Process Of Terminal Differentiation Research Articles

Immature Skeletal Myotubes Are an Effective Source for Improving the Terminal Differentiation of Skeletal Muscle.

Injured or atrophied adult skeletal muscles are regenerated through terminal differentiation of satellite cells to form multinucleated muscle fibers. Transplantation of satellite cells or cultured myoblasts has been used to improve skeletal muscle regeneration. Some of the limitations observed result from the limited number of available satellite cells that can be harvested and the efficiency of fusion of cultured myoblasts with mature muscle fibers (i.e., terminal differentiation) upon transplantation. However, the possible use of immature myotubes in the middle of the terminal differentiation process instead of satellite cells or cultured myoblasts has not been thoroughly investigated. Herein, myoblasts (Mb) or immature myotubes on differentiation day 2 (D2 immature myotubes) or 3 (D3 immature myotubes) were transferred to plates containing D2 or D3 immature myotubes as host cells. The transferred Mb/immature myotubes on the plates were further co-differentiated with host immature myotubes into mature myotubes in six conditions: Mb-to-D2, D2-to-D2, D3-to-D2, Mb-to-D3, D2-to-D3, and D3-to-D3. Among these six co-differentiation conditions, the D2-to-D3 co-differentiation condition exhibited the most characteristic myotube appearance and the greatest availability of Ca2+ for skeletal muscle contraction. Compared with non-co-differentiated control myotubes, D2-to-D3 co-differentiated myotubes presented increased MyoD and myosin heavy chain II (MyHC II) expression and increased myotube width, accompanied by parallel and swirling alignment. These increases correlated with functional increases in both electrically induced intracellular Ca2+ release and extracellular Ca2+ entry due to the increased expression of ryanodine receptor 1 (RyR1), sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1a (SERCA1a), and stromal interaction molecule 1 (STIM1). These increases were not detected in any of the other co-differentiation conditions. These results suggest that in vitro-cultured D2-to-D3 co-differentiated mature myotubes could be a good alternative source of satellite cells or cultured myoblasts for skeletal muscle regeneration.

A Comprehensive Review on Cell Death

A fundamental pathophysiological process, apoptosis is also a crucial stage in healthy growth and life. Despite the fact that a variety of triggers can cause cell death, the mode of cell death often follows distinct patterns. Necrosis is a non-physiological process that happens as a result of infection or injury. Apoptosis, also known as Type I cell death, and autophagy, often known as Type II cell death, are both types of planned cell death. Necrosis, which can take many various forms, is the death of cells brought on by outside forces like injury or infection. Necroptosis, a type of programmed necrosis, has recently been identified as an additional type of designed cell death. Necroptosis is thought to work as a cell-death fallback when apoptosis signaling is inhibited by endogenous or external elements like viruses or mutations. Cornification is a distinct process of terminal differentiation and programmed cell death that epidermal keratinocytes go through. The creation of the outermost skin barrier is a result of cornification. An overview of the various types of cell death and their mechanisms is provided in this review paper.

Impacts of autophagy on the formation of organelle-free zone during the lens development.

The thorough degeneration of organelles in the core of the lens is certainly a hallmark event during the lens development. Organelles degradation in the terminal differentiation process of lens fiber cells to form an organelle-free zone is critical for lens maturation and transparency. Several mechanisms have been proposed to expand our understanding of lens organelles degradation, including apoptotic pathways, the participation of ribozyme, proteolytic enzyme and phospholipase A and acyltransferase, and the newly discovered roles for autophagy. Autophagy is a lysosome-dependent degradation reaction during which the "useless" cellular components are degraded and recycled. These cellular components, such as incorrectly folded proteins, damaged organelles and other macromolecules, are first engulfed by the autophagosome before being further delivered to lysosomes for degradation. Although autophagy has been recognized involving in organelle degradation of the lens, the detailed functions remain to be discovered. Recent advances have revealed that autophagy not only plays a vital role in the intracellular quality control of the lens but is also involved in the degradation of nonnuclear organelles in the process of lens fiber cell differentiation. Herein, we first review the potential mechanisms of organelle-free zone formation, then discuss the roles of autophagy in intracellular quality control and cataract formation, and finally substantially summarize the potential involvement of autophagy in the development of organelle-free zone formation.

Peculiarities of epidermal proliferation and terminal differentiation in various histological types of seborrheic keratosis

Aim of the study is to determine the peculiarities of epidermal proliferation and terminal differentiation in various histological types of seborrheic keratosis (SK). Materials and methods. Pathomorphological and immunohistochemical analysis was performed on the skin biopsy material of 60 patients with SK, who constituted the study group, and on the material of 30 healthy skin samples, which were considered a control group. Results. Level of expression of Ki-67 marker in papillomatous and acanthotic types of SK is significantly lower (P < 0.005) compared to healthy skin samples, which indicates a low proliferative potential of tumors. While the expression of the marker of terminal differentiation between these groups of SK and healthy skin is significantly higher (P < 0.005). Comparative characterization of the association of the expression degree of the immunohistochemical marker of proliferation Ki-67 depending on the activity of terminal differentiation processes in acanthotic and papillomatous types of SK indicates a statistically significant inverse correlation of these pathogenetically determined processes. Thus, activation of terminal differentiation with increase of caspase-14 expression level inhibits proliferation in basal epidermal layers that is accompanied by decrease of Ki-67 level. Conclusions. Level of expression of Ki-67 as a marker of proliferative activity in patients with acanthotic and papillomatous types is significantly lower than in the control group, which may indicate no tendency to malignancy. The expression level of caspase-14 in acanthotic and papillomatous types is increased compared to healthy skin, which may indicate a violation of keratinization, which occurs during the terminal differentiation of epidermocytes. Statistical inverse conjugacy between caspase-14 and Ki-67 indicates inhibition of proliferation against the background of increased terminal differentiation activity and may justify the vectors of therapeutic action of topical agents.

Terminal differentiation of keratinocytes was damaged in type 2 diabetic mice.

Although skin manifestations are common in diabetic patients, its characteristics are poorly identified. This study explored the differentiation process of keratinocytes in type 2 diabetes mellitus (T2DM) in vivo. Back skin of T2DM model KKAy/TaJcl mice (KKAy) and C57BL/6JJcl mice (control) aged 8 and 12weeks was used. The mRNA expression of differentiation markers of keratinocytes was measured by quantitative real-time polymerase chain reaction (qRT-PCR). The expression of each marker in situ was examined immunohistochemically. KKAy mice showed hyperglycemia versus control mice. The histological findings showed increased thickness and structural impairment of epidermal tissue in KKAy mice. The qRT-PCR revealed that the expression of integrin beta 1 and keratin 14 in KKAy and control mice was identical. However, the expression of involucrin at 8 weeks, keratin 10 at 12 weeks, and filaggrin and loricrin at 8 and 12 weeks was decreased in KKAy mice. Immunohistochemical findings showed that filaggrin was markedly decreased in KKAy mice, though Ki-67 remained unchanged. The terminal differentiation process was impaired in the diabetic skin, while keratinocyte proliferation was preserved. Damaged terminal differentiation of keratinocytes may contribute to impairment of the skin barrier function in diabetic dermatoses.

Keratinization and Cornification are not equivalent processes but keratinization in fish and amphibians evolved into cornification in terrestrial vertebrates.

The present account offers a generalized view of the evolution of process of terminal differentiation in keratinocytes of the epidermis in anamniotes, indicated as keratinization, into a further differentiating process of cornification in the skin and appendages of terrestrial vertebrates. Keratinization indicates the prevalent accumulation of intermediate filaments of keratins (IFKs) and is present in most fish and amphibian epidermis and inner epithelia of all vertebrates. During land adaptation, terrestrial vertebrates evolved a process of cornification and keratinocytes became dead corneocytes by the addition of numerous others proteins to the IFKs framework, represented by keratin-associated proteins (KAPs) and corneous proteins (CPs). Most of genes coding for these types of proteins are localized in chromosomal loci different and un-related from those of IFKs, and CPs originated from a gene cluster indicated as epidermal differentiation complex. During the evolution of reptiles and birds, the epidermis and corneous derivatives such as scales, claws, beaks and feathers mainly accumulate a type of CPs that overcome IFKs and containing a 34 amino acid beta-sheet core indicated as corneous beta proteins, formerly known as beta-keratins. Mammals did not evolve a beta-sheet core in their CPs and KAPs but instead produced numerous cysteine-rich IFKs in their epidermis and specialized KAPs in hairs, claws, nails, hooves and horns.

The Immune Microenvironment of Hashimoto’s Thyroiditis Regulates the Glycosylation Modification of IgG

Objectives: Elevation of anti-thyroglobulin antibodies that are primarily IgG isotype is a hallmark of Hashimoto’s thyroiditis (HT). As for IgG,it bears two conserved repertoire of N-linked glycans attached to its crystallizable fragment (Fc) at the 297 asparagine residue (Asn297). In our previous study, we found that serum TgAb IgG from HT patients exhibits higher glycosylation levels than those observed from healthy controls. Previous studies confirmed that imbalance of Th1/Th2 and Th17/Treg leading to altered immune microenvironment with elevation of certain cytokines was found in the thyroid tissue of HT, including IFN-γ, TNF-α, IL-21, IL-17A, IL-6, BAFF, APRIL. Thus, the aim of our study was to investigate the influence of the elevated cytokines on the differentiation process of B cells and the glycosylation levels of IgG. Methods: We formed a two-phase culture system in vitro to promote B cells to differentiate to antibody-secreting cells (ASCs). In the process of cell culture, B cells were co-cultured with cytokines as followed: IFN-γ, TNF-α, IL-21, IL-17A, IL-6, BAFF and APRIL. Flow cytometry was performed to identify the percentage of plasmablasts (CD38+CD27high) and plasma cells (CD20-CD138+). ELISA was used to measure the yield of IgG in culture supernatants. The glycosylation levels of secreted IgG under different stimulation conditions were detected by lectin microarray. Results: We found that IL-21, TNF-α and BAFF can significantly promote the differentiation of B cells into ASCs in vitro culture system, and augment the production of IgG to over 4-fold. In addition, cytokines affected the glycosylation modification profile of IgG diversely: 1) IL-21, IL-17A, TNF-α, BAFF significantly increased the glycosylation level of sialic acid of total IgG; 2) IFN-γ significantly increased the level of galactose; 3) IL-21, IL-17A, IFN-γ, BAFF, and APRIL significantly increased the level of mannose; 4) IL-6 significantly decreased the level of sialic acid, galactose and mannose; 5) IL-17A, IFN-γ, TNF-α, BAFF significantly increased the level of GalNAc that was a component of O-Glycan,which only exists in the hinge region of IgG3 subclass. Conclusions: The abnormally elevated cytokines in microenvironment participated in the regulation of B cell terminal differentiation process and glycosylation level of IgG, thereby involving in the pathogenesis of AITD.

The Inhibitor of MyoD Family A Promotes Terminal Myeloid Differentiation

Abstract The differentiation of hematopoietic stem and progenitor cells is tightly regulated to maintain immune and blood homeostasis while also responding to injury and immunological insult. Myeloid lineage-committed progenitor cells are highly proliferative, but do not self-renew and must be continually replenished by the differentiation of hematopoietic stem cells. The process of terminal differentiation is controlled by various cell-intrinsic and - extrinsic factors, and the interactions between these factors remain not well understood. We hypothesize that the Inhibitor of MyoD Family A (I-mfa) protein is a novel regulator of myeloid lineage differentiation through its interaction with other cell-intrinsic factors. To examine this, mice which lack I-mfa expression (I-mfanull) were compared to wild type (WT) 129Sv age- and sex-matched controls. I-mfanull animals had reduced spleen size (both in weight and total cellularity), but had preserved relative proportions of B, T, and myeloid cells as analyzed by flow cytometry when compared to WT. Analysis of the bone marrow (BM) myeloid lineage-committed progenitors showed an accumulation of common myeloid progenitors (CMP) and granulocyte-macrophage progenitors (GMP) in I-mfanull animals when compared to WT. In serial colony forming unit assays I-mfanull BM produced fewer mature colonies and continued to produce colonies after 5 passages, suggesting aberrant self-renewal. Using myeloid cell lines lacking or overexpressing I-mfa, the molecular mechanism by which I-mfa acts is being elucidated. These results taken together suggest I-mfa acts to promote differentiation of myeloid cells, an effect which may be important in the treatment of myeloid neoplasms or bone marrow failure.

Expression pattern determines regulatory logic.

Large amounts of effort have been invested in trying to understand how a single genome is able to specify the identity of hundreds of cell types. Inspired by some aspects of Caenorhabditis elegans biology, we implemented an in silico evolutionary strategy to produce gene regulatory networks (GRNs) that drive cell-specific gene expression patterns, mimicking the process of terminal cell differentiation. Dynamics of the gene regulatory networks are governed by a thermodynamic model of gene expression, which uses DNA sequences and transcription factor degenerate position weight matrixes as input. In a version of the model, we included chromatin accessibility. Experimentally, it has been determined that cell-specific and broadly expressed genes are regulated differently. In our in silico evolved GRNs, broadly expressed genes are regulated very redundantly and the architecture of their cis-regulatory modules is different, in accordance to what has been found in C. elegans and also in other systems. Finally, we found differences in topological positions in GRNs between these two classes of genes, which help to explain why broadly expressed genes are so resilient to mutations. Overall, our results offer an explanatory hypothesis on why broadly expressed genes are regulated so redundantly compared to cell-specific genes, which can be extrapolated to phenomena such as ChIP-seq HOT regions.

MasterPATH: network analysis of functional genomics screening data

BackgroundFunctional genomics employs several experimental approaches to investigate gene functions. High-throughput techniques, such as loss-of-function screening and transcriptome profiling, allow to identify lists of genes potentially involved in biological processes of interest (so called hit list). Several computational methods exist to analyze and interpret such lists, the most widespread of which aim either at investigating of significantly enriched biological processes, or at extracting significantly represented subnetworks.ResultsHere we propose a novel network analysis method and corresponding computational software that employs the shortest path approach and centrality measure to discover members of molecular pathways leading to the studied phenotype, based on functional genomics screening data. The method works on integrated interactomes that consist of both directed and undirected networks – HIPPIE, SIGNOR, SignaLink, TFactS, KEGG, TransmiR, miRTarBase. The method finds nodes and short simple paths with significant high centrality in subnetworks induced by the hit genes and by so-called final implementers – the genes that are involved in molecular events responsible for final phenotypic realization of the biological processes of interest. We present the application of the method to the data from miRNA loss-of-function screen and transcriptome profiling of terminal human muscle differentiation process and to the gene loss-of-function screen exploring the genes that regulates human oxidative DNA damage recognition. The analysis highlighted the possible role of several known myogenesis regulatory miRNAs (miR-1, miR-125b, miR-216a) and their targets (AR, NR3C1, ARRB1, ITSN1, VAV3, TDGF1), as well as linked two major regulatory molecules of skeletal myogenesis, MYOD and SMAD3, to their previously known muscle-related targets (TGFB1, CDC42, CTCF) and also to a number of proteins such as C-KIT that have not been previously studied in the context of muscle differentiation. The analysis also showed the role of the interaction between H3 and SETDB1 proteins for oxidative DNA damage recognition.ConclusionThe current work provides a systematic methodology to discover members of molecular pathways in integrated networks using functional genomics screening data. It also offers a valuable instrument to explain the appearance of a set of genes, previously not associated with the process of interest, in the hit list of each particular functional genomics screening.

Comprehensive proteomic analysis of murine terminal erythroid differentiation

Murine-based cellular models have provided and continue to provide many useful insights into the fundamental mechanisms of erythropoiesis, as well as insights into the pathophysiology of inherited and acquired red cell disorders. Although detailed information on many aspects of these cell models is available, comprehensive proteomic data are lacking. This is a critical knowledge gap, as proteins are effectors of most biologic processes. To address this critical unmet need, proteomes of the murine cell lines Friend erythroleukemia (MEL), GATA1 erythroid (G1ER), and embryonic stem cell-derived erythroid progenitor (MEDEP) and proteomes of cultured murine marrow-derived erythroblasts at different stages of terminal erythroid differentiation were analyzed. The proteomes of MEDEP cells and primary murine erythroid cells were most similar, whereas those of MEL and G1ER cells were more distantly related. We demonstrated that the overall cellular content of histones does not decrease during terminal differentiation, despite strong chromatin condensation. Comparison of murine and human proteomes throughout terminal erythroid differentiation revealed that many noted transcriptomic changes were significantly dampened at the proteome level, especially at the end of the terminal differentiation process. Analysis of the early events associated with induction of terminal differentiation in MEDEP cells revealed divergent alterations in associated transcriptomes and proteomes. These proteomic data are powerful and valuable tools for the study of fundamental mechanisms of normal and disordered erythropoiesis and will be of broad interest to a wide range of investigators for making the appropriate choice of various cell lines to study inherited and acquired diseases of the erythrocyte.

Inhibition of Ihh Reverses Temporomandibular Joint Osteoarthritis via a PTH1R Signaling Dependent Mechanism.

The temporomandibular joint (TMJ), which is biomechanically related to dental occlusion, is often insulted by osteoarthritis (OA). This study was conducted to clarify the relationship between Indian hedgehog (Ihh) and parathyroid hormone receptor 1 (PTH1R) signaling in modulating the enhanced chondrocyte terminal differentiation in dental stimulated TMJ osteoarthritic cartilage. A gain- and loss-of-function strategy was used in an in vitro model in which fluid flow shear stress (FFSS) was applied, and in an in vivo model in which the unilateral anterior cross-bite (UAC) stimulation was adopted. Ihh and PTH1R signaling was modulated through treating the isolated chondrocytes with inhibitor/activator and via deleting Smoothened (Smo) and/or Pth1r genes in mice with the promoter gene of type 2 collagen (Col2-CreER) in the tamoxifen-inducible pattern. We found that both FFSS and UAC stimulation promoted the deep zone chondrocytes to undergo terminal differentiation, while cells in the superficial zone were robust. We demonstrated that the terminal differentiation process in deep zone chondrocytes promoted by FFSS and UAC was mediated by the enhanced Ihh signaling and declined PTH1R expression. The FFSS-promoted terminal differentiation was suppressed by administration of the Ihh inhibitor or PTH1R activator. The UAC-promoted chondrocytes terminal differentiation and OA-like lesions were rescued in Smo knockout, but were enhanced in Pth1r knockout mice. Importantly, the relieving effect of Smo knockout mice was attenuated when Pth1r knockout was also applied. Our data suggest a chondrocyte protective effect of suppressing Ihh signaling in TMJ OA cartilage which is dependent on PTH1R signaling.

Bone Marrow-Derived Macrophage Immortalization of LXR Nuclear Receptor-Deficient Cells.

Macrophages are professional phagocytic cells that play key roles in innate and adaptive immunity, metabolism, and tissue homeostasis. Lipid metabolism is tightly controlled at the transcriptional level, and one of the key players of this regulation in macrophages and other cell types is the LXR subfamily of nuclear receptors (LXRα and LXRβ). The use of LXR double knockout (LXR-DKO) macrophages in vitro has yielded extensive benefits in metabolism research, but this technique is hindered by primary macrophage cell expansion capability, which diminishes along terminal cell differentiation process. Here we detail a method to immortalize LXR double knockout bone marrow-derived macrophage cells at an early stage of differentiation, using a retroviral delivery of a combination of murine v-myc and v-raf oncogenes. This methodology enables the generation of autonomous self-renewing macrophages bearing an LXR-DKO genetic background, as a valuable tool for research in lipid metabolism and other LXR receptor-mediated effects.

The Transcription Factor Ets1 Suppresses T Follicular Helper Type 2 Cell Differentiation to Halt the Onset of Systemic Lupus Erythematosus

Single-nucleotide polymorphisms in ETS1 are associated with systemic lupus erythematosus (SLE). Ets1-/- mice develop SLE-like symptoms, suggesting that dysregulation of this transcription factor is important to the onset or progression of SLE. We used conditional deletion approaches to examine the impact of Ets1 expression in different immune cell types. Ets1 deletion on CD4+ Tcells, but not Bcells or dendritic cells, resulted in the SLE autoimmunity, and this was associated with the spontaneous expansion of T follicular helper type 2 (Tfh2) cells. Ets1-/- Tfh2 cells exhibited increased expression of GATA-3 and interleukin-4 (IL-4), which induced IgE isotype switching in B cells. Neutralization of IL-4 reduced Tfh2 cell frequencies and ameliorated disease parameters. Mechanistically, Ets1 suppressed signature Tfh and Th2 cell genes, including Cxcr5, Bcl6, and Il4ra, thus curbing the terminal Tfh2 cell differentiation process. Tfh2 cell frequencies in SLE patients correlated with disease parameters, providing evidence for the relevance of these findings to human disease.

STXBP4 regulates APC/C-mediated p63 turnover and drives squamous cell carcinogenesis

Levels of the N-terminally truncated isoform of p63 (ΔN p63), well documented to play a pivotal role in basal epidermal gene expression and epithelial maintenance, need to be strictly regulated. We demonstrate here that the anaphase-promoting complex/cyclosome (APC/C) complex plays an essential role in the ubiquitin-mediated turnover of ΔNp63α through the M-G1 phase. In addition, syntaxin-binding protein 4 (Stxbp4), which we previously discovered to bind to ΔNp63, can suppress the APC/C-mediated proteolysis of ΔNp63. Supporting the physiological relevance, of these interactions, both Stxbp4 and an APC/C-resistant version of ΔNp63α (RL7-ΔNp63α) inhibit the terminal differentiation process in 3D organotypic cultures. In line with this, both the stable RL7-ΔNp63α variant and Stxbp4 have oncogenic activity in soft agar and xenograft tumor assays. Notably as well, higher levels of Stxbp4 expression are correlated with the accumulation of ΔNp63 in human squamous cell carcinoma (SCC). Our study reveals that Stxbp4 drives the oncogenic potential of ΔNp63α and may provide a relevant therapeutic target for SCC.

NFκB regulates p21 expression and controls DNA damage-induced leukemic differentiation.

DNA damage exposure is a major modifier of cell fate in both normal and cancer tissues. In response to DNA damage, myeloid leukemia cells activate a poorly understood terminal differentiation process. Here, we show that the NFκB pathway directly activates expression of the proliferation inhibitor p21 in response to DNA damage in myeloid leukemia cells. In order to understand the role of this unexpected regulatory event, we ablated the NFκB binding site we identified in the p21 promoter, using CRISPR/Cas9-mediated genome editing. We found that NFκB-mediated p21 activation controls DNA damage-induced myeloid differentiation. Our results uncover a p53-independent pathway for p21 activation involved in controlling hematopoietic cell fate.

Relationship between nucleic acid structures and sequences on the expression of terminal differentiation (i.e., denucleation): alternative cell death pathway

Terminal differentiation (i.e., denucleation) is a type of cell death, similar to apoptosis and necrosis, and is considered an alternative cell death pathway. Denucleating secondary crystalline lens fiber cells, keratinocytes of the epidermis and erythrocytes use this cell death mechanism to maintain their normal cellular physiology. In all three cases, the overall process of organelle removal is somewhat understood morphologically and biochemically; however, the molecular biology is not fully known. Structural transitions of the canonical A‐RNA into alternative structures such as left‐handed Z‐RNA, and the formation of multistranded DNA's (e.g., quadruplex DNA) are important in cells. We are examining how structural polymorphisms play a role in regulating cell death in the three above mentioned tissues, namely, denucleation, and how it differs from apoptosis and necrosis. Immunohistochemistry was performed to study the distribution and specific amounts of single‐stranded (ss−) DNA, A‐RNA, Z‐RNA and quadruplex DNA during denucleation. Immunohistochemical staining of tissue sections were performed with anti‐ss‐DNA, anti‐A‐RNA, anti‐Z‐RNA and anti‐quadruplex DNA antibodies. We observed ss‐DNA, A‐RNA, Z‐RNA and quadruplex DNA immunoreactivity. Both negative, positive and isotype controls were used. ELISA data correlates well with our immunohistochemical results. Immunohistochemical staining of tissues for the demonstration and quantification of ss‐DNA, A‐RNA, Z‐RNA and G4‐quadruplex DNA, reveals different levels of decreasing and increasing immunoreactivity as dying cells progress thru denucleation. Immunohistochemistry shows different levels of gradually decreasing quadruplex DNA as cells progress thru denucleation, with precipitous declines in certain tissue zones at specific times [i.e., this correlates well with decrease in B‐DNA (conrtols)]. A‐RNA and mainly Z‐RNA content decreases during denucleation. A decline in the presence of quadruplex DNA also reveals how denucleation is eradicating different DNA structures. This gradual decline in quadruplex DNA represents chromatin fragmentation. A precipitous decline in quadruplex DNA occurs towards the end of denucleation, depending on tissue type. We believe that specific types of canonical (e.g. B‐DNA), alternative (e.g., Z‐DNA) and multistranded DNAs (e.g., triplex DNA and quadruplex DNA) regulate pro‐ and anti‐cell death factors (e.g., DNase IIβ, CDK1, and cyclins in lens fiber cells, respectively), which control programmed elimination of cellular organelles and DNA. Pro‐cell death factors allows the denucleated cells to remain viable for a certain period of time. Denucleating keratinocytes, secondary crystalline lens fibers and erythrocytes share some common initial characteristics with apoptosis and necrosis, however, the latter part of denucleation is completely different. Based on our research we believe that pro‐apoptotic proteins are involved in the early stages of denucleation, and then other mechanisms (e.g., anti‐apoptotic) take over the remaining processes of terminal differentiation. The gradual decrease in A‐RNA, Z‐RNA and quadruplex DNA during denucleation may play a role in regulating this alternative cell death pathway. Our results suggest that dissimilar nucleic acid structures cause nucleic acid fragmentation in denucleating cells, which differs from apoptosis and necrosis, in the sequence of events and the time course.Support or Funding InformationProject was supported by several NYIT ISRC and TriBeta Biology Honor Society grants.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Novel role for the testis-enriched HSPA2 protein in regulating epidermal keratinocyte differentiation.

HSPA2, a poorly characterized member of the HSPA (HSP70) chaperone family, is a testis-enriched protein involved in male germ cell differentiation. Previously, we revealed that HSPA2 is present in human stratified epithelia, including epidermis, however the contribution of this protein to epithelial biology remained unknown. Here, we show for the first time that HSPA2 is expressed in basal epidermal keratinocytes, albeit not in keratinocytes exhibiting features attributed to primitive undifferentiated progenitors, and participates in the keratinocyte differentiation process. We found that HSPA2 is dispensable for protection of HaCaT keratinocytes against heat shock-induced cytotoxicity. We also shown that lentiviral-mediated shRNA silencing of HSPA2 expression in HaCaT cells caused a set of phenotypic changes characteristic for keratinocytes committed to terminal differentiation such as reduced clonogenic potential, impaired adhesiveness and increased basal and confluency-induced expression of differentiation markers. Moreover, the fraction of undifferentiated cells that rapidly adhered to collagen IV was less numerous in HSPA2-deficient cells than in the control. In a 3D reconstructed human epidermis model, HSPA2 deficiency resulted in accelerated development of a filaggrin-positive layer. Collectively, our results clearly show a link between HSPA2 expression and maintenance of keratinocytes in an undifferentiated state in the basal layer of the epidermis. It seems that HSPA2 could retain keratinocytes from premature entry into the terminal differentiation process. Overall, HSPA2 appears to be necessary for controlling development of properly stratified epidermis and thus for maintenance of skin homeostasis.

Trabectedin Overrides Osteosarcoma Differentiative Block and Reprograms the Tumor Immune Environment Enabling Effective Combination with Immune Checkpoint Inhibitors.

Purpose: Osteosarcoma, the most common primary bone tumor, is characterized by an aggressive behavior with high tendency to develop lung metastases as well as by multiple genetic aberrations that have hindered the development of targeted therapies. New therapeutic approaches are urgently needed; however, novel combinations with immunotherapies and checkpoint inhibitors require suitable preclinical models with intact immune systems to be properly tested.Experimental Design: We have developed immunocompetent osteosarcoma models that grow orthotopically in the bone and spontaneously metastasize to the lungs, mimicking human osteosarcoma. These models have been used to test the efficacy of trabectedin, a chemotherapeutic drug utilized clinically for sarcomas and ovarian cancer.Results: Trabectedin, as monotherapy, significantly inhibited osteosarcoma primary tumor growth and lung metastases by both targeting neoplastic cells and reprogramming the tumor immune microenvironment. Specifically, trabectedin induced a striking differentiation of tumor cells by favoring the recruitment of Runx2, the master genetic regulator of osteoblastogenesis, on the promoter of genes involved in the physiologic process of terminal osteoblast differentiation. Differentiated neoplastic cells, as expected, showed reduced proliferation rate. Concomitantly, trabectedin enhanced the number of tumor-infiltrating T lymphocytes, with local CD8 T cells, however, likely post-activated or exhausted, as suggested by their high expression of the inhibitory checkpoint molecule PD-1. Accordingly, the combination with a PD-1-blocking antibody significantly increased trabectedin efficacy in controlling osteosarcoma progression.Conclusions: These results demonstrate the therapeutic efficacy of trabectedin in osteosarcoma treatment, unveiling its multiple activities and providing a solid rationale for its combination with immune checkpoint inhibitors. Clin Cancer Res; 23(17); 5149-61. ©2017 AACR.

TET2- and TDG-mediated changes are required for the acquisition of distinct histone modifications in divergent terminal differentiation of myeloid cells.

The plasticity of myeloid cells is illustrated by a diversity of functions including their role as effectors of innate immunity as macrophages (MACs) and bone remodelling as osteoclasts (OCs). TET2, a methylcytosine dioxygenase highly expressed in these cells and frequently mutated in myeloid leukemias, may be a key contributor to this plasticity. Through transcriptomic and epigenomic analyses, we investigated 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) and gene expression changes in two divergent terminal myeloid differentiation processes, namely MAC and OC differentiation. MACs and OCs undergo highly similar 5hmC and 5mC changes, despite their wide differences in gene expression. Many TET2- and thymine-DNA glycosylase (TDG)-dependent 5mC and 5hmC changes directly activate the common terminal myeloid differentiation programme. However, the acquisition of differential features between MACs and OCs also depends on TET2/TDG. In fact, 5mC oxidation precedes differential histone modification changes between MACs and OCs. TET2 and TDG downregulation impairs the acquisition of such differential histone modification and expression patterns at MAC-/OC-specific genes. We prove that the histone H3K4 methyltransferase SETD1A is differentially recruited between MACs and OCs in a TET2-dependent manner. We demonstrate a novel role of these enzymes in the establishment of specific elements of identity and function in terminal myeloid differentiation.