at Ser307, which is inhibitory to IRS-1 function, was promoted by TNFa through the activation of JNK. IRS-1 is activated by insulin and has a critical role in adipogenesis [16]. Accordingly, we examined by Western blotting whether the promotion of adipogenesis by JNK inhibition was accompanied by a decrease in Ser307phosphorylation of IRS-1 (Fig. 4). The Ser307 phosphorylation was reduced by SP600125 at 5 and 10 h after induction of differentiation. This result shows a possible link between the function of JNK in adipogenesis and phosphorylation of IRS-1 at Ser307.
Discussion Information was scarce about the signaling pathways involved in the determination of multipotent stem cells into the adipogenic lineage as well as the early stages of adipogenesis. In this study, we investigated the contributions of MAP kinases to the adipogenesis and osteogenesis from hMSCs. Inhibition of ERK signaling with PD98059 promoted adipogenesis, without affecting osteogenesis. A p38 inhibitor, SB203580, blocked both osteogenesis and adipogenesis, whereas a JNK inhibitor, SP600125, promoted adipogenesis, while suppressing osteogenesis. Thus, JNK signaling seemed to affect the divergence of adipogenic and osteogenic lineages from hMSCs, by changing the ratio of cells directed into these lineages. SP600125 and a dominant negative JNK enhanced CRE-dependent transcriptional activation, suggesting that the promotion of adipogenesis by JNK inhibition is at least in part through the activation of CREB. This
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was consistent with the increased expression of C/EBPb that is positioned downstream of CREB [14]. Increased C/EBPb activity would in turn lead to enhanced expression of PPARc and C/EBPa, both key regulators of adipogenesis, and then the downstream adipocyte markers [13]. In the present study, the activation of CREB by JNK inhibition was not accompanied by the increase in the phosphorylation level of CREB at Ser133 (data not shown). There is a precedent for phosphorylation-independent modification of CREB function in the process of adipocyte differentiation [17]. It was shown that the CREB activity is regulated by not only phosphorylation but also the association of another regulatory protein, transducers of regulated CREB (TORC), to the DNA-binding domain of CREB [18,19]. Thus, the activation of CREB by JNK inhibition may be due to another mechanism than phosphorylation. We also observed a decrease in the level of Ser307 phosphorylation of IRS-1 by the JNK inhibitor. IRS-1, a key regulatory factor of adipogenesis [16], is activated by insulin, and this effect is antagonized by phosphorylation at Ser307 [15]. Hence, the enhancement of adipogenesis by JNK inhibition is also possibly in part through decreasing Ser307 phosphorylation of IRS-1. Recently, ERK inhibition was reported to block the osteogenic conversion of hMSCs, resulting instead in the adipocyte formation in the absence of adipogenic hormone treatment [4]. These results may suggest that the ERK signaling pathway is involved in the divergence of adipogenic and osteogenic lineages from hMSCs. In the present study, however, neither the inhibition of osteogenesis nor promotion of adipogenesis was observed with PD98059, if the cells were cultured under the osteogenic conditions. Moreover, the effect of PD98059 was smaller than SP600125, even in the cells induced for adipogenesis. Difference in the origins (or donors) of hMSCs as well as the history of cells may affect the results. It was reported [20] that JNK activity was abnormally elevated in obese mice. Gene disruption of JNK1, a principal JNK isoform, alleviated dietary as well as genetic obesity, and protected the animals from the development of obesity-induced insulin resistance. The authors suggested that the major action of JNK in their study was the interruption of insulin signals by Ser307 phosphorylation of IRS-1. A similar mechanism was also implicated in the obesity and type2 diabetes induced by endoplasmic reticulum stress [21]. On the other hand, in the culture models including hMSCs, it is expected that the inhibition of insulin signaling would lead to decreased adipogenesis, because insulin is an essential adipogenic hormone in these experimental systems [22]. The complex regulatory circuit in the whole animals may result in an apparently opposite function of JNK.
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Acknowledgments We thank Dr. E. Nishida for the expression vectors of wild-type and dominant negative JNK, and Drs. I. Morita and T. Kamata for helpful suggestions. References
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Negative regulation of adipogenesis from human mesenchymal stem cells by Jun N-terminal kinase Sachiko Tominagaa, Tomohiro Yamaguchia, Shin-Ichiro Takahashib, Fumiko Hirosea, Takashi Osumia,* a
Graduate School of Life Science, Himeji Institute of Technology, University of Hyogo, 3-2-1 Koto, Kamigori, Hyogo 678-1297, Japan b Department of Animal Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Received 1 November 2004 Available online 20 November 2004
Abstract Human mesenchymal stem cells (hMSCs) are capable of differentiating into several cell types including adipocytes, osteoblasts, and chondrocytes, under appropriate culture conditions. We found that SP600125, an inhibitor of Jun N-terminal kinase (JNK), promoted adipogenesis whereas it repressed osteogenesis from hMSCs. SP600125 increased the expression of adipogenic transcription factors, CCAAT/enhancer-binding proteins a and b as well as peroxisome proliferator-activated receptor c2, which suggested that the chemical acted on the early steps of transcriptional regulatory cascade in adipogenesis. A gene reporter assay showed that SP600125 and a dominant negative JNK promoted a transcriptional activity dependent on the cAMP-response element (CRE). Thus, JNK represses adipogenesis from hMSCs probably by, at least in part, inhibiting the transactivating function of CRE-binding protein. Another action of JNK, phosphorylation at Ser307 of insulin receptor substrate-1, was also predicted to contribute to the repression of adipogenesis. 2004 Elsevier Inc. All rights reserved. Keywords: Human mesenchymal stem cells; Adipogenesis; Osteogenesis; Jun N-terminal kinase; cAMP-response element-binding protein; Insulin receptor substrate-1; Inhibitor; SP600125
Adipocytes are derived from mesenchymal stem cells that are also potent to differentiate into myocytes, osteoblasts, and chondrocytes. Adipogenesis is a complex process including commitment of the multipotent cells to the adipogenic lineage, proliferation of the preadipocytes, and terminal differentiation. Although the mechanism of terminal differentiation into adipocytes has been intensively studied using the established murine preadipocyte lines such as 3T3-L1 and F442A, key information is lacking on the commitment of multipotent precursor cells into individual cell lineages including preadipocytes. This was in part because of the difficulty in obtaining appropriate multipotent stem cells. In this *
Corresponding author. Fax: +81 791 58 0193. E-mail address: [email protected] (T. Osumi).
0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.11.056
regard, human mesenchymal stem cells (hMSCs) seem to be promising tools, because they have multipotency to differentiate into several cell types such as adipocytes, osteoblasts, and chondrocytes, under appropriate culture conditions [1]. In fact, successful adipogenesis from hMSCs was reported [2]. MAP kinases are a group of serine/threonine kinases conserved from yeast to mammals, being involved in versatile cellular processes, such as cell growth, apoptosis, and differentiation [3]. The classic MAP kinase, extracellular signal-regulated protein kinase (ERK), is a ubiquitous component of signal transduction pathways and activated by diverse extracellular stimuli. In animals two other major members of the MAP kinase family, Jun N-terminal kinase (JNK), also known as stress-activated protein kinase, and p38/reactivating
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kinase (p38) have been described. They are activated by a variety of cytokines, environmental stress, as well as ultraviolet and ionizing radiation. Using hMSCs, it was shown that inhibition of the ERK signaling pathway blocked osteogenic conversion, resulting instead in adipocyte formation [4]. This study suggested that hMSCs would be suitable for characterizing the early processes of adipogenesis and osteogenesis. In this study, we investigated the contribution of MAP kinases to adipogenesis and osteogenesis from hMSCs, using specific inhibitors. JNK signaling seemed to regulate osteogenesis positively, whereas adipogenesis negatively by inhibiting the function of an adipogenic transcription factor, cAMP-response element (CRE)binding protein (CREB).
Materials and methods Materials. PD98059 was purchased from Sigma, SP600125 from ALEXIS, and SB203580 from BIOMOL, respectively. Polyclonal antiC/EBPb (sc-150) and anti-C/EBPa (sc-9314) antibodies were obtained from Santa Cruz and phospho-specific anti-IRS-1 (Ser307) antibody was from Upstate. Polyclonal anti-insulin receptor substrate (IRS)-1 antibody was as described [5]. Cell culture. hMSCs were purchased from BioWhittaker. The cells were maintained in DulbeccoÕs modified EagleÕs medium (DMEM)high glucose (4.5 g glucose/L) containing 10% fetal bovine serum (FBS) (maintenance medium), in 5% CO2 at 37 C. They were passaged every 3 days at 70% confluence. For adipogenesis experiments, 2.1 · 104 hMSCs in 0.2–0.3 ml of maintenance medium per cm2 were seeded in 12-well plates or 35 mm dishes. After confluence, the cells were treated for 3 days with a hormone cocktail containing 1 lM dexamethasone (Dex), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.2 mM indomethacin, and 10 lg/ml insulin [6]. The cocktail was then removed, and the cells were cultured in maintenance medium containing 10 lg/ml insulin for further 6 days. For osteogenesis experiments, 3.1 · 103 cells in 0.2–0.3 ml of maintenance medium per cm2 were used. Cells were kept for 4–24 h in maintenance medium at 37 C in 5% CO2 to allow adhering to the bottom of plates. The medium was then replaced with maintenance medium supplemented with 0.1 lM Dex, 0.05 mM ascorbic acid, and 10 mM glycerol-2-phosphate (osteogenic medium) [6]. The cells were cultured for 8 days by changing the medium every 3 days. For simultaneous induction of adipocytes and osteoblasts, we optimized the culture conditions by preliminary experiments with regard to the cell density as well as the composition of supplements. In this protocol, the cells were plated as in the adipogenesis experiment. After confluence, the cells were treated for 3 days with the maintenance medium containing 0.1 lM Dex, 0.5 mM IBMX, 0.2 mM indomethacin, 10 lg/ml insulin, 0.05 mM ascorbic acid, and 10 mM glycerol-2phosphate. The cocktail was then removed, and the cells were cultured in the osteogenic medium for 6 days. In the experiments using MAP kinase inhibitors, PD98059 was used at 20 lM, whereas SB203580 and SP600125, 10 lM. The inhibitors were dissolved in dimethyl sulfoxide (DMSO), and the same amount of vehicle was added to the control samples. Estimation of differentiation. Glycerol-3-phosphate dehydrogenase (GPDH) activity and triglyceride content were measured as described previously [7]. Alkaline phosphatase (ALP) was assayed using an Alkaline Phospha K-test Wako kit (Wako Pure Chemicals) according to the manufacturerÕs protocol. Extracellularly deposited calcium was stained by the von Kossa method [8]. Assays were performed in trip-
licate, and the averages ± SD are given. Statistical significance was analyzed by StudentÕs t test and presented as: *P < 0.05; **P < 0.01; ***P < 0.001 and NS, not significant. Determination of the adipocyte marker gene expression. Total RNA was prepared with a RNeasy Mini Kit (Qiagen). RT-PCR was performed as described previously [7], using a ribosomal protein gene, 36B4, as a standard for the amount of RNA. The sequences of primers were: PPARc, forward (5 0 -gaggaattcatgggtgaaactctgggagattc-3 0 ) and reverse (5 0 -ccactggatctgttcttgtg-3 0 ); aP2, forward (5 0 -tactgggccaggaatttgac-3 0 ) and reverse (5 0 -taaactcttgtggaagtcacg-3 0 ); LPL, forward (5 0 cctgtacaagagagaacctgac-3 0 ) and reverse (5 0 -gcttctccaatgttgcatcctg-3 0 ); and 36B4, forward (5 0 -ttcgtgttcaccaaggaggac-3 0 ) and reverse (5 0 -atgatcagcccgaaggagaag-3 0 ). Western blot analysis was carried out as described previously [9].
Results To clarify the key signaling pathways in adipogenesis and osteogenesis from hMSCs, we investigated the contributions of MAP kinases, using specific inhibitors. When the cells were treated under the adipogenic conditions with PD98059, an ERK kinase (MEK1) inhibitor, the specific activity of GPDH and triglyceride content were increased as compared with the control cells (Fig. 1A). A JNK inhibitor, SP600125 [10], increased both parameters even more significantly, whereas a p38 inhibitor, SB203580, severely repressed them. These results suggest that all three MAP kinases are involved in adipogenesis from hMSCs, ERK and JNK acting negatively, whereas p38 positively. Next, we examined the effect of each inhibitor on osteogenic conversion of hMSCs. Under the osteogenic conditions, PD98059 did not affect the expression of ALP, an osteoblast marker (Fig. 1B). On the other hand, both SB203580 and SP600125 significantly decreased the ALP specific activity. Under these culture conditions, a small number of cells exhibited a round-shaped adipocyte-like morphology for the samples treated with SP600125, but not with PD98059. These results suggest that p38 and JNK participate in osteogenesis and adipogenesis from hMSCs, p38 positively regulating both processes, whereas JNK regulating positively the former and negatively the latter. We were particularly interested in JNK, because it seemed to affect the balance of forming adipogenic and osteogenic lineages from hMSCs. We asked whether the inhibitors exerted similar effects under the conditions that allowed simultaneous osteogenesis and adipogenesis. We established a protocol by which both types of cells differentiated, as confirmed by the increase in triglyceride content and ALP specific activity (see below). The lipid-containing cells were negative for the histochemical staining of ALP, and vice versa (data not shown), indicating that the differentiated adipocytes and osteoblasts expressed their own marker genes mutually exclusively. Under these conditions, PD98059 did not affect the ALP specific activity significantly, but SB203580 and SP600125
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Fig. 1. Effects of MAP kinase inhibitors on the adipogenesis and osteogenesis from hMSCs. (A) Specific activity of GPDH (top) and triglyceride content (bottom) of the cells after induction of adipogenesis. Cells were treated and cultured under the adipogenic conditions for 9 days in total, in the presence or absence of MAP kinase inhibitors. (B) Specific activity of ALP of the cells induced for osteogenesis. Cells were cultured for 8 days under the osteogenic conditions in the presence or absence of inhibitors. (C) Specific activity of ALP (top) and triglyceride content (bottom) of the cells induced for adipogenesis and osteogenesis simultaneously (see Materials and methods). (D) Staining of calcium deposition. Cells were cultured under the conditions of (C) and stained by the von Kossa method. Cells were cultured in the absence (left panels) and presence (right panels) of SP600125. Upper panels show the staining of whole culture dishes and lower panels the phase-contrast micrograms.
decreased it (Fig. 1C). On the other hand, PD98059 slightly increased the triglyceride accumulation, whereas SP600125 increased it 2.5-fold as compared with the control. In contrast, SB203580 severely repressed the triglyceride accumulation. As another measure of osteogenesis, we examined calcium deposition by von Kossa staining. On day 8 of post-confluence under the conditions for inducing both adipogenesis and osteogenesis, the SP600125-treated cells exhibited a reduced level of calcium staining as compared with the control (Fig. 1D, upper panels). Increase in the number of adipocytes by SP600125 was in parallel with the decrease in calcium deposition, which was mainly observed in the areas devoid of adipocytes microscopically (dark deposits in Fig. 1D, lower
panels). These results supported the notion that JNK might regulate adipogenesis negatively and osteogenesis positively, in the early stages of differentiation. We next investigated by RT-PCR the effect of SP600125 on the expression of adipocyte markers during the differentiation of hMSCs into adipocytes (Fig. 2A). In the presence of SP600125, the expression of lipoprotein lipase (LPL) and peroxisome proliferated activated receptor (PPAR) c, early to middle adipocytemarkers [11], was increased. The expression of aP2 was not affected significantly by SP600125. We failed to detect the expression of osteopontin, an osteoblast marker [12], in any sample (data not shown). We also studied the expression of CCAAT/enhancer-binding protein (C/EBP) a and b by Western blotting (Fig. 2B). In the
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Fig. 2. Effect of SP600125 on the expression of adipocyte markers. Total RNA and cell lysates were prepared on the days indicated from the cells induced for adipogenesis in the presence of SP600125 or the vehicle. (A) RT-PCR of mRNA. Duplicate samples were analyzed. (B) Immunoblot analysis of C/EBPb and C/EBPa. Arrowheads indicate the isoforms of C/EBPb and C/EBPa, together with the sizes in kiloDalton. Lactate dehydrogenase (LDH) was taken as a control that was not affected by differentiation. The primary antibodies were used at 1:1000 dilutions.
absence of SP600125, expression of C/EBPb increased at day 1 and then decreased. In the presence of SP600125, on the other hand, the expression of C/EBPb also once decreased after the initial increase at day 1, though to a smaller extent than the control, and again increased at day 7. On the other hand, expression of the two C/EBPa isoforms peaked at days 3–5, which was strongly promoted by SP600125. In a transcriptional cascade of adipocyte differentiation, C/EBPb has been suggested to play a key role, by inducing PPAEc and C/EBPa [13]. Thus, SP600125 seems to promote adipogenesis from hMSCs by acting on the early steps of the differentiation cascade. A transcription factor, CREB, was reported to be involved in the expression of key transcription factors including C/EBPb during the induction of adipogenesis [14]. Accordingly, the effect of JNK inhibition on the transactivating function of CREB was examined by a gene reporter assay, in the presence or absence of IBMX, an agent expected to increase the cellular cAMP level. With a reporter plasmid containing four copies of CRE (pCRE-Luc), an increase in the reporter expression was observed with SP600125, in both the presence and absence of IBMX (Fig. 3A). To confirm that the activating effect of SP600125 was due to the inhibition of JNK, we employed a dominant negative
Fig. 3. Effect of JNK inhibition on the CRE-mediated transcriptional activation. A CRE-containing vector, pCRE-Luc (Stratagene) (filled bar), or its CRE-deleted version (gray bar) was used as a reporter. (A) Effect of SP600125. Trypsinized hMSCs (1 · 106 cells) were suspended in 100 ll phosphate-buffered saline and mixed with 10 lg of a reporter plasmid and 1 lg of a Renila luciferase expression vector, pRL-tk (Promega). Electroporation was performed in a cuvette with electrodes 2 mm apart, at the settings of 100 V, 1000 lF, and 72 X, with an electroporator, Model BT-600 (BTX). Transfected cells were plated in 24-well plates, cultured for 12 h, and then treated with the adipogenic hormone cocktail, with or without IBMX and SP600125. Luciferase activity was measured after additional 20 h. (B) Effect of dominant negative (dn) JNK. Trypsinized hMSCs (6 · 105 cells) were suspended in 100 ll of the Human MSC Nucleofector solution (Amaxa). The cell suspension was mixed with 0.4 lg of a reporter plasmid, 1.2 lg of a wild-type (WT) or dn JNK expression plasmid (gift from E. Nishida), 0.2 lg of a GFP-expression vector, phGFP(105)-C1 [23], and 0.2 lg of a b-galactosidase expression vector, pCMVb. Electroporation was performed in a Nucleofector (Amaxa) as recommended by the manufacturer, using a program C-17. Luciferase activity was normalized with Renila luciferase or b-galactosidase activity for transfection efficiency. Activities are shown as relative values, taking as 1 the activity of CRE-deleted reporter in the absence of both IBMX and a JNK expression vector. Inset, Western blot examining for the expression of WT and dn JNK.
JNK in the reporter assay, and observed an increase in the reporter activity (Fig. 3B), despite a lower expression level as compared with the wild-type JNK (Fig. 3B, inset). In these experiments, the effect of IBMX was slight, possibly due to the elevated level
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Fig. 4. Effect of SP600125 on the level of phosphorylation at Ser307 of IRS-1. Cells were induced for adipogenesis in the presence or absence of SP600125 and harvested at the periods as indicated. Samples were immunoblotted with an anti-phospho Ser307 IRS-1 antibody at a dilution of 1:1500 and an anti-IRS-1 antibody at a dilution of 1:200.
of basal CREB activation. Consistent with the increase in the CRE-dependent transcriptional activity by JNK inhibition in the absence of IBMX, SP600125 also promoted adipogenesis in the absence of IBMX, as assessed by GPDH specific activity and expression of adipocyte markers including aP2 (data not shown). These results suggest that SP600125 increases CREB activity through the inhibition of JNK, acting apparently in a similar manner to IBMX. It was reported [15] that phosphorylation of IRS-1 at Ser307, which is inhibitory to IRS-1 function, was promoted by TNFa through the activation of JNK. IRS-1 is activated by insulin and has a critical role in adipogenesis [16]. Accordingly, we examined by Western blotting whether the promotion of adipogenesis by JNK inhibition was accompanied by a decrease in Ser307phosphorylation of IRS-1 (Fig. 4). The Ser307 phosphorylation was reduced by SP600125 at 5 and 10 h after induction of differentiation. This result shows a possible link between the function of JNK in adipogenesis and phosphorylation of IRS-1 at Ser307.
Discussion Information was scarce about the signaling pathways involved in the determination of multipotent stem cells into the adipogenic lineage as well as the early stages of adipogenesis. In this study, we investigated the contributions of MAP kinases to the adipogenesis and osteogenesis from hMSCs. Inhibition of ERK signaling with PD98059 promoted adipogenesis, without affecting osteogenesis. A p38 inhibitor, SB203580, blocked both osteogenesis and adipogenesis, whereas a JNK inhibitor, SP600125, promoted adipogenesis, while suppressing osteogenesis. Thus, JNK signaling seemed to affect the divergence of adipogenic and osteogenic lineages from hMSCs, by changing the ratio of cells directed into these lineages. SP600125 and a dominant negative JNK enhanced CRE-dependent transcriptional activation, suggesting that the promotion of adipogenesis by JNK inhibition is at least in part through the activation of CREB. This
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was consistent with the increased expression of C/EBPb that is positioned downstream of CREB [14]. Increased C/EBPb activity would in turn lead to enhanced expression of PPARc and C/EBPa, both key regulators of adipogenesis, and then the downstream adipocyte markers [13]. In the present study, the activation of CREB by JNK inhibition was not accompanied by the increase in the phosphorylation level of CREB at Ser133 (data not shown). There is a precedent for phosphorylation-independent modification of CREB function in the process of adipocyte differentiation [17]. It was shown that the CREB activity is regulated by not only phosphorylation but also the association of another regulatory protein, transducers of regulated CREB (TORC), to the DNA-binding domain of CREB [18,19]. Thus, the activation of CREB by JNK inhibition may be due to another mechanism than phosphorylation. We also observed a decrease in the level of Ser307 phosphorylation of IRS-1 by the JNK inhibitor. IRS-1, a key regulatory factor of adipogenesis [16], is activated by insulin, and this effect is antagonized by phosphorylation at Ser307 [15]. Hence, the enhancement of adipogenesis by JNK inhibition is also possibly in part through decreasing Ser307 phosphorylation of IRS-1. Recently, ERK inhibition was reported to block the osteogenic conversion of hMSCs, resulting instead in the adipocyte formation in the absence of adipogenic hormone treatment [4]. These results may suggest that the ERK signaling pathway is involved in the divergence of adipogenic and osteogenic lineages from hMSCs. In the present study, however, neither the inhibition of osteogenesis nor promotion of adipogenesis was observed with PD98059, if the cells were cultured under the osteogenic conditions. Moreover, the effect of PD98059 was smaller than SP600125, even in the cells induced for adipogenesis. Difference in the origins (or donors) of hMSCs as well as the history of cells may affect the results. It was reported [20] that JNK activity was abnormally elevated in obese mice. Gene disruption of JNK1, a principal JNK isoform, alleviated dietary as well as genetic obesity, and protected the animals from the development of obesity-induced insulin resistance. The authors suggested that the major action of JNK in their study was the interruption of insulin signals by Ser307 phosphorylation of IRS-1. A similar mechanism was also implicated in the obesity and type2 diabetes induced by endoplasmic reticulum stress [21]. On the other hand, in the culture models including hMSCs, it is expected that the inhibition of insulin signaling would lead to decreased adipogenesis, because insulin is an essential adipogenic hormone in these experimental systems [22]. The complex regulatory circuit in the whole animals may result in an apparently opposite function of JNK.
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Acknowledgments We thank Dr. E. Nishida for the expression vectors of wild-type and dominant negative JNK, and Drs. I. Morita and T. Kamata for helpful suggestions. References
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