Rapamycin and bafilomycin A1 alter autophagy and megakaryopoiesis.

Abstract Autophagy is an effective strategy for cell development by recycling cytoplasmic constituents. Genetic deletion of autophagy mediator Atg7 in hematopoietic stem cells (HSCs) can lead to failure of megakaryopoiesis and enhanced autophagy has been implicated in various hematological disorders such as immune thrombocytopenia and myelodysplastic syndrome. Here, we examined the hypothesis that optimal autophagy is essential for megakaryopoiesis and thrombopoiesis by altering autophagy using pharmacological approaches. When autophagy was induced by rapamycin or inhibited by bafilomycin A1 in fetal liver cells, we observed a significant decrease in high ploidy megakaryocytes, a reduction of CD41 and CD61 co-expressing cells, and less proplatelet or platelet formation. Additionally, reduced cell size was shown in megakaryocytes derived from rapamycin, but not bafilomycin A1-treated mouse fetal liver cells. However, when autophagy was altered in mature megakaryocytes, we observed no significant change in proplatelet formation, which was consistent with normal platelet counts, megakaryocyte numbers, and ploidy in Atg7flox/flox PF4-Cre mice with megakaryocyte- and platelet-specific deletion of autophagy-related gene Atg7. Therefore, our findings suggest that either induction or inhibition of autophagy in the early stage of megakaryopoiesis suppresses megakaryopoiesis and thrombopoiesis.


Introduction
Thrombosis and hemostasis are dependent on functional platelets derived from mature megakaryocytes which are differentiated from hematopoietic stem cells (HSCs) classically thought to follow a stepwise process including early common myeloid progenitor (CMP), common megakaryocyte-erythroid progenitor (MEP), early megakaryocyte burst-forming unit (BFU-MK), megakaryocyte colony-forming unit (CFU-MK), and megakaryoblasts [1][2][3][4]. Megakaryocytes are final differentiated cells that do not divide, but undergo DNA replication, termed endomitosis, which is important to enable the megakaryocytes to develop sufficiently for thrombopoiesis [5]. This process may occur at the interface between the bone marrow sinusoids and vascular space in response to fluid blood shear stress [6]. Accordingly, the bone marrow microenvironment plays an essential role in the regulation of megakaryopoiesis and thrombopoiesis, which are promoted by an array of cytokines including thrombopoietin (TPO), granulocyte-macrophage colony-stimulation factor (GM-CSF), interleukins, etc. [7][8][9].
Accumulating evidence shows that cell death is involved in the regulation of megakaryopoiesis and thrombopoiesis [10,11]. As an example, apoptosis is shown to facilitate the modulation of the cytoskeleton, which mediates proplatelet formation and platelet shedding through the pro-survival protein Bcl-xL [12,13]. Autophagy, another type of programmed cell death, has been proved as an effective mechanism for digestion and recycling of intracellular substrates through vesicular trafficking [14]. Microtubule-associated protein 1 light chain 3 (LC3) is an established hallmark of autophagy due to its coincident lipidation profile with autophagic flux [15]. Being a unique catabolic process, autophagy is capable of adapting cells to various stress conditions [16,17]. Of note, autophagy has recently been reported to be essential for the development of megakaryocyte-platelet lineage. Mortensen et al. showed that mice with conditional deletion of the autophagy-related gene Atg7 in hematopoietic cells failed to maintain normal hematopoiesis and showed substantially decreased numbers of megakaryocyte progenitors [18]. Further studies in these mice revealed disrupted megakaryopoiesis and thrombopoiesis with mitochondrial dysfunction and cell cycle retardation [19]. Moreover, Madhu et al. showed that autophagy defect due to megakaryocyte and platelet-specific deletion of Atg7 also impaired platelet function [20].
Despite its important role in megakaryopoiesis and thrombopoiesis, how autophagy regulates the development of megakaryocytes and production of platelets remains largely unclear. Using a pharmacological inducer (rapamycin) and inhibitor (bafilomycin A1) of autophagy, we found that either rapamycin or bafilomycin A1 in fetal liver cells impeded megakaryopoiesis. However, mice with conditional deletion of autophagy in megakaryocytic lineage retained normal megakaryopoiesis and platelet counts. These findings suggest that a complete basal autophagic flux is important for the development of megakaryocytes from the hematopoietic stem cell stage. PF4-Cre mice (Stock No: 008535, Bar Harbor, ME USA) were purchased from the Jackson Laboratory. Atg7 flox/flox mice and PF4-Cre mice were paired to obtain Atg7 flox/flox PF4-Cre mice that harbor megakaryocytic lineage specific deletion of autophagy. Atg7 flox/flox mice served as the controls. All animal procedures were performed according to the institutional protocols on animal welfares and approved by the Ethics Committee of Soochow University, China.

In vitro stimulation and inhibition of autophagy
Cultured mouse fetal liver cells and purified fetal liver derived megakaryocytes were treated with rapamycin (200nM, Cell Signaling Technology, cat No.9904, USA), bafilomycin A1 (10nM, Sigma Aldrich, cat No.B1793, USA) or both drugs for five days and 6 hours, respectively. Cells treated with solvent solution containing 0.2% DMSO for the same durations served as the controls (Ctrl).

Quantification of proplatelet-forming megakaryocytes (PPF-MKs)
Mouse fetal liver cells were allowed to sediment in a single-step gradient solution (1.5%/3% bovine serum albumin) for 50 minutes to isolate megakaryocytes, which were then seeded at a density of 5 × 10 5 cells per well in 24-well plates. Quantification of PPF-MKs was determined by the number of megakaryocytes with more than two pseudopods per well examined under bright field microscopy. All experiments were repeated at least three times.

Assessment of platelet production
We assessed the production of platelets from infused megakaryocytes using the allogeneic jugular vein infusion model and flow cytometry [22]. Briefly, induced megakaryocytes from wild type donor mice were infused into the jugular vein of recipient mice homozygously expressing human integrin αIIb (hαIIb) instead of mouse integrin αIIb (mαIIb). About 3 × 10 5 donor megakaryocytes were resuspended in 200 µl of phosphate buffered saline (PBS) (cat No. E404, AMRESCO, OH) for each recipient. The rate of infusion was tuned to avoid lethal occlusion. Subsequently, recipient mice underwent retro-orbital bleeding to collect blood, which was then double-stained with FITC-conjugated rat-antihuman CD41 (integrin αIIb) antibody (BD Bioscience, cat No.340929, USA) and rat anti-mouse CD41 antibody (BD Biosciences, clone:MWReg30, USA) labeled with Alexa Fluor 647 (Invitrogen) for 40 min. The released platelets from the megakaryocytes were defined as the mαIIb + hαIIb − population according to flow cytometry and counted at 0, 1.5, 5, and 24 hours after megakaryocyte infusion.

Western blot
Purified megakaryocytes were lysed in 1 × RIPA lysis buffer (Beijing Solarbio Science & Technology, cat No.R0010, Beijing, China) supplemented with a fresh protease and phosphatase inhibitor cocktail. The protein concentration was determined using a protein assay kit (Beyotime Biotechnology, cat No.P0010, Haimen, China). Total proteins were separated using SDS-PAGE electrophoresis and then transferred to nitrocellulose membranes. After being blocked with 5% non-fat milk, membranes were incubated with specific primary antibodies at 4°C overnight, followed by one hour of further incubation with goat anti-rabbit IRDye 800CW or goat anti-mouse IRDye 800CW secondary antibodies (LI-COR Biosciences, Lincoln, NE) at room temperature. The membranes were washed with TBS and visualized with Infrared Imagine System (LI-COR Biosciences, Lincoln, NE). The following primary antibodies were used in this study: rabbit-anti-mouse LC3 (1:1000, Cell Signaling, cat No.4108, USA), rabbit-anti-P-Myosin light chain (1:500, Cell Signaling, cat No.3671, USA), and mouse-anti-mouse β-actin (1:1000, Beyotime, cat No.AA128, Haimen, China). The densitometry measurements of scanned blots were performed using Image J software (NIH, Maryland, USA).

Flow cytometry
The size of megakaryocytes was determined by the mean value of forward side scatter (FSC) in each group. For ploidy analysis, 75% cold ethanol pre-fixed megakaryocytes were treated with 0.02 mg/ml RNase A (Beyotime, cat No.ST576, Haimen, China) for 30 minutes at 37°C and then double-stained with 0.01 mg/ml propidium iodide (Sigma Aldrich, cat No.P4170, USA) and FITC-conjugated rat anti-mouse CD41 antibody (BD Biosciences, cat No.553848, USA) for 30 minutes at room temperature. CD41 positive cells were selected to assess ploidy. To evaluate cell differentiation, megakaryocytes were double-stained with FITC-conjugated rat anti-mouse CD41 (BD Biosciences, cat No.553848, USA) and Armenian hamster anti-mouse CD61 PE (eBioscience, cat No.12-0611, USA) antibodies for 30 minutes at room temperature. To quantify apoptosis, megakaryocytes were labeled with APC-conjugated anti-Annexin V antibody (BD Biosciences, cat No.561012, USA) and propidium iodide (Sigma Aldrich, cat No.P4170, USA) for 30 minutes. A FACS Calibur (BD Biosciences) flow cytometer was used for the flow cytometric analysis.

Live differential interference contrast microscopy
Mouse fetal liver cells or purified megakaryocytes were seeded in semisolid media and transferred to 24-well culture plates. Pictures were taken at 40-minute interval from five separate views for each well under 20× magnification. CD41 positive cells were tracked and visualized using differential interference contrast imaging. Pictures were stored for further morphological analysis.

Spleen and bone marrow histology
Spleens and femurs of 8-week old Atg7 flox/flox PF4-Cre and Atg7 flox/flox mice were fixed overnight in 4% paraformaldehyde/ PBS. Ten percent EDTA (pH 7.8) was used to decalcify the bones for 21 days, with EDTA exchanged every two days. Sections of paraffin-embedded tissues were stained using H&E kit (Beyotime, cat No. C0105, Haimen, China) followed by histological analysis and quantification. Briefly, a total of 5 mice from each strain were used and over 5 sections from the similar location of each femur were obtained for histochemistry studies. For each section, seven random views were selected for microscopic examination and quantification.

Statistical analysis
Data are presented as mean ± standard error of the mean (SEM). Data analysis was performed using Prism 5 software package (Graphpad Inc.). One-way analysis of variance (1-way ANOVA) and two-way analysis of variance (2-way ANOVA) followed by Bonferroni post-hoc test were used to compare the difference among groups. A P value of <0.05 was considered statistically significant.

Results
Decreased ploidy and co-expression of CD41 and CD61 in megakaryocytes derived from mouse fetal liver cells treated with rapamycin or bafilomycin A1 The maturation of megakaryocytes is characterized by endomitosis [23]. To ask whether altered autophagy affects endomitosis, mouse fetal liver cells were treated with rapamycin or bafilomycin A1 along with TPO for five days to allow megakaryocyte differentiation. Cells were then analyzed for ploidy to determine endomitosis. Using propidium iodide as a marker for nuclear content, we found that both induction (Rap 54.8 ± 0.6%) and inhibition (BafA 50.4 ± 3.8%) of autophagy significantly decreased the proportions of high ploidy (≥8N) population in megakaryocytes compared to the control group (Ctrl 71.1 ± 2.0%) (P < 0.05) (Figures 1A and 1B).
CD41 and CD61 are markers for megakaryocyte lineage specificity and commitment from hematopoietic stem cells [24]. To determine the effect of altered autophagy on megakaryocyte differentiation, we measured the expression of CD41 and CD61 in megakaryocytes derived from fetal liver cells. We found that the percentages of cells co-expressing CD41 and CD61 in fetal liver cells treated by either rapamycin (Rap 1.0 ± 0.1%) or bafilomycin A1 (BafA 0.7 ± 0.0%) were dramatically reduced compared with the control group (Ctrl 5.2 ± 0.1%) (P < 0.001) (Figures 1C and  1D). Besides, successful induction of autophagy by rapamycin was confirmed by significantly increased expression of LC3-II (Rap 1.0 ± 0.1), which was further augmented by the addition of bafilomycin A1 (Rap+BafA 1.7 ± 0.0) compared to the control group (Ctrl 0.2 ± 0.0) (P < 0.001). However, inhibition of autophagic flux by bafilomycin A1 alone did not elicit any significant change in LC3-II expression (Figures 1E and 1F). Furthermore, the percentages of cells with positive Annexin V and negative PI staining were not significantly different among all groups, suggesting a minimal role of apoptosis in megakaryopoiesis at the concentrations of drugs that we used (Figures 1G  and 1H).
Reduced proplatelet formation in megakaryocytes derived from mouse fetal liver cells treated with rapamycin or bafilomycin A1 The production of platelets is preceded by the formation of proplatelets from megakaryocytes. To evaluate proplatelet formation, differential interference contrast microscopy was employed to examine the geometry of megakaryocytes. The results showed that the numbers of protruding barbell-like structures in induced megakaryocytes were significantly decreased by rapamycin or bafilomycin A1 treatment of mouse fetal liver cells compared to the control group (Figure 2A). Further quantification demonstrated a significant decrease in the numbers of PPF-MKs derived from mouse fetal liver cells treated with rapamycin (Rap 4.4 ± 0.6 × 10 4 /well) or bafilomycin A1 (BafA 4.9 ± 0.7 × 10 4 /well) compared with the control group (Ctrl 8.5 ± 0.5 × 10 4 /well) (P < 0.001) ( Figure 2B).

Decreased cell size of megakaryocytes derived from mouse fetal liver cells treated with rapamycin but not bafilomycin A1
The maturation of cytoplasm and nucleus, marked by cell size, is a priming step of platelet production in megakaryocytes. Thus, we assessed megakaryocyte size using flow cytometry. The forward side scatter height (FSC-H), indicative of cell size, was significantly decreased by rapamycin treatment of fetal liver cells compared with vehicle treatment (Rap 414.0 ± 8.6, Ctrl 873.7 ± 20.3, P < 0.001) (Figures 2C and 2D). The reduction of cell size was further supported by dynamic surface area monitoring using bright field microscopy, which displayed significant smaller megakaryocytes derived from rapamycin treated fetal liver cells from 24 hours (Rap 398.2 ± 42.3, Ctrl 1653 ± 240.2, P < 0.01) to 48 hours (Rap 702.0 ± 44.8, Ctrl 2814 ± 485.8, P < 0.001) (Figures 2E and 2F). However, in megakaryocytes derived from bafilomycin A1 treated fetal liver cells, neither FSC-H nor cell surface area was significantly different compared with the control group.

Reduced platelet release in megakaryocytes derived from mouse fetal liver cells treated with rapamycin or bafilomycin A1
To determine platelet release, we measured the proportions of mαIIb-positive platelets produced from exogenous megakaryocytes using the allogeneic jugular vein infusion model. Briefly, megakaryocytes derived from rapamycin-, bafilomycin A1-or solvent-treated mouse fetal liver cells were infused through the jugular vein into transgenic mice expressing hαIIb instead of mαIIb on platelets [22]. Thus, mαIIb-positive donor megakaryocyte-released platelets were easily recognized by flow cytometry when marked with specific fluorescent-labeled antibodies. In mice infused with megakaryocytes derived from fetal liver cells treated with rapamycin (Rap 0.4 ± 0.3%) or bafilomycin A1 (BafA 0.7 ± 0.1%), the percentage of mαIIb-positive platelets was significantly lower compared with those infused with megakaryocytes derived from vehicle treated (Ctrl 10.0 ± 4.1%) fetal liver cells (P < 0.05). The maximum differences were observed at 1.5 hours after the megakaryocyte infusion ( Figures 3A and 3B), when the peak level of platelet release occurred [22], although no significant changes in platelet production were observed at 5 hours or 24 hours after the infusion.
Normal platelet counts, megakaryocyte ploidy, and numbers in Atg7 flox/flox PF4-Cre mice To recapitalize the effect of autophagy on proplatelet formation in mature megakaryocytes, we generated the Atg7 flox/flox PF4-Cre mice with megakaryocyte and platelet deficiency of autophagy by lineage-specific deletion of Atg7 ( Figure 4D). The ablation of autophagy was verified by abolished expression of LC3-II ( Figure 4E). Quantitative hematologic studies and ploidy analysis showed that both platelet counts ( Figure 4F) and ploidy of megakaryocytes ( Figure 4G and 4H) in Atg7 flox/ flox PF4-Cre mice were not significantly different from Atg7 flox/flox littermates. Moreover, the numbers of megakaryocytes in spleen ( Figure 4I) and bone marrow ( Figure 4J) from these mice were also similar to control littermates as indicated by the quantitative histochemistry studies.

Discussion
Altered autophagy has been suggested in a number of hematological disorders [25][26][27][28][29]. In this study, we found that pharmacologically altered autophagy in hematopoietic stem cells leads to abnormal megakaryopoiesis and thrombopoiesis, which did not occur when autophagy was altered in mature megakaryocytes or in the animal model with megakaryocyte-and platelet-specific autophagy deficiency.
During autophagy, Atg7, an E1 ligase-like protein, plays a crucial role in autophagosome expansion and completion by activating LC3-I and Atg12 [30]. Conditional knockout of Atg7 gene has been shown to abrogate autophagy according to a number of studies [18,[31][32][33][34]. Microtubule-Associate Protein 1 Light Chain 3 (LC3) is the mammalian orthologue of Atg8, which is another protein essential for autophagy [35]. During autophagy, LC3 is cleaved by Atg4 into the cytosolic version named LC3-I, which is then activated by Atg7 and transferred to Atg3 [36]. After that, it undergoes lipidation to yield the membrane bound LC3-II [37]. This conversion, being an essential step of membrane reorganization during the formation of autophagosomes, provides a hallmark for the quantification of autophagy [38]. Accordingly, we showed diminished expression of LC3-II in the platelets from Atg7 flox/flox PF4-Cre mice, and increased LC3-II expression in cells treated with rapamycin.
So far, only sporadic studies have looked into the potential role of enhanced autophagy in megakaryopoiesis. For example, in the chronic myelogenous leukemia (CML) cell line K562, co-treatment with PMA and the p38 MAPK inhibitor promotes autophagy flux and blocks autophagic degradation, leading to megakaryocytic differentiation [39]. Autophagic morphological changes induced in the same cell line by Lapatinib, a tyrosine kinase inhibitor, are associated with megakaryocytic differentiation [40]. In this light, we treated hematopoietic stem cells with rapamycin, which directly induces autophagy by targeting mTORC [41], to investigate the effect of enhanced autophagy on megakaryopoiesis. Consistently, megakaryocytes derived from mouse fetal liver cells treated with rapamycin exhibited increased autophagy. Nevertheless, surprisingly, we observed reductions of megakaryocyte ploidy and CD41/CD61 expression and decreases in proplatelet formation and platelet release, which is different from previous reports in K562 cells. These discrepancies in megakaryopoiesis and thrombopoiesis are probably associated with cell types or stimulants. Fetal liver cells and megakaryocytes are primary cells retaining more physiological features and their autophagy machinery may function differently from that of cancer cell lines. In addition to the regulation of autophagy, different reagents initiate their own signaling pathways that may also affect megakaryopoiesis. For example, PKCα is recently proved to negatively regulate proplatelet formation and endomitosis [23], which can possibly explain the promegakaryopoiesis effect of PMA in K562 cells.
On the other hand, we found that impeding autophagy by bafilomycin A1 from early stage of megakaryopoiesis also inhibited megakaryocyte development. Bafilomycin A1 (BafA1) is a vacuolar ATPase (V-ATPase) inhibitor which is commonly used to impede autophagic flux [42]. In our case, we did not observe an increase in LC3-II level in megakaryocytes derived from fetal liver cells treated with bafilomycin A1. However, application of bafilomycin A1 along with rapamycin increased the levels of LC3-II in megakaryocytes. These results suggest a low level of basal autophagic flux in megakaryocytes, which can be blocked by bafilomycin A1 without causing significant increase of autophagosomes. In spite of similar LC3-II level as the control group, the bafilomycin A1 treated cells displayed reduced polyploidization, CD41 and CD61 expression, and platelet production. Therefore, impeding autophagy by bafilomycin A1 from early stage of megakaryopoiesis also inhibits megakaryocyte development independently of autophagosome formation. This suggests that the integrity of autophagic flux is essential for normal megakaryopoiesis.
Although clinical evidence implicates the potential association between autophagy and megakaryocytes, the stage in which altered autophagy regulates megakaryopoiesis remains unclear. Recently, deletion of the essential autophagy gene Atg7 in hematopoietic stem cells has been shown to impair hematopoiesis and cause excessive myeloproliferation by inducing oxidative stress, cell cycle arrest and mitochondrial dysfunction. Additionally, these mice also exhibit insufficient hematopoietic reconstitution after receiving lethal dosage of ionizing radiation [18,19]. These are consistent with our finding that megakaryopoiesis is suppressed by bafilomycin A1 mediated autophagy inhibition in fetal liver cells. Interestingly, similar phenotypes are readily observed in patients with MDS [43]. However, whether the later stage of megakaryopoiesis is the major target of autophagic regulation as well is unknown. To dissect the temporal regulation of megakaryopoiesis by autophagy, we treated differentiated megakaryocytes with rapamycin or bafilomycin A1, and found neither drugs altered proplatelet formation. In addition, state-of-the-art Atg7 flox/flox PF4-Cre mice provided the favorable model to elucidate this question. In contrast to the macrothrombocytopenia previously reported in Atg7 flox/flox Vav-Cre mice with hematopoietic deletion of autophagy, we showed that lineage specific deletion of autophagy in Atg7 flox/flox PF4-Cre mice exhibited normal platelet counts as well as megakaryocyte ploidy and numbers. Thus, autophagy exhibited an important role in the derivation of megakaryocytess from fetal liver cells but a minor role in vivo as seen in the Atg7 flox/flox PF4-Cre mice. These differential phenotypes may be explained by a canonical role of autophagy during the early stage rather than the late phase of megakaryopoiesis.
In summary, our data indicate that either upregulated or inhibited autophagy at early stage of megakaryocyte development impairs megakaryopoiesis. Due to the limitations of in vitro based studies, result interpretation needs to be cautious. For example, rapamycin may activate auxiliary signaling pathways in addition to autophagy induction. The other limitation involves that our in vitro and ex vivo approaches may underestimate the involvement of bone marrow microenvironment. Further studies using transgenic mice with conditional overexpression of autophagy-related genes may facilitate the understanding of comprehensive roles of autophagy in megakaryopoiesis. Nevertheless, our findings suggest that targeting autophagy may provide therapeutic potential for dysmegakaryopoiesis and thrombocytopenia