s2fieldsofstudy: Biology Medicine

text:
The effect of salusin-β on expression of pro- and anti-inflammatory cytokines in human umbilical vein endothelial cells (HUVECs)

BACKGROUND Atherosclerosis is one of the predominant causes of cardiovascular disease (CVD). Several studies indicated the significant pathophysiological role of salusin-β in atherosclerosis. Cytokines are involved in all stages of atherosclerosis. Therefore, we aimed to assess the effect of salusin-β on interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 18 (IL-18) (as inflammatory cytokines) and interleukin 1Ra (IL-1Ra) (as anti-inflammatory cytokines) levels in human umbilical vein endothelial cells (HUVECs). METHODS The HUVECs were cultured in HUVEC completed medium and treated with different doses of salusin-β for 6 and 12 hours. For the investigation of nuclear factor ƙβ (NF-ƙβ) signaling pathway involvement, cells were treated in the presence or absence of Bay 11-7082 (as NF-ƙβ inhibitor). The mRNA expression and protein level of cytokines were measured by a real-time polymerase chain reaction (PCR) system and enzyme-linked immunosorbent assay (ELISA) method, respectively. RESULTS Salusin-β increased mRNA expression and protein level of IL-6, IL-8 and IL-18. This protein decreased mRNA and protein level of IL-1Ra in HUVECs. NF-ƙβ signaling pathway was involved in the up-regulatory effect of salusin-β on mRNA expression of pro-inflammatory cytokines. The down-regulatory effect of salusin-β on IL-1Ra expression could not be influenced by Bay 11-7082 pre-treatment. CONCLUSION It seems that salusin-β may participate in a cascade pathway in vascular inflammation. Our findings suggested that salusin-β has potential use as a therapeutic target for atherosclerosis.

Introduction
Atherosclerosis is one of the predominant causes of cardiovascular disease (CVD). More than 17.5 million people die each year because of CVDs. 1 Due to the global growing rate of diabetes and obesity, it is expected that morbidity and mortality of CVD will increase. 2 Therefore, it is of great significance to understand the precise mechanisms which are involved in atherosclerosis. Atherosclerosis is now regarded as a chronic inflammatory disorder of large and medium arteries. 3 Cytokines are particularly significant in inflammatory processes, 4 and many of them are believed to be complicated in atherogenesis. 5 Several cytokines are detected in atherosclerotic plaque, on the other hand, all the cells involved in the disease can produce cytokines. 6 These proteins are involved in initial stages of atherosclerosis, recruitment and activation of leukocytes, foam cell and fatty streak formation, development of complex lesions, plaque stability and rupture. 2,4 It is noted that some cytokines have an anti-atherosclerotic effect. 2 Several signaling pathways are involved in cytokines expression, among which nuclear factor ƙβ (NF-ƙβ)

Original Article
Salusin-β: pro-& anti-inflammatory cytokines 2 ARYA Atheroscler 2018; Volume 14; Issue 1 transcription factor is an important signaling pathway inducing the expression of some cytokines. 7 Salusin-β (with 20 amino acids) is a novel peptide which is discovered via bioinformatics analysis of a human full-length cDNA library. 8 This protein is synthesized from pre-pro-salusin which is expressed at a great level in human vascular smooth muscle cells (VSMCs) and endothelial cells. 8 It is indicated that salusin-β is released greatly from cell lines such as THP-1 (a human monocytic cell line) and U937 (a model cell line for the study of behavior and differentiation of monocytes), at the time of stimulation to be differentiated into macrophages. 9 Several data indicated that this protein has a pathophysiological role in atherosclerosis via overexpression of acyl-CoA, cholesterol acyltransferase-1 (ACAT-1) and increase in macrophage foam cell formation (an important step in atherosclerosis), 10 stimulation of the proliferation of VSMCs and fibroblasts and induction of the expression of c-myc, c-fos. 8 Also in vivo and in vitro studies have shown that Salusin-β accelerates inflammatory responses in vascular endothelial cells. 11 As noted, salusin-β contributes to the pathogenesis of atherosclerosis, but little is known about the effect of this protein on pro-and antiinflammatory cytokines expression. We aimed to assess the effect of salusin-β on interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 18 (IL-18) (as inflammatory cytokines) and interleukin 1Ra (IL-1Ra) (as anti-inflammatory cytokines) levels in human umbilical vein endothelial cells (HUVECs). Further, we assessed the probable involvement of NF-ƙβ signaling pathway in salusin-β effect on cytokines.
The cell viability was assessed by the reduction of MTT to its insoluble formazan. 12 HUVECs (5000 cells/well) were seeded in 96-well microplates. After overnight incubation, the cells were treated with different concentrations of salusin-β (1, 3, 10, 30, 90 and 180 nM) for 24 hours. Afterward, the cells were incubated at 37 °C with 15 µl of MTT (5 mg/ml) in phosphate-buffered saline for 4 hours. Then, the medium was removed and 150 µl of dimethyl sulfoxide (DMSO) was added and the absorbance at 570 nm and 630 nm was measured by an enzyme-linked immunosorbent assay (ELISA) reader.
After RNA extraction by TRIzol® Reagent (Thermo Fisher Scientific, Cat No: 15596-026, USA) according to manufacturer's protocol, RNA concentration and purity were determined by microspectrophotometer (A & E Lab, UK). RNA was converted to first-strand cDNA by Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Cat No: K-1622, USA). The mRNA expression of IL-6, IL-8, IL-18 and IL-1Ra were measured by real-time polymerase chain reaction (PCR) system (BioRAD CFX 96) and Syber® Premix Ex TaqTMII (Takara, Cat.RR820L, Japan). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control gene. Real-time PCR data were quantified by 2 -ΔΔCT method, 13 it should note that the PCR efficiency of the target gene was similar to the internal control gene. All samples and no template controls were examined in duplicates. All amplicons (Table 1) were confirmed by sequencing (sequencing service-Bioneer, South Korea).
The IL-6 level was determined by a human IL-6 ELISA kit, Affymetrix eBioscience (catalog No: 88-7066, USA) sensitivity ˂ 2 pg/ml. The protein level of IL-8 was measured by Human IL-8 Elisa kit Affymetrix eBioscience (catalog No: 88-8086, USA), sensitivity 2 pg/ml. These ELISA kits were specifically engineered for accurate and precise measurement protein levels of IL-6 and IL-8, respectively. The protein level of IL-18 was quantified with Human IL-18 Elisa kit, Boster Bio (catalog No: EK0864, USA), with a sensitivity ˂ 1 pg/ml. The protein level of IL-1Ra was determined by human IL-1Ra Elisa kit, Boster Bio (catalog No: EK0782, USA) with a sensitivity less than 2 pg/ml. These ELISA kits were specific for natural and recombinant IL-18 and IL-1Ra, respectively. The procedures were done according to manufacturer's instructions. All cytokine level was measured in cell culture supernatant treated with salusin-β after 6 and 12-hour treatments. All samples were analyzed in duplicates.
Data were presented as the mean ± standard deviation (SD). Statistical analyses were performed using one-way analysis of variance (ANOVA) test. Bonferroni post-hoc test was done to access statistical differences between groups. The normality test was controlled by Kolmogorov-Smirnov test. The results supported normality of our variables. P ˂ 0.050 was considered as statistically significant. The statistical analyses were accomplished by SPSS software (version 24, IBM Corporation, Armonk, NY, USA).

Results
The effect of salusin-β on HUVECs: HUVECs viability was assessed by the MTT assay. The results indicated that salusin-β had no cytotoxic effect in HUVECs in the concentration range of 1-180 nM.

Discussion
Endothelial cells have a fundamental function in the inflammatory response. These cells can synthesize various pro-inflammatory cytokines in response to different stimuli. 14 The present study, for the first time, demonstrated that salusin-β can increase mRNA and protein level of pro-inflammatory cytokines including IL-6, IL-8 and IL-18 and decrease mRNA and protein level of IL-1Ra in HUVECs. Several studies confirmed that salusin-β has a pro-atherogenic effect, 15,16 considering the prominent role of cytokines in key pathogenic events in atherosclerosis, it is worth to recognize the relationship between salusin-β and cytokines.
IL-8 is an atherogenic chemokine. The high level of this protein has been reported in the arterial atherosclerotic wall, atherosclerotic plaques and macrophages. 17 IL-8 is involved in firm adhesion of monocytes to vascular endothelium under flow condition, a crucial step in atherosclerosis initiation. 18 Also, IL-8 can promote monocytes and neutrophils activation. 19 Because of specific biochemical properties of IL-8, this cytokine is a perfect choice for sites of inflammation. 20 IL-8 is a mitogenic and chemotactic factor for VSMCs, which up-regulates mRNA expression and production of matrix metallopeptidase 2 (MMP2) and matrix metallopeptidase 9 (MMP9) in endothelial cells. 21 It also inhibited tissue inhibitor of metalloproteinase 1 (TIMP-1) expression which leads to an imbalance between matrix metalloproteinase and TIMP-1; consequently, resulting in atherosclerotic plaque rupture and thrombosis. 22 Therefore, IL-8 can accelerate the initiation, progression and plaque destabilization.
Very recent studies revealed that salusin-β can elevate migration and intimal hyperplasia of VSMCs via reactive oxygen species (ROS)/NF-ƙβ/MMP-9 pathway. 23 Our results indicated that salusin-β increases mRNA and protein level of IL-8 in HUVECs. As noted above, salusin-β accelerates oxidative stress leading to activation of NF-ƙβ signaling pathway in endothelial cells. 11 Furthermore, salusin-β induces mRNA expression of IL-1β. 11 It is well known that these factors are involved in IL-8 expression. 24,25 IL-18 is an important player in atherosclerotic processes. This cytokine is highly expressed in macrophages, endothelial cells and smooth muscle cells (SMCs) of atherosclerotic lesions. Recombinant IL-18 accelerates atherogenesis and increases cytokines level such as IL-1β, IL-8, IL-6, 26-28 also intensify adhesion molecules expression such as vascular cell adhesion protein 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) in endothelial cells and fibroblasts. 29 Thus, IL-18 results in endothelial dysfunction. 28 IL-18 has a close association with interferon gamma (IFN-γ) induction and it is proposed that pro-atherogenic effect of IL-18 is mediated by this cytokine. IFN-γ has a key role in atherosclerosis especially in foam cell formation via up-regulation of scavenger receptor for phosphatidylserine and oxidized lipoprotein (SR-PSOX). 30 IFN-γ inhibits collagen synthesis by SMCs, thereby can modulate collagen content of atherosclerotic plaques. 31 IL-18 deficiency is associated with decreased recruitment of macrophages. 2 Apoptosis of endothelial cells and acceleration of migration and proliferation of SMCs is mediated by IL-18. 32,33 In vivo studies indicated that endogenous inhibitor of IL-18 impeded fatty streak development in the thoracic aorta and has slowing effect on progression of advanced atherosclerotic plaque. 34 IL-18 is a part of IL-1 superfamily; therefore, this cytokine has structural and functional similarity with IL-1β. It also has the identical signaling cascade in common with IL-1β. 35 Salusin-β increased mRNA expression of IL-1β and this study showed that salusin-β increased mRNA and protein level of IL-18 in HUVECs, suggesting a crosstalk between salusin-β, IL-1β and IL-18.
IL-6 is expressed in human atherosclerotic plaque and increases inflammatory cascade. 36,37 IL-6 is involved in the expression of acute phase proteins in SMCs, also in migration and differentiation of activated macrophages. 38 IL-6 accelerates the proliferation of VSMCs and enhances the permeability of endothelial cells. 14 These events are included in onset and development of atherosclerosis. Animal studies have shown that recombinant IL-6 caused atherosclerotic lesion development. 39 IL-6 is a procoagulant cytokine, 36 which can activate the tissue factor production. 40 Therefore, IL-6 has a role in plaque stability and rupture. It is demonstrated that endothelial cells can express ICAM-1 when are exposed to several inflammatory cytokines such as IL-6. 39 The studies indicated that salusin-β can increase mRNA level of ICAM-1. This study revealed that salusin-β increased mRNA and protein level of IL-6 in HUVECs. In line with our results, in vivo and in vitro studies indicated that salusin-β increased protein level of IL-6; however, these studies did not measure mRNA expression of IL-6. 41 IL-1 is an influential pro-inflammatory cytokine in vascular hemostasis. This cytokine induces the production of some cytokines and chemokines, 42 and stimulates adhesion molecule expression which accelerates monocyte recruitment and permeation into the arterial wall. 43 Also, IL-1 participates in the development of tissue damage via inducing cell proliferation and matrix metalloproteinases release. 43 IL-1Ra is a negative regulator of IL-1 signaling and has a role in maintaining vascular hemostasis. 44,45 It is proved that treatment of apolipoprotein E (ApoE) / mice with recombinant IL-1Ra is an impressive therapy for atherosclerosis. 45 Several lines of evidence indicated that IL-1Ra has an anti-atherosclerotic effect. 5,46 Animal studies indicated that lack of IL-1β causes less atherosclerotic lesions development; 42 On the other hand, partial deficiency of IL-1Ra changes the composition of atherosclerotic plaques with a higher level of membrane cofactor protein 1 (MCP-1), ICAM-1 and VCAM-1 mRNA and accelerates vascular inflammation. 42 Salusin-β increased mRNA level of MCP-1, ICAM-1, VCAM-1 and IL-1β in HUVECs. 11 Our results indicated that salusin-β decreased mRNA/ protein level of IL-1Ra in HUVECs. Because the balance between IL-1 and IL-1Ra may have a role in atherogenesis development, 42 we suggested the other role for salusin-β via disturbance in IL-1 and IL-1Ra equilibrium.
NF-ƙβ is an important transcription factor in inflammatory processes. 47 This transcriptional factor regulates transcriptions of several genes with a well-known function in atherosclerosis including cytokines, chemokines and adhesion molecules. 48 The promoters of IL-6 and IL-8 genes have functional NF-ƙβ binding sites which have been proven to be essential for the transcriptional activation of these genes. 14 Also, several studies confirmed the involvement of NF-ƙβ signaling pathway in IL-18 expression. [48][49][50] The studies demonstrated that salusin-β accelerated vascular inflammatory responses via NF-ƙβ signaling pathway. 11 We found that this complex protein involved in the up-regulatory effect of salusin-β on pro-inflammatory cytokines.
This study was performed with some limitations. We only studied mRNA expression of cytokines in the involvement of NF-ƙβ signaling pathway and further studies are necessary to confirm these results. Also, we could not study another cytokineassociated signaling pathway. Regarding no involvement of NF-ƙβ signaling pathway on the down-regulatory effect of salusin-β on IL-1Ra, it seems that the other cytokine-associated signaling pathways must be investigated.

Conclusion
Primary prevention of atherosclerosis, which is important for the management of atherosclerotic CVD, requires comprehensive assessment and modification of molecular cardiovascular risk factors. Cytokines are a central player in atherosclerotic processes. To the best of our knowledge, this is the first study to indicate the association of salusin-β with pro-and antiinflammatory cytokines. This work indicated that salusin-β increased mRNA/protein level of IL-6, IL-8 and IL-18 as pro-inflammatory cytokines and decreased mRNA/protein level of IL-1Ra as an anti-inflammatory cytokine in HUVECs. The previous studies demonstrated the accelerator effect of salusin-β on vascular inflammation. It seems that salusin-β may participate in a cascade pathway in vascular inflammation ( Figure 6). Our novel results can help to open up a new vista into the potential use of salusin-β as a therapeutic target for the prevention of atherosclerosis. Prospective studies to determine the mechanisms latent down-regulatory effect of salusin-β on IL-1Ra and in vivo researches are recommended.


extfieldsofstudy: Medicine Biology

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TAFA2 Induces Skeletal (Stromal) Stem Cell Migration Through Activation of Rac1‐p38 Signaling

Abstract Understanding the mechanisms regulating recruitment of human skeletal (stromal or mesenchymal) stem cells (hMSC) to sites of tissue injury is a prerequisite for their successful use in cell replacement therapy. Chemokine‐like protein TAFA2 is a recently discovered neurokine involved in neuronal cell migration and neurite outgrowth. Here, we demonstrate a possible role for TAFA2 in regulating recruitment of hMSC to bone fracture sites. TAFA2 increased the in vitro trans‐well migration and motility of hMSC in a dose‐dependent fashion and induced significant morphological changes including formation of lamellipodia as revealed by high‐content‐image analysis at single‐cell level. Mechanistic studies revealed that TAFA2 enhanced hMSC migration through activation of the Rac1‐p38 pathway. In addition, TAFA2 enhanced hMSC proliferation, whereas differentiation of hMSC toward osteoblast and adipocyte lineages was not altered. in vivo studies demonstrated transient upregulation of TAFA2 gene expression during the inflammatory phase of fracture healing in a closed femoral fracture model in mice, and a similar pattern was observed in serum levels of TAFA2 in patients after hip fracture. Finally, interleukin‐1β was found as an upstream regulator of TAFA2 expression. Our findings demonstrate that TAFA2 enhances hMSC migration and recruitment and thus is relevant for regenerative medicine applications. Stem Cells 2019;37:407–416

INTRODUCTION
Skeletal (also known as bone marrow stromal or mesenchymal) stem cells (MSC) are nonhematopoietic cells residing in the bone marrow stroma and are capable of differentiation to mesodermal cell types such as osteoblasts, adipocytes, and chondrocytes [1,2]. MSC are recruited to the bone formation sites during bone remodeling and after bone fracture, where they differentiate into osteoblastic bone-forming cells [3]. Insufficient or defective recruitment of MSC to bone formation sites has been suggested as a mechanism mediating impaired bone formation in osteoporosis and in nonunion or poorly healing fractures [4,5].
MSC are being tested in an increasing number of clinical trials for enhancing tissue regeneration, including bone tissue regeneration, where direct transplantation of MSC cultured on scaffolds has been tested with good preliminary results [6][7][8]. Systemic (intravenous) infusion of MSC is an attractive route of administration because of its ease and clinical feasibility [6][7][8]. Also hematopoietic stem cell (HSC) transplantation has demonstrated the ability of HSC to home to their bone marrow niche after intravenous infusion and to exert their biological functions [9], suggesting that MSC may be equipped with similar functional capacity.
Previous studies have demonstrated the presence of MSC in the circulation and that they can be recruited to injured tissues (for review, please see [10]). MSC can adhere to endothelial cells and exhibit leukocyte-like trans-endothelial migration [11]. However, few cells are recruited to fracture sites after systemic infusion of MSC [12,13]. Interestingly, some studies have reported enhanced tissue regeneration despite the presence of few engrafted MSC [14][15][16][17]. To increase the number of MSC homed to the bone, a number of approaches have been tried, for example, modifying CD44 expression by MSC [18] or surface conjugation of MSC with a synthetic peptidomimetic ligand with high affinity for activated α4β1 integrin to target MSC to the skeleton [19] or to precondition MSC with small molecules before systemic infusion in order to boost their skeletal homing properties [12].
In order to identify secreted factors with capacity for enhancing recruitment of MSC to the skeleton, we examined a number of factors known to regulate migration and motility of adult stem cells in a number of tissues. We identified TAFA2 in a screening experiment using chemoattraction of MSC as an endpoint. TAFA2 (also known as FAM19A2, family with sequence similarity 19 member A2) is a member of FAM19A family consisting of five highly homologous genes (TAFA1-5) encoding small secreted proteins [20]. TAFA proteins are broadly expressed with highest expression in the brain and function as neurokines [20]. Despite lack of functional studies about biological roles of TAFA2, genome-wide association studies have revealed an association between TAFA2 gene polymorphism and mental retardation and insulin sensitivity [21,22]. Here, we report a novel role for TAFA2 in recruiting human MSC (hMSC) to the site of skeletal fracture. Using cellbased and in vivo studies, we show that TAFA2 stimulates hMSC migration through activation of Rac1-p38 signaling. TAFA2 overexpression at the site of skeletal fracture increased the number of recruited hMSC to the injured tissue. TAFA2 levels were differentially regulated during the time course of fracture healing in a closed femoral fracture model in mice, and a similar pattern was observed in TAFA2 serum levels in patients with hip fractures.

MSC Culture
As a model for primary hMSC, we used our well-characterized, immortalized human MSC line (hereafter referred to as hMSC), established by ectopic expression of the catalytic subunit of human telomerase as previously described [40]. hMSC maintain all typical characteristics of primary hMSC [41]. Primary bone marrow-derived hMSC (hereafter referred to as hpMSC) were established from fresh bone marrow aspirates as previously described, from three young healthy donors after informed consent [42]. The cells were cultured in a standard growth medium containing minimal essential medium (MEM; Gibco, Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Biochrom, Germany) and 1% penicillin/streptomycin (Gibco, UK) at 37 C in a humidified atmosphere containing 5% CO 2 . The medium was changed every third day until the cells became 90% confluent.

Boyden Chamber Chemotaxis Trans-Well Migration Assay
The migration ability of hMSC toward a number of chemoattractant factors identified by NeuroSearch A/S, Copenhagen, Denmark, and provided by Dr. Per Horn was measured in a 48-well microchemotaxis Boyden chamber-based cell migration assay (8 μm pores, polycarbonate membrane, NeuroProbe, Inc., USA). The membrane was coated for 1 hour at 37 C with migration media (low glucose Dulbecco's MEM [LG-DMEM, Gibco]), supplemented with 0.2% FBS (Gibco) containing 5 μg/ml fibronectin and 10 μg/ml rat tail collagen I. hMSC were serum starved in migration media 24 hours before migration assay. The chemotaxis and control media (27 μl) were placed in the lower chamber and covered by the coated membrane and fixed by the upper chamber. hMSC (1.25 × 10 4 ) suspended in 50 μl migration media were loaded into each well of the upper chamber. The migration was carried out for 4 hours in 37 C. Nonmigrated cells on the upper side of the membrane were scraped and rinsed with cold phosphate buffered saline (PBS). Migrated cells on the lower side of the membrane were fixed and stained with hemacolor staining kit (Merck, Germany). The migrated cells were scanned and photos were taken at ×20 magnification by a computer-assisted cell-counting system (Visiopharm, Denmark). Images were captured of the entire wells and counted by using ImagJ software. Results were expressed as mean number of cells per at least six replicates and three independent experiments. For inhibition of the signaling pathways, the cells were incubated 30 minutes before the seeding in the chambers, with p38 inhibitor SB203580 (Sigma) or dimethyl sulfoxide (DMSO) (Sigma, USA) used as vehicle.

Oris Cell Motility Assay
The assay was performed according to the manufacturer's instructions (Platypus, Madison, WI). Briefly, 96-well cell culture plates were coated with fibronectin (10 μg/ml) in PBS for 2 hours at 37 C, rinsed twice with PBS. After washing, the Oris cell seeding stoppers were inserted in the wells. hMSC were trypsinized, washed twice with MEM, and resuspended in 0.2% serum medium, seeded and permitted to attach at a density of 2.5 × 10 3 cells in each well. Thereafter, the stoppers were gently lifted from all wells excluding the designated "reference" wells (n = 6). Then, cells were washed with migration media (LG-DMEM, supplemented with 0.2% FBS) and permitted to migrate during 24 hours in fresh migration media supplemented with chemoattractant in a humidified chamber (37 C, 5% CO 2 ). The cells were washed and fixed for 10 minutes in 4% paraformaldehyde/PBS, permeabilized with 0.1% Triton X-100 in PBS and stained with tetramethylrhodamine (TRITC)-conjugated phalloidin solution for 40 minutes followed by 4 0 ,6-diamidino-2-phenylindole (DAPI; 1 μg/ml). The mean fluorescence intensity of the migrated cells was analyzed by an enzyme-linked immunosorbent assay (ELISA) microtiter plate reader (FLUOstar Omega, BMG LABTECH, Ortenberg, Germany).

Quantitation of Cell Spreading and Immunofluorescent Intensity
Ninety-six-well cell culture plates (Perkin Elmer, Denmark) were coated with fibronectin (10 μg/ml) in PBS for 2 hours at 37 C, rinsed twice with PBS, and blocked with 1% BSA for 1 hour. hMSC were trypsinized, washed twice with MEM media, and resuspended in serum-free medium. Cells were stimulated with 10 μg/ml TAFA2 (R&D systems, Minneapolis, MN) for 30 minutes at 37 C and then replated onto fibronectin-coated plates in standard culture medium. Cells were fixed in 4% paraformaldehyde for 10 minutes, washed with PBS, and stained for F-actin with phalloidin (TRITC-labeled, Sigma) and DAPI for nuclear staining. Fluorescent images were analyzed by Operetta high-contentimaging system (Perkin Elmer) at ×20 magnification using Harmony High Content Imaging and Analysis Software (PerkinElmer).

Western Blotting Analysis
hMSC were cultured until 80% confluent, then starved in migration media for 24 hours before treatment with TAFA2 at 10 μg/ml or 10% FBS. Protein samples were harvested at 0, 10, 30, 60, 120, and 240 minutes after treatment. Briefly, cells were washed in PBS and lysed in RIPA buffer (Sigma, USA) supplemented with protease and phosphatase inhibitors (Roche, Germany). After 30 minutes incubation at 4 C, samples were centrifuged for 10 minutes at 12,000 rpm, 4 C. Protein concentration was determined with Pierce Coomassie Plus Bradford assay (Thermo Fisher Scientific, USA), and equal amounts of proteins were loaded on a 10% polyacrylamide gel (Novex, Life technologies, USA). Blotted nitrocellulose membranes were incubated overnight with antibodies against P-p38, p38, P-AKT, AKT, P-ERK, ERK2 (Cell Signaling, USA), and α-tubulin (Sigma, USA), overnight at 4 C. Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology, USA) for 45 minutes at room temperature, and protein bands were visualized with Amersham ECL chemiluminescence detection system (GE Healthcare, UK).

In Vivo Studies of Fracture Healing
The ethical approval for femur fracture in mice was granted by the Danish National Animal Experiment Inspectorate (2012-15-2934-00559). Female immunodeficient NOD.CB17-Prkdc scid /J (NOD-scid, 12 weeks old) and C57BL/6J (10 weeks old) mice were anesthetized by administration of intraperitoneal ketamine hydrochloride (100 mg/kg body weight) and xylazine hydrochloride (10 mg/kg). The closed femur fracture procedure was carried out as described [45]. Briefly, an incision was made just medial to the patella, and the fracture was fixed by inserting a 0.5 mm needle between the condyles and drilled in a retrograde fashion into the medullary cavity of the femur. Afterward, a standardized reproducible closed fracture was induced using a bluntimpact guillotine fracture apparatus. A radiograph was acquired after fracture induction (using Faxitron MX-20; Faxitron, USA) to confirm the fracture induction and needle position.

Immunohistochemical Staining
Tissues were harvested at different time points and fixed in 4% formalin for 3 days. Bones were decalcified in formic acid. The tissue was paraffin embedded, sectioned, and stained for human TAFA2 protein expression. Briefly, the staining was performed using DAKO En Vision+ and PowerVision according to manufacturer's instructions. The tissue sections were demasked using TEG solution (Tris 10 mM, EGTA 0.5 mM, pH 9.0) (15 minutes) and incubated 1 hour with sheep anti-human TAFA2 antibodies (R&D Systems, Abingdon, U.K.) diluted in ChemMate Antibody diluent (1:30 Dako, Glostrup, Denmark). After washing, the sections were incubated with secondary antibody (rabbit anti-goat antibody, cell signaling, USA) for 20 minutes. The slides were processed on an automatic slide processor (Techmate500, Dako, Glostrup, Denmark). Analysis was carried out on an IX50 Olympus microscope using Olympus DP Software v3.1 (Olympus, Essex, U.K.) or a Leica DM4500 (Leica, Wetzlar, Germany) using the Surveyor Turboscan Mosaic acquisition imaging analysis system v5.04.01 (Objective Imaging Ltd, Cambridge, U.K.).

Antibody Labeling and Flow Cytometry Analysis
The cells were incubated with antibodies against cell surface proteins for 20 minutes on ice, and two washes with PBE (PBS containing 2 mM EDTA and 0.5% FBS) were done to remove unbound antibody. For intracellular staining of TAFA2, the cells were fixed and permeabilized with BD-fixation and permeabilization kit (BD Biosciences, USA), and the cells were stained with 2 μg anti-TAFA2 antibodies and incubated for 1 hour on ice in dark. Cells were washed, fixed in 2% paraformaldehyde, and cell acquisition was performed with flow cytometer BD FACS LSRII. Kaluza 1.2 software program was used for analysis of the data. Mean fluorescence intensity (MFI) value of a relevant isotype control staining was subtracted from the corresponding MFI of the specific staining. The flow cytometer was standardized by using positive and negative controls and flow check beads each time to reduce day-to-day variation.

RNA Extraction and Quantitative Reverse Transcription Polymerase Chain Reaction
RNA was isolated from organs or cultured cells according to previously published method. [43] After cDNA synthesis, quantitative reverse transcription polymerase chain reaction was performed using a comparative Ct method [(1/ (2 ΔCt )] formula, and data were represented as relative expression to the HPRT1 reference gene.

Osteoblast Differentiation and ALP Activity Assay
Osteogenic induction was performed using MEM supplemented with 10 mM β-glycerophosphate (Calbiochem-Merck, Germany), 50 μg/ml L-ascorbic acid-2-phosphate (Wako Chemicals GmbH, Germany), 10 nM dexamethasone (Sigma-Aldrich, Denmark), and 10 nM calcitriol (1.25-dihydroxy vitamin-D3) kindly provided by Leo Pharma, Ballerup, Denmark. Medium was changed every third day during induction. The induction was performed with and without addition of different concentrations of rhTAFA2 protein. ALP activity was measured at day 6 of induction. Simultaneously, the numbers of viable cells were determined on the day of ALP activity measurement by adding the CellTiter-Blue solution (Promega, USA) to the wells. The cells were then rinsed with TBS (20 mM Trizma base, 150 mM NaCl at pH = 7.5) and fixed in 3.7% formaldehyde-90% ethanol for 30 seconds at room temperature. A reaction mixture containing 100 μl 50 mM NaHCO 3 , 1 mM MgCl 2 (Sigma, USA), and 1 mg/ml of p-nitrophenyl phosphate (Sigma) was added into each well and incubated 20 minutes at 37 C. The reaction was stopped by adding 50 μl of 3 M NaOH. The absorbance at 405 nm was determined using an ELISA microtiter plate reader (FLUOstar Omega, BMG LABTECH, Ortenberg, Germany). ALP enzymatic activity was normalized to the number of viable cells. After osteoblastic induction at day 15, assessment of hydroxyapatite deposition was performed using the OsteoImage Bone Mineralization Assay (Lonza, USA) according to manufacturer's instructions. Data were analyzed, and noninduced cells were compared with induced and induced cells supplemented with rhTAFA2 (10 μg/ml). (Sigma-Aldrich, Denmark), 500 μM 1-methyl-3-isobutylxanthine (IBMX, Sigma-Aldrich, USA), 1 μM rosiglitazone (BRL49653, Cayman Chemical, Ann Arbor, MI), and 5 μg/ml insulin (Sigma-Aldrich, USA) for 15 days. Media was changed every third day, and rhTAFA2 (10 μg/ml) was added to induced cells. Oil Red O staining and quantification was performed as previously described [43].

ELISA Measurement of TAFA2 Concentrations in Clinical Samples
ELISA measurements of TAFA2 concentrations in human serum samples were performed using an established ELISA procedure (Mybiosource, San Diego, USA) according to the manufacturer's instructions. The human serum samples used in this study were from patients with traumatic hip fracture admitted to the Orthopaedic Department at Herlev Hospital. The experimental protocol was approved by the regional scientific ethical committee in Copenhagen (Number KA 05081). All patients received oral and written information and participated under signed informed consent. Collection and handling of the serum samples was previously published [44].

TAFA2 Enhanced Migration and Motility of hMSC
To identify novel regulators of hMSC recruitment to injured tissues, we screened a number of newly identified chemoattractant neurokines, for their ability to enhance in vitro trans-well migration of hMSC using a three-dimensional-Boyden chamber assay. The members of TAFA family, TAFA2, 4, and 5, exhibited the highest chemoattractant activity for hMSC (Fig. 1A). Next, we used the Oris assay to confirm the trans-well migration-stimulating activity of TAFA proteins and found that TAFA2 is the most potent stimulator of hMSC motility (Fig. 1B, 1C). TAFA2 also stimulated cell motility in cultures of human primary bone marrow-derived MSC (hpMSC; Fig. 1D). To specify whether TAFA2-induced cell migration is directional (chemotaxis) or random (chemokinesis), TAFA2 was added to both upper and lower compartments of the Boyden chamber and we observed a significant reduction in number of migrating hMSC (Fig. 1E) and hpMSC (Fig. 1F). TAFA2 exhibited a dose-dependent chemoattractant effect on the trans-well migration of hMSC (Fig. 1G), and a similar effect was observed in hpMSC (Fig. 1H, 1I). In addition, combining TAFA2 with different concentrations of anti-human TAFA2 antibody reduced the chemotactic activity of TAFA2 (Fig. 1J).

TAFA2 Stimulates hMSC Migration Through Activation of Rac1-p38 Signaling
Formation of lamellipodia is an early event initiating migration of various cell types such as fibroblasts and leukocytes [23]. We used the Operetta high-content-imaging system to perform quantitative analysis of TAFA2-induced morphological changes in cultured hMSC at a single-cell resolution. We noted that TAFA2 induced formation of lamellipodia (cell protrusions that are needed for cell movement) in cultured hMSC ( Fig. 2A) that was associated with increased cell area (Fig. 2B). We then examined the activation status of Rac1 GTPase that is required for formation of lamellipodia and cell migration. Analysis of lysates from TAFA2-stimulated hMSC showed significantly increased levels of Rac1 activity at 10, 15, and 30 minutes after TAFA2 stimulation, which returned to the initial levels after 60 minutes (Fig. 2C). To examine the role of Rac1 in TAFA2-induced hMSC migration, we performed small interfering RNA (siRNA)-mediated knockdown of Rac1, which decreased Rac1 expression in hMSC cultures (Fig. 2D) and abolished the TAFA2-induced hMSC trans-well migration (Fig. 2E). To identify the downstream signaling pathways involved in TAFA2-mediated hMSC motility, we examined the activation status of several signaling factors involved in cell migration: p38, AKT, and ERK. We found increased levels of P-p38 within the initial 2 hours after TAFA2 stimulation ( Fig. 2F; Supporting Information Fig. S1). We did not detect changes in P-Akt and P-ERK levels in TAFA2-stimulated hMSC cultures (Fig. 2F). Interestingly, inhibition of p38 signaling using SB203580 abolished TAFA2-induced hMSC migration in a dose-dependent manner (Fig. 2G, 2H).

TAFA2 Regulates Proliferation, but Not Differentiation of hMSC
To investigate other possible functions of TAFA2 in hMSC biology, we tested whether TAFA2 could exert a regulatory effect on hMSC proliferation or differentiation. Adding TAFA2 to hMSC cultures in the presence of normal culture media increased cell proliferation as shown by increased cell number and Ki67 levels (Fig. 3A, 3B). Next, we examined TAFA2 expression during ex vivo differentiation of hMSC toward osteoblastic and adipocytic lineages. TAFA2 mRNA expression increased during osteoblast and adipocyte differentiation of hMSC (Fig. 3C, 3D). However, TAFA2 did not affect osteoblast differentiation as shown by quantitation of alkaline phosphatase (ALP) activity (Fig. 3E, 3F) and mineralized matrix formation assessed by measuring deposition of hydroxyapatite (Fig. 3G, 3H). Similarly, TAFA2 did not affect adipocyte differentiation of hMSC evidenced by measuring the number of mature lipidfilled adipocytes (Fig. 3I, 3J).

TAFA2 Levels Are Differentially Regulated During Fracture Healing
To determine the relevance of TAFA2 to pathophysiology of fracture healing, we examined the pattern of TAFA2 expression at different phases of fracture healing in the mouse femoral fracture model. TAFA2 was highly expressed at the site of fracture 48 hours post induction. TAFA2 immunoreactivity was present in the fracture line-adjacent bone marrow and in the injured muscle tissue above the fracture line, where the blunt impact from a dropped weight induced the mid-diaphysis fracture (Fig. 4A). Interestingly, TAFA2 protein expression was already increased 2 hours after the fracture and expression peaked during the first week of fracture (Fig. 4B). TAFA2 mRNA and protein expression was also differentially regulated, with an initial increase in the early phase of fracture healing followed by downregulation (Fig. 4C, 4D). A similar pattern of expression was observed for the pro-inflammatory cytokine interleukin-1β (IL-1β) (Fig. 4E). To investigate the role of IL-1β as an upstream regulator of TAFA2 expression, we added different doses of IL-1β to hMSC cultures and observed an enhancing effect on TAFA2 mRNA and protein levels (Fig. 4F, 4H). We then determined the cellular map of TAFA2 expression within the bone marrow during fracture healing. Interestingly, intracellular expression of TAFA2 in different leukocyte subpopulations was detectable with high levels of TAFA2 expression in monocytes and T cells at day 1 postfracture that was downregulated by day 3 (Fig. 4I), while monocyte cell frequency was not altered, and T-cell frequency was increased on day 3 postfracture compared to day 1 (Fig. 4J). To extrapolate these findings to human physiology, we assessed the kinetics of TAFA2 levels in consecutive serum samples from patients after hip fracture. TAFA2 serum levels were high at the first day after hip fracture and decreased gradually in the subsequent measurements during a 21 day observation period (Fig. 4K).

DISCUSSION
In the present study, we identified the neurokine TAFA2 as a novel chemoattractant factor for hMSC that is expressed during the inflammatory phase of bone fracture healing and enhances in vitro transmigration of hMSC. Because of the limited number of factors known to exert specific effects on MSC migration and recruitment capacity, studying the role of TAFA2 in MSC biology is relevant for better understanding the biological processes of bone remodeling and fracture healing as well as possible use for enhancing MSC recruitment to injured sites in regenerative medicine protocols.
TAFA2 is a member of the TAFA gene family comprising newly described chemoattractant neurokines with high expression in the central nervous system. The TAFA family is composed of five highly homologous genes encoding small secreted proteins that contain conserved cysteine residues at fixed positions [20]. The TAFA family is related to the chemokine, macrophage inflammatory protein-1α (MIP1a/CCL3) that plays a role in granulocyte recruitment to sites of neuroinflammation [24]. Several C-C chemokines ligands, such as CCL3 [24], CCL4/MIP-β [25], and CCL2/Monocyte chemoattractant protein-1 (MCP-1) [26], are known for their modulatory functions on the migration of endogenous or transplanted neural progenitors toward damaged areas of the brain in ischemic stroke.
We observed that in vitro treatment of hMSC with TAFA2 resulted in enhanced trans-well migration as well as significant changes in cell morphology and cytoskeletal remodeling as defined by rearrangement of F-actin to form cell protrusions (lamellipodia) that are needed for cell movement [27]. These responses are similar to changes in cell shape and cytoskeleton induced by chemokines known to affect cell motility [28]. Also, previous studies have demonstrated that MSC can respond to chemoattractant proteins by reorganizing their actin cytoskeletons and become polarized in the direction of the chemoattractant gradient [29,30]. Indeed, a disorganization of cytoskeletal F-actin has been associated with impaired migration of not only MSC but also epidermal cells and dendritic cells [31][32][33]. Formation of lamellipodia and membrane polarization at the leading edge of a migrating cell enables directional cell movement [27]. TAFA2 treatment of hMSC in two dimensional (2D) cultures resulted in formation of lamellipodia in a nondirectional fashion. It is plausible that restoration of Rac1 activity to initial levels at 60 minutes after treatment with TAFA2 represents a 2D-culture phenomenon because of the absence of directional migration. In addition, the chemoattractant effect of TAFA2 was associated with increased activity of the p38 signaling which is known to mediate the migration of stem cells by several chemotactic factors including: IL-33 [34], stromal cellderived factor 1 alpha (SDF-1α) [35,36], and transforming growth factor beta (TGF-β) [37]. It is plausible that p38 signaling is induced after TAFA2 interaction with a cognate receptor that remains to be determined.
We observed that TAFA2 is constitutively expressed in the bone marrow by leukocyte and MSC, and its expression is upregulated after in vitro osteoblast differentiation of MSC. In contrast to several other factors, TAFA2 does not affect differentiation capacity of the cells but enhances their proliferation, which is consistent with a role in enhancing cell recruitment to sites where bone formation is needed. In vivo, TAFA2 expression was upregulated in the callus area during the inflammatory phase of fracture healing and also in the newly formed chondrocytes. This expression pattern is similar to what has been reported for other members of the C-C family. For example, it has been reported that CCL3 and CCL4 (MIP1-β) are expressed by the callus chondrocytes during fracture healing [38]. The stimulating effects of TAFA2 on MSC in vitro transmigration, the increased expression of TAFA2 during fracture healing, and the absence of its direct effects of MSC differentiation suggest a possible role for in vivo chemotactic effects on MSC during fracture healing. Previous studies have shown that the number of osteoprogenitor cells in systemic circulation is increased after bone fracture [39]. It is thus plausible that the presence of high levels of TAFA2 inside the bone fracture microenvironment during the inflammatory phase of fracture healing results in recruitment of osteoprogenitor cells needed for subsequent bone formation (Fig. 4L). Future studies are needed to determine whether local expression of TAFA2 at the fracture site could be used to increase recruitment of osteoprogenitors and to enhance the fracture healing.

CONCLUSION
We demonstrate that during the inflammatory phase of fracture healing, TAFA2 levels are increased at the site of fracture that leads to recruitment of osteogenic cells to the fracture site through activation of Rac1-p38 signaling.


extfieldsofstudy: Medicine Chemistry

text:
Hypoxia potentiates gemcitabine-induced stemness in pancreatic cancer cells through AKT/Notch1 signaling

Background Profound chemoresistance remains an intractable obstacle in pancreatic cancer treatment. Pancreatic cancer stem cells (CSCs) and the ubiquitous hypoxic niche have been proposed to account for drug resistance. However, the mechanism involved requires further exploration. This study investigated whether the hypoxic niche enhances gemcitabine-induced stemness and acquired resistance in pancreatic cancer cells by activating the AKT/Notch1 signaling cascade. The therapeutic effects of blockading this signaling cascade on gemcitabine-enriched CSCs were also investigated. Methods The expression levels of CSC-associated markers Bmi1 and Sox2 as well as those of proteins involved in AKT/Notch1 signaling were measured by Western blot analysis. The expression level of the pancreatic CSC marker CD24 was measured by flow cytometry. Change in gemcitabine sensitivity was evaluated by the MTT assay. The ability of sphere formation was tested by the sphere-forming assay in stem cell medium. The ability of migration and invasion was detected by the transwell migration/invasion assay. A mouse xenograft model of pancreatic cancer was established to determine the effect of Notch1 inhibition on the killing effect of gemcitabine in vivo. The ability of metastasis was investigated by an in vivo lung metastasis assay. Results Gemcitabine promoted pancreatic cancer cell stemness and associated malignant phenotypes such as enhanced migration, invasion, metastasis, and chemoresistance. The AKT/Notch1 signaling cascade was activated after gemcitabine treatment and mediated this process. Blockading this pathway enhanced the killing effect of gemcitabine in vivo. However, supplementation with hypoxia treatment synergistically enhanced the AKT/Notch1 signaling pathway and collaboratively promoted gemcitabine-induced stemness. Conclusions These findings demonstrate a novel mechanism of acquired gemcitabine resistance in pancreatic cancer cells through induction of stemness, which was mediated by the activation of AKT/Notch1 signaling and synergistically aggravated by the ubiquitous hypoxic niche. Our results might provide new insights for identifying potential targets for reversing chemoresistance in patients with pancreatic cancer. Electronic supplementary material The online version of this article (10.1186/s13046-018-0972-3) contains supplementary material, which is available to authorized users.

Background
Pancreatic cancer is one of the most lethal cancers worldwide, with a 5-year survival rate that has remained at less than 10% for the past few decades [1,2]. For many patients, there is little choice other than chemotherapy, especially in the advanced stage [3]. However, gemcitabine, a first-line anticancer drug for pancreatic cancer, provides a limited survival advantage in treated patients [4]. Numerous strategies have been proposed to improve the therapeutic effect of gemcitabine, but the prognosis for patients with pancreatic cancer remains disappointing [5]. Therefore, identifying new chemotherapeutic agents or adjuvant therapies is necessary to enhance the effectiveness of chemotherapy and reduce tumor recurrence.
Accumulating evidence indicates that tumors harbor a subpopulation of cells, termed cancer stem cells (CSCs), that are responsible for initiating tumor growth and driving relapse after chemotherapy [6,7]. CSC-associated markers Bmi1 and Sox2 sufficiently enhance self-renewal and dedifferentiation and endow pancreatic cancer cells with stemness [8,9]. Further, the pancreatic CSC marker CD24 increases the ability of cells to migrate and invade and has a close correlation with a poor prognosis [10][11][12]. Our previous results suggested that gemcitabine can enhance the stemness of pancreatic cancer cells [13]; however, the exact mechanism remains to be determined. Clarifying the mechanism involved in this process will help identify adjuvant agents for enhancing the killing effect of gemcitabine chemotherapy.
A hypoxic microenvironment has been verified in many malignances, including pancreatic cancer; it plays a critical role in the resistance of cancer cells to anticancer drugs [14,15]. Recent studies have shown that, under hypoxic conditions, pancreatic cancer cells exhibit substantial apoptosis resistance induced by gemcitabine [14]; however, the mechanism remains elusive. Hypoxia signaling has a close correlation with the induction and maintenance of stemness phenotypes, such as self-renewal, undifferentiated condition, sphere-forming ability [16][17][18]. In a preliminary study, we also found that hypoxia promotes the expression of CSC-associated markers Bmi1 and Sox2 in pancreatic cancer cells. We, therefore, speculate that the hypoxic niche might synergistically enhance the acquired gemcitabine chemoresistance through stemness induction.
Notch signaling is evolutionarily conserved and critically implicated in cell fate, including proliferation, differentiation, and apoptosis [19,20]. Mounting evidence indicates that the release of the Notch intracellular domain (NICD) from its ligands leads to aberrant activation of Notch signaling in a variety of malignancies [21][22][23]. Reports concerning the correlation between Notch1 signaling and CSC phenotype have increased in recent times. It has been suggested that aberrant activation of Notch1 helps cells acquire epithelialmesenchymal transition and CSC self-renewal properties and is associated with pancreatic cancer treatment failure [24,25]. However, the role and mechanism of Notch1 signaling in acquired gemcitabine resistance remain elusive. PI3K/AKT signaling is extensively activated in many tumors, including pancreatic cancers [26,27]. AKT inhibition induces apoptosis of pancreatic cancer cells and enhances the killing effect of gemcitabine [28]. Moreover, there exists a reciprocal regulation between the Notch1 and AKT signaling pathways [29], by which both of them interactively regulate chemoresistance and maintenance of stemness.
In this study, we verified that the hypoxic niche synergistically enhances gemcitabine-induced stemness and acquired resistance in pancreatic cancer cells by activating the AKT/ Notch1 signaling cascade. Furthermore, a chemotherapeutic combination involving the blockade of such a signaling pathway weakens the gemcitabine-enriched CSC population, providing a new therapeutic strategy against acquired chemoresistance.

Cell culture and treatments
The human pancreatic cancer cell line PANC-1 was obtained from the American Type Culture Collection (Manassas, VA, USA). The Patu8988 cell line was purchased from Nanjing KeyGen Biotech. Co. Ltd. (Nanjing, China). Both cell lines were cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin, in a humidified incubator with 5% CO 2 at 37°C. After reaching a 60-80% confluence level, the cells were treated with different concentrations of gemcitabine (Selleck, Houston, TX, USA) for 24 h. To examine the role of the Notch1 or AKT signaling pathway in enhancing stemness, the pancreatic cancer cells were pretreated with 10 μM DAPT (γ-secretase inhibitor; Selleck) for 24 h or 20 μM LY294002 (AKT inhibitor; Beyotime Biotechnology, Shanghai, China) for 2 h before gemcitabine treatment. To clarify the effect of hypoxia on pancreatic cancer cell stemness, the cells were treated with 1% O 2 for different time intervals or with various doses of CoCl 2 (Sigma-Aldrich, St. Louis, MO, USA) for 24 h. To test the synergistic effect of hypoxia and gemcitabine, the cells were co-treated with optimal doses of gemcitabine and CoCl 2 (as indicated in the pertinent figure legends) for 24 h.

Western blot analysis
Western blot analysis was performed as previously described [13]. In brief, total cell lysates were electrophoresed in a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred onto polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA). The membranes were blocked with 5% skim milk and incubated overnight with primary antibodies. After washing, the membranes were incubated with secondary antibodies conjugated with horseradish peroxidase, and the proteins were visualized by adding an enhanced chemiluminescence substrate (Thermo Fisher Scientific, Waltham, MA, USA). Antibodies against Bmi1, Notch1, NICD1, AKT, p-AKT (phosphorylated AKT), and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) were purchased from Cell Signaling Technology (Danvers, MA, USA), and those against Sox2 and HIF-1α (hypoxia-inducible factor-1α) were purchased from Abcam (Boston, MA, USA).

Transwell migration/invasion assay
Migration and invasion assays were performed in 24-well Transwell chambers (Corning, Fisher Scientific). For the transwell invasion assay, the upper compartment of the chamber was precoated with Matrigel (Sigma-Aldrich). Equal amounts of approximately 10 × 10 4 cells were seeded into each upper chamber. The upper and lower chambers were filled with culture medium containing 0.1 and 30% FBS, respectively. After about 24 h, the migratory and invasive cells on the lower surface of the membrane were fixed, stained with 0.1% crystal violet, and then counted in five random fields under a light microscope.

MTT assay
The MTT assay was performed as previously described [30]. After different treatments, the pancreatic cancer cells were seeded into 96-well plates and further incubated with various concentrations of gemcitabine (Selleck) for 48 h. Then, 20 μL of MTT solution (5 mg/mL; Sigma-Aldrich) was added to each well. The plates were incubated for 4 h, after which the medium was replaced with 150 μL of dimethyl sulfoxide (Sigma-Aldrich). The optical density was detected at 490 nm. Each concentration of gemcitabine was set up in five replicate wells.

Flow cytometry analysis
Flow cytometry analysis was performed as previously described [13]. Anti-CD24-FITC antibody was purchased from BD Pharmingen (San Diego, CA, USA).

Sphere-forming ability assay
The sphere-forming ability assay was performed in stem cell medium (SCM) as previously described [13]. Briefly, after different treatments, the pancreatic cancer cells were washed three times and suspended in SCM, which consisted of Dulbecco's modified Eagle's medium/F12 medium supplemented with bovine serum albumin (0.4%; Sigma-Aldrich), Insulin-Transferrin-Selenium (ITS; 1×; Sigma-Aldrich), basic fibroblast growth factor (10 ng/mL; PeproTech, Rocky Hill, NJ, USA), and epidermal growth factor (20 ng/mL; Pepro-Tech). Approximately 1 × 10 4 cells per well were seeded into ultralow-attachment 6-well plates (Corning), and the medium was changed every 3 days. After 15 to 20 days, the formed spheres (diameter ≥ 50 μm) were counted under a light microscope. The efficiency of sphere formation was calculated on the basis of the ratio of number of spheres to total number of cells.

Tumor xenografts
Xenografts were formed by subcutaneously injecting PANC-1 cancer cells into the right flank of 3-to 4-week-old athymic mice (2 × 10 6 cells per 100 μL per mouse) (HFK Bioscience Co., Beijing, China). Approximately 6 days after subcutaneous implantation, the mice were randomly separated into the control, GEM (gemcitabine), GEM+DAPT, and DAPT groups (n = 5 per group). Gemcitabine (20 mg/ kg) and DAPT (10 mg/kg) were intraperitoneally injected every 3 days and every day, respectively. Tumor volume was measured periodically by using the following formula: Volume = 0.5 × length × width 2 . The experimental protocol complied with the "Guide for the Care and Use of Animals in Wuhan University".
In vivo lung metastasis assay PANC-1 cells were separated into four groups (control, GEM, GEM+DAPT, and GEM+LY294002) and treated as indicated above. After treatment, approximately 4 × 10 6 cells suspended in 0.2 mL phosphate-buffered saline were injected into the lateral tail vein of 7-to 8-week-old nude mice (HFK Bioscience Co.; n = 5 per group). After about 4 weeks, the mice were euthanized, and the lungs were completely resected and photographed. For hematoxylin and eosin (H&E) staining, the lungs were fixed with 4% paraformaldehyde and cut into 5-μm sections. The specimens were then stained with H&E, and the number of metastases was detected microscopically. All mice were handled in accordance with the protocols approved by the "Guide for the Care and Use of Animals in Wuhan University".

Statistical analysis
The data in our study were expressed as mean ± standard deviation. Student's t-test was used to compare differences between two groups. Values were considered statistically significant at P < 0.05.

Gemcitabine promotes Notch1 activation and pancreatic cancer cell stemness
In our previous study, we had shown that low-dose gemcitabine treatment can enhance the stemness of pancreatic cancer cell lines SW1990 and BxPC-3 [13]. In the present study, we further analyzed whether gemcitabine has a similar effect on other pancreatic cancer cell lines such as PANC-1 and Patu8988. Our results revealed that low-dose gemcitabine treatment (1-5 μM) for 24 h, which has a minimal killing effect on pancreatic cancer cells (Fig. 1a), induced the expression of stemness-associated molecules Bmi1 and Sox2 as well as the CSC marker CD24 (Fig. 1b-e). In line with these changes, gemcitabine treatment also enhanced the sphere-forming ability of the evaluated cell lines, which exhibited a greater number of cell spheres and larger microsphere size after treatment ( Fig. 1f-h). Although Notch1 signaling has been reported to play an important role in maintaining the stemness and self-renewal ability of CSCs [31], studies on the correlation between gemcitabine and Notch1 signaling are still lacking. Our results revealed that low-dose gemcitabine treatment promoted the expression of both Notch1 and NICD1 in a dose-dependent manner ( Fig. 1b and c). Together, our results suggest that low-dose gemcitabine treatment activates Notch1 signaling and induces stemness in pancreatic cancer cells.

Notch1 signaling mediates gemcitabine-induced stemness
To further confirm the role of Notch1 in gemcitabine-enhanced stemness, we pretreated pancreatic cancer cells with 10 μM γ-secretase inhibitor DAPT for 24 h before gemcitabine treatment. The Western blot findings showed that pretreatment with DAPT abolished gemcitabine-induced NICD1 expression (Fig. 2a). Further, Notch1 inhibition dramatically impaired the upregulation of Bmi1, Sox2, and CD24 expression (Fig. 2a-c). In addition, we observed a relative decrease in the number and size of spheres after Notch1 inhibition ( Fig. 2d-f). It has been established that the properties of CSCs are connected with enhanced migration and invasion [32]. In the present study, we detected such changes after Notch1 inhibition. Our results showed that gemcitabine treatment increased the migratory and invasive abilities of pancreatic cancer cells, whereas suppression of Notch1 significantly abolished these increases (Additional file 1: Figure S1a-d). Moreover, pretreatment with DAPT dramatically reversed the gemcitabine-induced chemoresistance (Additional file 1: Figure S1e). These results show that gemcitabine promotes pancreatic cancer cell stemness and associated migration, invasion, and chemoresistance partly through Notch1 activation.

Notch1 inhibition enhances the killing effect of gemcitabine and suppresses metastasis in vivo
Because Notch1 activation was revealed to play a role in gemcitabine-induced stemness and associated malignant traits, we next investigated the effect of supplementation with Notch1 inhibition on chemosensitivity in vivo. As shown in Fig. 3a and b, DAPT treatment significantly reduced the tumor growth rate and size relative to the control at 38 days post-treatment. When combined with gemcitabine chemotherapy, DAPT treatment also synergistically strengthened the killing effect of gemcitabine in pancreatic cancer cells. We further examined the changes in Bmi1 and Sox2 expression and CD24 + cell population at the end of treatment. As shown in Fig. 3c-e, gemcitabine chemotherapy increased the expression levels of Bmi1 and Sox2 as well as the proportion of CD24 + cells, while combination treatment with DAPT abolished these enrichments. CSCs have an inherent potential for metastasis [33,34]. Our results, too, revealed an enhanced ability of the cells for lung metastasis after gemcitabine treatment, which was attenuated when combined with DAPT treatment (Additional file 2: Figure  S2a-c). These results show that Notch1 inhibition synergistically potentiates the killing effect of gemcitabine and suppresses metastasis in vivo.
AKT promotes pancreatic cancer cell stemness partly by mediating Notch1 activation AKT is commonly activated in pancreatic cancer and participates in gemcitabine chemoresistance, and inhibition of AKT could enhance the killing effect of gemcitabine [35]. Our results revealed that gemcitabine treatment promoted the expression of p-AKT (serine 473) in PANC-1 and Patu8988 cell lines (Fig. 4a). To determine the role of AKT in gemcitabine-induced stemness, we pretreated the pancreatic cancer cells with 20 μM LY294002 (an AKT inhibitor) for 2 h before gemcitabine treatment. As indicated in Fig. 4a, AKT inhibition significantly suppressed gemcitabine-induced AKT activation. Subsequently, the expression of Bmi1, Sox2, and CD24 was significantly impaired (Fig. 4a and  b). Further, LY294002 pretreatment attenuated the gemcitabine-induced sphere-forming ability of the pancreatic cancer cells (Fig. 4c-e). We further examined the role of AKT in Notch1 activation after gemcitabine treatment. Our results demonstrated that LY294002 attenuated gemcitabine-induced NICD1 expression in both cancer cell lines (Fig. 4a). Then, we analyzed the changes in the stemness-related metastatic, migratory, and invasive abilities of cancer cells after AKT Charts showing the data on sphere number and diameter. The data are derived from three independent assays. Scale bar, 50 μm. *P < 0.05; **P < 0.01; ***P < 0.001 inhibition. Our results showed that pretreatment with LY294002 markedly attenuated gemcitabine-enhanced metastasis in vivo (Additional file 2: Figure S2a-c). It also weakened the migratory and invasive abilities of pancreatic cancer cells (Additional file 3: Figure  S3a-d). In total, our results suggest that AKT plays a role in promoting gemcitabine-induced Notch1 activation and stemness.

Hypoxia synergistically enhances gemcitabine-induced stemness
It is well known that hypoxia is a prominent feature of the microenvironment in pancreatic cancer and that it enhances the chemoresistance against gemcitabine [14]. We, therefore, analyzed the effect of hypoxia on stemness. As shown in Fig. 5a, treatment of pancreatic cancer cells under hypoxic (1%) conditions for 6-12 h significantly promoted the expression of HIF-1α, an important marker in hypoxia response. In addition, stemness-associated molecules Bmi1 and Sox2 were upregulated after hypoxia treatment. We also treated pancreatic cancer cells with CoCl 2 , a chemical which stabilizes HIF-1α, to mimic a hypoxic microenvironment. CoCl 2 treatment for 24 h, too, promoted Bmi1 and Sox2 expression in a dose-dependent manner in both evaluated cells (Fig. 5b). To further verify the synergistic effect of hypoxia and gemcitabine treatment on stemness induction, we co-treated pancreatic cancer cells with gemcitabine and CoCl 2 for 24 h. The Western blot findings showed that combination treatment with CoCl 2 further reinforced the gemcitabine-inductive effect on Bmi1 and Sox2 (Fig. 5c). In accord, CoCl 2 treatment also enhanced the number and size of gemcitabine-induced spheres (Fig. 5d-f). Our results suggest that hypoxia synergistically enhances gemcitabine-induced stemness.
AKT/Notch1 signaling mediates the synergistic enhancement of the stemness induced by gemcitabine and hypoxia co-treatment Because Notch1 has been demonstrated to mediate gemcitabine-induced stemness, we next analyzed the changes on the basis of hypoxic status. The Western blot findings revealed that both hypoxia and CoCl 2 treatment increased the expression of NICD1 ( Fig. 6a and b). The synergistically inductive effect of these two treatments was more obvious when combined with gemcitabine treatment (Fig. 6c). We also pretreated pancreatic cancer cells with DAPT before co-treatment with gemcitabine and CoCl 2 . The results showed that DAPT significantly attenuated the synergistic enhancement of Bmi1 and Sox2 expression (Fig. 6d). In addition, Notch1 inhibition significantly suppressed the synergistic enhancement of sphere number and size ( Fig. 6e-g). These results suggested that Notch1 activation plays a role in the synergistic enhancement of stemness induced by combination treatment with gemcitabine and CoCl 2 .
Hypoxia has been reported to activate AKT expression [14]. Our results also demonstrated a similar effect in pancreatic cancer cells ( Fig. 6a and b). In addition, synergistically augmented p-AKT expression was more evident after gemcitabine and CoCl 2 co-treatment (Fig. 6c), whereas AKT suppression significantly suppressed the collaboratively inductive effect of the co-treatment on Bmi1 and Sox2 expression (Fig. 6d). In line with these changes, the enhanced sphere-forming ability of the cells was also dramatically reduced after AKT inhibition ( Fig. 6e-g). Moreover, the induction of NICD1 expression by gemcitabine and CoCl 2 co-treatment was significantly abolished by pretreatment with an AKT inhibitor in both evaluated cells (Fig. 6d), which suggests the role of AKT regulation in Notch1 activation in cells treated with gemcitabine and CoCl 2 . Collectively, these results suggest that AKT/Notch1 signaling plays a role in promoting the synergistic induction of stemness by gemcitabine and hypoxia.

Discussion
Resistance to chemotherapy is an intractable problem in pancreatic cancer treatment. Gemcitabine is a standard first-line chemotherapeutic agent. However, its clinical benefit has remained dismal during the past decades. It has been suggested that a hypoxic microenvironment enhances the resistance of cells to gemcitabine chemotherapy. CSCs, or tumor-initiating cells, are a small subset of cancer cells that display high tumorigenicity and great resistance to drugs [36,37]. In this study, we showed that low-dose gemcitabine treatment promotes the stemness of pancreatic cancer cells, and this effect is synergistically enhanced by the hypoxic niche. Moreover, our results suggested that the AKT/Notch1 signaling cascade partly mediates this process (Fig. 7). Additionally, our in vivo findings demonstrated that pharmaceutical inhibition of Notch1 signaling enhances the killing effect of gemcitabine in pancreatic cancer cells. Together, our data put forth a new potential mechanism of gemcitabine resistance and offer potential targets to enhance the chemosensitivity of cells to gemcitabine. The Notch1 signaling pathway plays an essential role in the maintenance and self-renewal of CSCs in a variety of malignancies [38,39]. It has been suggested that a fraction of pancreatic CSCs are enriched as a consequence of the killing effect of gemcitabine treatment, with a small residual cells exhibiting an activated Notch1 signaling pathway [25]. In our study, gemcitabine (a relatively low dose) might not have had a cytotoxic effect because of the high rate of cell survival after gemcitabine treatment. We found that gemcitabine treatment increased Notch1 and NICD1 expression in a dose-dependent manner. Therefore, we believe that the activation of Notch1 resulted mainly from the "inductive effect" of gemcitabine and not just from the "enrichment" of CSCs. In addition, pretreatment with the γ-secretase inhibitor DAPT significantly inhibited the molecules and behaviors (such as migration, invasion, and chemoresistance) associated with gemcitabine-induced stemness. Further, combination treatment with DAPT enhanced the killing effect of gemcitabine and inhibited gemcitabine-induced metastasis in vivo. Our results suggest that induction of Notch1 signaling plays an important role in gemcitabine-enhanced stemness.
AKT activation plays an important role in the migration, invasion, metastasis, chemoresistance, and CSC-like phenotype of pancreatic cancer cells, and it is closely correlated with the prognosis [27,[40][41][42]. AKT activation overlaps in function and entails mutual cooperation with Notch1 [29,43]. In our study, we showed (See figure on previous page.) Fig. 6 AKT/Notch1 signaling mediates the synergistic enhancement of stemness induced by gemcitabine and hypoxia. (a) PANC-1 and Patu8988 cells were treated under hypoxic conditions (1%) for different durations, and the expression levels of NICD1, p-AKT, and AKT were determined by Western blot analysis. (b) After treatment with different concentrations of CoCl 2 , the changes in NICD1, p-AKT, and AKT expression levels were determined by Western blot analysis. (c) Two cell lines were co-treated with gemcitabine and CoCl 2 , and the protein expression levels were determined by Western blot analysis. (d) Two pancreatic cancer cell lines were pretreated with 10 μM DAPT or 20 μM LY294002 and then treated with a combination of gemcitabine and CoCl 2 . Then, the expression levels of Bmi1, Sox2, and NICD1 were determined by Western blot analysis. (e-g) After this treatment, the two cell lines were cultured with stem cell medium, and the ability of the cells for sphere formation was investigated by the sphere-forming assay: (e) Representative image of spheres after treatment; (f, g) Charts showing the data on sphere number and size. All data shown are from three independent experiments. Scale bar, 50 μm. *P < 0.05; **P < 0.01; ***P < 0.001 Fig. 7 Schematic illustration demonstrating how hypoxia potentiates gemcitabine-induced stemness and acquired resistance in pancreatic cancer cells through AKT/Notch1 signaling that low-dose gemcitabine can facilitate AKT activation, which is in line with our findings on the enhanced stemness of pancreatic cancer cells. Further, AKT inhibition significantly attenuated gemcitabine-activated Notch1 expression as well as the consequent CSC-like behaviors of the cells. Our results suggest a potentially adverse collaboration between Notch1 and AKT, in which the two signaling pathways are intertwined in increasing the stemness of pancreatic cancer cells upon gemcitabine chemotherapy. Therefore, our results are consistent with previous findings and suggest the role of AKT signaling in promoting gemcitabine-induced Notch1 activation and pancreatic cancer cell stemness.
Hypoxia is a frequent condition in pancreatic cancer. It contributes greatly to the apoptosis resistance of cells to chemotherapy [14]. However, the exact mechanism remains obscure. Increasing evidence has demonstrated that the hypoxic niche plays an important role in maintaining the self-renewal property and undifferentiated condition of various CSCs [18,44]. In our study, we also showed that hypoxia synergistically promotes the gemcitabine-induced CSC-like phenotype, with an increased expression of stemness-associated molecules and enhancement of sphere-forming ability, which might explain the persistence of gemcitabine resistance in pancreatic cancer. Hypoxia activates Notch-responsive promoters and promotes the expression of downstream genes [45]. Our study also demonstrated an increase in both NICD1 expression and AKT activation after hypoxia treatment. In addition, inhibition of AKT abolished Notch1 activation and pancreatic cell stemness induced by co-treatment with gemcitabine and hypoxia. Therefore, our results demonstrate that hypoxia collaboratively promotes gemcitabine-induced stemness through the AKT/Notch1 signaling pathway.

Conclusions
In conclusion, our data show a novel mechanism of acquired gemcitabine resistance in pancreatic cancer through stemness induction, which is aggravated by the ubiquitous hypoxic niche in cancer cells. Thus, strategies aimed at eliminating pancreatic CSCs might present a promising approach for overcoming gemcitabine resistance and developing effective treatments for pancreatic cancer. Moreover, our results highlight the important role of the AKT/Notch1 signaling pathway in mediating this process. We provide evidence that combination treatment with adjuvant drugs targeting such signaling pathways offers a better therapeutic benefit against pancreatic cancer in vitro and in vivo, suggesting AKT/ Notch1 as attractive targets for eliminating pancreatic CSCs. Altogether, our study provides new insights into strategies for reversing chemoresistance in patients with pancreatic cancer.

Additional files
Additional file 1: Figure S1. Notch1 inhibition abolishes gemcitabineenhanced migration, invasion, and chemoresistance. PANC-1 and Patu8988 cells were pretreated with 10 μM DAPT for 24 h and then treated with gemcitabine. (a, b) The transwell migration assay was performed to examine the change in the migratory ability of the cells, and the relative migratory ability was calculated by counting the number of stained cells migrating to the lower chamber. (c, d) The invasive ability of the cells was measured by the transwell invasion assay. (e) After treatment, the MTT assay was performed to test the change in the chemosensitivity of pancreatic cancer cells to gemcitabine. The graphs show the results of three independent experiments. Scale bar, 100 μm. *P < 0.05; **P < 0.01. (TIF 2082 kb) Additional file 2: Figure S2. AKT/Notch1 inhibition abolishes gemcitabine-induced metastasis. Non-treated (control) and treated (GEM, GEM+DAPT, and GEM+LY294002) PNAC-1 cells were injected into the tail vein of nude mice.