Chronic stress induces CD99, suppresses autophagy, and affects spontaneous adipogenesis in human bone marrow stromal cells
© The Author(s). 2017
Received: 25 October 2016
Accepted: 9 March 2017
Published: 18 April 2017
Bone marrow-derived mesenchymal stromal cells (MSCs) are multipotent cells with a high constitutive level of autophagy and low expression of CD99. Under certain conditions, MSCs may develop tumorigenic properties. However, these transformation-induced conditions are largely unknown. Recently, we have identified an association between Hsp70, a main participant in cellular stress response and tumorigenesis, and CD99. Preliminary observations had revealed upregulation of both proteins in stressed long-term cultured MSCs. And so we hypothesized that CD99 is implicated in stress-induced mechanisms of cellular transformation in MSCs. Hence, we investigated the effects of prolonged stress on MSCs and the role of CD99 and autophagy in their survival.
Human telomerase reverse transcriptase (hTERT) overexpressing immortalized MSCs and primary bone marrow stromal cells were used to investigate the influence of long-term serum deprivation and hypoxia on growth and differentiation of MSCs. Cell proliferation and apoptosis were evaluated using flow cytometry, differentiation capabilities of MSCs were assessed by immunohistochemical staining followed by microscopic examination. CD99, Hsp70 expression were analyzed using flow cytometry, western blotting, and reverse transcriptase polymerase chain reaction. Autophagy was explored with specific inhibitors using cell morphology examination and western blotting.
Chronic stress factors are able to change the morphology of MSCs and to inhibit spontaneous differentiation into adipocyte lineage. Furthermore, CD99 elevation and downregulation of p53 and p21 accompanied defective autophagy, which is usually associated with tumor formation. We found that inhibition of autophagy by chloroquine promoted cell detachment and modulated CD99 expression level whereas incorporation of CD99 recombinant protein into the cells suppressed autophagy.
Obtained results provide a model for chronic stress-induced transformation of MSCs via CD99 and may therefore be highly relevant to mesenchymal tumorigenesis.
KeywordsBone marrow stromal cells CD99 Stress MSC Autophagy
Bone marrow-derived mesenchymal stromal cells (MSCs) are spindle-shaped nucleated cells with multilineage differentiation potential and immune-modulating ability . They may develop into adipocytes, chondrocytes, and osteoblasts [1, 2]. Long-term survival of MSCs is one of their most significant characteristics. As a main strategy to maintain self-renewal and differentiation properties, MSCs use autophagy. This conserved catabolic mechanism to avoid cellular damage is a constitutive process in human MSCs where it also supports “stemness” properties . On the other hand, MSCs are characterized by elevated transformation propensity . Long-term expansion is under debate to lead to spontaneous transformation of MSCs [1, 4]. Among the main features of MSC transformation are changes in morphology, such as increased nucleus-to-cytoplasm ratio, disruption of cell cycle mechanism by p53 pathway(s) inhibition, and autophagy perturbation [5, 6]. Understanding the circumstances leading to the transformation of MSCs could be essential for therapeutic approaches.
MSCs have no specific surface markers and are usually recognized by co-expression of CD73, CD90, CD105, CD106, and CD146, and absence of hematopoietic markers such as CD34 and CD45 . Previously, MSCs have been shown to be negative or only slightly positive in CD99, which otherwise is a widely expressed multifunctional surface molecule [7, 8]. Recently, however, we have identified an association between CD99 and Hsp70 – a main participant of cellular stress response . This observation allowed us to hypothesize that CD99 is implicated in stress-induced signaling. MSCs are in the center of many studies investigating tumorigenesis under stress conditions, such as hypoxia and serum starvation. They are of special interest also because they are suggested to be the origin of Ewing’s tumors cells [7, 9], a highly aggressive pediatric malignancy characterized by high levels of CD99, presence of EWS-Fli1 rearrangement in most cases, and low levels of p53 and p21 [10, 11].
To investigate stress factors that influence stromal cells we mainly used a human bone marrow mesenchymal cell line immortalized with telomerase reverse transcriptase (TERT) . These cells, like primary normal mesenchymal cells, maintain their growth rate, general functions, have a normal karyotype and phenotype, and are pluripotent. Advantages of using hTERT-positive MSCs are their long life span without senescence and the absence of donor-dependent heterogeneity. Previously we had used these cells to maintain survival of primary B cell precursor acute lymphoblastic leukemia (BCP-ALL) cells . In the present study we found that prolonged stress conditions induce morphological transformation of MSCs, suppress autophagy, inhibit p53-p21 pathway, and upregulate CD99 – features which resemble well-described changes associated with neoplastic transformation of MSCs. Primary human bone marrow stromal cells were used in some experiments to verify the results obtained with the MSC cell line.
Human bone marrow stroma cell line immortalized by telomerase reverse transcriptase (TERT) overexpression (hMSCs)  was kindly provided by Dr. Giuseppe Gaipa (Centro Ricerca M. Tettamanti, Monza, Italy) and Dr. Dario Campana (St. Jude Children’s Research Hospital, Memphis, TN, USA). Primary bone marrow stroma cells were obtained from patients in remission after chemotherapy. Patients were recruited to the study (reference number 1500/2014), which was approved by the review board Ethikkommission der Universität Wien (University of Vienna Ethics Commission), and informed consent was obtained from parents or legal guardians for sampling procedures and subsequent laboratory investigations. Cells were maintained in RPMI medium (Fisher Scientific Austria, Vienna, Austria), supplemented with 10% fetal calf serum (FCS) (PAA Laboratories, Pasching, Austria), 2 mM glutamine and 100 U/ml antibiotic-antimycotic mix (both from LifeTech Austria, Vienna, Austria) at 37 °C and 5% CO2. To induce starvation, cells were washed with phosphate-buffered saline (PBS) and incubated in RPMI medium without FCS. For hypoxia experiments, cells were placed into an incubator with 1% oxygen for the required period of time. For the long-term assays, cells were plated in six-well plates and allowed to grow for 21 to 42 days, with the media changed every 3 days.
Cells were monitored in regular intervals by PCR to exclude mycoplasma infection.
Antibodies, materials and reagents
The following anti-CD99 monoclonal antibodies (mAbs) were used for CD99 detection: DN16 (Scipac, Sittingbourne, UK) for western blotting (WB), phycoerythrin (PE)-conjugated 3B2 mAb (Caltag, Hamburg, Germany) for FACS, and CD99-FITC, and CD99-PE were from Becton Dickinson (BD) (San Jose, CA, USA). CD99 recombinant protein was from Abcam (Cambridge, UK). Hsp70 (W27) un-conjugated and PE-conjugated antibodies were from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), anti-BECN1, anti-p53, and anti-p21mAbs were from Santa Cruz, anti-microtubule-associated protein 1 light chain 3 (LC3), and anti-ATG13 from Sigma-Aldrich (Steinheim, Germany), and anti-PARP from Cell Signaling Technology (Leiden, The Netherlands). Goat anti-mouse IgG DYLight 800 and goat anti-rabbit IgG DY Light 800 antibodies were from THP Medical Products (Vienna, Austria). PE-conjugated goat anti-mouse IgG was purchased from Szabo-Scandic (Vienna, Austria). FITC- and APC-conjugated Annexin V, and amino-actinomycin D (7AAD) were from BD. Chloroquine (CQ), bafilomycin A1, pifithrin-alpha, Ponceau S-10, dexamethasone, isobutylmethylxanthine, insulin, indomethacin, ascorbic acid, β-glycerol phosphate, L-thyroxine, L-proline, insulin-transferrin-sodium selenite (ITS), sodium pyruvate, Oil Red O (ORO), Alizarin Red S (ARS), and Toluidine Blue (TB) all were from Sigma-Aldrich. PageRuler Prestained Protein Ladder was from Thermo Fisher Scientific Biosciences (St. Leon-Rot, Germany).
Images were taken and processed using the Axiovert 40C inverted microscope (Zeiss, Göttingen, Germany) equipped with a MegaPixel FireWire PixeLink camera (PixeLink, Ottawa, ON, Canada).
Stained cells were acquired with a FACS Calibur flow cytometer (BD). Data were analyzed using the CellQuest™ software (BD). Expression of cell antigens was quantified on the basis of mean fluorescence intensity (MFI) values as previously reported by us, using the CellQuest™ software . Cytoplasmatic staining for CD99 and Hsp70 was performed with the Fixation/Permeabilization kit purchased from Invitrogen (Lofer, Austria) according to the manufacturer’s recommendations.
Apoptosis, cell growth, cell cycle
Cells were scraped and resuspended in Annexin V binding buffer to incubate with Annexin V and 7AAD at room temperature (RT) for 20 minutes. Detection and quantification of apoptotic/necrotic cells was done by the flow cytometric analysis of labeled cells. The assay was performed according to the manufacturer’s instructions. Briefly, cells were collected from culture and then suspended in 100 μl Annexin V binding buffer (10 mM HEPES, pH7.4, 2.5 mM CaCl2, 140 mM NaCl) containing 1 μg/ml Annexin V. After 15 minutes incubation in the dark at RT, 7AAD (1 μl/tube) was added and apoptotic and dead cells were quantified on the flow cytometer.
Cell cycle analysis was assessed with the Cycletest Plus DNA Reagent kit (BD) according to the manufacturer’s protocol.
For autophagy assay, cells were plated in six-well plates for 3 days in RPMI medium with or without FCS and 48 hours later were treated with or without CQ (75 μM and 150 μM) for 24 hours. Next, morphological examination and WB analysis were performed.
Cells were washed with PBS, harvested, and centrifuged at 2000 rpm for 10 minutes. Laemmli sample buffer (4% SDS, 20% glycerol, 120 mM Tris-HCl, pH 6.8, 3% beta-mercaptoethanol, bromophenol blue) was used to prepare whole cell lysates. Total proteins were subsequently separated by 8.5 or 10% SDS-PAGE and transferred onto a nitrocellulose membrane (VWR, Radnor, PA, USA). After the transfer the membrane was evaluated in Ponceau S-10, washed with H2O and blocked in Western Blocking Reagent (Roche Diagnostics, Burgess Hill, UK) for 1 hour at RT. Immunoblot analysis was performed with the indicated antibodies and visualized by Odysseys Infrared Imager (Li-COR Biosciences, Lincoln, NE, USA).
RT-PCR for CD99 and hsp70
Total RNA was isolated using RNeasy Micro Kit (Qiagen, Vienna, Austria). Single-strand cDNA was synthesized and hsc70/hsp70 and CD99 type I and type II were PCR amplified using the following specific primers: hsc70, forward: 5’-TGTGGCTTCCTTCGTTATTGG-3’ and reverse: 5’-GCCAGCATCATTCACCACCAT-3’; hsp70, forward: 5’-AGAGCCGAGCCGACAGAG-3’ and reverse: 5’-CACCTTGCCGTGTTGGAA-3’; GAPDH, forward: 5’-CCACTCCTCCACCTTTGAC-3’ and reverse: 5’-ACCCTGTTGCTGTAGCCA-3’  (all from Eurofins MWG Operon, Ebersberg, Germany). CD99 type I and CD99 type II primers (all from VBS-Genomics, Vienna, Austria.), forward: 5’-GTGCGGCTAGCACCATGGCCCGCGGGGCTG-3’; CD99 type I reverse: 5’-CTAGTCTCGAGCTGGTAAGCAATGAAGCTAG-3’; CD99 type II reverse: 5’-GCTCTAGACCCTAGGTCTTCAGCCAT-3’ . PCR products were separated on 2% agarose gel and visualized with GelStar Stain (Lonza, Rockland, ME, USA).
Cells were incubated in 2 ml of the appropriate StemPro differentiation medium from Gibco Carlsbad, CA, USA (StemPro Adipogenesis Kit; StemPro Chondrogenesis Kit; StemPro Osteogenesis Kit) in six-well plates for 14 days. After 10 days, 500 μl of fresh medium were administrated to re-feed cell cultures.
Adipocyte induction and detection
Cells were grown in six-well plates (VWR) in RPMI medium till they reached subconfluence. For long-term culture, we prepared an adipocyte-inducing medium based on Dulbecco’s modified Eagle’s medium (DMEM)-LC (LifeTech Austria) containing 1 μM dexamethasone, 500 μM isobutylmethylxanthine, 1 μg/ml insulin, and 100 μM indomethacin with supplementation. Before detection, cells were washed with PBS w/out Ca2+, Mg2+, fixed with 10% neutral-buffered formalin in PBS for 30–60 minutes and washed with water. Cells were incubated with 60% isopropanol for 5 minutes at RT. Afterward, isopropanol was removed and cells were stained with ORO working solution for 15 minutes at RT. After cell washing, hematoxylin solution was added for 1 minute. The washing step was repeated and cells were analyzed under an Axiovert 40C microscope.
Osteocyte induction and detection
Osteocyte-induced differentiation medium was prepared using DMEM-LC containing 1 μM (10 nM) dexamethasone, 50 μg/ml ascorbic acid, 10 mM β-glycerol phosphate, and L-thyroxine with supplementation. Osteoblast detection was performed according to the standard protocol with some modification. Briefly, fixed cells were stained with 1 ml/well ARS working solution for 45 minutes, under gentle shaking, at RT, in the dark. Then unincorporated dye was aspirated, cells were washed with water, PBS was added, and the cells were analyzed under an inverted microscope.
Chondrocyte induction and detection
For long-term chondrocyte induction we used Iscove’s modified Dulbecco’s medium (IMDM; LifeTech Austria) with 100 nM dexamethasone, 50 μg/ml ascorbic acid, 40 μg/ml L-proline, ITS, and 1 mM sodium pyruvate with supplementation. Cells were permeabilized with 0,5% Triton X-100 for 5 minutes, washed with water, and stained with TB working solution for 1–2 minutes. After washing, cells were microscopically analyzed.
All numerical data including error bars represent the mean ± SEM. Statistical analysis was performed using Dunnett’s test and two-tailed Student’s t test.
Serum starvation, hypoxia, and their combination change MSC phenotype
Stress factors induce Hsp70
Chronic stress upregulates CD99
Stress inhibits autophagy in MSCs
Autophagy inhibitor promotes detachment of MSCs and modulates CD99
CD99 modulation inhibits autophagy in MSCs
Hypoxia and starvation suppress differentiation potential of hMSCs
Interest in MSCs as a source of stem cells for regenerative medicine arises due to their ability to differentiate into multiple lineages. Many efforts are currently focusing on the investigation of the transformation potentials of MSCs, either primary or immortalized. Conflicting results show, on the one hand, the stable genotype and phenotype of MSCs in long-term (12 weeks) cultures , and on the other, spontaneous neoplastic transformation of MSCs . Even cell lines immortalized in the same way – by overexpression of hTERT, for example – are reported to be either genomically instable  and transformed  or stable , with unclear transforming potential. Generally, hTERT overexpression and also loss of p53 and p21 are on the top of the list of events shown to be the most relevant for induction of MSC transformation [5, 20–22]. The circumstances that induce these mutations and lead to the critical changes in MSCs are, however, still unknown. According to some hypotheses they may be promoted by external factors.
Previously, different stress stimuli were tested for their effects on MSCs. It was shown that stressed MSCs lose their phenotype stability and change differentiation potentials: heat shock enhanced osteoblast differentiation of hTERT-immortalized human MSCs ; hypoxia increased ectodermal differentiation into neuron-like cells , but reduced adipogenic and osteogenic differentiation of primary human MSCs [15, 24]; serum starvation caused upregulation of myocardial markers on human bone marrow-derived MSC  and epithelial markers on adipose stem cells ; exposure to electric field or to mechanical strain improved hMSCs osteogenic differentiation  and pulsed electromagnetic fields induced chondrogenesis ; high cell density initiated commitment of hMSCs to endothelial cells . To find conditions which may cause in vitro transformation of MSCs, we decided to examine the influence of typical stress factors, like starvation and hypoxia, on a hTERT-positive MSC cell line.
It is well known that oxygen distribution in different tissues is uneven, with 1% to 7% gradient across bone marrow, for example. However, survival of MSCs after transplantation is not supported by conditions associated with hypoxia and starvation, like ischemia. Taking into account that usual in vitro cell cultures are performed under 21% oxygen is an important issue to investigate characteristics of MSC cells over prolonged periods of time [15, 24].
First, we observed that long-term stressed MSCs exhibited drastic changes in morphology, especially in cultures which underwent starvation or combined H/S conditions: they showed increased nucleus-to-cytoplasm ratio in round cells or flat polyangular cells in contrast to stretched normoxic cells. The latter proliferated faster than others but at day 21 cells reached confluence, and by day 42 their proliferation was strongly suppressed as was seen from cell cycle analyses. Normoxic cells exhibited the lowest level of apoptosis, starved cells, the highest (around 30% more). As expected, apoptosis was not elevated by hypoxia and was less visible when hypoxia was combined with starvation .
As stress-induced morphological changes in MSCs were observed, we looked for further evidences of possible transformation: altered differentiation, changes in metabolism, or stress-dependent protein modulation.
We found that multipotent hMSCs underwent spontaneous adipogenic differentiation after a few weeks in normoxic but not in stressed cultures. This could, of course, also be caused by the aging of very dense cells, similar to the influence of chemotherapy or irradiation on bone marrow stromal cells [30, 31]. However, activity of autophagy and cell cycle machinery, which are shown to be age-related [32, 33], as well as Hsp70 and CD99 expression levels, were unchanged. Hsp70 is involved in cellular transformation (reviewed in ref. ) and recently has been recognized as a negative regulator of autophagy . CD99 expression depends on the differentiation stage of many cell types [13, 36–38], serves by itself as inhibitor of differentiation [36, 37], and modulates survival of different cells [13, 39]. That is why the obtained results are probably indicative of amplification of cell density-dependent signals directing MSCs along adipocyte lineage, on the one hand, and “stemness-saving” signals of some stress stimuli [15, 24], on the other hand. Indeed, high cell density is required for MSCs differentiation into each of the three lineages (adipocytes, osteocytes, or chondrocytes) , but spontaneous differentiation just into adipocyte lineage results from a combined effect of cell density and choice of medium. Moreover, the requirement of autophagy for adipogenesis has been described recently, linking LC3 expression and lipid droplets formation (reviewed in refs. [41, 42]). However, autophagy is also involved in osteogenesis  and chondrogenesis  and, therefore, should be one of the key regulators of mesenchymal development. Here we found that autophagy, which is a constitutive process in MSCs , was dramatically reduced by stress.
It is widely known that tumorigenesis may be promoted either by suppression of autophagy or by autophagy induction (reviewed in ref. ). This duality can be explained by the indispensable role of autophagy for normal cell homeostasis where deregulation of balance leads to cell transformation. Here we found that inhibition of autophagy by chloroquine in a dose- and time-dependent manner induced morphological changes of the MSCs, followed by detachment and cell death, thus mimicking stress effects. However, CQ upregulated p53 as well as downregulated CD99 and Hsp70. The discrepancy between pro-apoptotic CQ effects and upregulation of CD99 and Hsp70 in chronically stressed cells suggests involvement of both proteins into a pro-survival adaptation strategy of MSCs. Indeed, we found that overexpression of CD99 promoted proliferation of MSCs. It has been shown that hypoxia induces upregulation of both CD99  and Hsp70 . These two proteins may be regulated by the hypoxia-inducible factor (HIF) system. Increased expression of HIF-alpha in hypoxic MSCs , Hsp70-HIF-1alpha interactions under hypoxic conditions , as well as CD99 targeting by von Hippel-Lindau tumor suppressor gene (VHL) through HIF-alpha subunits , have been described in the literature. Additionally, we found that treatment with bafilomycin A1, another autophagy inhibitor, revealed not only inhibition of autophagy detected by LC3 but induction of apoptosis machinery too (Additional file 6: Figure S6).
Furthermore, stress-induced changes of MSC phenotype and molecular pathways correlated with CD99 upregulation. At each examined time point, the CD99 level in normoxic cells was lower than in stressed cells. RT-PCR and WB showed that this effect was due to stabilization of CD99 under stress-like hypoxia and under starvation due to its upregulation. In parallel, induction of Hsp70 was observed. To find a direct link between CD99 and MSCs transformation, CD99 recombinant protein was administered to the cell cultures and the level of autophagy was examined. We observed CD99-dependent autophagy delay (suppression of p53, p21, and LC3) paralleled by cell cycle promotion. A similar effect, which is a hallmark of transformation (inhibition of p53 and p21), was seen under stress. Hence, our results demonstrate the interplay between CD99 and autophagy. Recently, we have already shown that CD99 is a functional partner for Hsp70 , and Hsp70 has been suggested to be a negative regulator of autophagy . Direct delivery of Hsp70 into MSCs protected the cells from hypoxia-induced apoptosis . We speculate that upregulation of CD99 and inhibition of adipocyte differentiation under stress might be linked to Sp1 transcriptional activity. Sp1 was shown to be a negative regulator of adipogenesis in MSCs , and a positive regulator of CD99 .
Altogether, the presented data demonstrate the ability of chronic stress to transform the morphology of MSCs, to suppress differentiation, to alter cell proliferation, to inhibit autophagy, and to induce apoptosis. We also showed that CD99 and Hsp70 elevation and disappearance of p53 and p21 accompany defected autophagy. While short-term inhibition of autophagy by chloroquine downregulates CD99 expression in MSCs and induces p53, on the one hand, long-term stress in MSCs leads to CD99 upregulation and p53 suppression, on the other. Thus, we hypothesize that CD99 may serve as a pro-survival molecule in stressed MSCs, which in cooperation with Hsp70 adapts them to unfavorable conditions. This suggestion is supported by the experiment in which modulation of CD99 level by CD99 recombinant protein inhibits LC3, an autophagy marker, and blocks the p53-p21 pathway (Fig. 7c).
Our results provide a model of spontaneous adipocyte differentiation of long-term cultured hTERT-immortalized MSCs as well as primary stromal cells and show chronic stress to be a differentiation blocking mechanism potentially leading to transformation of MSCs along with autophagy inhibition in a CD99-dependent manner. These circumstances should be taken into account, among others , in the current efforts for standardization of MSC therapy.
Alizarin Red S
B cell precursors acute lymphoblastic leukemia
Dulbecco’s modified Eagle’s medium
Fetal calf serum
Human bone marrow mesenchymal stromal cells
Human telomerase reverse transcriptase
Iscove’s modified Dulbecco’s medium
Microtubule-associated protein 1 light chain 3
Oil Red O
We are thankful to K. Mühlbacher for technical assistance. We wish to thank U. Pötschger for help with statistical analysis. We also thank M. Zavadil for secretarial assistance.
This study was supported by the Children’s Cancer Research Institute (CCRI), Vienna, Austria.
Availability of data and materials
All relevant data are included within the article and supporting information files.
ZH designed the study, performed experiments, analyzed and interpreted data, and wrote the manuscript. MD participated in the interpretation of the data and edited the manuscript. All authors read and approved the final manuscript.
1. Dr. Zvenyslava Husak, PhD, staff scientist.
2. Univ. Doz. Dr. Michael Dworzak, MD. Associate professor of pediatrics, senior pediatric oncologist, study chair pedAML/CML/MDS/SAA Austria, IBFM representative Austria, ITCC representative Austria, co-ordinator iBFM FLOW Network, section editor PedOnc MEMO Journal.
The authors declare that they have no competing interests.
Consent for publication
All authors agreed to the submission.
Ethical approval and consent to participate
Patients were recruited to the study (reference number 1500/2014), which was approved by the review board Ethikkommission der Universität Wien (University of Vienna Ethics Commission) and informed consent was obtained from parents or legal guardians for sampling procedures and subsequent laboratory investigations.
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