Correlation of CDC42 Activity with Cell Proliferation and Palmitate-Mediated Cell Death in Human Umbilical Cord Wharton's Jelly Derived Mesenchymal Stromal Cells

Abstract

RHO GTPases regulate cell migration, cell-cycle progression, and cell survival in response to extracellular stimuli. However, the regulatory effects of RHO GTPases in mesenchymal stromal cells (MSCs) are unclear. Herein, we show that CDC42 acts as an essential factor in regulating cell proliferation and also takes part in lipotoxic effects of palmitate in human umbilical cord Wharton's jelly derived MSCs (hWJ-MSCs). Cultured human bone marrow, adipose tissue, and hWJ-MSC derived cells had varying pro-inflammatory cytokine secretion levels and cell death rates when treated by palmitate. Strikingly, the proliferation rate of these types of MSCs correlated with their sensitivity to palmitate. A glutathione-S-transferase pull-down assay demonstrated that hWJ-MSCs had the highest activation of CDC42, which was increased by palmitate treatment in a time-dependent manner. We demonstrated that palmitate-induced synthesis of pro-inflammatory cytokines and cell death was attenuated by shRNA against CDC42. In CDC42 depleted hWJ-MSCs, population-doubling levels were notably decreased, and phosphorylation of ERK1/2 and p38 MAPK was reduced. Our data therefore suggest a mechanistic role for CDC42 activity in hWJ-MSC proliferation and identified CDC42 activity as a promising pharmacological target for ameliorating lipotoxic cell dysfunction and death.

Introduction

Mesenchymal stromal cells (MSCs) can be isolated from various sources, including bone marrow, fat, and umbilical cord among others. MSCs serve as precursors for the generation of mesodermal tissues [1,2]. In addition to their inherent regenerative capacity, MSCs also regulate localized and systemic immune responses [3].

Quantitative and qualitative changes in plasma lipid levels are often observed in patients with type 2 diabetes mellitus (T2DM). The saturated fatty acid palmitate has been shown to induce apoptosis and stimulate secretion of pro-inflammatory cytokines in a variety of cell types, including pancreatic β-cells [4]. Indeed, our previously published study demonstrated that palmitate results in human bone marrow–derived MSC (hBM-MSC) exhaustion [5]. As widely demonstrated, the upregulation of pro-inflammatory cytokines is a consequence of cells undergoing apoptosis [6]. Furthermore, the stimulation of pro-inflammatory cytokine secretion by palmitate in hBM-MSCs has also been recently demonstrated [7,8]. However, to date, the effects of palmitate on other types of human MSCs have not been investigated. In addition, many studies have shown that different types of MSC share similar features, including the expression of similar cell surface antigens and common morphologic features [9,10]. Significant biological differences have also been observed between subtypes, with respect to their proliferation, differentiation capacity, and immunomodulatory effects [11].

To analyze whether palmitate mediates different effects in human bone, adipose tissue (hAD-MSCs), and umbilical cord Wharton's jelly derived MSCs (hWJ-MSCs), the cell death rates and levels of pro-inflammatory cytokines were investigated in the palmitate-treated three subtypes of MSCs. Recent studies have suggested that MSC cytotherapy is a promising therapeutic modality for the treatment of diabetes [12]. Defined lipotoxic effects in three subtypes of MSC may provide guidelines to select the most optimal cell types in MSC therapy for T2DM treatment.

Small GTPases, which exist in both active (GTP-bound) and inactive (GDP-bound) states, regulate a variety of functions, including cellular migration, cell cycle checkpoint, and survival in response to diverse extracellular stimuli [13]. Recent findings have suggested that palmitate may regulate RHO GTPases, Rac1, in β-cells [14]. Another study also provided evidence that CDC42 and Rac1 are important components of palmitate-stimulated JNK pathway in hepatocytes [15]. Herein, we show the diverse effects of palmitate on three subtypes of human MSCs and further analyze the relationship of CDC42 activity with cell proliferation rates and palmitate lipotoxic effects in hWJ-MSCs.

Materials and Methods

Reagents

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, and penicillin/streptomycin were from Hyclone; palmitate (sodium salt, low endotoxin), 4′ 6-diamidino-2-phenylindole (DAPI), bovine serum albumin (BSA) (fatty acid free), and anti-β-Actin were purchased from Sigma-Aldrich; SB203580, U0126, anti-phospho-ERK1/2 (T202/Y204), -p38 (T180/Y182), -JNK (T183/Y185), and total ERK1/2, p38, and JNK antibodies, active CDC42 and Rac1 detection kits, anti-Na, K-ATPase, and secondary anti-mouse, -rabbit IgG antibodies, coupled to horseradish peroxidase, were purchased from Cell Signaling Technology, Inc.; anti-BrdU was purchased from Abcam; FuGENE HD transfection reagent was purchased from Promega; and SYBR Green PCR Master Mix was purchased from Roche. Total RNA Kit was obtained from Omega Bio-Tek.

Isolation and characterization of human MSCs

Normal hBM-MSCs were isolated from six healthy adult donors (four males and two females), aged between 36 and 51 years. hBM-MSCs were isolated as previously described [5,16]. Subcutaneous adipose tissue was obtained from five patients undergoing elective surgical procedures (one male and four females), aged between 32 and 45 years. hAD-MSCs were extracted according to the method described by Katz et al. [17]. hWJ-MSCs were extracted from umbilical cords (eight samples) according to the method described by Wang et al. [18]. All cell isolation was carried out after receiving informed consent. The isolation and use of human MSCs were approved by the Fuzhou General Hospital IRB, China, with written consent.

As per standard practice at our center, we performed cell surface marker analysis (Becton Dickinson; FACSAria) and showed that hBM-MSCs, hAD-MSCs, and hWJ-MSCs were positive for MSC-like cell surface markers (CD13, CD29, CD73, CD90, and CD105), negative for hematopoietic markers (CD14, CD19, CD34, and CD45), endothelial cell marker (CD31), or human leukocyte antigen (locus) DR (HLA-DR) (Becton Dickinson). The differentiation potential was evaluated by culturing hBM-MSCs, hAD-MSCs, and hWJ-MSCs in StemPro MSC SFM XenoFree medium (Thermo Fisher Scientific) for 14 days and staining them with alizarin red S, oil red O, and alcian blue for osteocytes, adipocytes, and chondrocytes, respectively. Supplementary Figures S1 and S2 (Supplementary Data are available online at www.liebertpub.com/scd) show the fluorescence-activated cell sorting and differentiation results of hWJ-MSCs.

Palmitate-BSA preparation

Palmitate-BSA was prepared similarly to the previous study [5]. A 20 mM solution of sodium palmitate in 0.01 M NaOH was incubated at 70°C for 30 min. Then, 330 μL of 30% BSA and 400 μL of sodium palmitate/NaOH mixture were mixed together and filter-sterilized with 20 mL of DMEM. The approximate molar ratio of fatty acids to BSA was 6:1 with 0.4 mM palmitate-BSA. The addition of BSA or palmitate-BSA mixture has not been previously shown to affect the pH of the media. A mixture of 0.1, 0.2, or 0.3 mM palmitate-BSA was prepared by mixing 0.4 mM palmitate-BSA and BSA solution (BSA control) at ratio of 1:3, 1:1, or 3:1, respectively. All solutions, containing 0.5% BSA, were prepared fresh for each experiment.

Quantitative reverse transcription-polymerase chain reaction

Quantitative reverse transcription-polymerase chain reaction was performed as previously described [19] with specific primers for human interleukin (IL)-1β, IL-6, IL-7, IL-8, IL-10, IL-18, tumor necrosis factor-α (TNF-α), MCP1, transforming growth factor-β (TGF-β), Rac1, CDC42, and Actin; sequences are provided in Supplementary Table S1.

GTPase activation assay

Activation of CDC42 and Rac1 was analyzed using CDC42 or Rac1 Activation Assay Kit from Cell Signaling Technology according to the manufacturer's instructions.

Lentivirus-mediated gene knockdown

Lentiviral shRNA vectors for the human genes used in this study were obtained from Open Biosystem (Rac1: clone ID TRCN0000004870; CDC42: clone ID TRCN0000047630). One shRNA sequence, which had the highest reducing efficiency, was chosen from five sequences for each gene, respectively. SHC002, a pLKO.1-puro Non-Mammalian shRNA Control Plasmid, was obtained from Sigma-Aldrich. For virus packaging, the nontargeting sequence control or shRNA constructs were cotransfected with pMD2.G and psPAX2 into 293T cells using FuGENE HD. The hWJ-MSCs were infected with virus containing media.

Isolation of cytoplasmic and membrane proteins

Cytoplasmic and membrane proteins of palmitate-BSA–treated MSCs were extracted by a Subcellular Protein Fractionation Kit (Beyotime) according to the manufacturer's instructions.

Western blot

Western blot was prepared similarly to a previous study [5]. Primary antibody dilution buffer contained anti-phospho-ERK1/2 (T202/Y204), -p38 (T180/Y182), -JNK (T183/Y185), total ERK1/2, p38, JNK, Rac1, Na, K-ATPase antibodies (1:1,000), CDC42 antibody (1:300), or β-Actin (1:5,000). Results were quantified by densitometric scan of X-ray films with a densitometer. Antibodies are provided in Supplementary Table S2.

Enzyme linked immunosorbent assay

Cells were stimulated with palmitate-BSA or BSA alone and then incubated with serum-free medium for 24 h. IL-6 and IL-8 were measured in cell-free culture supernatant by highly specific ELISA Kits (R&D) according to the manufacturer's instructions. The results were corrected according to cell number, which was analyzed by WST-1 (Roche Diagnostics) assay.

Determination of apoptosis

Apoptotic cell death was assessed by flow cytometry using the Annexin V-FITC Apoptosis Detection Kit (KeyGEN) following the manufacturer's instructions.

Cumulative population doubling

Cells (3 × 10³ cells/cm²) were plated in growth medium. After 3 days, cells were harvested and counted. Cumulative population doublings were calculated using the formula: x = [log10 (NH) − log10 (NI)]/log10, where NH is the cell harvest number, and NI is the inoculum cell number. To generate the cumulative population doubling level, the population doubling for passage 3 and passage 4 was calculated and then added to the population doubling levels of the previous passages.

BrdU staining

For cell BrdU experiments, cells were pulsed with 10 μM BrdU for 6 h, fixed in 4% polyparaformaldehyde. Cells were labeled with anti-BrdU at 4°C overnight and then washed and treated with anti-mouse Alexa Fluor 488, DAPI. Photomicrographs were captured using an Olympus IX81 microscope (Olympus).

Endotoxin test

Bacterial endotoxin tests quantified endotoxins from Gram-negative bacteria using an Endotoxin Assay Kit (Houshiji).

Statistics

The results of the quantitative data are expressed as mean ± standard deviation. Statistical differences between the groups were analyzed with the Student's t-test or one-way analysis of variance followed by t-test with the Bonferroni correction for multiple comparisons. A statistically significant difference was set at P < 0.05.

Results

Palmitate increases pro-inflammatory cytokine production in hWJ-MSCs

Circulating free fatty acid (FFA) concentrations are <0.7 mM in the immediate postabsorptive state and rise to >1 mM after consumption of a high-fat meal or in a fasted state. Palmitate is the most abundant saturated FFA in plasma (20%–35%) [20]. In this study, we chose a concentration range of palmitate between 0.1 and 0.4 mM to determine effects on MSCs. Palmitate is insoluble in aqueous solution and requires binding to BSA to solubilize [21]. Therefore, we sought to examine whether palmitate had the ability to induce pro-inflammatory responses in hWJ-MSCs by exposing cells to palmitate conjugated to fatty-acid-free BSA (palmitate-BSA, endotoxin tested <0.25 EU/mL, pH = 7.26) and BSA control at equivalent concentrations (BSA, endotoxin tested <0.25 EU/mL, pH = 7.15) for 12 h before the triggering of apoptosis. As shown in Fig. 1A, IL-6 and IL-8 mRNA levels treated with palmitate-BSA (0.2, 0.3, and 0.4 mM palmitate-BSA) were increased (P < 0.05) compared to BSA controls. Furthermore, mRNA levels of IL-1β and TNF-α were also slightly increased at the concentration of 0.4 mM (P < 0.05) palmitate-BSA compared to controls. The expression of IL-7, IL-10, IL-18, MCP1, and TGF-β did not show any significant increases compared to controls. Treatment with palmitate-BSA significantly increased both IL-6 and IL-8 concentrations in a dose-dependent manner, with a maximum response observed at 0.4 mM (Fig. 1A). We also examined whether there were any significant changes between BSA alone and a non-BSA control on the expression of pro-inflammatory cytokines. However, we did not demonstrate any relevant differences. In parallel with the increase in mRNA levels, there were significant increases in IL-6 and IL-8 secretion concentrations (Fig. 1B). These results indicate that exposure to palmitate-BSA in hWJ-MSCs results in production of pro-inflammatory cytokines.

FIG. 1.

Palmitate increases pro-inflammatory cytokine gene expression. (A) hWJ-MSCs were treated with BSA (white bar) and a final concentration of 0.1, 0.2, 0.3, or 0.4 mM palmitate-BSA (black bars). The mRNA levels of IL-1β, IL-6, IL-7, IL-8, IL-10, IL-18, TNF-α, MCP1, and TGF-β1 were analyzed by quantitative RT-PCR. Error bars indicate the SD of six biological replicates. For all genes, data are shown relative to the BSA-treated control. (*P < 0.05). (B) hWJ-MSCs were treated with BSA and different concentrations of palmitate-BSA for 12 h. Secretion of IL-6 and IL-8 was measured by ELISA in three separate experiments. P values were calculated compared with BSA-treated control (*P < 0.05). hWJ-MSCs, human umbilical cord Wharton's jelly derived mesenchymal stem cells; IL, interleukin; TNF-α, tumor necrosis factor-α; TGF-β1, transforming growth factor-β1; RT-PCR, reverse transcription-polymerase chain reaction; BSA, bovine serum albumin; SD, standard deviation; ELISA, enzyme linked immunosorbent assay.

Palmitate augments MAPK signaling in hWJ-MSCs

The MAPK pathway is thought to be a mediator of palmitate-induced apoptosis, pro-inflammatory responses, and insulin resistance [5,22,23]. Figure 2A shows that palmitate-BSA treatment stimulated ERK1/2 and p38 phosphorylation in hWJ-MSCs. These results are consistent with hBM-MSCs in our previous study. In addition to the ability of ERK1/2 or p38 inhibitors to attenuate cell death rates, the inhibitors also reduced IL-6 and IL-8 expression in this study (Fig. 2B, C).

FIG. 2.

ERK1/2 and p38 MAPK phosphorylation has been linked with induction of inflammatory cytokines. (A) hWJ-MSCs were incubated in palmitate-BSA for 1.5, 3, 6, and 12 h. Immunoblot for ERK1/2 (T202/Y204 and total), p38 (T180/Y182 and total), and JNK (T183/Y185 and total). The result is representative of three separate experiments. (B) hWJ-MSCs were pretreated with p38 inhibitor SB203580 (20 μM) or ERK1/2 inhibitor U0126 (10 μM) for 1 h and then treated with palmitate-BSA (0.4 mM) for 12 h. Secretion of IL-6 and IL-8 was measured by ELISA analysis in three separate experiments. P values are indicated (*P < 0.05). (C) hWJ-MSCs were pretreated with p38 or ERK1/2 inhibitor for 1 h and then treated with palmitate (0.4 mM) for 24 h. Cell apoptosis were determined by flow cytometry after staining with FITC-conjugated Annexin V. The result is representative of three separate experiments. SB: SB203580, U: U0126. FITC, fluorescein isothiocyanate.

Comparison of palmitate-mediated cell death rates and pro-inflammatory cytokine release levels among subtypes of MSCs

To assess palmitate-induced apoptosis rates and pro-inflammatory cytokine levels in human MSCs expanded from tissues other than bone marrow, hWJ-MSCs, hAD-MSCs, and hBM-MSCs were treated with palmitate-BSA. A cell death assay showed that hWJ-MSCs exhibited the highest levels of cell death, followed by hBM-MSCs and, to a lesser extent, hAD-MSCs, 24 h after 0.4 mM palmitate-BSA treatment (Fig. 3A). In all MSC subtypes, which were treated with BSA alone, apoptosis levels were <8% (Supplementary Fig. S3). Upon measuring release of IL-6 and IL-8, similar trend to cell death rates was apparent in all three cell types (Fig. 3B). Figure 3C shows that palmitate-BSA trigged ERK1/2 and p38 phosphorylation in hBM-MSCs and hWJ-MSCs, whereas it did not affect MAPK activation in hAD-MSCs.

FIG. 3.

Effect of palmitate on three types of MSC. (A) hBM-MSCs, hWJ-MSCs, and hAD-MSCs were treated with 0.4 mM palmitate-BSA for 24 h. Flow cytometry was used to determine cell apoptosis after staining with FITC-conjugated Annexin V. The result is representative of three separate experiments. (B) hBM-MSCs, hWJ-MSCs, and hAD-MSCs were treated with BSA or 0.4 mM palmitate-BSA for 12 h, respectively. Secretion of IL-6 and IL-8 was measured by ELISA in three separate experiments. Due to large differences in the expression of the cytokines between the different MSC sources, the graphs are represented as fold production. *P < 0.05. (C) Phosphorylated ERK1/2 and p38 were immunoblotted from extracts of hBM-MSCs, hWJ-MSCs, and hAD-MSCs treated with 0.4 mM palmitate-BSA for 3 h. The result is representative of three separate experiments. BM: hBM-MSCs, WJ: hWJ-MSCs, AD: hAD-MSCs; hBM, human bone marrow; hAD, human adipose tissue.

Comparison of cell proliferation rates among subtypes of MSCs

Cell proliferation and apoptosis have been demonstrated to be interlinked [24,25] with previous studies suggesting that proliferation rates of MSC vary between subtypes [26]. We decided to examine whether cell proliferation differences relate to palmitate-induced apoptosis rates in three subtypes of MSC. Analysis of the proliferation capacity of MSCs derived from three tissues showed that hAD-MSCs possessed the lowest population doubling numbers (Fig. 4A). hWJ-MSCs displayed the highest doubling numbers at passage 3 and passage 4. Figure 4B demonstrates that hWJ-MSCs also had the highest level of BrdU inclusion. The results were compared with palmitate-treated apoptosis levels of the three other sources of MSC.

FIG. 4.

Cell proliferation levels of human MSCs derived from the three kinds of tissues. (A) Population doubling was measured at passage 3 and passage 4. Data are expressed as the mean ± SD. *P < 0.05. (B) Levels of BrdU staining. For BrdU the level is determined as percent positive staining obtained using the proportion of pixels at intensities meeting or exceeding a threshold value above background. *P < 0.05. BM: hBM-MSCs, WJ: hWJ-MSCs, AD: hAD-MSCs.

Role of CDC42 in hWJ-MSC proliferation

GTPases have been shown to induce both proliferation and apoptosis [13,27]. CDC42 and Rac1, which regulate the cell cycle in many cells, have been shown to mediate palmitate triggered cell apoptosis [15]. Therefore, we hypothesized that one or more GTPases, which are essential for MSC proliferation, may also play important roles in palmitate-BSA induced cell apoptosis in MSCs.

Knockdown experiments were used to assay the impact of CDC42 or Rac1 effect on cell viability. As hWJ-MSCs have the highest cell proliferation rates among the three sources of MSC, hWJ-MSCs were transfected with shRNA specific to CDC42, shRNA specific to Rac1, or a nonsilencing control shRNA (SHC002). Stable gene knocked-down hWJ-MSCs can be enriched through the addition of puromycin to culture medium, whereas stable gene knockdown hBM-MSCs and hAD-MSCs cannot be obtained due to significant cell death after being selected by puromycin. Figure 5A shows that population doubling numbers in CDC42 knockdown cells was significantly attenuated. Phosphorylation of ERK1/2 and p38 MAPK was also decreased. In contrast, proliferation rates were unchanged in Rac1 knockdown cells, as well as phosphorylation of ERK1/2 and p38 MAPK, (Fig. 5B). BrdU staining demonstrated reduced staining in CDC42 knockdown cells, whereas staining was unchanged in Rac1 knockdown cells (Fig. 5C). Flow cytometry analysis of CDC42 knockdown cells following propidium iodide staining showed that 85.6% of the cells were in the G1/G0 phase of the cell cycle (Fig. 5D). These results imply that CDC42 is a key regulator in hWJ-MSC proliferation.

FIG. 5.

Activation of CDC42 involved in human MSC proliferation. (A) Population doubling levels in Rac1 and CDC42 knockdown cells. (B) hWJ-MSCs were transfected and grown under puromycin selection for 1 day. Immunoblot for Rac1 or CDC42, ERK1/2 (T202/Y204 and total), and p38 (T180/Y182 and total). The result is representative of three separate experiments. (C) hWJ-MSCs were transfected and grown under puromycin selection for 3 days. Cells were incubated with 10 μM BrdU for 6 h and processed for immunofluorescence. (D) Representative charts for cell cycle distribution in Rac1 and CDC42 knockdown and control cells. (E) The mRNA levels of Rac1 and CDC42 in three MSC types were detected by quantitative RT-PCR. N.S., not significant, compared with hAD-MSC group. (F) The activation of Rac1 and CDC42 was analyzed by Activation Assay Kit and immunoblot. *P < 0.05, compared with hAD-MSC group. AD: hAD-MSCs, BM: hBM-MSCs, WJ: hWJ-MSCs, NC: SHC002 control.

In addition, we examined mRNA levels and the activation of CDC42 and Rac1 in the selected three subtypes of MSCs. As shown in Fig. 5E, there was no significant difference in mRNA levels of CDC42 among the three cell lines. A glutathione-S-transferase-PBD (Rac1/Cdc42-binding domain of PAK) pull-down assay that specifically recognizes the activated form of GTP-bound GTPase was used to examine CDC42 and Rac1 activity. Analysis revealed that hWJ-MSCs had the highest CDC42 activity among the three subtypes of MSCs (Fig. 5F).

Role of CDC42 in palmitate-mediated pro-inflammatory cytokine release and cell death in hWJ-MSCs

Our data demonstrated that the activation of CDC42 is involved in palmitate-mediated lipotoxic responses. Palmitate-BSA treatment led to activation of CDC42 in a time-dependent manner in hWJ-MSCs, with activation at 15 min and lasting up to 30 min (Fig. 6A). In contrast, palmitate-BSA treatment did not significantly increase Rac1 activity. It has been shown previously that the activation of CDC42 and Rac1 is associated with translocation of their respective protein products to the cell membrane [15,28]. We subsequently performed subcellular fractionation to determine the distribution of Rac1 and CDC42 by western blot analysis. As shown in Fig. 6B, the translocation of CDC42 was increased in response to palmitate-BSA treatment, whereas there was no significant change in the location of Rac1 within the cell. Following western blot experiments, knockdown experiments were used to examine whether CDC42, Rac1, or both had an effect on palmitate-mediated lipotoxicity. Transfected hWJ-MSCs were selected by puromycin for 72 h, and CDC42 and Rac1 expression were examined using western blot (Fig. 6C).

FIG. 6.

Activation of CDC42 involved in palmitate-mediated lipotoxic effects. (A) hWJ-MSCs were incubated in palmitate-BSA for 0, 15, 30, and 60 min. The activation of GTP-bound Rac1 and CDC42 was measured by immunoblot. The result is representative of three separate experiments. (B) The membrane translocation levels of Rac1 and CDC42 were immunoblotted from extracts of hWJ-MSCs treated with BSA or palmitate-BSA for 30 min. The result is representative of three separate experiments. mem: membrane; cyto: cytoplasm. (C) Rac1 or CDC42 knockdown hWJ-MSCs and control cells were treated with BSA or PA for 3 h. Total cell extracts were immunoblotted with indicated antibodies. The result is representative of three separate experiments. (D) Secretion of IL-6 and IL-8 was measured in three separate experiments. P values were calculated compared with PA-treated SHC002-NC group (*P < 0.05). (E) Rac1 or CDC42 knockdown hWJ-MSCs and control cells were treated with 0.4 mM palmitate-BSA for 24 h. Flow cytometry was used to determine cell apoptosis after staining with FITC-conjugated Annexin V. The result is representative of three separate experiments. NC: SHC002 control.

CDC42 and Rac1 have been established as critical regulators of p38 and JNK MAPK pathways in palmitate exposure [15,29]. Therefore, we examined the role of CDC42 and Rac1 in palmitate-BSA–induced ERK1/2 and p38 phosphorylation and found that phosphorylation of ERK1/2 and p38 MAPK was attenuated in CDC42 knockdown cells (Fig. 6C), implying that CDC42 is part of a palmitate stimulated signaling pathway that regulates ERK1/2 and p38 in hWJ-MSCs. As the incidence of apoptosis in palmitate-BSA treated hWJ-MSCs was more prevalent, we reduced treatment time to 12 h to examine cytokine secretion. We demonstrated that knockdown of CDC42 partially decreased palmitate-BSA–induced IL-6 and IL-8 production (Fig. 6D). The level of cell death in CDC42 knockdown cells was partially ameliorated (Fig. 6E). The reduction in cytokine secretion and cell death in Rac1 knockdown cells was lower compared with CDC42 knockdown cells. Together, these data demonstrate that CDC42 is an important regulator for palmitate-BSA–mediated lipotoxic effects in hWJ-MSCs.

Discussion

The work presented in this study demonstrates a significant difference in the response to palmitate among human MSC cell lines derived from bone marrow, adipose tissue, and umbilical cord Wharton's jelly. We found that palmitate-induced cell death was associated with the activation of CDC42, an essential factor for both cellular proliferation and lipotoxic effects in hWJ-MSCs. ERK1/2 and p38 MAPK underwent phosphorylation induced by CDC42 when the cells were treated with palmitate-BSA. Gene knockdown of CDC42 resulted in a significant inhibition of hWJ-MSC proliferation and led to a reduction in palmitate-induced cytokine production, as well as cell death.

CDC42 is a regulator in hematopoietic stem cells (HSCs) cell cycle regulation [30]. Aging has been shown to result in an increase in CDC42 activity in HSCs, which is through to thereby regulate the constitutional aging of HSCs [31,32]. Recent studies have suggested that CDC42 plays a vital role in extracellular stress responses, including mediation of apoptosis [15,28]. In the present study, we have, for the first time, demonstrated that CDC42 is a signal coordinator in hWJ-MSC. Furthermore, CDC42 promotes cell cycle progression and plays an important role in palmitate-induced cytotoxic stress in hWJ-MSCs. The activation of CDC42 in hWJ-MSCs leads to the activation of the MAPK cascade. MAPKs act as signal coordinators in MSCs, which are utilized to regulate cell functions, including gene expression, cell proliferation, differentiation, and stress response [33 –35]. Identification of the differences among subtypes of MSC will lead to a success of MSC-based therapies within rational optimization of therapeutic strategies. In our study, we demonstrated that treatment with palmitate increased a higher level of pro-inflammatory cytokine secretion, as well as the rate of apoptosis in hWJ-MSCs, which had the highest CDC42 activity, compared to hBM-MSCs and hAD-MSCs, suggesting that diverse CDC42 activity may correlate with the varying degrees of lipotoxic effects in the three subtypes of human MSCs examined in this study. However, when CDC42 was blocked in hWJ-MSCs, lipotoxic effects were not reduced completely in comparison to controls; therefore, other factors may have a potential role in the palmitate-mediated effects observed. Furthermore, as we were unable to generate stable gene knockdown cell lines in hBM-MSCs and hAD-MSCs, it cannot possibly draw any conclusions regarding the role of CDC42 in these types of human MSC. Lentivirus transduction resulted in faster senescence of bone marrow- and adipose-derived MSC, a finding similar to that reported in other studies [36]. We observed that hWJ-MSCs were more sensitive to the lipotoxic effects of palmitate than either subtype MSCs. Nevertheless, hWJ-MSCs also showed their advantages, such as higher cell proliferation capability and accessibility to genetic manipulation.

The results are obtained using in vitro approaches in this study, but we believe that exposure to high concentrations of saturated FFAs in vivo may result in deterioration of hWJ-MSC function. A meta-analysis study has previously shown that infusion of umbilical cord derived MSCs achieved better outcome in T1DM patients, compared to hBM-MSCs, but provided no significant effects in T2DM patients [12]. In this study, we show that palmitate stimulation induced lipotoxic effects in hWJ-MSCs, possibly accounting for the reason hWJ-MSCs are unsuitable to be used in the treatment of T2DM.

In conclusion, this study highlights the function of CDC42 in maintaining hWJ-MSC proliferation and in influencing palmitate-induced lipotoxic effects.

Footnotes

Acknowledgments

The authors thank all the study participants. This work was supported, in part, by the National Natural Science Foundation of China (no. 81370948 and no. 81570748).

Author Disclosure Statement

All authors had full access to the data and take responsibility for its integrity. All authors have read and agreed with the article as written. There are no conflicts of interest among the authors.

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