FcγR-dependent apoptosis regulates tissue persistence of mucosal and connective tissue mast cells.

Rodent mast cells can be divided into two major subtypes: the mucosal mast cell (MMC) and the connective tissue mast cell (CTMC). A decade-old observation revealed a longer lifespan for CTMC compared with MMC. The precise mechanisms underlying such differential tissue persistence of mast cell subsets have not been described. In this study, we have discovered that mast cells expressing only one receptor, either FcγRIIB or FcγRIIIA, underwent caspase-independent apoptosis in response to IgG immune complex treatment. Lower frequencies of CTMC in mice that lacked either FcγRIIB or FcγRIIIA compared with WT mice were recorded, especially in aged mice. We proposed that this paradigm of FcγR-mediated mast cell apoptosis could account for the more robust persistence of CTMC, which express both FcγRIIB and FcγRIIIA, than MMC, which express only FcγRIIB. Importantly, we reproduced these results using a mast cell engraftment model, which ruled out possible confounding effects of mast cell recruitment or FcγR expression by other cells on mast cell number regulation. In conclusion, our work has uncovered an FcγR-dependent mast cell number regulation paradigm that might provide a mechanistic explanation for the long-observed differential mast cell subset persistence in tissues.

HIPK2 phosphorylates HDAC3 for NF-κB acetylation to ameliorate colitis-associated colorectal carcinoma and sepsis.

Although inflammation is critical for the clearance of pathogens, uncontrolled inflammation also contributes to the development of multiple diseases such as cancer and sepsis. Since NF-κB-mediated transactivation in the nucleus is pivotal downstream of various stimuli to induce inflammation, searching the nuclear-localized targets specifically regulating NF-κB activation will provide important therapeutic application. Here, we have identified that homeodomain-interacting protein kinase 2 (HIPK2), a nuclear serine/threonine kinase, increases its expression in inflammatory macrophages. Importantly, HIPK2 deficiency or overexpression could enhance or inhibit inflammatory responses in LPS-stimulated macrophages, respectively. HIPK2-deficient mice were more susceptible to LPS-induced endotoxemia and CLP-induced sepsis. Adoptive transfer of Hipk2+/- bone marrow cells (BMs) also aggravated AOM/DSS-induced colorectal cancer. Mechanistically, HIPK2 bound and phosphorylated histone deacetylase 3 (HDAC3) at serine 374 to inhibit its enzymatic activity, thus reducing the deacetylation of p65 at lysine 218 to suppress NF-κB activation. Notably, the HDAC3 inhibitors protected wild-type or Hipk2-/- BMs-reconstituted mice from LPS-induced endotoxemia. Our findings suggest that the HIPK2-HDAC3-p65 module in macrophages restrains excessive inflammation, which may represent a new layer of therapeutic mechanism for colitis-associated colorectal cancer and sepsis.

Mouse hygiene status-A tale of two environments for mast cells and allergy.

Animal models, including those employing the use of house mice (Mus musculus), are crucial in elucidating mechanisms in human pathophysiology. However, it is evident that the impreciseness of using laboratory mice maintained in super-hygienic barrier facilities to mirror relevant aspects of human physiology and pathology exists, which is a major limitation in translating mouse findings to inferring human medicine. Interestingly, free-living wild mice are found to be substantially different from laboratory-bred, specific pathogen-free mice with respect to various immune system compartments. Wild mice have an immune system that better reflects human immunity. In this review article, we discuss recent experimental findings that address the so-called "wild immunology", which reveals the contrasting immune features between laboratory-raised mice and their wild companions as well as laboratory mice that have been exposed to a natural rodent habitat. A particular focus will be given to the development of pulmonary mast cells and its possible impact on the use of "naturalized" or "rewilded" laboratory mice as experimental asthma models.

Role of B Lymphocytes in the Pathogenesis of NAFLD: A 2022 Update.

Non-alcoholic fatty liver disease and its related complications are becoming one of the most important health problems globally. The liver functions as both a metabolic and an immune organ. The crosstalk between hepatocytes and intrahepatic immune cells plays a key role in coordinating a dual function of the liver in terms of the protection of the host from antigenic overload as a result of receiving nutrients and gut microbiota antigenic stimulation via facilitating immunologic tolerance. B cells are the most abundant lymphocytes in the liver. The crucial role of intrahepatic B cells in energy metabolism under different immune conditions is now emerging in the literature. The accumulating evidence has demonstrated that the antibodies and cytokines produced by B cells in the microenvironment play key and distinct roles in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Herein, we have aimed to consolidate and update the current knowledge about the pathophysiological roles of B cells as well as the underlying mechanisms in energy metabolism. Understanding how B cells can exacerbate and suppress liver damage by exploiting the antibodies and cytokines they produce will be of great importance for designing B-cell targeting therapies to treat various liver diseases.

Induction of the apoptosis, degranulation and IL-13 production of human basophils by butyrate and propionate via suppression of histone deacetylation.

Allergic diseases are caused by dysregulated Th2 immune responses involving multiple effector cells including basophils. Short chain fatty acids (SCFAs), mainly acetate, propionate and butyrate, exert immunomodulatory functions via activation of its receptors GPR41 and GPR43, and inhibition of the histone deacetylases (HDACs) activity. In allergic diseases, SCFAs suppress the activity of mast cells, eosinophils and type 2 innate lymphoid cells (ILC2) but enhance the function of Th2 cells. Here, we aimed to elucidate the function of SCFAs on human basophils. Human basophils were purified from healthy donors by flow cytometric sorting. The surface proteins, apoptosis and degranulation of basophils were analyzed by flow cytometric analysis. The mRNA expression was assayed using real-time PCR. Interleukin 4 (IL-4) and IL-13 were measured by ELISA. Histone acetylation was examined by western blot. GPR41 was expressed by basophils and was enhanced by IL-3. Acetate induced intracellular calcium influx in basophils which was suppressed by blocking GPR41. Propionate and butyrate, but not acetate, induced the expression of CD69 and IL-13. In addition, propionate and butyrate enhanced IgE-mediated basophil degranulation but inhibited basophil survival and IL-4 secretion. Propionate and butyrate induced histone acetylation of basophils and suppression of HDACs activity mimicked the effects of propionate and butyrate on human basophils. Our findings demonstrate that propionate and butyrate may play a complex role in regulating basophil apoptosis, activation and degranulation via inhibiting HDACs activity. The in vivo effects of SCFAs on the regulation of basophil-associated allergic diseases need to be further explored.

The Shh/Gli signaling cascade regulates myofibroblastic activation of lung-resident mesenchymal stem cells via the modulation of Wnt10a expression during pulmonary fibrogenesis.

Lung-resident mesenchymal stem cells (LR-MSCs) are important regulators of lung repair and regeneration, and evidence suggests that this cell population also plays a vital role in fibrosis. Crosstalk between sonic hedgehog (Shh) signaling and wingless/integrated (Wnt) has been demonstrated in idiopathic pulmonary fibrosis (IPF). However, the underlying correlation between LR-MSCs and the Shh-Wnt signaling cascade remains poorly understood. Here, we identified Wnt10a as a key factor in pulmonary fibrosis. Using a bleomycin mouse model, we found that highly expressed Wnt10a was secreted by LR-MSCs undergoing myofibroblastic differentiation. LR-MSCs with myofibroblast characteristics isolated from fibrotic lungs exhibited increased Shh pathway activity, suggesting their role as Shh targets. In vitro, LR-MSCs responded to stimulation by recombinant Shh, acquiring a myofibroblast phenotype. We further demonstrated that the Shh/glioblastoma (Gli) system machinery regulated LR-MSC-to-myofibroblast transition and pulmonary fibrosis via manipulation of Wnt/ß-catenin signaling. Accordingly, inhibition of the Shh-Wnt signaling cascade prevented LR-MSC transformation into myofibroblasts and ameliorated pulmonary fibrotic lesions. Moreover, induction of Wnt10a expression and activation of Shh/Gli signaling were confirmed in human pulmonary fibrosis. In summary, this study linking the Shh-Wnt signaling cascade with LR-MSC fibrogenic activity furthered the current understanding of pulmonary fibrosis pathogenesis and might provide a new perspective in the development of treatment strategies for IPF.

Microcystin-leucine-arginine induces liver fibrosis by activating the Hedgehog pathway in hepatic stellate cells.

Microcystin-leucine-arginine (MC-LR), produced by cyanobacteria, accumulates in the liver through blood circulation. We investigated the impact of MC-LR on liver fibrosis. Mice received a daily injection of MC-LR at various concentrations for 14 consecutive days aa and then mouse liver was obtained for histopathological and immunoblot analysis. Next, a human hepatic stellate cell line (LX-2) was treated with MC-LR at various concentrations followed by measurement of cell viability, cell cycle and relevant protein expression levels. Our data confirmed the induction of mouse liver fibrosis after exposure to MC-LR at 15 µg/kg and 30 µg/kg. Furthermore, we demonstrated that LX-2 cells could uptake MC-LR, resulting in cell proliferation and differentiation through impacting the Hedgehog signaling after the treatment of MC-LR at 50 nM. Our data supported that MC-LR could induce liver fibrosis by modulating the expression of the transcription factor Gli2 in the Hedgehog signaling in hepatic stellate cells.

Suppression of p66Shc prevents hyperandrogenism-induced ovarian oxidative stress and fibrosis.

BACKGROUND: Rats with hyperandrogen-induced polycystic ovary syndrome (PCOS) have been shown to develop ovarian oxidative stress (OS) and fibrosis. The Sirt1 agonist, resveratrol, can reduce OS through inhibiting p66Shc in other models of OS. METHODS: We created a rat PCOS model with increased OS levels following treatment with one of the two androgens, dehydroepiandrosterone (DHEA) and dihydrotestosterone (DHT). The PCOS related features were determined by measurement of malondialdehyde (MDA) and superoxide dismutase (SOD) levels or by examining the reactive oxygen species (ROS) levels using the DCF-DA probe. The potential mechanisms by which p66Shc/Sirt1 mediates ovarian fibrosis were explored by western blotting, quantitative reverse transcription-PCR, immunofluorescence staining, and immunohistochemistry. RESULTS: Hyperandrogen dramatically augmented OS and activation of fibrotic factors in the ovary. Our data demonstrated that treatment with resveratrol enhanced Sirt1 and decreased ovarian OS as well as inhibited phosphorylation of p66Shc both in vivo and in vitro. The treatment suppressed fibrotic factor activation and improved ovarian morphology. Lentivirus- or siRNA-mediated p66Shc knockdown resulted in a dramatic enhancement of Sirt1 expression, down-regulation of ROS and suppression of fibrotic factors in granulosa cells. Moreover, p66Shc overexpression markedly increased the expression of fibrotic factors. Additionally, silencing Sirt1 induced a dramatic increase in p66Shc and enhanced activation of fibrotic factors. CONCLUSIONS: p66Shc may be a direct target of Sirt1 for inducing ROS and thus promoting fibrosis. Further exploration of the mechanisms of p66Shc in both fibrosis and OS may provide novel therapeutic strategies that will facilitate the improvement in PCOS symptoms and reproductive functions.

TNF-α-induced NF-κB activation promotes myofibroblast differentiation of LR-MSCs and exacerbates bleomycin-induced pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and irreversible lung disease of unknown cause. It has been reported that both lung resident mesenchymal stem cells (LR-MSCs) and tumor necrosis factor-α (TNF-α) play important roles in the development of pulmonary fibrosis. However, the underlying connections between LR-MSCs and TNF-α in the pathogenesis of pulmonary fibrosis are still elusive. In this study, we found that the pro-inflammatory cytokine TNF-α and the transcription factor nuclear factor kappa B (NF-κB) p65 subunit were both upregulated in bleomycin-induced fibrotic lung tissue. In addition, we discovered that TNF-α promotes myofibroblast differentiation of LR-MSCs through activating NF-κB signaling. Interestingly, we also found that TNF-α promotes the expression of ß-catenin. Moreover, we demonstrated that suppression of the NF-κB signaling could attenuate myofibroblast differentiation of LR-MSCs and bleomycin-induced pulmonary fibrosis which were accompanied with decreased expression of ß-catenin. Our data implicates that inhibition of the NF-κB signaling pathway may provide a therapeutic strategy for pulmonary fibrosis, a disease that warrants more effective treatment approaches.

M2 macrophages promote myofibroblast differentiation of LR-MSCs and are associated with pulmonary fibrogenesis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by the histopathological pattern of usual interstitial pneumonia and is associated with a high mortality rate. Recently, lung resident mesenchymal stem cells (LR-MSCs) have been identified as an important contributor to myofibroblast activation in pulmonary fibrosis. Macrophages are also believed to play a critical role in pulmonary fibrosis. However, the underlying connections between LR-MSCs and macrophages in the pathogenesis of pulmonary fibrosis are still elusive. METHODS: In this study, we investigated the interaction between LR-MSCs and macrophages using a bleomycin-induced mouse pulmonary fibrosis model and a coculture system. RESULTS: Here, we show that blocking pulmonary macrophage infiltration attenuated bleomycin-induced pulmonary fibrosis. In addition, as determined by flow cytometry, we discovered that the recruited macrophages in fibrotic lungs of bleomycin-treated mice were mainly M2 macrophages. In particular, we found that M2, rather than M1 macrophages, promoted myofibroblast differentiation of LR-MSCs. Moreover, we demonstrated that suppression of the Wnt/ß-catenin signaling pathway could attenuate myofibroblast differentiation of LR-MSCs induced by M2 macrophages and bleomycin-induced pulmonary fibrosis. Tissue samples from IPF patients confirmed the infiltration of M2 macrophages and activation of Wnt/ß-catenin signaling pathway. CONCLUSION: In summary, this study furthered our understanding of the pulmonary fibrosis pathogenesis and highlighted M2 macrophages as a critical target for treating pulmonary fibrosis.

Microcystin-leucine arginine inhibits gonadotropin-releasing hormone synthesis in mice hypothalamus.

Microcystin-leucine arginine (MC-LR) causes serum testosterone declines and male reproductive disorders. However, the molecular mechanisms underlying the pathological changes are still unclear. In the present study, we aimed to investigate the toxic effects of MC-LR on gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus. Our results demonstrated that MC-LR could enter GnRH neurons and inhibit GnRH synthesis, resulting in the decrease of serum GnRH and testosterone levels. The inhibitory effects of MC-LR on GnRH synthesis were identified to be associated with activation of the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP response element-binding protein (CREB)/c-Fos signaling pathway. With miRNA microarray analyses, we found that miR-329-3p was down-regulated most dramatically in MC-LR-treated GT1-7 cells. We then further identified that miR-329-3p regulated PRKAR1A and PRKACB expression and thus influenced GnRH synthesis. This is the first study to explore the molecular mechanism underlying the inhibitory effects of MC-LR on GnRH synthesis in the hypothalamus. Our data have provided a new perspective in the development of diagnosis and treatment strategies for male infertility as a result of dysfunction of the hypothalamic-pituitary-gonadal axis.

Inhibition of Wnt/ß-catenin signaling suppresses bleomycin-induced pulmonary fibrosis by attenuating the expression of TGF-ß1 and FGF-2.

Pulmonary fibrosis is a progressive lung disorder of unknown etiology, which is characterized by alterations in alveolar epithelium function, fibroblast activation, and increased extracellular matrix deposition. Recent studies have demonstrated that PF is associated with uncontrolled production of cytokines after lung injury. In the present study, we found that transforming growth factor-ß1 (TGF-ß1) and fibroblast growth factor 2 (FGF-2) were both upregulated in bleomycin-induced fibrotic lung tissue and primary murine alveolar epithelial Type II (ATII) cells treated with bleomycin. Furthermore, we discovered that TGF-ß1 could induce the differentiation of lung resident mesenchymal stem cells (LR-MSCs) into fibroblasts, which may play an essential role in PF. LR-MSCs incubated with FGF-2 showed modest alterations in the expression of α-SMA and Vimentin. Moreover, in our study, we found that Wnt/ß-catenin signaling was activated both in vitro and in vivo as a result of bleomycin treatment. Interestingly, we also found that suppression of the Wnt/ß-catenin signaling could significantly attenuate bleomycin-induced PF accompanied with decreased expression of TGF-ß1 and FGF-2 in vitro and in vivo. These results support that controlling the aberrant expression of TGF-ß1 and FGF-2 via inhibition of Wnt/ß-catenin signaling could serve as a potential therapeutic strategy for PF.

Antigen/IgG immune complex-primed mucosal mast cells mediate antigen-specific activation of co-cultured T cells.

Mast cells are proposed to be one of the targets for mucosal vaccine adjuvants. We previously demonstrated that mucosal adjuvants containing IgG immune complexes could activate connective tissue mast cells enhancing immune responses. Here we suggest that mucosal mast cells (MMC) may also contribute to augmentation of antigen-specific immune responses following treatment with antigens complexed with IgG. We demonstrated that both bone marrow-derived cultured MMC and tissue resident MMC incorporated ovalbumin (OVA) at a greater level in the presence of anti-OVA IgG. Co-culture of OVA/IgG-pulsed bone marrow-derived MMC with splenocytes from OT-II mice promoted OVA-specific activation and proliferation of T cells, a process known as cross-presentation. Furthermore, bone marrow-derived cultured MMC underwent apoptosis following treatment with IgG immune complexes, a feature that has been described as favouring phagocytosis of mast cells by professional antigen-presenting cells.

Role of Wnt/ß-Catenin Signaling in Epithelial Differentiation of Lung Resident Mesenchymal Stem Cells.

Accumulating evidence has demonstrated that stem cells have the ability to repair the lung tissue injuries following either injection of cultured cells or bone marrow transplantation. As a result, increasing attention has focused on the lung resident mesenchymal stem cells (LR-MSCs) for repairing damaged lung tissues. Meanwhile, some studies have revealed that Wnt/ß-catenin signaling plays an important role in the epithelial differentiation of mesenchymal stem cells (MSCs). In the current study, our aim was to explore the roles of Wnt/ß-catenin signaling on cell proliferation and epithelial differentiation of LR-MSCs. We have successfully isolated the stem cell antigen (Sca)-1(+) CD45(-) CD31(-) cells which were proposed to be LR-MSCs by magnetic-activated cell sorting (MACS). Furthermore, we demonstrated the expression of epithelial markers on LR-MSCs following indirect co-culture of these cells with alveolar epithelial type II (ATII) cells, confirming the epithelial phenotype of LR-MSCs following co-culture. In order to clarify the regulatory mechanisms of Wnt/ß-catenin signaling in epithelial differentiation of LR-MSCs, we measured the protein levels of several important members involved in Wnt/ß-catenin signaling in the presence or absence of some canonical activators and inhibitors of the ß-catenin pathways. In conclusion, our study demonstrated that Wnt/ß-catenin signaling may be an essential mechanism underlying the regulation of epithelial differentiation of LR-MSCs.

Roles of miRNAs in microcystin-LR-induced Sertoli cell toxicity.

Microcystin (MC)-LR, a cyclic heptapeptide, is a potent reproductive system toxin. To understand the molecular mechanisms of MC-induced reproductive system cytotoxicity, we evaluated global changes of miRNA and mRNA expression in mouse Sertoli cells following MC-LR treatment. Our results revealed that the exposure to MC-LR resulted in an altered miRNA expression profile that might be responsible for the modulation of mRNA expression. Bio-functional analysis indicated that the altered genes were involved in specific cellular processes, including cell death and proliferation. Target gene analysis suggested that junction injury in Sertoli cells exposed to MC-LR might be mediated by miRNAs through the regulation of the Sertoli cell-Sertoli cell pathway. Collectively, these findings may enhance our understanding on the modes of action of MC-LR on mouse Sertoli cells as well as the molecular mechanisms underlying the toxicity of MC-LR on the male reproductive system.

Important role for FcγRIIB on B lymphocytes for mucosal antigen-induced tolerance and Foxp3+ regulatory T cells.

FcγRIIB, the only FcγR expressed on B cells, is important in the maintenance of immunological tolerance to self-Ags. In this study, we investigated the role of FcγRIIB in Ag-specific CD4 T cell tolerance induced by mucosally administered Ag (OVA) coupled to cholera toxin B subunit (Ag/CTB) or given alone. We found that sublingual administration of Ag/CTB conjugate or intragastric administration of a >100-fold higher dose of Ag alone efficiently suppressed parenteral immunization-induced Ag-specific T cell proliferation and delayed-type hypersensitivity responses in FcγRIIB-expressing wild-type (WT), but not FcγRIIB(-/-), mice. Such mucosally induced tolerance (oral tolerance) associated with induction of Ag-specific Foxp3(+) regulatory T cells was restored in FcγRIIB(-/-) mice by adoptive transfer of either WT B cells or WT dendritic cells before the mucosal Ag/CTB treatment; it was even more pronounced in µMT mice that received FcγRIIB-overexpressing B cells before treatment. Furthermore, cell transfer in either WT or µMT mice of WT but not FcγRIIB(-/-) B cells pretreated for 1 h in vitro with Ag/CTB conjugate induced Ag-specific immunological tolerance, which was further enhanced by adoptive transfer of WT B cells pretreated with anti-Ag IgG immune complexed Ag/CTB. We conclude that FcγRIIB expression on B cells, in addition to dendritic cells, is important for mucosal induction of Ag-specific immune tolerance.

Epigenetic pattern changes in prenatal female Sprague-Dawley rats following exposure to androgen.

Androgen excess is generally considered to be one of the major characteristics of polycystic ovary syndrome (PCOS). Evidence from both clinical research and animal studies has revealed that this syndrome may have fetal origins, with epigenetics being proposed as the underlying mechanism. Our PCOS rat model induced by prenatal administration of 3mg testosterone from Embryonic Day (E) 16 to E19 showed polycystic ovaries, irregular oestrous cycles and endocrine disorders in adulthood. The methylation status of 16, 8 and 4 cytosine-phosphate-guanine (CpG) sites in the promoter regions of the androgen receptor (Ar), cytochrome P450 family 11, subfamily A, polypeptide 1 (Cyp11a1) and cytochrome P450, family 17, subfamily A, polypeptide 1 (Cyp17a1) genes, respectively, were measured by pyrosequencing. We identified three hypomethylated sites (CpG +58, +65 and +150) in Ar and one hypomethylated site (CpG +1016) in Cyp11a1 in peripheral blood cells of prenatally androgenised (PNA) rats. In ovarian tissue, five CpG sites of Ar (CpG +87, +91, +93, +98, +150) and one single CpG site in Cyp11a1 (CpG +953) were significantly hypomethylated in PNA rats, but the modified methylation of these two genes may not be sufficient to significantly alter levels of gene expression. Furthermore, tissue-specific methylation analysis revealed that both Ar and Cyp11a1 exhibited significant hypomethylation in testis in contrast with ovary and blood. PNA may lead to methylation pattern changes and the development of PCOS, but further studies are required to reveal causal relationships.

Inhibition of Wnt/ß-catenin signaling promotes epithelial differentiation of mesenchymal stem cells and repairs bleomycin-induced lung injury.

Idiopathic pulmonary fibrosis is a progressive lung disorder of unknown etiology. Previous studies have shown that aberrant activation of the Wnt/ß-catenin signaling cascade occurs in lungs of patients with idiopathic pulmonary fibrosis. Given the important roles of the Wnt/ß-catenin signaling pathway in the development of pulmonary fibrosis, we targeted this pathway for the intervention of pulmonary fibrosis with XAV939, a small molecule that specifically inhibits Tankyrase 1/2, eventually leading to the degradation of ß-catenin and suppression of the Wnt/ß-catenin signaling pathway. Our results demonstrated that XAV939 significantly inhibited the activation of Wnt/ß-catenin signaling and attenuated bleomycin-induced lung fibrosis in mice, and thus improved the survival of mice with lung injury. Interestingly, previous investigations have confirmed that endogenous and exogenous mesenchymal stem cells could be recruited to the injured lung, although the exact effects of these cells are debatable. To determine the effect of Wnt/ß-catenin signaling in the epithelial differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs), we established a coculture system that contains BM-MSCs and alveolar type II epithelial cells. The in vitro experiments demonstrated that XAV939 could promote the differentiation of BM-MSCs into an epithelium-like phenotype in the coculture system. We also found that XAV939 could inhibit the proliferation and myofibroblast differentiation of NIH/3T3 fibroblasts. This work supports that inhibition of the Wnt/ß-catenin signaling pathway may be exploited for the treatment of idiopathic pulmonary fibrosis for which effective treatment strategies are still lacking.

Regulation of microcystin-LR-induced toxicity in mouse spermatogonia by miR-96.

Microcystin (MC)-LR is a cyclic heptapeptide that acts as a potent reproductive system toxin, especially by decreasing sperm quality through affecting spermatogonia. However, the molecular mechanisms of MC-induced spermatogonial cytotoxicity still remain unclear. The present study was designed to investigate changes in microRNA (miRNA) profiles and their potential functions in spermatogonia (GC-1 cell line) following treatment with MC-LR. With microarray analysis, 101 miRNAs were identified to be significantly altered in GC-1 cells treated with MC-LR. Among the 25 miRNAs associated with spermatogenesis, miR-96 was down-regulated most dramatically and thus selected for further functional analysis. Deleted-in azoospermia-associated protein 2 (DAZAP2) was predicted to have a binding sequence for miR-96 within its 3'-untranslated region. Fluorescent reporter assay confirmed that DAZAP2 was the target gene of miR-96. The expression of DAZAP2 decreased significantly when miR-96 was up-regulated. Consistently, down-regulation of miR-96 significantly increased the level of DAZAP2. Up-regulation of miR-96 promoted cell viability in GC-1 cells as a result of exposure to MC-LR. Our study suggested a crucial role for miR-96 in the regulation of cytotoxic effects of MC-LR in spermatogonia, which provides new perspectives in the diagnosis and treatment strategies for MC-induced male infertility.