- Email: [email protected]
ROCK and cytoskeleton in the adipogenesis of human mesenchymal stem cells
Journal of Bioscience and Bioengineering VOL. 117 No. 5, 624e631, 2014 www.elsevier.com/locate/jbiosc
Role of p38, ERK1/2, focal adhesion kinase, RhoA/ROCK and cytoskeleton in the adipogenesis of human mesenchymal stem cells Baiyao Xu,1 Yang Ju,1, * and Guanbin Song2 Department of Mechanical Science and Engineering, Nagoya University, Nagoya 464-8603, Japan1 and Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400030, PR China2 Received 4 July 2013; accepted 23 October 2013 Available online 9 December 2013
Adipogenesis is important to health and is thought occurring in the two stages of mesenchymal stem cell commitment to a preadipocyte fate and terminal differentiation of the preadipocyte. However, the mechanism of adipogenesis is still not clear. In this study, the roles of p38, extracellular regulated protein kinases 1/2 (ERK1/2), focal adhesion kinase (FAK), RhoA/ROCK, and cytoskeleton in both of the two stages of adipogenesis were assayed. Our results showed that the treatments of SB203580 (the inhibitor of p38) and U0126 (the inhibitor of ERK1/2) suppressed the adipogenesis induced by differentiation medium, and the treatments of PF573228 (a specific inhibitor of FAK), Y27632 (a specific inhibitor of RhoA/ROCK) and cytochalasin D (an inhibitor of cytoskeletal organization) promoted the adipogenesis. The treatments of SB203580 and U0126 significantly inhibited the adipogenic differentiation of hMSCs cultured in differentiation medium in the presence of PF573228, Y27632 or cytochalasin D. Moreover, the treatments of PF573228, Y27632 and cytochalasin D promoted p38 and ERK1/2 phosphorylations, and the treatments of U0126 and SB203580 decreased p38 and ERK1/2 phosphorylations, respectively. These results demonstrated that p38 and ERK1/2 played crucial positive roles in adipogenesis, and FAK, RhoA/ROCK and cytoskeleton played negative roles. Furthermore, FAK, RhoA/ROCK and cytoskeleton affected adipogenesis by regulating the activities of p38 and ERK1/2 which interacted with each other in the process of adipogenesis. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: p38; ERK1/2; Focal adhesion kinase; RhoA/ROCK; Cytoskeleton; Adipogenesis]
Adipose tissue, which is primarily composed of adipocytes, is important for energy balance and metabolic homeostasis. Adipocytes are actively involved in the regulation of cell function by a complex signaling network of endocrine, paracrine, and autocrine signals. They are also associated with a lot of pathological disorders including diabetes, hypertension, cancer, atherosclerosis, etc. All adipocytes are derived from mesenchymal stem cells (MSCs) (1). Adipogenesis is thought occurring in two stages: the commitment of MSC to a preadipocyte fate and the terminal differentiation of the preadipocyte (2). Numerous works have focused on the study of adipogenesis mechanism based on using preadipocytes and achieved quite a lot. However, the adipogenesis mechanism of MSCs is still not clear. The investigation of the mechanism of adipogenesis is beneficial to finding therapeutic targets against metabolic diseases which are becoming a global epidemic. p38 and ERK1/2 are the members of mitogen-activated protein kinases (MAPK) family. They are intracellular signaling pathways and play a pivotal role in many essential cellular processes, such as proliferation and differentiation (3). A major function of p38 and ERK1/2 is to transduce signaling of cell surface receptors to transcription factors in nucleus, which consequently triggers cellular differentiation. Engelman et al. described a positive role of p38 in adipogenesis first time (4). They found that the treatment of
* Corresponding author. Tel.: þ81 52 789 4672; fax: þ81 52 789 3109. E-mail address: [email protected] (Y. Ju).
SB203580 in 3T3-L1 differentiation decreased adipocyte formation. However, other researchers found an opposite role of p38 and demonstrated that p38 prevented adipocyte differentiation (5). In the case of ERK1/2, Sale et al. showed that ERK1/2 was required for adipogenesis in cell model of 3T3-L1 (6). Nevertheless, it was blurred by the work that ERK1/2 activation sufficiently inhibited fat cell differentiation by suppressing the activation of peroxisome proliferator-activated receptor-g (PPARg) which was a crucial factor for adipogenesis (7). Therefore, further researches are needed to investigate the roles of p38 and ERK1/2 in both of the stages of adipogenesis. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase functionalized as an important mediator for signaling transduction and guiding cell fate (8,9). RhoA, a small G-protein in Rho family, transfers extracellular signals by coupling with its direct effector, ROCK. RhoA/ROCK plays a pivotal role in signaling transduction and triggering cell differentiation (10). As a cellular scaffolding or skeleton, cytoskeleton contained within a cell’s cytoplasm. Cytoskeleton defines cell shape and also participates in cell differentiation (11). Our previous works had demonstrated that FAK, RhoA/ ROCK and cytoskeleton interacted with each other, and they were positive regulators in mechanical stretch-induced tenogenic differentiation (12). These studies suggested that p38, ERK1/2, FAK, RhoA/ROCK and cytoskeleton play an important role in guiding cell fate including adipogenic differentiation. Moreover, the crosstalk of p38, ERK1/2, FAK, RhoA/ROCK and cytoskeleton may exist in mammalian cells
1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.10.018
VOL. 117, 2014
RELATED SIGNALING MOLECULES IN ADIPOGENESIS
625
FIG. 1. Actin microstructure of each group after culturing for 14 days. (A) Cultured in expansion medium; (B) cultured in AGDM; (C) cultured in AGDM and treated with SB203580; (D) cultured in AGDM and treated with U0126; (E) cultured in AGDM and treated with PF573228; (F) cultured in AGDM and treated with Y27632; (G) cultured in AGDM and treated with cytochalasin D. Scale bar: 100 mm.
626
XU ET AL.
J. BIOSCI. BIOENG.,
FIG. 2. Gene expressions of PPARg and formations of lipid droplets after culturing for 14 days. (A) Gene expressions of PPARg were normalized to GAPDH. Control group (Con) was cultured in expansion medium; induced group (Ind) was cultured in AGDM; SB203580 group (SB) was cultured in AGDM and treated with SB203580; U0126 group (U) was cultured in AGDM and treated with U0126; PF573228 group (PF) was cultured in AGDM and treated with PF573228; Y27632 group (Y) was cultured in AGDM and treated with Y27632; cytochalasin D group (CD) was cultured in AGDM and treated with cytochalasin D. (B) Oil red O staining of Con; (C) oil red O staining of Ind; (D) oil red O staining of SB; (E) oil red O staining of U; (F) oil red O staining of PF; (G) oil red O staining of Y; (H) oil red O staining of CD. Data represent means standard deviations of three separate experiments (n ¼ 3). Double asterisk (**) indicates p < 0.01 vs. Con. Number sign (#) and double number sign (##) indicate p < 0.05 and p < 0.01 vs. Ind, respectively. Scale bar: 500 mm.
(12,13). However, the roles of p38, ERK1/2, FAK, RhoA/ROCK and cytoskeleton in adipogenesis are still not clear, especially the role of FAK. Understanding the roles of p38, ERK1/2, FAK, RhoA/ROCK and cytoskeleton in adipogenesis will help us to elaborate the relation of these signaling molecules in adipogenesis and the mechanism of adipogenesis. In this study, we utilized primary hMSCs and preadipocytes as cellular models, which were closer to the physiological conditions than other cell lines, to investigate the roles of p38, ERK1/2, FAK, RhoA/ROCK and cytoskeleton in adipogenesis. Our results suggested that p38 and ERK1/2 were the positive regulators of adipogenesis, while FAK, RhoA/ROCK and cytoskeleton were the negative regulators. Moreover, FAK, RhoA/ROCK and
cytoskeleton played negative roles by regulating the phosphorylations of p38 and ERK1/2. To our knowledge, this is the first paper to show the relation among p38, ERK1/2, FAK, RhoA/ROCK and cytoskeleton in adipogenesis. MATERIALS AND METHODS Cell culture hMSCs (MSC-R53-1, Passage 2) were acquired from Riken Cell Bank (Tsukuba, Japan). They were cultured in expansion medium which is DMEM medium (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS, Invitrogen, CA, USA) and 1% penicillin/streptomycin (Invitrogen) in 25 cm2 culture flasks (Becton Dickinson Labware, MA, USA) for expansion without differentiation (14). Cells were kept in a humidified incubator at 37 C and supplemented with 5% CO2.
VOL. 117, 2014
RELATED SIGNALING MOLECULES IN ADIPOGENESIS
Unpassaged primary human preadipocytes were acquired from Lonza (NJ, USA) and cultured in preadipocyte growth medium (Lonza) in 25 cm2 culture flasks for expansion without differentiation. Cells were kept in a humidified incubator at 37 C and supplemented with 5% CO2. Adipogenesis of hMSCs and preadipocytes For the adipogenic differentiation of hMSCs, hMSCs of passage 4 were subcultured in expansion medium in 6 well plates. When the cells became confluent, the expansion medium was changed to adipogenic differentiation medium (AGDM, HyClone, UT, USA) and cultured for 14 days with medium changed every 3 days. As a control, hMSCs were cultured in expansion medium with medium changed every 3 days. In the case of preadipocytes, preadipocytes of passage 3 were subcultured in preadipocyte growth medium in 6 well plates. After confluent, adipocyte differentiation medium (ACDM, Lonza) was introduced to induce adipogenesis. Preadipocytes were induced for 14 days with medium changed every 3 days. As a control, preadipocytes were cultured in growth medium and the medium was changed every 3 days. Pharmacological inhibitors The following pharmacological inhibitors were employed: 10 mM SB203580 (Calbiochem, CA, USA), 10 mM U0126 (Calbiochem), 10 mM PF573228 (Tocris, MO, USA), 10 mM Y27632 (Calbiochem) and 0.02 mg/ml cytochalasin D (Calbiochem). When cells became confluent, the inhibitors were added simultaneously when the medium was changed at the beginning of the experiment. The inhibitors were added with the medium changed every 3 days. As a control, cells were cultured in the medium without any inhibitors and the medium was changed every 3 days. Immunofluorescence staining Cells were fixed in 4% paraformaldehyde (PFA) for 15 min and permeabilized with 0.5% Triton-X-100 in PBS for 10 min. They were pre-incubated in 200 ml of 0.1 mM FITC-conjugated phalloidin (Enzo Life Science, NY, USA) in primary blocking solution (1% BSA in PBS) at room temperature for 90 min. Then, the cells were incubated with 0.1 mg/ml DAPI (Calbiochem) in PBS for 5 min, after which mounting medium (10 ml) was dispensed on the cells. The cells were washed three times with PBS for 5 min after each step. A glass coverslip was placed on the slide and sealed with nail polish before observation. Finally, the slides were visualized with a confocal microscope (Nikon A1Rsi, Nikon, Tokyo, Japan). RNA isolation and real-time reverse transcription quantitative polymerase chain reaction (RT-PCR) Cells were lysed and total RNA was isolated using a RNeasy Mini Kit (Qiagen, Duesseldorf, Germany). The 260/280 absorbance ratio was measured for verification of the purity and concentration of the RNA. Reverse transcription was completed using a High Capacity RNA-to-cDNA Kit (ABI, CA, USA). The pre-designed minor groove binder (MGB) probes of glyceraldehydes 3-phosphate dehydrogenase (GAPDH), PPARg (ABI), as well as Taqman PCR Master Mix and a Light Cycler apparatus (ABI 7300, ABI), were used to analyze the gene expressions of the interested genes. The gene expressions of PPARg were calculated using standard curve method and normalized to GAPDH. Oil red O straining Cells were washed with PBS and fixed with 4% PFA for 20 min. After washing the cells twice with PBS, the cells were stained with oil red O solution (Sigma, MO, USA). Then the cells were washed with tap water flow for 20 s. Western blot After treating with or without inhibitors for 30 min, cells were washed with PBS and 100 ml of detergent-based lysis buffer (M-PER Mammalian Protein Extraction Reagent, Pierce, IL, USA), supplemented with protease inhibitor PMSF and a cocktail of phosphatase inhibitors (Pierce, 1:100 dilution) were added to each well for the collection of total cellular proteins. The protein (30 mg) of each sample was loaded onto a 10% SDSePAGE gel for gel electrophoresis. The separated proteins were transferred to a PVDF membrane (Bio-Rad, CA, USA) at 80 V for 120 min. The membranes were blocked in 5% BSA/TBS-Tween 20 solution for 1 h at room temperature, followed by the application of the monoclonal antibody specific for p-p38 (Bioworld Technology Inc., MN, USA), p38 (Bioworld Technology Inc., MN, USA), p-ERK1/2 (Epitomics, CA, USA), and ERK1/2 (Epitomics, CA, USA), at 1:1000 in 5% BSA/TBS-Tween 20. After incubating for 90 min with the primary antibodies at room temperature, the secondary antibody, anti-rabbit IgG-HRP (Cayman Chemical, MI, USA) at 1:10,000 in 5% BSA/TBS-Tween 20 was applied for 1 h at room temperature. The membrane was washed three times with 0.1% TBS/Tween 20 for 10 min after each antibody application. The proteins on the PVDF membrane were detected with an ECL detection system (Pierce), according to the manufacturer’s protocol. The proteins were quantified by volume summation of image pixels with a Fujifilm LAS-4000 (Fujifilm, Tokyo, Japan). Statistical analysis The means and standard deviations were reported for three single repeat samples. A paired Student’s t-test was performed, and a p-value of less than 0.05 was considered to be statistically significant. The p-value was calculated by the soft of SPSS 16.0.
RESULTS Cytoskeletal microstructure of hMSCs at different culture conditions To characterize the effects of SB203580, U0126, PF573228, Y27632 and cytochalasin D on the changes in cytoskeletal microstructure during adipogenic differentiation, cytoskeletal
627
microstructure was observed by confocal microscope after cultured for 14 days. The cytoskeletal microstructure of each group was somewhat different. Cells cultured in expansion medium were mostly well spread, elongated, and often triangular. The actin fibers were aligned along the long axis of the cells (Fig. 1A). After cultured in AGDM, cells changed to round. Actin fibers were shortened and a lot of punctuated fiber actins gathered in the edge of cells. The actin fibers distributed around nuclei were reduced (Fig. 1B). When cells were cultured in AGDM, and treated with SB203580 or U0126, shorten actin fibers became discontinuous. The actin fibers distributed around nuclei were further reduced and only a few distributed around the nuclei (Fig. 1C and D). PF573228 treatment caused cells to become larger and some of actin fibers were still aligned along the long axis of the cells (Fig. 1E). Treatment with Y27632 led to stellate-like cells with a lack of actin fibers (Fig. 1F). Cytochalasin D treatment resulted in the disruption and punctuate of actin fiber organization (Fig. 1G). Although some of actin fibers of the cells treated with PF573228, Y27632 or cytochalasin D were shortened, there were still a lot of actin fibers as long as before. In addition, the actin fibers distributed around nuclei were also reduced by the treatments of PF573228, Y27632 and cytochalasin D. These results indicated that AGDM gave rise to the cortical organization of cytoskeleton, and the treatments of the inhibitors resulted in the changes of cytoskeletal microstructure. Nevertheless, the treatments did not change the cortical organization significantly. By the way, in the experiments of this part, the time point of 14th day was chosen due to most of cells treated with PF573228, Y27632 or cytochalasin D formulated a lot lipid droplets. Gene expressions of PPARg and accumulations of lipid droplets in hMSCs with/without the treatments of inhibitors To evaluate the roles of p38, ERK1/2, FAK, RhoA/ ROCK and cytoskeleton on adipogenesis, hMSCs were cultured in AGDM with the treatments of SB203580, U0126, PF573228, Y27632 and cytochalasin D. After cultured for 14 days, the gene expressions of PPARg and the formations of lipid droplets were assessed by realtime RT-PCR and oil red staining. As shown in Fig. 2A, the inhibitors have significantly influenced on the gene expressions of PPARg. After cultured in AGDM, the gene expression of PPARg was increased about 8 times compared to that of control group. Treatments with SB203580 and U0126 attenuated the AGDM-induced increase in the gene expressions of PPARg. The gene expression of PPARg in the SB203580 group was much lower than that in the U0126 group (p < 0.01). However, when cells were cultured in AGDM and treated with PF573228, Y27632 or cytochalasin D, the gene expressions of PPARg were significantly increased compared to that of induced group (p < 0.05). The gene expression of PPARg of cytochalasin D group was the highest one among these groups. As shown in Fig. 2BeH, the inhibitors had a significant influence on the formations of lipid droplets. After cultured in AGDM for 14 days, approximately 50% cells were stained with oil red O (Fig. 2C), while cells cultured in expansion medium were negative to oil red O staining (Fig. 2B). When cultured in AGDM and treated with SB203580, they were also negative to oil red O staining (Fig. 2D). When cells were cultured in AGDM and treated with U0126, only approximately 25% of them were positive to the staining and the ratio was much lower than the cells cultured in AGDM only (Fig. 2E). On the other hand, when cells were cultured in AGDM and treated with PF573228, Y27632 or cytochalasin D, the positive staining ratios of the cells were much higher than that of the cells cultured in AGDM only (Fig. 2FeH). The positive staining ratio of the cells treated with PF573228 was approximately 70% and lower than that of the cells treated with Y27632 or cytochalasin D. There was no significant difference between the ratios of the cells treated
628
XU ET AL.
J. BIOSCI. BIOENG.,
with Y27632 and cytochalasin D. Consistent with the results of hMSCs, the inhibitors had the same effect on lipid droplets formations of human preadipocytes (Fig. S1). The effects of PF573228, Y27632 and cytochalasin D on the gene expressions of PPARg and the formations of lipid droplets in hMSCs cultured in expansion medium were further investigated. The treatments of PF573228, Y27632 and/or cytochalasin D markedly increased the gene expressions of PPARg when cells were cultured in expansion medium without any adipogenic factors and only a few cells were positive to oil red O staining (Fig. S2). Although the treatments of PF573228, Y27632 and/or cytochalasin D without any adipogenic factors increased the gene expressions of PPARg, they cannot induce abundant accumulations of lipid droplets. These results suggested that p38 and ERK1/2 were positive regulators, while FAK, RhoA/ROCK and cytoskeleton were negative regulators in the adipogenic differentiation of hMSCs. Phosphorylations of p38 and ERK1/2 by the treatments of cytochalasin D, Y27632 and PF573228 The effects of cytochalasin D, Y27632 and PF573228 on the phosphorylations of p38 and ERK1/2 in hMSCs were investigated. As shown in Fig. 3, the treatments of cytochalasin D, Y27632 and PF573228 significantly increased the phosphorylations of p38 and ERK1/2. It meant that the inhibitions of the polymerization of cytoskeleton or the activities of RhoA/ROCK and FAK, promoted p38 and ERK1/2 phosphorylations. Interestingly, the treatments of SB203580 and U0126 resulted in the decrease in the phosphorylations of ERK1/2 and p38, respectively. However, the change in the phosphorylation of ERK1/2 by the treatments of SB203580 did not reach statistical significance. These results were consistent with the conclusion that p38 and ERK1/2 were positive regulators, while FAK, RhoA/ ROCK and cytoskeleton were negative regulators in adipogenic differentiation of hMSCs. Moreover, these findings suggested that the crosstalk between p38 and ERK1/2 existed in the process of adipogenic differentiation of hMSCs. Gene expressions of PPARg in hMSCs treated with PF573228, Y27632 or cytochalasin D in the presence of SB203580 or U0126 To further study the roles of p38, ERK1/2, FAK, RhoA/ ROCK and cytoskeleton in the process of adipogenic differentiation, the effects of SB203580 and U0126 on the gene expressions of PPARg in hMSCs which were cultured in AGDM and treated with PF573228, Y27632 or cytochalasin D were investigated. As shown in Fig. 4, the gene expression of PPARg in hMSCs cultured in AGDM
FIG. 4. Gene expressions of PPARg in hMSCs treated with PF573228, Y27632 or cytochalasin D in the presence of SB203580 or U0126. The gene expressions of PPARg were normalized to GAPDH. Control group (Con) was cultured in expansion medium; induced group (Ind) was cultured in AGDM; SB203580 plus PF573228 group (SB þ PF) was cultured in AGDM treated with SB203580 and PF573228; SB203580 plus Y27632 group (SB þ Y) was cultured in AGDM treated with SB203580 and Y27632; SB203580 plus cytochalasin D group (SB þ CD) was cultured in AGDM treated with SB203580 and cytochalasin D; U0126 plus PF573228 group (U þ PF) was cultured in AGDM treated with U0126 and PF573228; U0126 plus Y27632 group (U þ Y) was cultured in AGDM treated with U0126 and Y27632; U0126 plus cytochalasin D group (U þ CD) was cultured in AGDM treated with U0126 and cytochalasin D; data represent means standard deviations of three separate experiments (n ¼ 3). Double asterisk (**) indicates p < 0.01 vs. Con.
was around 7.5 times compared to that of control group. However, the treatments of SB203580 and U0126 inhibited the gene expressions of PPARg in all groups. In SB þ PF, SB þ Y and SB þ CD groups, the gene expressions of PPARg were around 4 times compared to that in control group; in U þ PF, U þ Y and U þ CD groups, the gene expressions of PPARg were around 5 times (Fig. 4). Compared to the gene expressions of PPARg in hMSCs cultured by AGDM and treated with PF573228, Y27632 or cytochalasin D shown in Fig. 2A, the gene expressions of PPARg in the SB þ PF, SB þ Y, SB þ CD, U þ PF, U þ Y and U þ CD groups were significantly reduced by the treatments of SB203580 and U0126. Consistent with the results of gene expressions, the
FIG. 3. Phosphorylations of p38 and ERK1/2 by the treatments of cytochalasin D, Y27632, PF573228, U0126 and SB203580. (A) Phosphorylation of p38 by the treatments of cytochalasin D, Y27632 and PF573228; (B) phosphorylation of ERK1/2 by the treatments of cytochalasin D, Y27632 and PF573228; (C) phosphorylation of p38 by the treatment of U0126; (D) phosphorylation of ERK1/2 by the treatment of SB203580. Panels a, b, c and d indicate the results of panels A, B, C and D, respectively. Control group (Con) was cultured in medium without any inhibitor; cytochalasin D group (CD) was treated with 0.02 mg/ml cytochalasin D; Y27632 group (Y) was treated with 10 mM Y27632; PF573228 group (PF) was treated with 10 mM PF573228; U0126 group (U) was treated with 10 mM U0126; SB203580 group (SB) was treated with 10 mM SB203580. Data represent means standard deviations of three separate experiments (n ¼ 3). Asterisk (*) indicates p < 0.05 vs. Con.
VOL. 117, 2014
RELATED SIGNALING MOLECULES IN ADIPOGENESIS
treatments of SB203580 and U0126 resulted in sharply decreasing in the accumulations of lipid droplets (Fig. S3).
DISCUSSION p38 and ERK1/2 are the members of MAPK which coordinately regulate proliferation, differentiation and so on (15). Engelman et al. described a positive role of adipogenesis utilizing 3T3-L1 cell line firstly (4,16). Aouadi et al. also found that SB203580 incubation reduced the accumulation of lipid and p38 activity was required for human primary adipocyte differentiation (17). Coincides with the report of Aouadi et al. (17), our results showed that the treatment of SB203580 sharply suppressed the gene expression of PPARg which acted as a crucial regulator of adipogenesis program (18), and it completely inhibited the formation of lipid droplets. Although the gene expression of PPARg was sharply inhibited by the treatment of SB203580, it was still four times higher than the cells cultured in expansion medium. It meant that the increase in the gene expression of PPARg was not only regulated by p38. Some other signaling molecules may be activated to up-regulate the gene expression of PPARg. Furthermore, the formation of lipid droplets was completely suppressed by the inhibition of p38. It implied that p38 did not only regulate the gene expression of PPARg, but also played an important role in gene expressions which regulated the formation of lipid droplets, such as the gene expression of C/EBPa. These results suggested that the increase in the gene expression of PPARg was not the only precondition to induce the differentiation of hMSCs into matured adipocytes, and p38 activity was necessary to the adipogenesis of hMSCs. However, the role of p38 in adipogenesis was disputed due to the opposing reports. Some reports showed that p38 activity inhibited adipocyte differentiation (5,19). Aouadi et al. (20) reported that the inhibition of p38 increased adipogenesis from embryonic to adult stages. The reason of opposite results about the role of p38 in adipogenesis may be due to the role of p38 depending on the stage of adipogenesis, differentiation condition and cellular model. Regarding to the role of ERK1/2 in adipogenesis, apparent contradictory results were reported recently. Ling et al. (7) reported that ERK1/2 activation sufficiently inhibited fat cell differentiation by suppressing the activation of PPARg. Nevertheless, Sale et al. (6) showed that ERK1/2 was required for the adipogenesis in the cell model of 3T3-L1. Some researches indicated that ERK1/2 had multiple functions in adipogenesis (21e24). In consistent with the report by Sale et al. (6), our findings showed that the treatment of U0126 dramatically suppressed the gene expression of PPARg and partly inhibited the formation of lipid droplets which suggested that ERK1/2 was a positive regulator in the process of adipogenic differentiation of hMSCs. FAK, RhoA/ROCK and cytoskeleton play important roles in guiding MSCs fate. Here we showed that the inhibition of FAK activity significantly increased the gene expression of PPARg and promoted the formation of lipid droplets. Moreover, the inhibition of FAK activity up-regulated the gene expression of PPARg in the cells cultured without any adipogenic factor. These findings manifested that FAK was a negative regulator in adipogenesis. This consequence could be used to explain that FAK was cleaved gradually during adipocyte differentiation (25), FAK stabilized cytoskeleton, and the inhibition of FAK resulted in lower cell stiffness occurred during the process of adipogenesis (26). For the roles of RhoA/ROCK and cytoskeleton in adipogenesis, they were represented as similar as the role of FAK. The inhibitions of RhoA/ROCK activity and the polymerization of cytoskeleton increased the gene expressions of PPARg, even though the cells were cultured without any adipogenic factor. These results demonstrated that RhoA/ROCK and cytoskeleton were negative
629
regulators of adipogenesis. In agreement with this interpretation, RhoA inhibits adipogenesis by the regulation of cytoskeletal tension through RhoA-Rho kinase (ROCK) signaling pathway (27). It was well known that Rho family small GTPases regulated cell shape through modulating cytoskeleton, and RhoA/ROCK had been implicated in the lineage commitment of MSCs (28e32). The changes in the activity of RhoA/ROCK and the remodeling of cytoskeleton were concurrent with a modulation of cellular mechanical properties (33). For osteogenesis, cells typically become stiffer, while cells become more compliant in adipogenesis (26). The inhibition of RhoA/ROCK and the remodeling of cytoskeleton result in the reduction of pre-existing inner cellular force and lower stiffness. Furthermore, our results also showed that adipogenic differentiation resulted in shortened and punctuated actin fibers which would cause the reduction of pre-existing inner cellular force and Young’s modulus. Therefore, similar to FAK, the mechanisms of RhoA/ROCK and cytoskeleton exerting influence on adipogenesis may be conjectured that the inhibitions of RhoA/ROCK and cytoskeleton reduced pre-existing inner cellular force which was involved in adipogenesis. In the process of adipogenic differentiation of hMSCs, the inhibitions of cytoskeleton, RhoA/ROCK and FAK resulted in the increase of the phosphorylations levels of p38 and ERK1/2. It implied that cytoskeleton, RhoA/ROCK and FAK regulated adipogenic differentiation by acting upon the activity of p38 or ERK1/2. Moreover, our results showed that inhibiting the phosphorylations of p38 and ERK1/2, blocked the adipogenic differentiation of hMSCs which was promoted by blocking the activity of FAK, RhoA/ROCK or the polymerization of cytoskeleton. These results demonstrated that FAK, RhoA/ROCK and cytoskeleton played a negative role in adipogenesis by regulating the activities of p38 and ERK1/2. Besides, our findings showed that the treatments of SB203580 and U0126 resulted in slight decrease in the phosphorylations of ERK1/2 and p38, respectively. It suggested that p38 and ERK1/2 interacted with each other in the process of adipogenesis. Crosstalk between branches of p38 and ERK1/2 depends on protein phosphatase (34). Wang et al. reported that inhibiting p38 and ERK1/2 leads to increase in phosphorylations of ERK1/2 and p38 in corneal epithelial cells, respectively (35). The result was different from ours. It implied that the crosstalk between p38 and ERK1/2 may depend on cell type or growth condition. In addition, FAK, RhoA/ROCK and cytoskeleton interacted with each other in a lot of cellular activities (12,36,37). They may interact with each other in the process of adipogenesis as negative regulators. FAK and RhoA/ROCK stabilized the cytoskeleton and the inhibition of FAK or RhoA/ROCK made cell soft (38). The remodeling of cytoskeleton also affected the activities of FAK and RhoA/ROCK (12,39). Moreover, cell shape can regulate RhoA activity, and this activity mediates the shape-dependent control of hMSCs lineage commitment to adipocytes (27,40,41). Our previous study had demonstrated that FAK, RhoA/ROCK and cytoskeleton, as positive regulators, act upon each other in the process of tenogenic differentiation of MSCs (12). These studies inferred that the crosstalk among FAK, RhoA/ ROCK and cytoskeleton existed in the process of adipogenesis. Signaling transduction is a complex network. For the hMSC lineage commitment, a lot of signaling pathways communicate with each other to control the targeted gene expressions. In this study, the different roles of p38, ERK1/2, FAK, RhoA/ROCK and cytoskeleton in adipogenesis were depicted. p38, ERK1/2, FAK, RhoA/ROCK and cytoskeleton did not work alone, but interacted with each other to compose a network to regulate the process of adipogenesis. The roles of p38, ERK1/2, FAK, RhoA/ROCK and cytoskeleton in adipogenesis are shown in Fig. 5: (i) p38 and ERK1/ 2 were positive regulators of adipogenesis; (ii) crosstalk between p38 and ERK1/2 existed in adipogenesis; (iii) FAK, RhoA/ROCK and cytoskeleton involved in adipogenesis by acting upon ERK1/2 and
630
XU ET AL.
J. BIOSCI. BIOENG.,
FIG. 5. Proposed schematic diagram of related signaling molecules in the adipogenesis of hMSCs. p38 and ERK1/2 are positive regulators in the adipogenesis, while FAK, RhoA/ROCK and cytoskeleton are negative regulators by acting upon p38 and ERK1/2.
p38; (iv) FAK, RhoA/ROCK and cytoskeleton, as negative regulators, may interact with each other in adipogenesis. In summary, our findings demonstrated that p38 and ERK1/2 were positive regulators, and FAK, RhoA/ROCK and cytoskeleton were negative regulators by regulating the activities of p38 and ERK1/2 in adipogenesis. p38, ERK1/2, FAK, RhoA/ROCK and cytoskeleton composed a network to regulate adipogenesis. To our knowledge, this is the first study which suggests that FAK, RhoA/ ROCK and cytoskeleton play negative roles in adipogenesis by regulating activity of p38 and ERK1/2. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2013.10.018. ACKNOWLEDGMENTS This work was supported by JSPS KAKENHI Grant numbers 23246024 and 24656085. We thank Dr. Kato (Hiroshima University) for the supply of hMSCs. References 1. Gesta, S., Tseng, Y., and Kahn, C.: Developmental origin of fat: tracking obesity to its source, Cell, 131, 242e256 (2007). 2. Cristancho, A. and Lazar, M.: Forming functional fat: a growing understanding of adipocyte differentiation, Nat. Rev. Mol. Cell Biol., 12, 722e734 (2011). 3. Bost, F., Aouadi, M., Caron, L., and Binetruy, B.: The role of MAPKs in adipocyte differentiation and obesity, Biochimie, 87, 51e56 (2005). 4. Engelman, J., Lisanti, M., and Scherer, P.: Specific inhibitors of p38 mitogenactivated protein kinase block 3T3-L1 adipogenesis, J. Biol. Chem., 273, 32111e32120 (1998). 5. Wang, X. and Ron, D.: Stress-induced phosphorylation and activation of the transcription factor CHOP (GADD153) by p38 MAP kinase, Science, 272, 1347e1349 (1996). 6. Sale, E., Atkinson, P., and Sale, G.: Requirement of MAP kinase for differentiation of fibroblasts to adipocytes, for insulin activation of p90 S6 kinase and for insulin or serum stimulation of DNA synthesis, EMBO J., 14, 674e684 (1995). 7. Ling, H., Wen, G., Feng, S., Tuo, Q., and Ou, H.: MicroRNA-375 promotes 3T3L1 adipocyte differentiation through modulation of extracellular signal-regulated kinase signalling, Clin. Exp. Pharmacol. Physiol., 38, 239e246 (2011).
8. Guan, J.: Integrin signaling through FAK in the regulation of mammary stem cells and breast cancer, IUBMB Life, 62, 268e276 (2010). 9. Xu, B., Song, G., and Ju, Y.: Effect of focal adhesion kinase on the regulation of realignment and tenogenic differentiation of human mesenchymal stem cells by mechanical stretch, Connect. Tissue Res., 52, 373e379 (2011). 10. Sun, Y., Chen, C., and Fu, J.: Forcing stem cells to behave: a biophysical perspective of the cellular microenvironment, Annu. Rev. Biophys., 41, 519e542 (2012). 11. Treiser, M., Yang, E., Gordonov, S., Cohen, D., and Androulakis, I.: Cytoskeleton-based forecasting of stem cell lineage fates, Proc. Natl. Acad. Sci. USA, 107, 610e615 (2010). 12. Xu, B., Song, G., Ju, Y., Li, X., and Song, Y.: RhoA/ROCK, cytoskeletal dynamics, and focal adhesion kinase are required for mechanical stretch-induced tenogenic differentiation of human mesenchymal stem cells, J. Cell Physiol., 227, 2722e2729 (2012). 13. Chaturvedi, L., Marsh, H., and Basson, M.: Role of RhoA and its effectors ROCK and mDia1 in the modulation of deformation-induced FAK, ERK, p38, and MLC motogenic signals in human Caco-2 intestinal epithelial cells, Am. J. Physiol. Cell Physiol., 301, C1224eC1238 (2011). 14. Tsutsumi, S., Shimazu, A., Miyazaki, K., Pan, H., and Koike, C.: Retention of multilineage differentiation potential of mesenchymal cells during proliferation in response to FGF, Biochem. Biophys. Res. Commun., 288, 413e419 (2001). 15. Cargnello, M. and Roux, P.: Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases, Microbiol. Mol. Biol. Rev., 75, 50e83 (2011). 16. Engelman, J., Berg, A., Lewis, R., Lin, A., and Lisanti, M.: Constitutively active mitogen-activated protein kinase kinase 6 (MKK6) or salicylate induces spontaneous 3T3-L1 adipogenesis, J. Biol. Chem., 274, 35630e35638 (1999). 17. Aouadi, M., Jager, J., Laurent, K., Gonzalez, T., and Cormont, M.: p38MAP kinase activity is required for human primary adipocyte differentiation, FEBS Lett., 581, 5591e5596 (2007). 18. Tang, Q. and Lane, M.: Adipogenesis: from stem cell to adipocyte, Annu. Rev. Biochem., 81, 715e736 (2012). 19. Yang, T., Xiong, Q., Enslen, H., Davis, R., and Chow, C.: Phosphorylation of NFATc4 by p38 mitogen-activated protein kinases, Mol. Cell. Biol., 22, 3892e3904 (2002). 20. Aouadi, M., Laurent, K., Prot, M., Le Marchand-Brustel, Y., Binetruy, B., and Bost, F.: Inhibition of p38MAPK increases adipogenesis from embryonic to adult stages, Diabetes, 55, 281e289 (2006). 21. Font de Mora, J., Porras, A., Ahn, N., and Santos, E.: Mitogen-activated protein kinase activation is not necessary for, but antagonizes, 3T3-L1 adipocytic differentiation, Mol. Cell. Biol., 17, 6068e6075 (1997). 22. Kim, S., Muise, A., Lyons, P., and Ro, H.: Regulation of adipogenesis by a transcriptional repressor that modulates MAPK activation, J. Biol. Chem., 276, 10199e10206 (2001). 23. Lim, S., Jang, H., Park, E., Kim, J., and Kim, J.: Wedelolactone inhibits adipogenesis through the ERK pathway in human adipose tissue-derived mesenchymal stem cells, J. Cell. Biochem., 113, 3436e3445 (2012). 24. Than, A., Cheng, Y., Foh, L., Leow, M., and Lim, S.: Apelin inhibits adipogenesis and lipolysis through distinct molecular pathways, Mol. Cell. Endocrinol., 362, 227e241 (2012). 25. Li, J. and Xie, D.: Cleavage of focal adhesion kinase (FAK) is essential in adipocyte differentiation, Biochem. Biophys. Res. Commun., 357, 648e654 (2007). 26. Gonzalez-Cruz, R., Fonseca, V., and Darling, E.: Cellular mechanical properties reflect the differentiation potential of adipose-derived mesenchymal stem cells, Proc. Natl. Acad. Sci. USA, 109, E1523eE1529 (2012). 27. McBeath, R., Pirone, D., Nelson, C., Bhadriraju, K., and Chen, C.: Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment, Dev. Cell, 6, 483e495 (2004). 28. Chen, Z., Wang, X., Shao, Y., Shi, D., and Chen, T.: Synthetic osteogenic growth peptide promotes differentiation of human bone marrow mesenchymal stem cells to osteoblasts via RhoA/ROCK pathway, Mol. Cell. Biochem., 358, 221e227 (2011). 29. Xu, T., Wu, M., Feng, J., Lin, X., and Gu, Z.: RhoA/Rho kinase signaling regulates transforming growth factor-beta1-induced chondrogenesis and actin organization of synovium-derived mesenchymal stem cells through interaction with the Smad pathway, Int. J. Mol. Med., 30, 1119e1125 (2012). 30. Meyers, V., Zayzafoon, M., Douglas, J., and McDonald, J.: RhoA and cytoskeletal disruption mediate reduced osteoblastogenesis and enhanced adipogenesis of human mesenchymal stem cells in modeled microgravity, J. Bone Miner. Res., 20, 1858e1866 (2005). 31. Shi, Y., Li, H., Zhang, X., Fu, Y., and Huang, Y.: Continuous cyclic mechanical tension inhibited Runx2 expression in mesenchymal stem cells through RhoAERK1/2 pathway, J. Cell. Physiol., 226, 2159e2169 (2011). 32. Etienne-Manneville, S. and Hall, A.: Rho GTPases in cell biology, Nature, 420, 629e635 (2002). 33. Titushkin, I. and Cho, M.: Modulation of cellular mechanics during osteogenic differentiation of human mesenchymal stem cells, Biophys. J., 93, 3693e3702 (2007).
VOL. 117, 2014 34. Rice, A., Ingram, J., and Bonner, J.: p38 mitogen-activated protein kinase regulates growth factor-induced mitogenesis of rat pulmonary myofibroblasts, Am. J. Respir. Cell Mol. Biol., 27, 759e765 (2002). 35. Wang, Z., Yang, H., Tachado, S. D., Capo-Aponte, J., and Bildin, V.: Phosphatase-mediated crosstalk control of ERK and p38 MAPK signaling in corneal epithelial cells, Invest. Ophthalmol. Vis. Sci., 47, 5267e5275 (2006). 36. Sen, B., Guilluy, C., Xie, Z., Case, N., and Styner, M.: Mechanically induced focal adhesion assembly amplifies anti-adipogenic pathways in mesenchymal stem cells, Stem Cells, 29, 1829e1836 (2011). 37. Torsoni, A., Marin, T., Velloso, L., and Franchini, K.: RhoA/ROCK signaling is critical to FAK activation by cyclic stretch in cardiac myocytes, Am. J. Physiol. Heart Circ. Physiol., 289, H1488eH1496 (2005).
RELATED SIGNALING MOLECULES IN ADIPOGENESIS
631
38. Fabry, B., Klemm, A., Kienle, S., Schaffer, T., and Goldmann, W.: Focal adhesion kinase stabilizes the cytoskeleton, Biophys. J., 101, 2131e2138 (2011). 39. Gao, L., McBeath, R., and Chen, C.: Stem cell shape regulates a chondrogenic versus myogenic fate through Rac1 and N-cadherin, Stem Cells, 28, 564e572 (2010). 40. Feng, T., Szabo, E., Dziak, E., and Opas, M.: Cytoskeletal disassembly and cell rounding promotes adipogenesis from ES cells, Stem Cell Rev., 6, 74e85 (2010). 41. Noguchi, M., Hosoda, K., Fujikura, J., Fujimoto, M., and Iwakura, H.: Genetic and pharmacological inhibition of Rho-associated kinase II enhances adipogenesis, J. Biol. Chem., 282, 29574e29583 (2007).
Role of p38, ERK1/2, focal adhesion kinase, RhoA/ROCK and cytoskeleton in the adipogenesis of human mesenchymal stem cells Baiyao Xu,1 Yang Ju,1, * and Guanbin Song2 Department of Mechanical Science and Engineering, Nagoya University, Nagoya 464-8603, Japan1 and Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400030, PR China2 Received 4 July 2013; accepted 23 October 2013 Available online 9 December 2013
Adipogenesis is important to health and is thought occurring in the two stages of mesenchymal stem cell commitment to a preadipocyte fate and terminal differentiation of the preadipocyte. However, the mechanism of adipogenesis is still not clear. In this study, the roles of p38, extracellular regulated protein kinases 1/2 (ERK1/2), focal adhesion kinase (FAK), RhoA/ROCK, and cytoskeleton in both of the two stages of adipogenesis were assayed. Our results showed that the treatments of SB203580 (the inhibitor of p38) and U0126 (the inhibitor of ERK1/2) suppressed the adipogenesis induced by differentiation medium, and the treatments of PF573228 (a specific inhibitor of FAK), Y27632 (a specific inhibitor of RhoA/ROCK) and cytochalasin D (an inhibitor of cytoskeletal organization) promoted the adipogenesis. The treatments of SB203580 and U0126 significantly inhibited the adipogenic differentiation of hMSCs cultured in differentiation medium in the presence of PF573228, Y27632 or cytochalasin D. Moreover, the treatments of PF573228, Y27632 and cytochalasin D promoted p38 and ERK1/2 phosphorylations, and the treatments of U0126 and SB203580 decreased p38 and ERK1/2 phosphorylations, respectively. These results demonstrated that p38 and ERK1/2 played crucial positive roles in adipogenesis, and FAK, RhoA/ROCK and cytoskeleton played negative roles. Furthermore, FAK, RhoA/ROCK and cytoskeleton affected adipogenesis by regulating the activities of p38 and ERK1/2 which interacted with each other in the process of adipogenesis. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: p38; ERK1/2; Focal adhesion kinase; RhoA/ROCK; Cytoskeleton; Adipogenesis]
Adipose tissue, which is primarily composed of adipocytes, is important for energy balance and metabolic homeostasis. Adipocytes are actively involved in the regulation of cell function by a complex signaling network of endocrine, paracrine, and autocrine signals. They are also associated with a lot of pathological disorders including diabetes, hypertension, cancer, atherosclerosis, etc. All adipocytes are derived from mesenchymal stem cells (MSCs) (1). Adipogenesis is thought occurring in two stages: the commitment of MSC to a preadipocyte fate and the terminal differentiation of the preadipocyte (2). Numerous works have focused on the study of adipogenesis mechanism based on using preadipocytes and achieved quite a lot. However, the adipogenesis mechanism of MSCs is still not clear. The investigation of the mechanism of adipogenesis is beneficial to finding therapeutic targets against metabolic diseases which are becoming a global epidemic. p38 and ERK1/2 are the members of mitogen-activated protein kinases (MAPK) family. They are intracellular signaling pathways and play a pivotal role in many essential cellular processes, such as proliferation and differentiation (3). A major function of p38 and ERK1/2 is to transduce signaling of cell surface receptors to transcription factors in nucleus, which consequently triggers cellular differentiation. Engelman et al. described a positive role of p38 in adipogenesis first time (4). They found that the treatment of
* Corresponding author. Tel.: þ81 52 789 4672; fax: þ81 52 789 3109. E-mail address: [email protected] (Y. Ju).
SB203580 in 3T3-L1 differentiation decreased adipocyte formation. However, other researchers found an opposite role of p38 and demonstrated that p38 prevented adipocyte differentiation (5). In the case of ERK1/2, Sale et al. showed that ERK1/2 was required for adipogenesis in cell model of 3T3-L1 (6). Nevertheless, it was blurred by the work that ERK1/2 activation sufficiently inhibited fat cell differentiation by suppressing the activation of peroxisome proliferator-activated receptor-g (PPARg) which was a crucial factor for adipogenesis (7). Therefore, further researches are needed to investigate the roles of p38 and ERK1/2 in both of the stages of adipogenesis. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase functionalized as an important mediator for signaling transduction and guiding cell fate (8,9). RhoA, a small G-protein in Rho family, transfers extracellular signals by coupling with its direct effector, ROCK. RhoA/ROCK plays a pivotal role in signaling transduction and triggering cell differentiation (10). As a cellular scaffolding or skeleton, cytoskeleton contained within a cell’s cytoplasm. Cytoskeleton defines cell shape and also participates in cell differentiation (11). Our previous works had demonstrated that FAK, RhoA/ ROCK and cytoskeleton interacted with each other, and they were positive regulators in mechanical stretch-induced tenogenic differentiation (12). These studies suggested that p38, ERK1/2, FAK, RhoA/ROCK and cytoskeleton play an important role in guiding cell fate including adipogenic differentiation. Moreover, the crosstalk of p38, ERK1/2, FAK, RhoA/ROCK and cytoskeleton may exist in mammalian cells
1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.10.018
VOL. 117, 2014
RELATED SIGNALING MOLECULES IN ADIPOGENESIS
625
FIG. 1. Actin microstructure of each group after culturing for 14 days. (A) Cultured in expansion medium; (B) cultured in AGDM; (C) cultured in AGDM and treated with SB203580; (D) cultured in AGDM and treated with U0126; (E) cultured in AGDM and treated with PF573228; (F) cultured in AGDM and treated with Y27632; (G) cultured in AGDM and treated with cytochalasin D. Scale bar: 100 mm.
626
XU ET AL.
J. BIOSCI. BIOENG.,
FIG. 2. Gene expressions of PPARg and formations of lipid droplets after culturing for 14 days. (A) Gene expressions of PPARg were normalized to GAPDH. Control group (Con) was cultured in expansion medium; induced group (Ind) was cultured in AGDM; SB203580 group (SB) was cultured in AGDM and treated with SB203580; U0126 group (U) was cultured in AGDM and treated with U0126; PF573228 group (PF) was cultured in AGDM and treated with PF573228; Y27632 group (Y) was cultured in AGDM and treated with Y27632; cytochalasin D group (CD) was cultured in AGDM and treated with cytochalasin D. (B) Oil red O staining of Con; (C) oil red O staining of Ind; (D) oil red O staining of SB; (E) oil red O staining of U; (F) oil red O staining of PF; (G) oil red O staining of Y; (H) oil red O staining of CD. Data represent means standard deviations of three separate experiments (n ¼ 3). Double asterisk (**) indicates p < 0.01 vs. Con. Number sign (#) and double number sign (##) indicate p < 0.05 and p < 0.01 vs. Ind, respectively. Scale bar: 500 mm.
(12,13). However, the roles of p38, ERK1/2, FAK, RhoA/ROCK and cytoskeleton in adipogenesis are still not clear, especially the role of FAK. Understanding the roles of p38, ERK1/2, FAK, RhoA/ROCK and cytoskeleton in adipogenesis will help us to elaborate the relation of these signaling molecules in adipogenesis and the mechanism of adipogenesis. In this study, we utilized primary hMSCs and preadipocytes as cellular models, which were closer to the physiological conditions than other cell lines, to investigate the roles of p38, ERK1/2, FAK, RhoA/ROCK and cytoskeleton in adipogenesis. Our results suggested that p38 and ERK1/2 were the positive regulators of adipogenesis, while FAK, RhoA/ROCK and cytoskeleton were the negative regulators. Moreover, FAK, RhoA/ROCK and
cytoskeleton played negative roles by regulating the phosphorylations of p38 and ERK1/2. To our knowledge, this is the first paper to show the relation among p38, ERK1/2, FAK, RhoA/ROCK and cytoskeleton in adipogenesis. MATERIALS AND METHODS Cell culture hMSCs (MSC-R53-1, Passage 2) were acquired from Riken Cell Bank (Tsukuba, Japan). They were cultured in expansion medium which is DMEM medium (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS, Invitrogen, CA, USA) and 1% penicillin/streptomycin (Invitrogen) in 25 cm2 culture flasks (Becton Dickinson Labware, MA, USA) for expansion without differentiation (14). Cells were kept in a humidified incubator at 37 C and supplemented with 5% CO2.
VOL. 117, 2014
RELATED SIGNALING MOLECULES IN ADIPOGENESIS
Unpassaged primary human preadipocytes were acquired from Lonza (NJ, USA) and cultured in preadipocyte growth medium (Lonza) in 25 cm2 culture flasks for expansion without differentiation. Cells were kept in a humidified incubator at 37 C and supplemented with 5% CO2. Adipogenesis of hMSCs and preadipocytes For the adipogenic differentiation of hMSCs, hMSCs of passage 4 were subcultured in expansion medium in 6 well plates. When the cells became confluent, the expansion medium was changed to adipogenic differentiation medium (AGDM, HyClone, UT, USA) and cultured for 14 days with medium changed every 3 days. As a control, hMSCs were cultured in expansion medium with medium changed every 3 days. In the case of preadipocytes, preadipocytes of passage 3 were subcultured in preadipocyte growth medium in 6 well plates. After confluent, adipocyte differentiation medium (ACDM, Lonza) was introduced to induce adipogenesis. Preadipocytes were induced for 14 days with medium changed every 3 days. As a control, preadipocytes were cultured in growth medium and the medium was changed every 3 days. Pharmacological inhibitors The following pharmacological inhibitors were employed: 10 mM SB203580 (Calbiochem, CA, USA), 10 mM U0126 (Calbiochem), 10 mM PF573228 (Tocris, MO, USA), 10 mM Y27632 (Calbiochem) and 0.02 mg/ml cytochalasin D (Calbiochem). When cells became confluent, the inhibitors were added simultaneously when the medium was changed at the beginning of the experiment. The inhibitors were added with the medium changed every 3 days. As a control, cells were cultured in the medium without any inhibitors and the medium was changed every 3 days. Immunofluorescence staining Cells were fixed in 4% paraformaldehyde (PFA) for 15 min and permeabilized with 0.5% Triton-X-100 in PBS for 10 min. They were pre-incubated in 200 ml of 0.1 mM FITC-conjugated phalloidin (Enzo Life Science, NY, USA) in primary blocking solution (1% BSA in PBS) at room temperature for 90 min. Then, the cells were incubated with 0.1 mg/ml DAPI (Calbiochem) in PBS for 5 min, after which mounting medium (10 ml) was dispensed on the cells. The cells were washed three times with PBS for 5 min after each step. A glass coverslip was placed on the slide and sealed with nail polish before observation. Finally, the slides were visualized with a confocal microscope (Nikon A1Rsi, Nikon, Tokyo, Japan). RNA isolation and real-time reverse transcription quantitative polymerase chain reaction (RT-PCR) Cells were lysed and total RNA was isolated using a RNeasy Mini Kit (Qiagen, Duesseldorf, Germany). The 260/280 absorbance ratio was measured for verification of the purity and concentration of the RNA. Reverse transcription was completed using a High Capacity RNA-to-cDNA Kit (ABI, CA, USA). The pre-designed minor groove binder (MGB) probes of glyceraldehydes 3-phosphate dehydrogenase (GAPDH), PPARg (ABI), as well as Taqman PCR Master Mix and a Light Cycler apparatus (ABI 7300, ABI), were used to analyze the gene expressions of the interested genes. The gene expressions of PPARg were calculated using standard curve method and normalized to GAPDH. Oil red O straining Cells were washed with PBS and fixed with 4% PFA for 20 min. After washing the cells twice with PBS, the cells were stained with oil red O solution (Sigma, MO, USA). Then the cells were washed with tap water flow for 20 s. Western blot After treating with or without inhibitors for 30 min, cells were washed with PBS and 100 ml of detergent-based lysis buffer (M-PER Mammalian Protein Extraction Reagent, Pierce, IL, USA), supplemented with protease inhibitor PMSF and a cocktail of phosphatase inhibitors (Pierce, 1:100 dilution) were added to each well for the collection of total cellular proteins. The protein (30 mg) of each sample was loaded onto a 10% SDSePAGE gel for gel electrophoresis. The separated proteins were transferred to a PVDF membrane (Bio-Rad, CA, USA) at 80 V for 120 min. The membranes were blocked in 5% BSA/TBS-Tween 20 solution for 1 h at room temperature, followed by the application of the monoclonal antibody specific for p-p38 (Bioworld Technology Inc., MN, USA), p38 (Bioworld Technology Inc., MN, USA), p-ERK1/2 (Epitomics, CA, USA), and ERK1/2 (Epitomics, CA, USA), at 1:1000 in 5% BSA/TBS-Tween 20. After incubating for 90 min with the primary antibodies at room temperature, the secondary antibody, anti-rabbit IgG-HRP (Cayman Chemical, MI, USA) at 1:10,000 in 5% BSA/TBS-Tween 20 was applied for 1 h at room temperature. The membrane was washed three times with 0.1% TBS/Tween 20 for 10 min after each antibody application. The proteins on the PVDF membrane were detected with an ECL detection system (Pierce), according to the manufacturer’s protocol. The proteins were quantified by volume summation of image pixels with a Fujifilm LAS-4000 (Fujifilm, Tokyo, Japan). Statistical analysis The means and standard deviations were reported for three single repeat samples. A paired Student’s t-test was performed, and a p-value of less than 0.05 was considered to be statistically significant. The p-value was calculated by the soft of SPSS 16.0.
RESULTS Cytoskeletal microstructure of hMSCs at different culture conditions To characterize the effects of SB203580, U0126, PF573228, Y27632 and cytochalasin D on the changes in cytoskeletal microstructure during adipogenic differentiation, cytoskeletal
627
microstructure was observed by confocal microscope after cultured for 14 days. The cytoskeletal microstructure of each group was somewhat different. Cells cultured in expansion medium were mostly well spread, elongated, and often triangular. The actin fibers were aligned along the long axis of the cells (Fig. 1A). After cultured in AGDM, cells changed to round. Actin fibers were shortened and a lot of punctuated fiber actins gathered in the edge of cells. The actin fibers distributed around nuclei were reduced (Fig. 1B). When cells were cultured in AGDM, and treated with SB203580 or U0126, shorten actin fibers became discontinuous. The actin fibers distributed around nuclei were further reduced and only a few distributed around the nuclei (Fig. 1C and D). PF573228 treatment caused cells to become larger and some of actin fibers were still aligned along the long axis of the cells (Fig. 1E). Treatment with Y27632 led to stellate-like cells with a lack of actin fibers (Fig. 1F). Cytochalasin D treatment resulted in the disruption and punctuate of actin fiber organization (Fig. 1G). Although some of actin fibers of the cells treated with PF573228, Y27632 or cytochalasin D were shortened, there were still a lot of actin fibers as long as before. In addition, the actin fibers distributed around nuclei were also reduced by the treatments of PF573228, Y27632 and cytochalasin D. These results indicated that AGDM gave rise to the cortical organization of cytoskeleton, and the treatments of the inhibitors resulted in the changes of cytoskeletal microstructure. Nevertheless, the treatments did not change the cortical organization significantly. By the way, in the experiments of this part, the time point of 14th day was chosen due to most of cells treated with PF573228, Y27632 or cytochalasin D formulated a lot lipid droplets. Gene expressions of PPARg and accumulations of lipid droplets in hMSCs with/without the treatments of inhibitors To evaluate the roles of p38, ERK1/2, FAK, RhoA/ ROCK and cytoskeleton on adipogenesis, hMSCs were cultured in AGDM with the treatments of SB203580, U0126, PF573228, Y27632 and cytochalasin D. After cultured for 14 days, the gene expressions of PPARg and the formations of lipid droplets were assessed by realtime RT-PCR and oil red staining. As shown in Fig. 2A, the inhibitors have significantly influenced on the gene expressions of PPARg. After cultured in AGDM, the gene expression of PPARg was increased about 8 times compared to that of control group. Treatments with SB203580 and U0126 attenuated the AGDM-induced increase in the gene expressions of PPARg. The gene expression of PPARg in the SB203580 group was much lower than that in the U0126 group (p < 0.01). However, when cells were cultured in AGDM and treated with PF573228, Y27632 or cytochalasin D, the gene expressions of PPARg were significantly increased compared to that of induced group (p < 0.05). The gene expression of PPARg of cytochalasin D group was the highest one among these groups. As shown in Fig. 2BeH, the inhibitors had a significant influence on the formations of lipid droplets. After cultured in AGDM for 14 days, approximately 50% cells were stained with oil red O (Fig. 2C), while cells cultured in expansion medium were negative to oil red O staining (Fig. 2B). When cultured in AGDM and treated with SB203580, they were also negative to oil red O staining (Fig. 2D). When cells were cultured in AGDM and treated with U0126, only approximately 25% of them were positive to the staining and the ratio was much lower than the cells cultured in AGDM only (Fig. 2E). On the other hand, when cells were cultured in AGDM and treated with PF573228, Y27632 or cytochalasin D, the positive staining ratios of the cells were much higher than that of the cells cultured in AGDM only (Fig. 2FeH). The positive staining ratio of the cells treated with PF573228 was approximately 70% and lower than that of the cells treated with Y27632 or cytochalasin D. There was no significant difference between the ratios of the cells treated
628
XU ET AL.
J. BIOSCI. BIOENG.,
with Y27632 and cytochalasin D. Consistent with the results of hMSCs, the inhibitors had the same effect on lipid droplets formations of human preadipocytes (Fig. S1). The effects of PF573228, Y27632 and cytochalasin D on the gene expressions of PPARg and the formations of lipid droplets in hMSCs cultured in expansion medium were further investigated. The treatments of PF573228, Y27632 and/or cytochalasin D markedly increased the gene expressions of PPARg when cells were cultured in expansion medium without any adipogenic factors and only a few cells were positive to oil red O staining (Fig. S2). Although the treatments of PF573228, Y27632 and/or cytochalasin D without any adipogenic factors increased the gene expressions of PPARg, they cannot induce abundant accumulations of lipid droplets. These results suggested that p38 and ERK1/2 were positive regulators, while FAK, RhoA/ROCK and cytoskeleton were negative regulators in the adipogenic differentiation of hMSCs. Phosphorylations of p38 and ERK1/2 by the treatments of cytochalasin D, Y27632 and PF573228 The effects of cytochalasin D, Y27632 and PF573228 on the phosphorylations of p38 and ERK1/2 in hMSCs were investigated. As shown in Fig. 3, the treatments of cytochalasin D, Y27632 and PF573228 significantly increased the phosphorylations of p38 and ERK1/2. It meant that the inhibitions of the polymerization of cytoskeleton or the activities of RhoA/ROCK and FAK, promoted p38 and ERK1/2 phosphorylations. Interestingly, the treatments of SB203580 and U0126 resulted in the decrease in the phosphorylations of ERK1/2 and p38, respectively. However, the change in the phosphorylation of ERK1/2 by the treatments of SB203580 did not reach statistical significance. These results were consistent with the conclusion that p38 and ERK1/2 were positive regulators, while FAK, RhoA/ ROCK and cytoskeleton were negative regulators in adipogenic differentiation of hMSCs. Moreover, these findings suggested that the crosstalk between p38 and ERK1/2 existed in the process of adipogenic differentiation of hMSCs. Gene expressions of PPARg in hMSCs treated with PF573228, Y27632 or cytochalasin D in the presence of SB203580 or U0126 To further study the roles of p38, ERK1/2, FAK, RhoA/ ROCK and cytoskeleton in the process of adipogenic differentiation, the effects of SB203580 and U0126 on the gene expressions of PPARg in hMSCs which were cultured in AGDM and treated with PF573228, Y27632 or cytochalasin D were investigated. As shown in Fig. 4, the gene expression of PPARg in hMSCs cultured in AGDM
FIG. 4. Gene expressions of PPARg in hMSCs treated with PF573228, Y27632 or cytochalasin D in the presence of SB203580 or U0126. The gene expressions of PPARg were normalized to GAPDH. Control group (Con) was cultured in expansion medium; induced group (Ind) was cultured in AGDM; SB203580 plus PF573228 group (SB þ PF) was cultured in AGDM treated with SB203580 and PF573228; SB203580 plus Y27632 group (SB þ Y) was cultured in AGDM treated with SB203580 and Y27632; SB203580 plus cytochalasin D group (SB þ CD) was cultured in AGDM treated with SB203580 and cytochalasin D; U0126 plus PF573228 group (U þ PF) was cultured in AGDM treated with U0126 and PF573228; U0126 plus Y27632 group (U þ Y) was cultured in AGDM treated with U0126 and Y27632; U0126 plus cytochalasin D group (U þ CD) was cultured in AGDM treated with U0126 and cytochalasin D; data represent means standard deviations of three separate experiments (n ¼ 3). Double asterisk (**) indicates p < 0.01 vs. Con.
was around 7.5 times compared to that of control group. However, the treatments of SB203580 and U0126 inhibited the gene expressions of PPARg in all groups. In SB þ PF, SB þ Y and SB þ CD groups, the gene expressions of PPARg were around 4 times compared to that in control group; in U þ PF, U þ Y and U þ CD groups, the gene expressions of PPARg were around 5 times (Fig. 4). Compared to the gene expressions of PPARg in hMSCs cultured by AGDM and treated with PF573228, Y27632 or cytochalasin D shown in Fig. 2A, the gene expressions of PPARg in the SB þ PF, SB þ Y, SB þ CD, U þ PF, U þ Y and U þ CD groups were significantly reduced by the treatments of SB203580 and U0126. Consistent with the results of gene expressions, the
FIG. 3. Phosphorylations of p38 and ERK1/2 by the treatments of cytochalasin D, Y27632, PF573228, U0126 and SB203580. (A) Phosphorylation of p38 by the treatments of cytochalasin D, Y27632 and PF573228; (B) phosphorylation of ERK1/2 by the treatments of cytochalasin D, Y27632 and PF573228; (C) phosphorylation of p38 by the treatment of U0126; (D) phosphorylation of ERK1/2 by the treatment of SB203580. Panels a, b, c and d indicate the results of panels A, B, C and D, respectively. Control group (Con) was cultured in medium without any inhibitor; cytochalasin D group (CD) was treated with 0.02 mg/ml cytochalasin D; Y27632 group (Y) was treated with 10 mM Y27632; PF573228 group (PF) was treated with 10 mM PF573228; U0126 group (U) was treated with 10 mM U0126; SB203580 group (SB) was treated with 10 mM SB203580. Data represent means standard deviations of three separate experiments (n ¼ 3). Asterisk (*) indicates p < 0.05 vs. Con.
VOL. 117, 2014
RELATED SIGNALING MOLECULES IN ADIPOGENESIS
treatments of SB203580 and U0126 resulted in sharply decreasing in the accumulations of lipid droplets (Fig. S3).
DISCUSSION p38 and ERK1/2 are the members of MAPK which coordinately regulate proliferation, differentiation and so on (15). Engelman et al. described a positive role of adipogenesis utilizing 3T3-L1 cell line firstly (4,16). Aouadi et al. also found that SB203580 incubation reduced the accumulation of lipid and p38 activity was required for human primary adipocyte differentiation (17). Coincides with the report of Aouadi et al. (17), our results showed that the treatment of SB203580 sharply suppressed the gene expression of PPARg which acted as a crucial regulator of adipogenesis program (18), and it completely inhibited the formation of lipid droplets. Although the gene expression of PPARg was sharply inhibited by the treatment of SB203580, it was still four times higher than the cells cultured in expansion medium. It meant that the increase in the gene expression of PPARg was not only regulated by p38. Some other signaling molecules may be activated to up-regulate the gene expression of PPARg. Furthermore, the formation of lipid droplets was completely suppressed by the inhibition of p38. It implied that p38 did not only regulate the gene expression of PPARg, but also played an important role in gene expressions which regulated the formation of lipid droplets, such as the gene expression of C/EBPa. These results suggested that the increase in the gene expression of PPARg was not the only precondition to induce the differentiation of hMSCs into matured adipocytes, and p38 activity was necessary to the adipogenesis of hMSCs. However, the role of p38 in adipogenesis was disputed due to the opposing reports. Some reports showed that p38 activity inhibited adipocyte differentiation (5,19). Aouadi et al. (20) reported that the inhibition of p38 increased adipogenesis from embryonic to adult stages. The reason of opposite results about the role of p38 in adipogenesis may be due to the role of p38 depending on the stage of adipogenesis, differentiation condition and cellular model. Regarding to the role of ERK1/2 in adipogenesis, apparent contradictory results were reported recently. Ling et al. (7) reported that ERK1/2 activation sufficiently inhibited fat cell differentiation by suppressing the activation of PPARg. Nevertheless, Sale et al. (6) showed that ERK1/2 was required for the adipogenesis in the cell model of 3T3-L1. Some researches indicated that ERK1/2 had multiple functions in adipogenesis (21e24). In consistent with the report by Sale et al. (6), our findings showed that the treatment of U0126 dramatically suppressed the gene expression of PPARg and partly inhibited the formation of lipid droplets which suggested that ERK1/2 was a positive regulator in the process of adipogenic differentiation of hMSCs. FAK, RhoA/ROCK and cytoskeleton play important roles in guiding MSCs fate. Here we showed that the inhibition of FAK activity significantly increased the gene expression of PPARg and promoted the formation of lipid droplets. Moreover, the inhibition of FAK activity up-regulated the gene expression of PPARg in the cells cultured without any adipogenic factor. These findings manifested that FAK was a negative regulator in adipogenesis. This consequence could be used to explain that FAK was cleaved gradually during adipocyte differentiation (25), FAK stabilized cytoskeleton, and the inhibition of FAK resulted in lower cell stiffness occurred during the process of adipogenesis (26). For the roles of RhoA/ROCK and cytoskeleton in adipogenesis, they were represented as similar as the role of FAK. The inhibitions of RhoA/ROCK activity and the polymerization of cytoskeleton increased the gene expressions of PPARg, even though the cells were cultured without any adipogenic factor. These results demonstrated that RhoA/ROCK and cytoskeleton were negative
629
regulators of adipogenesis. In agreement with this interpretation, RhoA inhibits adipogenesis by the regulation of cytoskeletal tension through RhoA-Rho kinase (ROCK) signaling pathway (27). It was well known that Rho family small GTPases regulated cell shape through modulating cytoskeleton, and RhoA/ROCK had been implicated in the lineage commitment of MSCs (28e32). The changes in the activity of RhoA/ROCK and the remodeling of cytoskeleton were concurrent with a modulation of cellular mechanical properties (33). For osteogenesis, cells typically become stiffer, while cells become more compliant in adipogenesis (26). The inhibition of RhoA/ROCK and the remodeling of cytoskeleton result in the reduction of pre-existing inner cellular force and lower stiffness. Furthermore, our results also showed that adipogenic differentiation resulted in shortened and punctuated actin fibers which would cause the reduction of pre-existing inner cellular force and Young’s modulus. Therefore, similar to FAK, the mechanisms of RhoA/ROCK and cytoskeleton exerting influence on adipogenesis may be conjectured that the inhibitions of RhoA/ROCK and cytoskeleton reduced pre-existing inner cellular force which was involved in adipogenesis. In the process of adipogenic differentiation of hMSCs, the inhibitions of cytoskeleton, RhoA/ROCK and FAK resulted in the increase of the phosphorylations levels of p38 and ERK1/2. It implied that cytoskeleton, RhoA/ROCK and FAK regulated adipogenic differentiation by acting upon the activity of p38 or ERK1/2. Moreover, our results showed that inhibiting the phosphorylations of p38 and ERK1/2, blocked the adipogenic differentiation of hMSCs which was promoted by blocking the activity of FAK, RhoA/ROCK or the polymerization of cytoskeleton. These results demonstrated that FAK, RhoA/ROCK and cytoskeleton played a negative role in adipogenesis by regulating the activities of p38 and ERK1/2. Besides, our findings showed that the treatments of SB203580 and U0126 resulted in slight decrease in the phosphorylations of ERK1/2 and p38, respectively. It suggested that p38 and ERK1/2 interacted with each other in the process of adipogenesis. Crosstalk between branches of p38 and ERK1/2 depends on protein phosphatase (34). Wang et al. reported that inhibiting p38 and ERK1/2 leads to increase in phosphorylations of ERK1/2 and p38 in corneal epithelial cells, respectively (35). The result was different from ours. It implied that the crosstalk between p38 and ERK1/2 may depend on cell type or growth condition. In addition, FAK, RhoA/ROCK and cytoskeleton interacted with each other in a lot of cellular activities (12,36,37). They may interact with each other in the process of adipogenesis as negative regulators. FAK and RhoA/ROCK stabilized the cytoskeleton and the inhibition of FAK or RhoA/ROCK made cell soft (38). The remodeling of cytoskeleton also affected the activities of FAK and RhoA/ROCK (12,39). Moreover, cell shape can regulate RhoA activity, and this activity mediates the shape-dependent control of hMSCs lineage commitment to adipocytes (27,40,41). Our previous study had demonstrated that FAK, RhoA/ROCK and cytoskeleton, as positive regulators, act upon each other in the process of tenogenic differentiation of MSCs (12). These studies inferred that the crosstalk among FAK, RhoA/ ROCK and cytoskeleton existed in the process of adipogenesis. Signaling transduction is a complex network. For the hMSC lineage commitment, a lot of signaling pathways communicate with each other to control the targeted gene expressions. In this study, the different roles of p38, ERK1/2, FAK, RhoA/ROCK and cytoskeleton in adipogenesis were depicted. p38, ERK1/2, FAK, RhoA/ROCK and cytoskeleton did not work alone, but interacted with each other to compose a network to regulate the process of adipogenesis. The roles of p38, ERK1/2, FAK, RhoA/ROCK and cytoskeleton in adipogenesis are shown in Fig. 5: (i) p38 and ERK1/ 2 were positive regulators of adipogenesis; (ii) crosstalk between p38 and ERK1/2 existed in adipogenesis; (iii) FAK, RhoA/ROCK and cytoskeleton involved in adipogenesis by acting upon ERK1/2 and
630
XU ET AL.
J. BIOSCI. BIOENG.,
FIG. 5. Proposed schematic diagram of related signaling molecules in the adipogenesis of hMSCs. p38 and ERK1/2 are positive regulators in the adipogenesis, while FAK, RhoA/ROCK and cytoskeleton are negative regulators by acting upon p38 and ERK1/2.
p38; (iv) FAK, RhoA/ROCK and cytoskeleton, as negative regulators, may interact with each other in adipogenesis. In summary, our findings demonstrated that p38 and ERK1/2 were positive regulators, and FAK, RhoA/ROCK and cytoskeleton were negative regulators by regulating the activities of p38 and ERK1/2 in adipogenesis. p38, ERK1/2, FAK, RhoA/ROCK and cytoskeleton composed a network to regulate adipogenesis. To our knowledge, this is the first study which suggests that FAK, RhoA/ ROCK and cytoskeleton play negative roles in adipogenesis by regulating activity of p38 and ERK1/2. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2013.10.018. ACKNOWLEDGMENTS This work was supported by JSPS KAKENHI Grant numbers 23246024 and 24656085. We thank Dr. Kato (Hiroshima University) for the supply of hMSCs. References 1. Gesta, S., Tseng, Y., and Kahn, C.: Developmental origin of fat: tracking obesity to its source, Cell, 131, 242e256 (2007). 2. Cristancho, A. and Lazar, M.: Forming functional fat: a growing understanding of adipocyte differentiation, Nat. Rev. Mol. Cell Biol., 12, 722e734 (2011). 3. Bost, F., Aouadi, M., Caron, L., and Binetruy, B.: The role of MAPKs in adipocyte differentiation and obesity, Biochimie, 87, 51e56 (2005). 4. Engelman, J., Lisanti, M., and Scherer, P.: Specific inhibitors of p38 mitogenactivated protein kinase block 3T3-L1 adipogenesis, J. Biol. Chem., 273, 32111e32120 (1998). 5. Wang, X. and Ron, D.: Stress-induced phosphorylation and activation of the transcription factor CHOP (GADD153) by p38 MAP kinase, Science, 272, 1347e1349 (1996). 6. Sale, E., Atkinson, P., and Sale, G.: Requirement of MAP kinase for differentiation of fibroblasts to adipocytes, for insulin activation of p90 S6 kinase and for insulin or serum stimulation of DNA synthesis, EMBO J., 14, 674e684 (1995). 7. Ling, H., Wen, G., Feng, S., Tuo, Q., and Ou, H.: MicroRNA-375 promotes 3T3L1 adipocyte differentiation through modulation of extracellular signal-regulated kinase signalling, Clin. Exp. Pharmacol. Physiol., 38, 239e246 (2011).
8. Guan, J.: Integrin signaling through FAK in the regulation of mammary stem cells and breast cancer, IUBMB Life, 62, 268e276 (2010). 9. Xu, B., Song, G., and Ju, Y.: Effect of focal adhesion kinase on the regulation of realignment and tenogenic differentiation of human mesenchymal stem cells by mechanical stretch, Connect. Tissue Res., 52, 373e379 (2011). 10. Sun, Y., Chen, C., and Fu, J.: Forcing stem cells to behave: a biophysical perspective of the cellular microenvironment, Annu. Rev. Biophys., 41, 519e542 (2012). 11. Treiser, M., Yang, E., Gordonov, S., Cohen, D., and Androulakis, I.: Cytoskeleton-based forecasting of stem cell lineage fates, Proc. Natl. Acad. Sci. USA, 107, 610e615 (2010). 12. Xu, B., Song, G., Ju, Y., Li, X., and Song, Y.: RhoA/ROCK, cytoskeletal dynamics, and focal adhesion kinase are required for mechanical stretch-induced tenogenic differentiation of human mesenchymal stem cells, J. Cell Physiol., 227, 2722e2729 (2012). 13. Chaturvedi, L., Marsh, H., and Basson, M.: Role of RhoA and its effectors ROCK and mDia1 in the modulation of deformation-induced FAK, ERK, p38, and MLC motogenic signals in human Caco-2 intestinal epithelial cells, Am. J. Physiol. Cell Physiol., 301, C1224eC1238 (2011). 14. Tsutsumi, S., Shimazu, A., Miyazaki, K., Pan, H., and Koike, C.: Retention of multilineage differentiation potential of mesenchymal cells during proliferation in response to FGF, Biochem. Biophys. Res. Commun., 288, 413e419 (2001). 15. Cargnello, M. and Roux, P.: Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases, Microbiol. Mol. Biol. Rev., 75, 50e83 (2011). 16. Engelman, J., Berg, A., Lewis, R., Lin, A., and Lisanti, M.: Constitutively active mitogen-activated protein kinase kinase 6 (MKK6) or salicylate induces spontaneous 3T3-L1 adipogenesis, J. Biol. Chem., 274, 35630e35638 (1999). 17. Aouadi, M., Jager, J., Laurent, K., Gonzalez, T., and Cormont, M.: p38MAP kinase activity is required for human primary adipocyte differentiation, FEBS Lett., 581, 5591e5596 (2007). 18. Tang, Q. and Lane, M.: Adipogenesis: from stem cell to adipocyte, Annu. Rev. Biochem., 81, 715e736 (2012). 19. Yang, T., Xiong, Q., Enslen, H., Davis, R., and Chow, C.: Phosphorylation of NFATc4 by p38 mitogen-activated protein kinases, Mol. Cell. Biol., 22, 3892e3904 (2002). 20. Aouadi, M., Laurent, K., Prot, M., Le Marchand-Brustel, Y., Binetruy, B., and Bost, F.: Inhibition of p38MAPK increases adipogenesis from embryonic to adult stages, Diabetes, 55, 281e289 (2006). 21. Font de Mora, J., Porras, A., Ahn, N., and Santos, E.: Mitogen-activated protein kinase activation is not necessary for, but antagonizes, 3T3-L1 adipocytic differentiation, Mol. Cell. Biol., 17, 6068e6075 (1997). 22. Kim, S., Muise, A., Lyons, P., and Ro, H.: Regulation of adipogenesis by a transcriptional repressor that modulates MAPK activation, J. Biol. Chem., 276, 10199e10206 (2001). 23. Lim, S., Jang, H., Park, E., Kim, J., and Kim, J.: Wedelolactone inhibits adipogenesis through the ERK pathway in human adipose tissue-derived mesenchymal stem cells, J. Cell. Biochem., 113, 3436e3445 (2012). 24. Than, A., Cheng, Y., Foh, L., Leow, M., and Lim, S.: Apelin inhibits adipogenesis and lipolysis through distinct molecular pathways, Mol. Cell. Endocrinol., 362, 227e241 (2012). 25. Li, J. and Xie, D.: Cleavage of focal adhesion kinase (FAK) is essential in adipocyte differentiation, Biochem. Biophys. Res. Commun., 357, 648e654 (2007). 26. Gonzalez-Cruz, R., Fonseca, V., and Darling, E.: Cellular mechanical properties reflect the differentiation potential of adipose-derived mesenchymal stem cells, Proc. Natl. Acad. Sci. USA, 109, E1523eE1529 (2012). 27. McBeath, R., Pirone, D., Nelson, C., Bhadriraju, K., and Chen, C.: Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment, Dev. Cell, 6, 483e495 (2004). 28. Chen, Z., Wang, X., Shao, Y., Shi, D., and Chen, T.: Synthetic osteogenic growth peptide promotes differentiation of human bone marrow mesenchymal stem cells to osteoblasts via RhoA/ROCK pathway, Mol. Cell. Biochem., 358, 221e227 (2011). 29. Xu, T., Wu, M., Feng, J., Lin, X., and Gu, Z.: RhoA/Rho kinase signaling regulates transforming growth factor-beta1-induced chondrogenesis and actin organization of synovium-derived mesenchymal stem cells through interaction with the Smad pathway, Int. J. Mol. Med., 30, 1119e1125 (2012). 30. Meyers, V., Zayzafoon, M., Douglas, J., and McDonald, J.: RhoA and cytoskeletal disruption mediate reduced osteoblastogenesis and enhanced adipogenesis of human mesenchymal stem cells in modeled microgravity, J. Bone Miner. Res., 20, 1858e1866 (2005). 31. Shi, Y., Li, H., Zhang, X., Fu, Y., and Huang, Y.: Continuous cyclic mechanical tension inhibited Runx2 expression in mesenchymal stem cells through RhoAERK1/2 pathway, J. Cell. Physiol., 226, 2159e2169 (2011). 32. Etienne-Manneville, S. and Hall, A.: Rho GTPases in cell biology, Nature, 420, 629e635 (2002). 33. Titushkin, I. and Cho, M.: Modulation of cellular mechanics during osteogenic differentiation of human mesenchymal stem cells, Biophys. J., 93, 3693e3702 (2007).
VOL. 117, 2014 34. Rice, A., Ingram, J., and Bonner, J.: p38 mitogen-activated protein kinase regulates growth factor-induced mitogenesis of rat pulmonary myofibroblasts, Am. J. Respir. Cell Mol. Biol., 27, 759e765 (2002). 35. Wang, Z., Yang, H., Tachado, S. D., Capo-Aponte, J., and Bildin, V.: Phosphatase-mediated crosstalk control of ERK and p38 MAPK signaling in corneal epithelial cells, Invest. Ophthalmol. Vis. Sci., 47, 5267e5275 (2006). 36. Sen, B., Guilluy, C., Xie, Z., Case, N., and Styner, M.: Mechanically induced focal adhesion assembly amplifies anti-adipogenic pathways in mesenchymal stem cells, Stem Cells, 29, 1829e1836 (2011). 37. Torsoni, A., Marin, T., Velloso, L., and Franchini, K.: RhoA/ROCK signaling is critical to FAK activation by cyclic stretch in cardiac myocytes, Am. J. Physiol. Heart Circ. Physiol., 289, H1488eH1496 (2005).
RELATED SIGNALING MOLECULES IN ADIPOGENESIS
631
38. Fabry, B., Klemm, A., Kienle, S., Schaffer, T., and Goldmann, W.: Focal adhesion kinase stabilizes the cytoskeleton, Biophys. J., 101, 2131e2138 (2011). 39. Gao, L., McBeath, R., and Chen, C.: Stem cell shape regulates a chondrogenic versus myogenic fate through Rac1 and N-cadherin, Stem Cells, 28, 564e572 (2010). 40. Feng, T., Szabo, E., Dziak, E., and Opas, M.: Cytoskeletal disassembly and cell rounding promotes adipogenesis from ES cells, Stem Cell Rev., 6, 74e85 (2010). 41. Noguchi, M., Hosoda, K., Fujikura, J., Fujimoto, M., and Iwakura, H.: Genetic and pharmacological inhibition of Rho-associated kinase II enhances adipogenesis, J. Biol. Chem., 282, 29574e29583 (2007).