Targeting ABCA12-controlled ceramide homeostasis inhibits breast cancer stem cell function and chemoresistance.

Cancer stem cells (CSCs) drive tumor growth, metastasis, and chemoresistance. While emerging evidence suggests that CSCs have a unique dependency on lipid metabolism, the functions and regulation of distinct lipid species in CSCs remain poorly understood. Here, we developed a stem cell factor SOX9-based reporter for isolating CSCs in primary tumors and metastases of spontaneous mammary tumor models. Transcriptomic analyses uncover that SOX9high CSCs up-regulate the ABCA12 lipid transporter. ABCA12 down-regulation impairs cancer stemness and chemoresistance. Lipidomic analyses reveal that ABCA12 maintains cancer stemness and chemoresistance by reducing intracellular ceramide abundance, identifying a CSC-associated function of ABCA subfamily transporter. Ceramide suppresses cancer stemness by inhibiting the YAP-SOX9 signaling pathway in CSCs. Increasing ceramide levels in tumors enhances their sensitivity to chemotherapy and prevents the enrichment of SOX9high CSCs. In addition, SOX9high and ABCA12high cancer cells contribute to chemoresistance in human patient-derived xenografts. These findings identify a CSC-suppressing lipid metabolism pathway that can be exploited to inhibit CSCs and overcome chemoresistance.

A SOX9-B7x axis safeguards dedifferentiated tumor cells from immune surveillance to drive breast cancer progression.

How dedifferentiated stem-like tumor cells evade immunosurveillance remains poorly understood. We show that the lineage-plasticity regulator SOX9, which is upregulated in dedifferentiated tumor cells, limits the number of infiltrating T lymphocytes in premalignant lesions of mouse basal-like breast cancer. SOX9-mediated immunosuppression is required for the progression of in situ tumors to invasive carcinoma. SOX9 induces the expression of immune checkpoint B7x/B7-H4 through STAT3 activation and direct transcriptional regulation. B7x is upregulated in dedifferentiated tumor cells and protects them from immunosurveillance. B7x also protects mammary gland regeneration in immunocompetent mice. In advanced tumors, B7x targeting inhibits tumor growth and overcomes resistance to anti-PD-L1 immunotherapy. In human breast cancer, SOX9 and B7x expression are correlated and associated with reduced CD8+ T cell infiltration. This study, using mouse models, cell lines, and patient samples, identifies a dedifferentiation-associated immunosuppression mechanism and demonstrates the therapeutic potential of targeting the SOX9-B7x pathway in basal-like breast cancer.

Heterochromatic silencing of immune-related genes in glia is required for BBB integrity and normal lifespan in drosophila.

Glia and neurons face different challenges in aging and may engage different mechanisms to maintain their morphology and functionality. Here, we report that adult-onset downregulation of a Drosophila gene CG32529/GLAD led to shortened lifespan and age-dependent brain degeneration. This regulation exhibited cell type and subtype-specificity, involving mainly surface glia (comprising the BBB) and cortex glia (wrapping neuronal soma) in flies. In accordance, pan-glial knockdown of GLAD disrupted BBB integrity and the glial meshwork. GLAD expression in fly heads decreased with age, and the RNA-seq analysis revealed that the most affected transcriptional changes by RNAi-GLAD were associated with upregulation of immune-related genes. Furthermore, we conducted a series of lifespan rescue experiments and the results indicated that the profound upregulation of immune and related pathways was not the consequence but cause of the degenerative phenotypes of the RNAi-GLAD flies. Finally, we showed that GLAD encoded a heterochromatin-associating protein that bound to the promoters of an array of immune-related genes and kept them silenced during the cell cycle. Together, our findings demonstrate a previously unappreciated role of heterochromatic gene silencing in repressing immunity in fly glia, which is required for maintaining BBB and brain integrity as well as normal lifespan.

Inhibition of the MEK/ERK pathway suppresses immune overactivation and mitigates TDP-43 toxicity in a Drosophila model of ALS.

TDP-43 is an important DNA/RNA-binding protein that is associated with age-related neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD); however, its pathomechanism is not fully understood. In a transgenic RNAi screen using Drosophila as a model, we uncovered that knockdown (KD) of Dsor1 (the Drosophila MAPK kinase dMEK) suppressed TDP-43 toxicity without altering TDP-43 phosphorylation or protein levels. Further investigation revealed that the Dsor1 downstream gene rl (dERK) was abnormally upregulated in TDP-43 flies, and neuronal overexpression of dERK induced profound upregulation of antimicrobial peptides (AMPs). We also detected a robust immune overactivation in TDP-43 flies, which could be suppressed by downregulation of the MEK/ERK pathway in TDP-43 fly neurons. Furthermore, neuronal KD of abnormally increased AMPs improved the motor function of TDP-43 flies. On the other hand, neuronal KD of Dnr1, a negative regulator of the Drosophila immune deficiency (IMD) pathway, activated the innate immunity and boosted AMP expression independent of the regulation by the MEK/ERK pathway, which diminished the mitigating effect of RNAi-dMEK on TDP-43 toxicity. Finally, we showed that an FDA-approved MEK inhibitor trametinib markedly suppressed immune overactivation, alleviated motor deficits and prolonged the lifespan of TDP-43 flies, but did not exhibit a lifespan-extending effect in Alzheimer disease (AD) or spinocerebellar ataxia type 3 (SCA3) fly models. Together, our findings suggest an important role of abnormal elevation of the MEK/ERK signaling and innate immunity in TDP-43 pathogenesis and propose trametinib as a potential therapeutic agent for ALS and other TDP-43-related diseases.

MLL3 loss drives metastasis by promoting a hybrid epithelial-mesenchymal transition state.

Phenotypic plasticity associated with the hybrid epithelial-mesenchymal transition (EMT) is crucial to metastatic seeding and outgrowth. However, the mechanisms governing the hybrid EMT state remain poorly defined. Here we showed that deletion of the epigenetic regulator MLL3, a tumour suppressor frequently altered in human cancer, promoted the acquisition of hybrid EMT in breast cancer cells. Distinct from other EMT regulators that mediate only unidirectional changes, MLL3 loss enhanced responses to stimuli inducing EMT and mesenchymal-epithelial transition in epithelial and mesenchymal cells, respectively. Consequently, MLL3 loss greatly increased metastasis by enhancing metastatic colonization. Mechanistically, MLL3 loss led to increased IFNγ signalling, which contributed to the induction of hybrid EMT cells and enhanced metastatic capacity. Furthermore, BET inhibition effectively suppressed the growth of MLL3-mutant primary tumours and metastases. These results uncovered MLL3 mutation as a key driver of hybrid EMT and metastasis in breast cancer that could be targeted therapeutically.

Uniparental parthenogenetic embryonic stem cell derivatives adaptable for bone and cartilage regeneration.

Cells with the desired phenotype and number are critical for regenerative medicine and tissue engineering. Uniparental parthenogenetic embryonic stem cells (pESCs) share fundamental properties with embryonic stem cells. This study aims to determine the viability of pESC-based tissue engineering for bone and cartilage reconstruction. The mouse pESCs were cultured in suspension to form embryoid bodies. An adherent cultivation approach was employed to obtain parthenogenetic embryonic mesenchymal stem cells (pMSCs) from the embryoid bodies. Then, the pMSCs were cultured in conditional media to differentiate into osteogenic and chondrogenic lineages. The pESC-derived osteoblasts and chondroblasts were seeded into coral and sodium alginate scaffolds, respectively. The cell-seeded scaffolds were implanted into dorsal subcutaneous pockets of nude mice to evaluate ectopic reconstruction of bone and cartilage. We demonstrated that pESCs display the capacity to differentiate into all three germ layers. The generated pMSCs were able to differentiate into osteogenic and chondrogenic lineages, which survived well after seeding into coral and alginate acid scaffolds. Six weeks after cell-scaffold implantation, gross inspection and histological examination revealed that ectopic bone and cartilage tissues had successfully regenerated in the specimen. According to the findings of this study, pESC derivatives have a high potential for bone and cartilage regeneration.

The Stimulation of Macrophages by Systematical Administration of GM-CSF Can Accelerate Adult Wound Healing Process.

Skin wound repair remains a major challenge in clinical care, and various strategies have been employed to improve the repair process. Recently, it has been reported that macrophages are important for the regeneration of various tissues and organs. However, their influence on wound repair is unclear. Here, we aimed to explore whether macrophages would participate in the wound healing process and to explore new possibilities of treatment for skin defects. We firstly created a mouse full-thickness skin defect model to observe the distribution of macrophages in the regenerating tissue and then detected the influence of macrophages on skin defect repair in both macrophage-depletion and macrophage-mobilization models. We found that the number of macrophages increased significantly after skin defect and persisted during the process of wound repair. The regeneration process was significantly prolonged in macrophage-depleted animals. RT-qPCR and ELISA assays further demonstrated that the expression of growth factors was perturbed in the regenerating tissue. The activation of macrophages by granulocyte-macrophage colony-stimulating factor (GM-CSF) injection could significantly improve wound healing, accompanied with an upregulation of the expression of various growth factors. In conclusion, the current study demonstrated that macrophages are critical for skin regeneration and that GM-CSF exhibited therapeutic potential for wound healing.

CHMP2B regulates TDP-43 phosphorylation and cytotoxicity independent of autophagy via CK1.

The ESCRT protein CHMP2B and the RNA-binding protein TDP-43 are both associated with ALS and FTD. The pathogenicity of CHMP2B has mainly been considered a consequence of autophagy-endolysosomal dysfunction, whereas protein inclusions containing phosphorylated TDP-43 are a pathological hallmark of ALS and FTD. Intriguingly, TDP-43 pathology has not been associated with the FTD-causing CHMP2BIntron5 mutation. In this study, we identify CHMP2B as a modifier of TDP-43-mediated neurodegeneration in a Drosophila screen. Down-regulation of CHMP2B reduces TDP-43 phosphorylation and toxicity in flies and mammalian cells. Surprisingly, although CHMP2BIntron5 causes dramatic autophagy dysfunction, disturbance of autophagy does not alter TDP-43 phosphorylation levels. Instead, we find that inhibition of CK1, but not TTBK1/2 (all of which are kinases phosphorylating TDP-43), abolishes the modifying effect of CHMP2B on TDP-43 phosphorylation. Finally, we uncover that CHMP2B modulates CK1 protein levels by negatively regulating ubiquitination and the proteasome-mediated turnover of CK1. Together, our findings propose an autophagy-independent role and mechanism of CHMP2B in regulating CK1 abundance and TDP-43 phosphorylation.

Deep Learning-Based Artificial Intelligence System for Automatic Assessment of Glomerular Pathological Findings in Lupus Nephritis.

Accurate assessment of renal histopathology is crucial for the clinical management of patients with lupus nephritis (LN). However, the current classification system has poor interpathologist agreement. This paper proposes a deep convolutional neural network (CNN)-based system that detects and classifies glomerular pathological findings in LN. A dataset of 349 renal biopsy whole-slide images (WSIs) (163 patients with LN, periodic acid-Schiff stain, 3906 glomeruli) annotated by three expert nephropathologists was used. The CNN models YOLOv4 and VGG16 were employed to localise the glomeruli and classify glomerular lesions (slight/severe impairments or sclerotic lesions). An additional 321 unannotated WSIs from 161 patients were used for performance evaluation at the per-patient kidney level. The proposed model achieved an accuracy of 0.951 and Cohen's kappa of 0.932 (95% CI 0.915-0.949) for the entire test set for classifying the glomerular lesions. For multiclass detection at the glomerular level, the mean average precision of the CNN was 0.807, with 'slight' and 'severe' glomerular lesions being easily identified (F1: 0.924 and 0.952, respectively). At the per-patient kidney level, the model achieved a high agreement with nephropathologist (linear weighted kappa: 0.855, 95% CI: 0.795-0.916, p < 0.001; quadratic weighted kappa: 0.906, 95% CI: 0.873-0.938, p < 0.001). The results suggest that deep learning is a feasible assistive tool for the objective and automatic assessment of pathological LN lesions.

Novel tissue-engineered skin equivalent from recombinant human collagen hydrogel and fibroblasts facilitated full-thickness skin defect repair in a mouse model.

Tissue-engineered skin equivalent (TESE) is an optimized alternative for the treatment of skin defects. Designing and fabricating biomaterials with desired properties to load cells is critical for the approach. In this study, we aim to develop a novel TESE with recombinant human collagen (rHC) hydrogel and fibroblasts to improve full-thickness skin defect repair. First, the bioactive effect of rHC on fibroblast proliferation, migration and phenotype was assayed. The results showed that rHC had good biocompatibility and could stimulate fibroblasts migration and secrete various growth factors. Then, rHC was cross-linked with transglutaminase (TG) to prepare rHC hydrogel. Rheometer tests indicated that 10% rHC/TG hydrogel could reach a oscillate stress of 251 Pa and remained stable. Fibroblasts were seeded into rHC/TG hydrogel to prepare TESE. Confocal microscope and scanning electronic microscope observation showed that seeded fibroblasts survived well in the hydrogel. Finally, the therapeutic effect of the newly prepared TESE was tested in a mouse full-thickness skin defect model. The results demonstrated that TESE could significantly improve skin defect repair in vivo. Conclusively, TESE prepared from rHC and fibroblasts in this study exhibits great potential for clinical application in the future.

Establishment and long-term culture of mouse mammary stem cell organoids and breast tumor organoids.

Mammary stem cells (MaSCs) contribute to mammary epithelium development and homeostasis. They have been proposed as cells of origin for breast cancer. Here, we describe an organoid culture protocol for ex vivo expansion of MaSCs from mouse tissues. These organoids maintain the self-renewal of gland-reconstituting MaSCs and can be used to model tumorigenesis by introducing patient-relevant cancer drivers and mutations. Similar organoid culture can be used for long-term expansion of luminal stem/progenitor cells from normal glands and tumor-initiating cells from mammary tumors. For complete details on the use and execution of this protocol, please refer to Christin et al. (2020) and Zhang et al. (2016).

Expression and Functional Characterization of c-Fos Gene in Chinese Fire-Bellied Newt Cynops Orientalis.

c-Fos is an immediate-early gene that modulates cellular responses to a wide variety of stimuli and also plays an important role in tissue regeneration. However, the sequence and functions of c-Fos are still poorly understood in newts. This study describes the molecular cloning and characterization of the c-Fos gene (Co-c-Fos) of the Chinese fire-bellied newt, Cynops orientalis. The full-length Co-c-Fos cDNA sequence consists of a 1290 bp coding sequence that encoded 429 amino acids. The alignment and phylogenetic analyses reveal that the amino acid sequence of Co-c-Fos shared a conserved basic leucine zipper domain, including a nuclear localization sequence and a leucine heptad repeat. The Co-c-Fos mRNA is widely expressed in various tissues and is highly and uniformly expressed along the newt limb. After limb amputation, the expression of Co-c-Fos mRNA was immediately upregulated, but rapidly declined. However, the significant upregulation of Co-c-Fos protein expression was sustained for 24 h, overlapping with the wound healing stage of C. orientalis limb regeneration. To investigate if Co-c-Fos participate in newt wound healing, a skin wound healing model is employed. The results show that the treatment of T-5224, a selective c-Fos inhibitor, could largely impair the healing process of newt's skin wound, as well as the injury-induced matrix metalloproteinase-3 upregulation, which is fundamental to wound epithelium formation. These data suggest that Co-c-Fos might participate in wound healing by modulating the expression of its potential target gene matrix metalloproteinase-3. Our study provides important insights into mechanisms that are responsible for the initiation of newt limb regeneration.

Glycogen synthase kinase-3ß: a promising candidate in the fight against fibrosis.

Fibrosis exists in almost all organs/tissues of the human body, plays an important role in the occurrence and development of diseases and is also a hallmark of the aging process. However, there is no effective prevention or therapeutic method for fibrogenesis. As a serine/threonine (Ser/Thr)-protein kinase, glycogen synthase kinase-3ß (GSK-3ß) is a vital signaling mediator that participates in a variety of biological events and can inhibit extracellular matrix (ECM) accumulation and the epithelial-mesenchymal transition (EMT) process, thereby exerting its protective role against the fibrosis of various organs/tissues, including the heart, lung, liver, and kidney. Moreover, we further present the upstream regulators and downstream effectors of the GSK-3ß pathway during fibrosis and comprehensively summarize the roles of GSK-3ß in the regulation of fibrosis and provide several potential targets for research. Collectively, the information reviewed here highlights recent advances vital for experimental research and clinical development, illuminating the possibility of GSK-3ß as a novel therapeutic target for the management of tissue fibrosis in the future.

Integrative Analysis of MicroRNAome, Transcriptome, and Proteome during the Limb Regeneration of Cynops orientalis.

Salamanders completely regenerate their limbs after amputation. Thus, these animals are unique models to investigate the mechanisms modulating regeneration in vertebrates. To investigate the influence of microRNAs (miRNAs) on newt limb regeneration, the miRNAs and mRNAs were simultaneously profiled using Illumina HiSeq 2500 System during limb regeneration of Cynops orientalis at 3, 7, 14, 30 and 42 days postamputation. A total of 203 miRNAs and 4230 mRNAs were identified to be differentially expressed. Together with the proteomic data obtained from our previous study, integrative analysis of multiple profiling data sets was performed to construct an interaction network of differentially expressed miRNAs, mRNAs and proteins. Results of GO and KEGG analyses showed that the differentially expressed miRNA targets were mainly directed to cytoskeletal remodeling and carbohydrate metabolism. The stage-specific regulation of miRNAs on their targets was analyzed by hierarchical clustering analysis and validated by qRT-PCR. The negative regulation of miR-223 and miR-133a on their targets was tested by performing dual luciferase reporter assay. The integration analysis will provide a powerful tool to identify the regulatory mechanisms of miRNAs and their targets. The results may have implications in understanding the complex mechanisms underlying newt limb regeneration.

Alterations in ß­catenin/E­cadherin complex formation during the mechanotransduction of Saos­2 osteoblastic cells.

Mechanical load application promotes bone formation, while reduced load leads to bone loss. However, the underlying mechanisms that regulate new bone formation are not fully understood. Wnt/ß­catenin signaling has an important role in bone formation, bone growth and remodeling. The aim of the present study was to investigate whether mechanical stimuli regulated bone formation through the Wnt/ß­catenin signaling pathway. Saos­2 osteoblastic cells were subjected to mechanical strain using a Flexcell strain loading system. The results demonstrated that 12% cyclical tensile stress significantly stimulated Saos­2 cell proliferation, increased the activity of alkaline phosphatase and promoted the formation of mineralized nodules, as determined by MTT and p­nitrophenyl phosphate assays and Alizarin Red S staining, respectively. Furthermore, western blot analysis demonstrated that, following mechanical strain, increased phosphorylation of glycogen synthase kinase­3ß and nuclear ß­catenin expression was observed in cells, compared with static control culture cells. Results of reporter gene and reverse transcription­polymerase chain reaction assays also demonstrated that mechanical strain significantly increased T­cell factor reporter gene activity and the mRNA expression of cyclooxygenase (COX)­2, cyclin D1, c­fos and c­Jun in Saos­2 cells. Co­immunoprecipitation analysis revealed that elongation mechanical strain activated Wnt/ß­catenin signaling and reduced ß­catenin and E­cadherin interaction in Saos­2 cells. In conclusion, the results of the current study indicate that mechanical strain may have an important role in the proliferation and differentiation of osteoblasts. The disassociation of the ß­catenin/E­cadherin complex in the osteoblast membrane under stretch loading and the subsequent translocation of ß­catenin into the nucleus may be an intrinsic mechanical signal transduction mechanism.

Novel hemostatic biomolecules based on elastin-like polypeptides and the self-assembling peptide RADA-16.

BACKGROUND: Safe and effective hemostatic materials are important for reducing mortality resulting from excessive hemorrhage. In this work, new biomaterials with hemostatic effects were created by fusing the gene coding for RADA-16, a self-assembling peptide with the sequence RADARADARADARADA, to the 3'-end of the open reading frame (ORF) encoding elastin-like polypeptides through gene recombination. RESULTS: The fusion proteins, termed 36R, 60R and 96R, were solubly over-expressed in Escherichia coli BL21 (DE3) based on genetic manipulation of the high-efficiency prokaryotic expression vector pET28a (+) and bacterial transformation. Western Blot analysis showed that the over-expressed proteins were the target fusion proteins. The target proteins 36R with 94.72% purity, 60R with 96.91% purity and 96R with 96.37% purity were prepared using an inverse phase transition cycle at 65 °C followed by His-tag affinity chromatography. The proliferation results of the mouse fibroblast cell line L929 and hippocampus neuron cell line HT22 indicated that the fusion proteins did not cause obvious cell toxicity. The lyophilized spongy film of the purified 36R, 60R and 96R could stop the hemorrhage of a 2 × 2 mm bleeding wound in the mouse liver after 27.21 ± 1.92 s, 18.65 ± 1.97 s and 15.85 ± 1.21 s, respectively. The hemostasis time was 21.23 ± 1.84 s for rat-tail collagen and 14.44 ± 1.33 s for RADA-16 lyophilized on gauze. The hemostatic time of three treated groups were all significantly superior to that of the negative control without any hemostasis treatment, which spontaneously stopped bleeding after 37.64 ± 1.34 s. Statistical analysis showed that the spongy film with purified 96R exhibited an exciting hemostatic effect that was superior to rat-tail collagen and close to that of RADA-16 lyophilized on gauze. CONCLUSIONS: These results revealed that the fusion proteins achieved by gene recombination technology could serve as a promising hemostatic material.

Influence of Mussel-Derived Bioactive BMP-2-Decorated PLA on MSC Behavior in Vitro and Verification with Osteogenicity at Ectopic Sites in Vivo.

Osteoinductive activity of the implant in bone healing and regeneration is still a challenging research topic. Therapeutic application of recombinant human bone morphogenetic protein-2 (BMP-2) is a promising approach to enhance osteogenesis. However, high dose and uncontrolled burst release of BMP-2 may introduce edema, bone overgrowth, cystlike bone formation, and inflammation. In this study, low-dose BMP-2 of 1 µg was used to design PLA-PD-BMP for functionalization of polylactic acid (PLA) implants via mussel-inspired polydopamine (PD) assist. For the first time, the binding property and efficiency of the PD coating with BMP-2 were directly demonstrated and analyzed using an antigen-antibody reaction. The obtained PLA-PD-BMP surface immobilized with this low BMP-2 dose can endow the implants with abilities of introducing strong stem cell adhesion and enhanced osteogenicity. Furthermore, in vivo osteoinduction of the PLA-PD-BMP-2 scaffolds was confirmed by a rat ectopic bone model, which is marked as the "gold standard" for the evidence of osteoinductive activity. The microcomputed tomography, Young's modulus, and histology analyses were also employed to demonstrate that PLA-PD-BMP grafted with 1 µg of BMP-2 can induce bone formation. Therefore, the method in this study can be used as a model system to immobilize other growth factors onto various different types of polymer substrates. The highly biomimetic mussel-derived strategy can therefore improve the clinical outcome of polymer-based medical implants in a facile, safe, and effective way.

Molecular cloning, characterization, and expression analysis of the three cysteine and glycine-rich protein genes in the Chinese fire-bellied newt Cynops orientalis.

The cysteine- and glycine-rich protein (CRP) family members, including the cysteine- and glycine-rich protein 1 (CSRP1), cysteine- and glycine-rich protein 2 (CSRP2), and the cysteine- and glycine-rich protein 3 (CSRP3), have exhibited various cellular functions during cell development and differentiation. However, the sequences of the three CSRP genes and their functions are still poorly understood in newts. In this study, we cloned the complete open reading frame (ORF) sequences of the three CSRP genes from the Chinese fire-bellied newt, Cynops orientalis (C. orientalis). The complete ORF sequences of Co-CSRP1, Co-CSRP2, and Co-CSRP3 were 582, 582, and 576bp, respectively, and encoded 193, 193, and 191 amino acids, respectively. The deduced amino acid sequences of the three CRP members showed high similarities with that of other species, particularly, with amphibians. Co-CSRP1 was highly expressed in the kidney, limb, and stomach, however, the expression was low in the spleen, heart, intestine, liver, and tail (P<0.05). The mRNA expression of Co-CSRP2 was higher in the kidney and heart than that in other organs (P<0.05). It was observed that Co-CSRP3 was only expressed in the heart, limb, and tail. The mRNA expression of Co-CSRP1 and Co-CSRP3 was lower in the digits in comparison to other limb segments. However, there was no significant difference of Co-CSRP2 mRNA expression in the four limb segments. The Co-CSRP1 and Co-CSRP2 mRNA expressions were significantly increased, whereas the expression of Co-CSRP3 was remarkably decreased during the limb regeneration. This study will provide useful information for further elucidating the role of Co-CSRP genes during newt limb regeneration.

Histone Variant MacroH2A1 Plays an Isoform-Specific Role in Suppressing Epithelial-Mesenchymal Transition.

Epithelial-Mesenchymal Transition (EMT) is a biological program that plays key roles in various developmental and pathological processes. Although much work has been done on signaling pathways and transcription factors regulating EMT, the epigenetic regulation of EMT remains not well understood. Histone variants have been recognized as a key group of epigenetic regulators. Among them, macroH2A1 is involved in stem cell reprogramming and cancer progression. We postulated that macroH2A1 may play a role in EMT, a process involving reprogramming of cellular states. In this study, we demonstrate that expression of macroH2A1 is dramatically reduced during EMT induction in immortalized human mammary epithelial cells (HMLE). Moreover, ectopic expression of the macroH2A1.1 isoform, but not macroH2A1.2, can suppress EMT induction and reduce the stem-like cell population in HMLE. Interestingly, macroH2A1.1 overexpression cannot revert stable mesenchymal cells back to the epithelial state, suggesting a stage-specific role of macroH2A1.1 in EMT. We further pinpointed that the function of macroH2A1.1 in EMT suppression is dependent on its ability to bind the NAD+ metabolite PAR, in agreement with the inability to suppress EMT by macroH2A1.2, which lacks the PAR binding domain. Thus, our work discovered a previously unrecognized isoform-specific function of macroH2A1 in regulating EMT induction.

Alterations of protein glycosylation in embryonic stem cells during adipogenesis.

The understanding of adipose tissue development is crucial for the treatment of obesity-related diseases. Adipogenesis has been extensively investigated at the gene and protein levels in recent years. However, the alterations in protein glycosylation during this process remains unknown, particularly that of parthenogenetic embryonic stem cells (pESCs), a type of ESCs with low immunogenicity and no ethical concerns regarding their use. Protein glycosylation markedly affects cell growth and development, cell-to-cell communication, tumour growth and metastasis. In the present study, the adipogenic potentials of J1 ESCs and pESCs were first compared and the results demonstrated that pESCs had lower adipogenic potential compared with J1 ESCs. Lectin microarray was then used to screen the alteration of protein glycosylation during adipogenesis. The results revealed that protein modification of GlcNAc and α-1-2-fucosylation increased, whereas α-1-6­fucosylation, α-2-6-sialylation and α-1-6-mannosylation decreased in J1 ESCs and pESCs during this process. In addition, α-1-3-mannosylation decreased only in pESCs. Lectin histochemistry and quantitative polymerase chain reaction of glycosyltransferase confirmed the results obtained by lectin microarray. Therefore, protein glycosylation of ESCs was significantly altered during adipogenesis, indicating that protein glycosylation analysis is not only helpful for studying the mechanism of adipogenesis, but may also be used as a marker to monitor adipogenic development.