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Cancer Susceptibility Candidate 15 (CASC15), which is a newly identified long noncoding RNA crucial for epigenetic regulation in human tumors, was found to be associated with poor prognosis of the patients with ovarian cancer by utilizing The Cancer Genome Atlas and Gene Expression Omnibus database. Therefore, the purpose of this paper was to explore the functional role and latent molecular mechanism of CASC15 in the progression of ovarian cancer.

Long noncoding RNAs (lncRNAs) are emerging as a new class of important regulators of signal transduction in tissue homeostasis and cancer development. Liquid-liquid phase separation (LLPS) occurs in a wide range of biological processes, while its role in signal transduction remains largely undeciphered. In this study, we uncovered a lipid-associated lncRNA, small nucleolar RNA host gene 9 (SNHG9) as a tumor-promoting lncRNA driving liquid droplet formation of Large Tumor Suppressor Kinase 1 (LATS1) and inhibiting the Hippo pathway. Mechanistically, SNHG9 and its associated phosphatidic acids (PA) interact with the C-terminal domain of LATS1, promoting LATS1 phase separation and inhibiting LATS1-mediated YAP phosphorylation. Loss of SNHG9 suppresses xenograft breast tumor growth. Clinically, expression of SNHG9 positively correlates with YAP activity and breast cancer progression. Taken together, our results uncover a novel regulatory role of a tumor-promoting lncRNA (i.e., SNHG9) in signal transduction and cancer development by facilitating the LLPS of a signaling kinase (i.e., LATS1).

Cyclic GMP-AMP synthase (cGAS) is one of the most-characterized cytoplasmic DNA sensors in humans and other mammals. However, knowledge about cGAS homologs in nonmammalian species remains limited. In this study, we report the molecular and functional identification of two cGAS homologs, namely, DrcGASa and DrcGASb, from a zebrafish (Danio rerio) model. DrcGASa and DrcGASb share the same overall conservative structural architectures and functional domains/residues to mammalian cGASs. Both homologs synthesized a 2'3'-cGAMP isomer but not a 3'3'-cGAMP isomer via oligomerization in response to DNA stimulation. Overexpression of DrcGASa/b in HEK293T cells and zebrafish embryos significantly activated NF-κB and IFN-I signaling pathways in a STING-dependent manner. Knockdown of DrcGASa or DrSTING impaired such activations, thereby reducing the host innate immunity against bacterial and viral infections. DrcGASa, but not DrcGASb, was involved in immunoglobulin Z-mediated mucosal immunity in gill-associated lymphoid tissue, suggesting differential functions between the two DrcGASs. This reaction was associated with the DrcGAS-DrSTING-IFNφ1 signaling axis in GALT's γδ T cells. Our findings provide experimental evidence that a modern cGAS-STING pathway that mainly participates in IFN-mediated immunity originated from teleost fish based on the functional constraint of cGAS and STING proteins during vertebrate evolution.

The NLR family pyrin domain containing 3 (NLRP3) inflammasome is one of the best-characterized inflammasomes in humans and other mammals. However, knowledge about the NLRP3 inflammasome in nonmammalian species remains limited. Here, we report the molecular and functional identification of an NLRP3 homolog (DrNLRP3) in a zebrafish (Danio rerio) model. We found that DrNLRP3's overall structural architecture was shared with mammalian NLRP3s. It initiates a classical inflammasome assembly for zebrafish inflammatory caspase (DrCaspase-A/-B) activation and interleukin 1β (DrIL-1β) maturation in an apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC)-dependent manner, in which DrNLRP3 organizes DrASC into a filament that recruits DrCaspase-A/-B by homotypic pyrin domain (PYD)–PYD interactions. DrCaspase-A/-B activation in the DrNLRP3 inflammasome occurred in two steps, with DrCaspase-A being activated first and DrCaspase-B second. DrNLRP3 also directly activated full-length DrCaspase-B and elicited cell pyroptosis in a gasdermin E (GSDME)-dependent but ASC-independent manner. These two events were tightly coordinated by DrNLRP3 to ensure efficient IL-1β secretion for the initiation of host innate immunity. By knocking down DrNLRP3 in zebrafish embryos and generating a DrASC-knockout (DrASC−/−) fish clone, we characterized the function of the DrNLRP3 inflammasome in anti-bacterial immunity in vivo. The results of our study disclosed the origin of the NLRP3 inflammasome in teleost fish, providing a cross-species understanding of the evolutionary history of inflammasomes. Our findings also indicate that the NLRP3 inflammasome may coordinate inflammatory cytokine processing and secretion through a GSDME-mediated pyroptotic pathway, uncovering a previously unrecognized regulatory function of NLRP3 in both inflammation and cell pyroptosis.

NLRP1 inflammasome is one of the best-characterized inflammasomes in humans and other mammals. However, the existence of this inflammasome in nonmammalian species remains poorly understood. In this study, we report the molecular and functional identification of an NLRP1 homolog, Danio rerio NLRP1 (DrNLRP1) from a zebrafish (D. rerio) model. This DrNLRP1 possesses similar structural architecture to mammalian NLRP1s. It can trigger the formation of a classical inflammasome for the activation of zebrafish inflammatory caspases (D. rerio Caspase [DrCaspase]–A and DrCaspase-B) and maturation of D. rerio IL-1β in a D. rerio ASC (DrASC)–dependent manner. In this process, DrNLRP1 promotes the aggregation of DrASC into a filament with DrASCCARD core and DrASCPYD cluster. The assembly of DrNLRP1 inflammasome depends on the CARD–CARD homotypic interaction between DrNLRP1 and DrASCCARD core, and PYD–PYD interaction between DrCaspase-A/B and DrASCPYD cluster. The FIIND domain in DrNLRP1 is necessary for inflammasome assembly. To understand the mechanism of how the two DrCaspases are coordinated in DrNLRP1 inflammasome, we propose a two-step sequential activation model. In this model, the recruitment and activation of DrCaspase-A/B in the inflammasome is shown in an alternate manner, with a preference for DrCaspase-A followed by a subsequent selection for DrCaspase-B. By using morpholino oligonucleotide–based knockdown assays, the DrNLRP1 inflammasome was verified to play important functional roles in antibacterial innate immunity in vivo. These observations demonstrate that the NLRP1 inflammasome originated as early as in teleost fish. This finding not only gives insights into the evolutionary history of inflammasomes but also provides a favorable animal model for the study of NLRP1 inflammasome-mediated immunology and diseases.

Macrophages are dominant innate immune cells. They demonstrate remarkable heterogeneity and plasticity that are essential for homeostasis and host defense. The heterogeneity of tissue macrophages is shaped by the ontogeny, tissue factors, and environmental signals, a pattern in a tissue-associated latitudinal manner. At the same time, macrophages have long been considered as mainly plastic cells. These cells respond to stimulation quickly and in a stimulus-specific way by utilizing a longitudinal cascaded activation, including coordination of signal transducer, epigenetic elements, and transcription factors, conclusively determine the macrophage phenotypes and functions. With the development of cutting-edge technologies, such as fate-mapping, single-cell transcriptomics, ipsc platform, nanotherapeutic materials, etc., our understanding of macrophage biology and the roles in the pathogenesis of diseases is much advanced. This review summarizes recent progress on the latitudinal and longitudinal regulation of tissue macrophages in inflammatory diseases. The latitudinal regulation covers the tissue macrophage origins, tissue factors, and environmental signals, reflecting the macrophage heterogeneity. The longitudinal regulation focuses on how multiple factors shape the phenotypes and functions of macrophage subsets to gain plasticity in inflammatory diseases (i.e., inflammatory bowel disease). In addition, how to target macrophages as a potential therapeutic approach and cutting edge-technologies for tissue macrophage study are also discussed in this review.

IgZ or its equivalent IgT is a newly discovered teleost specific Ig class that is highly specialized in mucosal immunity. However, whether this IgZ/IgT class participates in other biological processes remains unclear. In this study, we unexpectedly discovered that IgZ is highly expressed in zebrafish ovary, accumulates in unfertilized eggs, and is transmitted to offspring from eggs to zygotes. Maternally transferred IgZ in zygotes is found at the outer and inner layers of chorion, perivitelline space, periphery of embryo body, and yolk, providing different lines of defense against pathogen infection. A considerable number of IgZ+ B cells are found in ovarian connective tissues distributed between eggs. Moreover, pIgR, the transporter of IgZ, is also expressed in the ovary and colocalizes with IgZ in the zona radiata of eggs. Thus, IgZ is possibly secreted by ovarian IgZ+ B cells and transported to eggs through association with pIgR in a paracrine manner. Maternal IgZ in zygotes showed a broad bacteriostatic activity to different microbes examined, and this reactivity can be manipulated by orchestrating desired bacteria in water where parent fish live or immunizing the parent fish through vaccination. These observations suggest that maternal IgZ may represent a group of polyclonal Abs, providing protection against various environmental microbes encountered by a parent fish that were potentially high risk to offspring. To our knowledge, our findings provide novel insights into a previously unrecognized functional role of IgZ/IgT Ig in the maternal transfer of immunity in fish, greatly enriching current knowledge about this ancient Ig class.

The BTLA and HVEM are two well-characterized immune checkpoint inhibitors in humans and other mammalian species. However, the occurrence and functionality of these two molecules in non-mammalian species remain poorly understood. In the present study, we identified the BTLA and HVEM homologs from large yellow croaker (Larimichthys crocea), an economically important marine species of the perciform fish family. The Larimichthys crocea BTLA and HVEM (LcBTLA and LcHVEM) share conserved structural features to their mammalian counterparts, and they were expressed in various tissues and cells examined at different transcriptional levels, with particular abundance in immune-relevant tissues and splenic leukocytes. Immunofluorescence staining and flow cytometry analysis showed that LcHVEM and LcBTLA proteins were distributed on MHC-II+ APCs and CD4-2+ T cells, and a strong interaction between LcBTLA and LcHVEM was detected in splenic leukocytes in the mixed lymphocyte reaction (MLR). By blockade assays using anti-LcBTLA and anti-LcHVEM Abs as well as recombinant soluble LcBTLA and LcHVEM proteins in different combinations, it was found that LcBTLA-LcHVEM interactions play an important inhibitory role in the activation of alloreactive T cells using MLR as a model, and APC-initiated antigen-specific CD4-2+ T cells in response to A. hydrophila (A. h) stimulation. These observations highlight the extensive functional roles of LcBTLA and LcHVEM immune-checkpoint inhibitors in allogeneic T cell reactions, and CD4-2+ T cell-mediated adaptive immune responses in Larimichthys crocea. Thus, the BTLA-HVEM checkpoint may represent an ancient coinhibitory pathway, which was originated in fish and was conserved from fish to mammals throughout the vertebrate evolution.

Organelles entail specialized molecules to regulate their essential cellular processes. However, systematically elucidating the subcellular distribution of functional molecules such as long non-coding RNAs (lncRNAs) in tissue homeostasis and diseases has not been fully achieved. Here, we characterized the organelle-associated lncRNAs from mitochondria, lysosome, and endoplasmic reticulum (ER), respectively, and revealed the diverse and abundant distribution of lncRNAs. Among them, we identified mitochondrial lncRNA Growth-Arrest-Specific 5 (GAS5) as a tumor suppressor in maintaining cellular energy homeostasis. Mechanistically, energy stress-induced GAS5 modulated mitochondria TCA flux by declining metabolic tandem association of FH-MDH2-CS, the canonical members of the TCA cycle. Remarkably, the expression of GAS5 negatively related with levels of its associated mitochondrial metabolic enzymes and breast cancer development. Together with the detailed functional annotations, this subcellular lncRNA identification revealed the human cell’s inquisitively complex architecture, aiding in the development of new strategies for the clinical application of organelle-associated lncRNAs.

Inner ear hair cells are mechanoreceptors responsible for hearing. Pathogenic defects of hair cell-specific genes are one of the major causes of deafness. The BarH class homeobox gene Barhl1 is a deafness gene expressed in developing hair cells, yet the role of Barhl1 during hair cell development remains poorly understood. In the present study, we first established an in vitro differentiation system to efficiently obtain mouse embryonic stem cell (mESC)-derived hair cell-like cells. Subsequently, a mESC line carrying a targeted disruption of Barhl1 was generated using CRISPR/Cas9 technology and subjected to the established in vitro hair cell differentiation protocol. Targeted disruption of Barhl1 does not affect the induction of mESCs toward early primitive ectoderm-like (EPL) cells and otic progenitors but strongly inhibits the differentiation of hair cell-like cells. Using RNA-sequencing and bioinformatics, we further unravel the molecular mechanism underlying Barhl1-mediated hair cell development. Our data demonstrate the essential role of Barhl1 during hair cell development and provide a basis for the treatment of Barhl1 mutation-based deafness.

Scavenger receptor class A member 5 (SCARA5) and high-mobility group box 1 (HMGB1) protein have become increasingly attractive for their critical functions in innate inflammatory reactions and disorders. However, the functional relevance between these two molecules has never been described. This study discovered that SCARA5 is an HMGB1 recognition receptor that is negatively involved in HMGB1-mediated inflammation in pufferfish (Tetraodon nigroviridis) and zebrafish (Danio rerio) models. Hence, SCARA5 is added as a new member to the HMGB1 receptor family. Tetraodon HMGB1 (TnHMGB1) is a trafficking protein that can be secreted from the nucleus to the outside of cells upon CpG-oligodeoxynucleotide (ODN) stimulation. This protein exerts a strong synergistic effect on CpG-ODN–induced inflammation, as determined by the enhanced proinflammatory cytokine expression through coadministration of TnHMGB1 with CpG-ODN and impaired inflammatory responses through TnHMGB1 depletion. Tetraodon SCARA5 (TnSCARA5) is an inducible protein detected upon TnHMGB1 stimulation; this protein plays an inhibitory role in CpG-ODN–induced inflammation because TnSCARA5 overexpression suppresses cell responsiveness to CpG-ODN induction, whereas TnSCARA5 ablation intensifies the inflammatory reactions. TnSCARA5 can strongly associate with TnHMGB1 through the A and B boxes, depending on the redox state of the cysteine residues, but T box inhibits the association. TnSCARA5 mediates the endocytosis of TnHMGB1 into lysosomes. Results suggest that TnSCARA5 inhibits the CpG-ODN–mediated inflammation via the clearance of HMGB1 mediator for CpG-ODN stimulant. The above findings highlight a novel regulatory mechanism underlying innate inflammation and provide new insights into the clinical treatment of HMGB1-mediated diseases.

TIM-1 and TIM-4 proteins have become increasingly attractive for their critical functions in immune modulation, particularly in CD4+ Th2 cell activation. Thus, these proteins were hypothesized to regulate adaptive humoral immunity. However, further evidence is needed to validate this hypothesis. This study describes the molecular and functional characteristics of TIM-1 and TIM-4 homologs from a zebrafish (Danio rerio) model (D. rerio TIM [DrTIM]-1 and DrTIM-4). DrTIM-1 and DrTIM-4 were predominantly expressed in CD4+ T cells and MHC class II+ APCs under the induction of Ag stimulation. Blockade or knockdown of both DrTIM-1 and DrTIM-4 significantly decreased Ag-specific CD4+ T cell activation, B cell proliferation, Ab production, and vaccinated immunoprotection against bacterial infection. This result suggests that DrTIM-1 and DrTIM-4 serve as costimulatory molecules required for the full activation of adaptive humoral immunity. DrTIM-1 was detected to be a trafficking protein located in the cytoplasm of CD4+ T cells. It can translocate onto the cell surface under stimulation by TIM-4–expressing APCs, which might be a precise regulatory strategy for CD4+ T cells to avoid self-activation before APCs stimulation. Furthermore, a unique alternatively spliced soluble DrTIM-4 variant was identified to exert a negative regulatory effect on the proliferation of CD4+ T cells. The above findings highlight a novel costimulatory mechanism underlying adaptive immunity. This study enriches the current knowledge on TIM-mediated immunity and provides a cross-species understanding of the evolutionary history of costimulatory systems throughout vertebrate evolution.

Sevoflurane has been commonly utilized in nonobstetric surgeries in pregnant women, and its impacts on fetal brain are still not completely known. Ectopic NR4A2 expression has been reported to be related with familial Parkinson disease, and through dual luciferase we found that NR4A2 is a target gene of microRNA-183 (miR-183). We proposed a hypothesis that miR-183 may participate in the process by targeting NR4A2 in neurons after sevoflurane anesthesia. To verify the effect of sevoflurane on hippocampal neural stem cells (NSCs) proliferation and differentiation, we conducted EdU assay and immunofluorescence staining. Next, for better understanding of the impact of miR-183, we altered the miR-183 expression using mimic and inhibitor. Meanwhile, the targeting relationship between miR-183 and NR4A2 was validated by a bioinformatics website and dual-luciferase reporter gene assay. Finally, expressions of miR-184, NR4A2, SRY (sex-determining region Y)-box 2 (Sox2), and brain-derived neurotrophic factor (BDNF) were determined and evaluated by reverse transcription quantitative polymerase chain reaction and western blot analysis. First, sevoflurane was determined a crucial factor in biological behaviors of hippocampal NSCs. Moreover, upregulated miR-183 expression by mimic inhibited the proliferation and differentiation of NSCs. Sevoflurane negatively regulated NR4A2 and Sox2 expressions but positively regulated miR-183 and BDNF expressions. Our findings revealed the underlying novel mechanism by which sevoflurane inhibits hippocampal NSC proliferation and differentiation through interaction with miR-183 and NR4A2. The study provides reliable reference for safe application of sevoflurane anesthesia in neonates.

Peroxiredoxin 1 (Prdx1/Prx1), a ubiquitous antioxidant enzyme involved in preventing oxidative damage and maintaining redox homeostasis, is essential for various cellular activities. Extracellular Prdx1 also plays important roles in innate immune responses. However, little is known about the regulatory functions of Prdx1 in adaptive immunity. In the present study, we report the stimulatory role of Prdx1 in the initiation of adaptive humoral immunity in a zebrafish model. Administration of Prdx1 protein to zebrafish significantly induced the expression of TNF-α, IL-1β, and IL-6 by the Toll-like-receptor-4a-mediated NF-κB signaling pathway and enhanced the activation of MHC-II+ antigen-presenting cells, CD4+ T cells, and mIgM+ B cells. Subsequently, increased production of antigen-specific IgM antibody was observed. Thus, Prdx1 can be used as a novel molecular adjuvant with great therapeutic value for the vaccination of fish diseases. Our study improved the understanding of the biology of Prdx1 family.

Peroxiredoxins (Prxs) are a ubiquitous family of antioxidant proteins that are known as thioredoxin peroxidases. Here we report that Prx1 proteins from Tetraodon nigroviridis and humans also possess a previously unknown catalase-like activity that is independent of Cys residues and reductants but dependent on iron. We identified that the GVL motif was essential to the catalase (CAT)-like activity of Prx1 but not to the Cys-dependent thioredoxin peroxidase (POX) activity, and we generated mutants lacking POX and/or CAT activities for individually delineating their functional features. We discovered that the TnPrx1 POX and CAT activities possessed different kinetic features in reducing H2O2. The overexpression of wild-type TnPrx1 and mutants differentially regulated the intracellular levels of reactive oxygen species and p38 phosphorylation in HEK-293T cells treated with H2O2. These observations suggest that the dual antioxidant activities of Prx1 may be crucial for organisms to mediate intracellular redox homeostasis.

Dendritic cells (DCs) are among the most important professional antigen-presenting cells (APCs) that participate in various biological activities in mammals. However, evidence of the existence of DCs in teleost fish and other lower vertebrates remains limited. In this study, phenotypic and functional characteristics of teleost DCs were described in a zebrafish model. An improved method to efficiently enrich DCs was established. Immunofluorescence staining revealed that the surface phenotypic hallmarks of mammalian DCs, including MHC-II, CD80/86, CD83, and CD209, were distributed on the surfaces of zebrafish DCs (DrDCs). Functional analysis results showed that DrDCs could initiate antigen-specific CD4(+) T cell activation, in which MHC-II, CD80/86, CD83, and CD209 are implicated. Hence, teleost DCs exhibit conserved immunophenotypes and functions similar to those of their mammalian counterparts. Our findings contributed to the current understanding of the evolutionary history of DCs and the DC-regulatory mechanisms of adaptive immunity.

The mechanism underlying how transcription factors regulate mesenchymal stem cell lineage commitment remains unclear. To determine the role of core-binding factor subunit beta (Cbfβ) in osteoblast lineage commitment, we generated three mouse models by deleting Cbfβ at different osteoblast lineage stages. We demonstrated that the Cbfβf/fPrx1-Cre, Cbfβf/fCol2α1-Cre, and Cbfβf/fOsx-Cre mice exhibited severe osteoporosis with substantial accumulation of marrow adipocytes resembling aged bone from enhanced adipogenesis, indicating that mesenchymal stem cells and osteoblasts can be programed and reprogramed, respectively, into adipocytes. Consistently, Cbfβ-deficient calvarial cells and bone marrow mesenchymal stem cells displayed strong adipogenic potential, with 5- to ∼70-fold increased adipocyte gene expression, which can be rescued by Cbfβ overexpression. Canonical Wnt signaling was impeded in the Cbfβ-deficient cells, with ∼80% decrease of Wnt10b expression. Accordingly, ChIP and luciferase assays demonstrated that Cbfβ/RUNX2 binds to Wnt10b promoter driving Wnt10b expression. Furthermore, Wnt3a suppressed adipogenesis but did not rescue osteoblastogenesis in Cbfβ-deficient cells. Notably, mixing culture of Cbfβ-deficient with normal cells demonstrates that Cbfβ functions not only through WNT paracrine pathway but also through endogenous signaling. Further analysis shows that Cbfβ/RUNX2 inhibits c/ebpα expression at transcriptional level. Our results show that, besides its osteogenic role, Cbfβ governs osteoblast−adipocyte lineage commitment both cell nonautonomously through enhancing β-catenin signaling and cell autonomously through suppressing adipogenesis gene expression to maintain osteoblast lineage commitment, indicating Cbfβ may be a therapeutic target for osteoporosis.

Core-binding factor β (Cbfβ) is a subunit of the Cbf family of heterodimeric transcription factors, which plays a critical role in skeletal development through its interaction with the Cbfα subunits, also known as Runt-related transcription factors (Runxs). However, the mechanism by which Cbfβ regulates cartilage and bone development remains unclear. Existing Cbfβ-deficient mouse models cannot specify the role of Cbfβ in skeletal cell lineage. Herein, we sought to specifically address the role of Cbfβ in cartilage and bone development by using a conditional knockout (CKO) approach. A mesenchymal-specific Cbfβ CKO mouse model was generated by using the Dermo1-Cre mouse line to specifically delete Cbfβ in mesenchymal stem cells, which give rise to osteoblasts and chondrocytes. Surprisingly, the mutant mice had under-developed larynx and tracheal cartilage, causing alveolus defects that led to death shortly after birth from suffocation. Also, the mutant mice exhibited severe skeletal deformities from defective intramembranous and endochondral ossification, owing to delayed chondrocyte maturation and impaired osteoblast differentiation. Almost all bones of the mutant mice, including the calvariae, vertebrae, tibiae, femurs, ribs, limbs and sternums were defective. Importantly, we showed that Cbfβ was expressed throughout the skeleton during both embryonic and postnatal development, which explains the multiple-skeletal defects observed in the mutant mice. Consistently, Cbfβ deficiency impaired both chondrocyte proliferation and hypertrophy zone hypertrophy during growth-plate development in the long bones of mutant mice. Notably, Cbfβ, Runx1 and Runx2 displayed different expression patterns in the growth plates of the wild-type mice, indicating that Cbfβ/Runx1 complex and Cbfβ/Runx2 complex may regulate chondrocyte proliferation and hypertrophy, respectively, in a spatial and temporal manner. Cbfβ deletion in the mesenchymal progenitors affected bone development by dramatically down-regulating Collagen X (Col X) and Osterix (Osx) but had a dispensable effect on osteoclast development. Collectively, the results demonstrate that Cbfβ mediates cartilage and bone development by interacting with Runx1 and Runx2 to regulate the expressions of Col X and Osx for chondrocyte and osteoblast development. These findings not only reveal a critical role for Cbfβ in cartilage and bone development but also facilitate the design of novel therapeutic approaches for skeletal diseases.

Recently, a unique population of progenitor cells was isolated from human menstrual blood. The human menstrual blood progenitor cells (MBPCs) possess many advantages, such as the noninvasive acquisition procedure, broad multipotency, a higher proliferative rate, and low immunogenicity, and have attracted extensive attention in regenerative medicine. Preclinical studies to test the safety and efficacy of MBPCs have been underway in several animal models. However, relevant studies in type 1 diabetes mellitus (T1DM) have not yet been proceeded. Herein, we studied the therapeutic effect of MBPCs and the mechanism of β-cell regeneration after MBPC transplantation in the T1DM model. Intravenous injection of MBPCs can reverse hyperglycemia and weight loss, prolong lifespan, and increase insulin production in diabetic mice. Histological and immunohistochemistry analyses indicated that T1DM mice with MBPC transplantation recovered islet structures and increased the β-cell number. We further analyzed in vivo distribution of MBPCs and discovered that a majority of MBPCs migrated into damaged pancreas and located at the islet, duct, and exocrine tissue. MBPCs did not differentiate into insulin-producing cells, but enhanced neurogenin3 (ngn3) expression, which represented endocrine progenitors that were activated. Ngn3(+) cells were not only in the ductal epithelium, but also in the islet and exocrine tissue. We analyzed a series of genes associated with the embryonic mode of β-cell development by real-time polymerase chain reaction and the results showed that the levels of those gene expressions all increased after cell transplantation. According to the results, we concluded that MBPCs stimulated β-cell regeneration through promoting differentiation of endogenous progenitor cells.

The long-held paradigm that B cells cannot uptake nonspecific particulate Ags for the initiation of primary adaptive immunity has been challenged by the recent discovery that teleost B cells have potent phagocytic and microbicidal abilities. This discovery provides preliminary clues that primitive B cells might act as initiating APCs in priming adaptive immunity. In this study, zebrafish B cells clearly showed a potent Ag-presenting ability to both soluble Ags and bacterial particles to prime naive CD4+ T cell activation. This finding demonstrates the innate-like nature of teleost B cells in the interface of innate and adaptive immunity, indicating that they might consist of a major population of initiating APCs whose performance is similar to that of dendritic cells. Given the functional similarities between teleost B cells and the mammalian B-1 subset, we hypothesize that B-1 lineage and teleost B cells might originate from a common ancestor with potent phagocytic and initiating APC capacities. In addition, CD80/86 and CD83 costimulatory signals were identified as being essential for B cell–initiated adaptive immunity. This result suggests that the costimulatory mechanism originated as early as the origin of adaptive immunity and is conserved throughout vertebrate evolution. In fish, only a single CD80/86 copy exists, which is similar to mammalian CD86 rather than to CD80. Thus, CD86 might be a more primordial B7 family member that originated from fish. This study provides valuable insights into the evolutionary history of professional APCs, B cell lineages, and the costimulatory mechanism underlying adaptive immunity as a whole.

Although epigenetic modulation is critical for a variety of cellular activities, its role in erythropoiesis remains poorly understood. Ten-eleven translocation (TET) molecules participate in methylcytosine (5mC) hydroxylation, which results in DNA demethylation in several biological processes. In this research, the role of TETs in erythropoiesis was investigated by using the zebrafish model, where three TET homologs were identified. These homologs share conserved structural domains with their mammalian counterparts. Zebrafish TETs mediate the conversion of 5mC to hydroxymethylcytosine (5hmC) in zebrafish embryos, and the deletion of TET2 inhibits erythropoiesis by suppressing the expression of the scl, gata-1, and cmyb genes. TET2-upregulated lineage-specific genes and erythropoiesis are closely associated with the occurrence of 5hmC and demethylation in the intermediate CpG promoters (ICPs) of scl, gata-1, cmyb, which frequently occur at specific regions or CpG sites of these ICPs. Moreover, TET2 regulates the formation and differentiation of erythroid progenitors, and deletion of TET2 leads to erythrocyte dysplasia and anemia. Here, we preliminarily proved that TET2 plays an essential role in erythrocyte development by regulating lineage-specific genes via DNA oxidative demethylation. This report is anticipated to broaden current information on hematopoiesis and pathogenesis of hematopoiesis-related diseases.

The suppressor of cytokine signaling 1 (SOCS-1) protein is a critical regulator in the immune systems of humans and mammals, which functions classically as an inhibitor of the IFN signaling pathways. However, data on functional characterisation of SOCS-1 in ancient vertebrates are limited. In this study, we report the function of teleost SOCS-1s in IFN signaling in fish models (zebrafish and Tetraodon) and human cells. Structurally, teleost SOCS-1s share conserved functional domains with their mammalian counterparts. Functionally, teleost SOCS-1s could be significantly induced upon stimulation with IFN stimulants and zebrafish IFNφ1. Overexpression of teleost SOCS-1s could dramatically suppress IFNφ1-induced Mx, Viperin and PKZ activation in zebrafish, and IFN-induced ISG15 activation in HeLa cells. Furthermore, a SOCS-1 variant that lacks the KIR domain was also characterised. This study demonstrates the conserved negative regulatory role of teleost SOCS-1s in IFN signaling pathways, providing perspective into the functional conservation of SOCS-1 proteins during evolution.

Despite years of research into bone formation, the mechanisms by which transcription factors specify growth plate development and trabecular bone formation remain unclear and the role of hypertrophic chondrocytes in trabeculae morphogenesis is controversial. To study the role of Core binding factor beta (Cbfβ) in postnatal cartilage development and endochondral bone formation, we generated chondrocyte-specific Cbfβ-deficient mice (Cbfβf/fCol2α1-Cre mice) using floxed alleles of Cbfβ (Cbfβf/f) and Cre driven by the Collagen 2α1 promoter (Col2α1-Cre). Cbfβf/fCol2α1-Cre mice evaded developmental and newborn lethality to survive to adulthood and displayed severe skeletal malformation. Cbfβf/fCol2α1-Cre mice had dwarfism, hypoplastic skeletons, defective bone mineralization, shortened limbs, shortened sternum bodies, and un-calcified occipital bones and hyoid bones. In the long bone cartilage, the resting zone was elongated, and chondrocyte proliferation and hypertrophy were impaired in Cbfβf/fCol2α1-Cre mice, which led to deformation of the growth plates. Primary spongiosa formation was delayed, diaphysis was shortened and trabecular bone formation was almost absent in the mutant mice. In addition, lamellar bone formation in the secondary spongiosa was also impaired. However, osteoclast formation in the trabecular bone was not affected. Cbfβ deficiency led to down-regulation of chondrocyte-regulating genes [i.e, patched (Ptc1), Cyclin D1 and Indian hedgehog (Ihh)] in the cartilage. Interestingly, the expression of Runx2 and Runx3 was not changed in the cartilage of the mutants. Collectively, the results revealed that Cbfβ is crucial for postnatal skeletal development and endochondral bone formation through its function in growth plate development and chondrocyte proliferation and differentiation. This study also revealed that chondrocyte maturation, mediated by Cbfβ, was critical to trabecular bone morphogenesis. Significantly, these findings provide insight into the role of Cbfβ in postnatal skeletogenesis, which may assist in the development of new therapies for osteoporosis.

Single Ig IL-1R–related molecule (SIGIRR, also called IL-1R8 or Toll/IL-1R [TIR]8), a negative regulator for Toll/IL-1R signaling, plays critical roles in innate immunity and various diseases in mammals. However, the occurrence of this molecule in ancient vertebrates and its function in liver homeostasis and disorders remain poorly understood. In this study, we identified a SIGIRR homology from zebrafish (Danio rerio [DrSIGIRR]) by using a number of conserved structural and functional hallmarks to its mammalian counterparts. DrSIGIRR was highly expressed in the liver. Ablation of DrSIGIRR by lentivirus-delivered small interfering RNA in the liver significantly enhanced hepatic inflammation in response to polyinosinic-polycytidylic acid [poly(I:C)] stimulation, as shown by the upregulation of inflammatory cytokines and increased histological disorders. In contrast, depletion of TIR domain–containing adaptor inducing IFN-β (TRIF) or administration of TRIF signaling inhibitor extremely abrogated the poly(I:C)-induced hepatic inflammation. Aided by the zebrafish embryo model, overexpression of DrSIGIRR in vivo significantly inhibited the poly(I:C)- and TRIF-induced NF-κB activations; however, knockdown of DrSIGIRR promoted such activations. Furthermore, pull-down and Duolink in situ proximity ligation assay assays showed that DrSIGIRR can interact with the TRIF protein. Results suggest that DrSIGIRR plays an inhibitory role in TRIF-mediated inflammatory reactions by competitive recruitment of the TRIF adaptor protein from its TLR3/TLR22 receptor. To our knowledge, this study is the first to report a functional SIGIRR homolog that existed in a lower vertebrate. This molecule is essential to establish liver homeostasis under inflammatory stimuli. Overall, the results will enrich the current knowledge about SIGIRR-mediated immunity and disorders in the liver.

The objective of this study was to investigate the expression of netrin-1 in placenta from patients with fetal growth restriction (FGR) and its effect on the viability and apoptosis of human placental microvascular endothelial cells. Thirty-three patients with FGR (including eighteen severe cases) and twenty-four normal late pregnant women were investigated. The expression of netrin-1 in placental tissues was detected by employing immunohistochemistry, real-time PCR, and Western blotting. Human placental microvascular endothelial cells were isolated and, after treatment with netrin-1, examined for their viability and apoptosis by using MTT assay and flow cytometry. We demonstrated that the netrin-1 was present in placenta. Netrin-1 was significantly reduced in pregnant women with FGR as compared with the controls. Furthermore, netrin-1 enhanced the viability of human placental microvascular endothelial cells and inhibited their apoptosis. Netrin-1 may regulate the development of placental vessels and plays a key role in the pathogenesis of FGR.

C1qs are key components of the classical complement pathway. They have been well documented in human and mammals, but little is known about their molecular and functional characteristics in fish. In the present study, full-length cDNAs of c1qA, c1qB, and c1qC from zebrafish (Danio rerio) were cloned, revealing the conservation of their chromosomal synteny and organization between zebrafish and other species. For functional analysis, the globular heads of C1qA (ghA), C1qB (ghB), and C1qC (ghC) were expressed in Escherichia coli as soluble proteins. Hemolytic inhibitory assays showed that hemolytic activity in carp serum can be inhibited significantly by anti-C1qA, -C1qB, and -C1qC of zebrafish, respectively, indicating that C1qA, C1qB, and C1qC are involved in the classical pathway and are conserved functionally from fish to human. Zebrafish C1qs also could specifically bind to heat-aggregated zebrafish IgM, human IgG, and IgM. The involvement of globular head modules in the C1q-dependent classical pathway demonstrates the structural and functional conservation of these molecules in the classical pathway and their IgM or IgG binding sites during evolution. Phylogenetic analysis revealed that c1qA, c1qB, and c1qC may be formed by duplications of a single copy of c1qB and that the C1q family is, evolutionarily, closely related to the Emu family. This study improves current understanding of the evolutionary history of the C1q family and C1q-mediated immunity.

Recent studies suggest that mesenchymal stem cells (MSCs) possess a greater differentiation potential than once thought and that they have the capacity to regenerate damaged tissues/organs. However, the evidence is insufficient, and the mechanism governing the recruitment and homing of MSCs to these injured sites is not well understood. We first examined the MSCs circulating in peripheral blood and then performed chemotaxis, wound healing and tubule-formation assays to investigate the migration capability of mouse bone marrow MSCs (mBM-MSCs) in response to liver-injury signals. In addition, BM-MSCs from donor enhanced green fluorescent protein transgenic male mice were transplanted into liver-injured co-isogenic female recipients, either by intra-bone marrow injection or through the caudal vein, to allow in vivo tracking analysis of the cell fate after transplantation. Donor-derived cells were analysed by in vivo imaging analysis, PCR, flow cytometry and frozen sections. Microarray and real-time PCR were used for chemokine/cytokine and receptor analyses. We successfully isolated circulating MSCs in peripheral blood of liver-injured mice and provided direct evidence that mBM-MSCs could be mobilized into the circulation and recruited into the liver after stimulation of liver injury. CCR9, CXCR4 and c-MET were essential for directing cellular migration towards the injured liver. The recruited mBM-MSCs may play different roles, including hepatic fate specification and down-regulation of the activity of hepatic stellate cells which inhibits over-accumulation of collagen and development of liver fibrosis. Our results provide new insights into liver repair involving endogenous BM-MSCs and add new information for consideration when developing clinical protocols involving the MSCs.

Mesenchymal stem cells were initially characterized as plastic adherent, fibroblastoid cells. In recent years, there has been an increasing focus on mesenchymal stem cells since they have great plasticity and are potential for therapeutic applications. Mesenchymal stem cells or mesenchymal stem cell-like cells have been shown to reside within the connective tissues of most organs. These cells can differentiate into osteogenic, adipogenic and chondrogenic lineages under appropriate conditions. A number of reports have also indicated that these cells possess the capacity to trans-differentiate into epithelial cells and lineages derived from the neuro-ectoderm, and in addition, mesenchymal stem cells can migrate to the sites of injury, inflammation, and to tumors. These properties of mesenchymal stem cells make them promising candidates for use in regenerative medicine and may also serve as efficient delivery vehicles in site-specific therapy.

Lipopolysaccharide (LPS), the endotoxin of Gram-negative bacteria, is capable of eliciting a wide variety of pathophysiological effects, including endotoxin shock, tissue injury and lethality in both humans and animals. It is also a potent stimulant to initiate the proliferation, differentiation and activation of B lymphocytes and macrophages, resulting in changes of inflammatory cytokines, such as TNF-alpha, IL1-beta, IL6, IL-8 and IL-12, and enhancement of immune responses. However, little is known about its effect on the induction of apoptosis in lymphocytes. In the present study, the lymphocytes from Carassius auratus were employed for this purpose. The cells were exposed to LPS at various doses for different time periods. By careful apoptotic characteristic analysis, such as condensation of nuclear chromatin, fragmentation of genomic DNA and formation of apoptotic bodies, it provided the first evidence that LPS had apoptotic-inducing effect on fish lymphocytes in a time- and dose-dependent manner. LPS exposure induced significant increase of intracellular reactive oxygen species (ROS), loss of mitochondrial transmembrane potential (DeltaPsi), depletion of ATP production, down-regulation of Bcl-2 expression, up-regulation of Bax and mitochondrial NO-synthase (mNOS) expression, and selective activation of caspase-9 rather than caspase-8. Each of these observations suggests that the LPS-induced apoptosis in C. auratus lymphocytes occurs largely via the mitochondrial apoptotic pathway. This observation was different from the mechanism behind the LPS-induced apoptosis in mammalian macrophages/thymocytes that occurs via the TNF-alpha-mediated death-receptor pathway. Our study suggested the existence of a possible novel role in the pathogenesis of Gram-negative bacterial infection in fish and even in mammals, which may contribute to the therapy of bacterial diseases. Also, it will help to gain more insights into the mechanisms of septic shock and of LPS-induced immunosuppression and autoimmunity.

Pycnodysostosis is a genetic bone disease featuring the unique bone homeostasis disorders of osteolysis and osteopetrosis in the same organism. The pathomechanism for pycnodysostosis has been largely unknown due to the unavailability of a pycnodysostosis mouse model with all the traits of the disease. We generated cathepsin K(-/-) mouse strains in the 129/Sv and C57BL/6J backgrounds and found that, only in the 129/Sv background, cathepsin K(-/-) mice exhibit many characteristics of the human pycnodysostosis-like phenotype. Our data indicated that 129/Sv cathepsin K(-/-) osteoclasts (OCs) lacked normal apoptosis and senescence and exhibited over-growth both in vitro and in vivo. These abnormalities resulted in an unusually high OC number, which is consistent with a recent case study of human pycnodysostosis. Our results show that cathepsin K function has different effects around the skeleton due to site-specific variations in bone homeostasis, such as phenotypes of osteopetrosis in tibiae and osteolysis in calvariae as a result of cathepsin K mutation. Our data demonstrated that the expression levels of p19, p53 and p21 were significantly reduced in 129/Sv cathepsin K(-/-) OCs and forced expression of cathepsin K in pre-OCs induced premature senescence and increased expression of p19, p53 and p21. This is the first evidence that cathepsin K plays a key role in OC apoptosis and senescence, revealing the importance of OC senescence in bone homeostasis. The finding of this novel cathepsin K function provides insight into the pathomechanism of pycnodysostosis and may provide new drug targets for diseases involved in OC-related abnormal bone homeostasis.

Recently it was shown that embryonic stem (ES) cells could differentiate into hepatocytes both in vitro and in vivo, however, prospective hepatic progenitor cells have not yet been isolated and characterized from ES cells. Here we presented a novel 4-step procedure for the differentiation of mouse ES cells into hepatic progenitor cells and then hepatocytes. The differentiated hepatocytes were identified by morphological, biochemical, and functional analyses. The hepatic progenitor cells were isolated from the cultures after the withdrawal of sodium butyrate, which was characterized by scant cytoplasm, ovoid nuclei, the ability of rapid proliferation, expression of a series of hepatic progenitor cell markers, and the potential of differentiation into hepatocytes and bile duct-like cells under the proper conditions that favor hepatocyte and bile epithelial differentiation. The differentiation of hepatocytes from hepatic progenitor cells was characterized by a number of hepatic cell markers including albumin secretion, upregulated transcription of glucose-6-phosphatase and tyrosine aminotransferase, and functional phenotypes such as glycogen storage. The results from our experiments demonstrated that ES cells could differentiate into a novel bipotential hepatic progenitor cell and mature into hepatocytes with typical morphological, phenotypic and functional characteristics, which provides an useful model for the studies of key events during early liver development and a potential source of transplantable cells for cell-replacement therapies.

The differentiation potential of adult stem cells has long been believed to be limited to the tissue or germ layer of their origin. However, recent studies have demonstrated that adult stem cells may encompass a greater potential than once thought. In the present study, we examined whether murine bone marrow derived stromal stem cells (BMSSCs) are able to differentiate into functional hepatocytes in vitro. BMSSCs were isolated from murine femora and tibiae, and the mesodermal multilineage differentiation potentials of these cells were functionally characterized. To effectively induce hepatic differentiation, we designed a novel protocol by using hepatocyte-conditioned medium. Hepatic differentiation from mouse BMSSCs was examined by a variety of assays at morphological and molecular levels. Morphologically, mouse BMSSCs became round and epithelioid, binucleated after induction. Differentiated cells were harvested on Days 0, 10, and 20 and subjected to examination of hepatocyte characteristics by reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry. We detected AFP, HNF-3β, CK19, CK18, ALB, TAT, and G-6-Pase at the mRNA and/or protein levels, hepatocyte-like cells by culture in conditioned medium further demonstrated in vitro functions characteristic of liver cells, including glycogen storage, and urea secretion. Moreover, transplantation of the differentiated cells into liver-injured mice partially restored serum albumin level and significantly suppressed transaminase activity. Our findings indicated the transdifferentiation potential of mouse BMSSCs developing into the functional hepatocyte-like cells by conditioned culture medium and, hence, may serve as a model system for the study of mechanisms involved in the transdifferentiation, and a cell source for cell therapy of hepatic diseases. J. Cell. Biochem. J. Cell. Biochem. 102: 52–63, 2007. © 2007 Wiley-Liss, Inc.

Embryonic stem (ES) cells, derived from blastocyst-stage of early mammalian embryos, have the potential to differentiate into derivatives of all three embryonic germ layers. Here we reported the first evidence that murine pluripotent ES cells could be induced to differentiate into cardiomyocytes by cyclic adenosine 3',5'-monophosphate (cAMP) in vitro. Spontaneously beating of cardiac cell clusters began to be observed within the outgrowths of embryoid bodies (EBs) as early as 2 days after the onset of differentiation. By days 5-8 after induction, a maximum level of cardiomyocyte differentiation could be achieved. Incubation of EBs with cAMP at concentrations ranging from 0.01 mg/L to 1 mg/L resulted in a significant elevation in differentiation rate, reaching a maximum value of 44.0 +/- 1.3% at 0.03 mg/L of exposure. At 0.03 mg/L concentration point, an approximately 8.1-fold increase in cardiomyocyte differentiation was observed in comparison with 5.4 +/- 0.9% of untreated controls. The differentiation rate induced by cAMP was shown to be similar to that of RA/DMSO treated controls, indicating that cAMP has the same inducing effect as RA/DMSO. However, no significant co-inducing effects between cAMP and RA/DMSO were seen. Cardiomyocytes were evident as they expressed cardiac cell specific genes and protein markers including GATA4, Nkx2.5, beta-MHC, atrial natriuretic factor (ANF) and alpha-actin when analyzed by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining. The results from the present study suggested a novel role of cAMP in cardiomyocyte differentiation and provided a new research model for the study of cardiac cell biology.

During the past few years multiple studies have revealed that adult stem cells, including BM origin stem cells, can be transdifferentiated into various cell types, including hepatocyte-like cells, under proper treatments or in a suitable microenvironment. However, little is known about the mechanism of the transdifferentiation, and the treatments employed seem to be very complicated and require simplification. It is important to determine the suitable conditions in which BM cells would be efficiently differentiated into hepatocytes.Mouse BM cells were isolated from femurs and tibias and cultured in IMDM supplemented with 10% FBS. Hepatic differentiation was induced in a differentiation medium containing 20 ng/mL HGF, 10 ng/mL FGF-4, 10 ng/mL Oncostatin M (OSM) and different concentrations of liver-injured mouse sera. The differentiated hepatic cells were characterized by the expression of liver-associated mRNA and proteins and morphologic and functional features.BM cell-derived polygonal cell colonies appeared after several days of culture, and these hepatocyte-like cells expressed AFP, HNF-3beta, CK19, CK18, ALB, TAT and G-6-Pase at mRNA and protein levels, and the cells also had some hepatic cellular functions, such as glycogen storage and urea production. Interestingly, suitable concentrations of sera from liver-injured mice added to this system showed strong stimulation on the in vitro transdifferentiation of mouse BM cells into hepatocytes.In the present study we have established an effective hepatic differentiation system by a combination of HGF, FGF-4, OSM and liver-injured mouse sera in vitro. Accordingly, it will be a useful resource not only for understanding the mechanisms of transdifferentiation but also for efficient amplification of hepatocyte progenitor cells of BM origin.

Atrazine is a widely used herbicide that was considered to be an endocrine disrupter capable of interfering with the synthesis and action of natural hormones. In the present study, we found that atrazine was able to cause apoptosis in grass carp (Ctenopharyngodon idellus) cells from cell line ZC7901. By fluorescent and transmission electron microscopy, the atrazine-incubated cells displayed a series of morphological changes, including condensation of the nucleus, margination of chromatin to form dense granular caps, and formation of apoptotic bodies. Moreover, DNA fragmentation was detected by the TUNEL reaction and agarose gel electrophoresis. These typical characteristics of cells undergoing apoptosis indicated the occurrence of apoptosis in ZC7901. Apoptosis induced by atrazine was dose- and time-dependent and was involved in mitochondrial membrane potential (ΔΨm) disruption, elevation in intracellular Ca2+, generation of reactive oxygen species, and intracellular ATP depletion. This study provides the first evidence that atrazine was able to induce apoptosis in fish cells, which indicated the existence of a novel cytotoxic mechanism caused by atrazine and may improve our understanding of the complex relationship between contaminants and aquatic organisms. © 2006 Wiley Periodicals, Inc. Environ Toxicol 21: 80–89, 2006.

Embryoid bodies, which are similar to post-implantation egg-cylinder stage embryos, provide a model for the study of embryo development and stem cell differentiation. We describe here a novel method for generating embryoid bodies from murine embryonic stem (ES) cells cultured on the STO feeder layer. The ES cells grew into compact aggregates in the first 3 days of coculture, then became simple embryoid bodies (EBs) possessing primitive endoderm on the outer layer. They finally turned into cystic embryoid bodies after being transferred to Petri dishes for 1-3 days. Evaluation of the EBs in terms of morphology and differentiating potential indicates that they were typical in structure and could generate cells derived from the three germ layers. The results show that embryoid bodies can form not only in suspension culture but also directly from ES cells cultured on the STO feeder layer.

Wnt signaling affects both bone modeling, which occurs during development, and bone remodeling, which is a lifelong process involving tissue renewal. Wnt signals are especially known to affect the differentiation of osteoblasts. In this review, we summarize recent advances in understanding the mechanisms of Wnt signaling, which is divided into two major branches: the canonical pathway and the noncanonical pathway. The canonical pathway is also called the Wnt/β-catenin pathway. There are two major noncanonical pathways: the Wnt-planar cell polarity pathway (Wnt-PCP pathway) and the Wnt-calcium pathway (Wnt-Ca2+ pathway). This review also discusses how Wnt ligands, receptors, intracellular effectors, transcription factors, and antagonists affect both the bone modeling and bone remodeling processes. We also review the role of Wnt ligands, receptors, intracellular effectors, transcription factors, and antagonists in bone as demonstrated in mouse models. Disrupted Wnt signaling is linked to several bone diseases, including osteoporosis, van Buchem disease, and sclerosteosis. Studying the mechanism of Wnt signaling and its interactions with other signaling pathways in bone will provide potential therapeutic targets to treat these bone diseases.

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg cells) are critical for the maintenance of peripheral tolerance, and the suppression of autoimmune diseases and even tumors. Although Treg cells are well characterized in humans, little is known regarding their existence or occurrence in ancient vertebrates. In the present study, we report on the molecular and functional characterization of a Treg-like subset with the phenotype CD4-2(+)CD25-like(+)Foxp3-like(+) from a pufferfish (Tetraodon nigroviridis) model. Functional studies showed that depletion of this subset produced an enhanced mixed lymphocyte reaction (MLR) and nonspecific cytotoxic cell (NCC) activity in vitro, as well as inflammation of the intestine in vivo. The data presented here will not only enrich the knowledge of fish immunology but will also be beneficial for a better cross-species understanding of the evolutionary history of the Treg family and Treg-mediated regulatory networks in cellular immunity.

Dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN/CD209) has become hot topic in recent studies because of its important roles in immune responses and immune escape. CD209 has been well characterized in humans and several other mammals, but little documentation exists about it in lower vertebrates. This is the first report on the identification and functional characterization of a fish DC-SIGN/CD209 molecule. The zebrafish DC-SIGN/CD209 cDNA translates into 343 aa organized into three domains structurally conserved among vertebrates. An EPN motif essential for interacting with Ca2+ and for recognizing mannose-containing motifs has been identified. Several conserved motifs crucial for internalization and signal transduction are also present within the cytoplasmic tail. Phylogenetic analysis supports the hypothesis that CD209 family members diverged from a common ancestor. The expression of DC-SIGN/CD209 in immune-related tissues can be significantly up-regulated by exogenous Ags and IL-4. This molecule associates with various APCs, including macrophages, B lymphocytes, and a possible dendritic cell-like (CD83+/CD80+CD209+) population. Functionally, T cell activation, Ab (IgM) production, and bacterial vaccination-elicited immunoprotection can be dramatically inhibited by a CD209 blockade after stimulation with keyhole limpet hemocyanin (KLH) in vivo or challenged with Aeromonas hydrophila, suggesting that DC-SIGN/CD209 in zebrafish is crucial for the initiation and development of adaptive immunity. Phagocytosis analysis showed that DC-SIGN/CD209 does not participate in the uptake of KLH Ag, suggesting that other mechanisms might exist that underlie DC-SIGN/CD209 involvement. We hope that the present study will contribute to a better cross-species understanding of the evolutionary history of the DC-SIGN/CD209 family.

Mesenchymal stem cells (MSCs) are considered to be one of the most promising therapeutic cell sources as they encompass a plasticity of multiple cell lineages. The challenge in using these cells lies in developing well-defined protocols for directing cellular differentiation to generate a desired lineage. In this study, we investigated the effect of 5-azacytidine, a DNA demethylating agent, on osteogenic differentiation of MSCs. The cells were exposed to 5-azacytidine in culture medium for 24 h prior to osteogenic induction. Osteogenic differentiation was determined by several the appearance of a number of osteogenesis characteristics, including gene expression, ALP activity, and calcium mineralization. Pretreatment of MSCs with 5-azacytidine significantly facilitated osteogenic differentiation and was accompanied by hypomethylation of genomic DNA and increased osteogenic gene expression. Taking dlx5 as a representative, methylation alterations of the “CpG island shore” in the promoter caused by 5-azacytidine appeared to contribute to osteogenic differentiation.

The pre-B cell colony-enhancing factor identified in mammals is an important cytokine involved in multiple functions, such as immunoregulation, cellular proliferation and differentiation. However, little is known about its existence and function in lower vertebrates; therefore, we investigated the characterization, expression and especially the biofunction of this factor in Tetraodon nigroviridis, a model organism of lower vertebrates. We focus on the question of whether the pre-B cell colony-enhancing factor of lower vertebrates contributes to the NAD mediated antioxidant activity by its involvement in the biosynthesis of NAD through pyridine nucleotide cycles. Experimental data demonstrated that by transforming fish pre-B cell colony-enhancing factor into Escherichia coli cells, the amounts of NAD and NADP significantly increased, and cellular antioxidant activity greatly improved. This is the first report about vertebrate pre-B cell colony-enhancing factor acting as a nicotinamide phosphoribosyltransferase to reconstruct a pyridine nucleotide cycle III pathway in E. coli and playing an important role in antioxidant stress by up-regulation of NAD biosynthesis. We hope that our observations may enrich the study of pre-B cell colony-enhancing factor, and contribute to a better understanding of the molecular and functional evolution of the pre-B cell colony-enhancing factor family in both lower vertebrates and mammals as a whole.

Bone morphogenetic proteins (BMPs) are multi-functional growth factors belonging to the transforming growth factor-beta superfamily. It has been demonstrated that BMPs had been involved in the regulation of cell proliferation, survival, differentiation and apoptosis. However, their hallmark ability is that play a pivotal role in inducing bone, cartilage, ligament, and tendon formation at both heterotopic and orthotopic sites. In this review, we mainly concentrate on BMP structure, function, molecular signaling and potential medical application.

The vacuolar H(+)-ATPase (V-ATPase) is a universal component of eukaryotic organisms, which is present in both intracellular compartments and the plasma membrane. In the latter, its proton-pumping action creates the low intravacuolar pH, benefiting many processes such as, membrane trafficking, protein degradation, renal acidification, bone resorption, and tumor metastasis. In this article, we briefly summarize recent studies on the essential and diverse roles of mammalian V-ATPase and their medical applications, with a special emphasis on identification and use of V-ATPase inhibitors.

The feasibility of transforming embryonic endoderm into different cell types is tightly controlled by mesodermal and septum transversumal signalings during early embryonic development. Here, an induction protocol tracing embryonic liver development was designed, in which, three growth factors, acid fibroblast growth factor, basic fibroblast growth factor and bone morphological protein-4 that secreted from pre-cardiac mesoderm and septum transversum mesenchyme, respectively, were employed to investigate their specific potency of modulating the mature hepatocyte proportion during the differentiation process. Results showed that hepatic differentiation took place spontaneously at a low level, however, supplements of the three growth factors gave rise to a significant up-regulation of mature hepatocytes. Bone morphological protein-4 highlighted the differentiation ratio to 40-55%, showing the most effective promotion, and also exhibited a synergistic effect with the other two fibroblast factors, whereas no similar phenomenon was observed between the other two factors, which was reported for the first time. Our study not only provides a high-performance system of embryonic stem cells differentiating into hepatocytes, which would supply a sufficient hepatic population for related studies, but also make it clear of the inductive effects of three important growth factors, which could support for further investigation on the mechanisms of mesodermal and septumal derived signalings that regulate hepatic differentiation.

Osteoclasts, the bone resorbing cells, play a key role both in normal bone remodeling and in the skeletal osteopenia of arthritis, osteoporosis, periodontal disease and certain malignancies. Osteoclast cellular commitment, differentiation and function depend upon the establishment of specific patterns of gene expression achieved through networks of transcription factors activated by osteoclastogenic cytokines. This review is an updated look at the various transcription factors and cytokines that have been demonstrated to play critical roles in osteoclast differentiation and function, along with their known animal models, such as: PU.1, Mcsf, RANKL, NF-kappaB, AP-1, NFATc1, Mitf, Myc, and Src. Further studies on these transcription factors and cytokines will not only expand our basic understanding of the molecular mechanisms of osteoclast differentiation, but will also aid our ability to develop therapeutic means of intervention in osteoclast-related diseases.

We investigated the role of apoptosis in malathion-induced cytotoxicity in the grass carp (Ctenopharyngodon idellus) cell line ZC-7901. Fish cells were treated with different concentrations of malathion (0.62-95 mg/L), and the IC(50) ranged from 37.94+/-1.93 mg/L for 12 h to 3.04+/-0.27 mg/L for 72 h by the MTT assay. Apoptosis was detected by confocal laser scanning microscopy, transmission electron microscopy, TUNEL reaction, DNA laddering and a flow cytometric PI staining assay. The results demonstrated that apoptosis was involved in the cytotoxic effect of malathion, and that malathion-induced apoptosis occurred in a dose- and time-dependent manner. In addition, the induction of apoptosis by malathion was accompanied by mitochondrial membrane potential (DeltaPsi(m)) disruption, intracellular Ca(2+) elevation, generation of reactive oxygen species (ROS) and ATP depletion. Our investigation suggested that malathion exerts its cytotoxic effects by the induction of apoptosis via a direct effect on the mitochondria.

BACKGROUND: Spontaneous and stress-induced germ cell apoptosis during spermatogenesis of multicellular organisms have been investigated broadly in mammals. Spermatogenetic process in urodele amphibians was essentially like that in mammals in spite of morphological differences; however, the mechanism of germ cell apoptosis in urodele amphibians remains unknown. The Chinese fire-belly newt, Cynops orientalis, was an excellent organism for studying germ cell apoptosis due to its sensitiveness to temperature, strong endurance of starvation, and sensitive skin to heavy metal exposure. METHODOLOGY/PRINCIPAL FINDINGS: TUNEL result showed that spontaneous germ cell apoptosis took place in normal newt, and severe stress-induced apoptosis occurred to spermatids and sperm in response to heat shock (40°C 2 h), cold exposure (4°C 12 h), cadmium exposure (Cd 36 h), and starvation stress. Quantitative reverse transcription polymerase chain reactions (qRT-PCR) showed that gene expression of Caspase3 or Caspase7 was obviously elevated after stress treatment. Apaf1 was not altered at its gene expression level, and p53 was significantly decreased after various stress treatment. Caspase assay demonstrated that Caspase-3, -8, -9 enzyme activities in newt testis were significantly elevated after heat shock (40°C 2 h), cold exposure (4°C 12 h), and cadmium exposure (Cd 36 h), while Caspase3 and Caspase8 activities were increased with Caspase9 significantly decreased after starvation treatment. CONCLUSIONS/SIGNIFICANCE: Severe germ cell apoptosis triggered by heat shock, cold exposure, and cadmium exposure was Caspase3 dependent, which probably involved both extrinsic and intrinsic pathways. Apaf1 may be involved in this process without elevating its gene expression. But starvation-induced germ cell apoptosis was likely mainly through extrinsic pathway. p53 was probably not responsible for stress-induced germ cell apoptosis in newt testis. The intriguing high occurrence of spermatid and sperm apoptosis probably resulted from the sperm morphology and unique reproduction policy of Chinese fire-belly newt, Cynops orientalis.