An H3F3A K27M‐mutation in a sonic hedgehog medulloblastoma

Medulloblastomas are malignant embryonal brain tumours that may harbour mutations in histone‐modifying genes, while mutations in histone genes have not been detected to date. We here describe the first SHH medulloblastoma with H3 K27M mutation. This may have diagnostic implications as H3 K27M mutations are the hallmark of diffuse midline gliomas, H3 K27M mutant, WHO grade IV. Medulloblastomas arise in midline structures and thus must not be mistaken for DMG when using an antibody detecting the H3 K27M mutation.     

Expression of WNT signalling pathway genes during chicken craniofacial development

A comprehensive expression analysis of WNT signalling pathway genes during several stages of chicken facial development was performed. Thirty genes were surveyed including: WNT1, 2B, 3A, 4, 5A, 5B, 6, 7A, 7B, 8B, 8C, 9A, 9B, 11, 11B, 16, CTNNB1, LEF1, FRZB1, DKK1, DKK2, FZD1‐8, FZD10. The strictly canonical WNTs (2B, 7A, 9B, and 16) in addition to WNT4 WNT6 (both canonical and non‐canonical) are epithelially expressed, whereas WNT5A, 5B, 11 are limited to the mesenchyme. WNT16 is limited to the invaginating nasal pit, respiratory epithelium, and lip fusion zone. Antagonists DKK1 and FRZB1 are expressed in the fusing primary palate but then are decreased at stage 28 when fusion is beginning. This suggests that canonical WNT signalling may be active during lip fusion. Mediators of canonical signalling, CTNNB1, LEF1, and the majority of the FZD genes are expressed ubiquitously. These data show that activation of the canonical WNT pathway is feasible in all regions of the face; however, the localization of ligands and antagonists confers specificity. Developmental Dynamics 238:1150–1165, 2009. © 2009 Wiley‐Liss, Inc.     

MiRNA profile of osteosarcoma with CD117 and stro-1 expression: miR-1247 functions as an onco-miRNA by targeting MAP3K9

microRNAs (miRNA) are regulators of gene expression, but little is known about miRNA expression profiles in stem cells of osteosarcoma (OS). C117 and Stro-1 are known stem cell markers of OS. In the study, CD117 and stro-1 positive (CD117⁺stro-1⁺) and CD117 and stro-1 negative (CD117^-stro-1^-) cells were isolated from MG63 cells CD117⁺stro-1⁺ cells showed more metastatic ability and stem cell formation rate than CD117^-stro-1^- ones. To find the difference between CD117⁺stro-1⁺ and CD117^-stro-1^- cells, the miRNA expression profile was examined using DNA microarray. MicroRNAs were differentially expressed in osteosarcoma cells with CD117⁺stro-1⁺ and CD117^-stro-1^-. The significant miRNAs included miR-15a, miR-302a, miR-423-5p, miR-1247, miR-1243 and others, which were confirmed by real time RT-PCR. The significant down-regulated miR-1247 was confirmed that was a potential tumor suppressor by targeting MAP3K9. Our results indicated that dysregulation of miRNAs is involved in osteosarcoma and miR-1247 plays an important role in progression of osteosarcoma.     

Allele-specific regulation of DISC1 expression by miR-135b-5p

Disrupted-in-schizophrenia-1 (DISC1) gene has been established as a risk factor for various neuropsychiatric phenotypes. Both coding and regulatory variants in DISC1 have been identified and associated with these phenotypes in genetic studies. MicroRNAs (miRNAs) are important regulators of protein coding genes. Since the miRNA-mRNA target recognition mechanism is vulnerable to disruption by DNA polymorphisms, we investigated whether polymorphisms in the DISC1 3′UTR affect binding of miRNAs and lead to allele-specific regulation of DISC1. We identified four predicted polymorphic miRNA target sites in the DISC1 3′UTR, and demonstrated that miR-135b-5p regulates the level of DISC1 mRNA. Moreover, DISC1 regulation by miR-135b-5p is allele specific: miR-135b-5p only binds to the major allele (A) of rs11122396, not to the minor allele (G). Thus, the G allele may be functionally related to the DISC1-associated phenotypes by abolishing regulation by miR-135b-5p, leading to elevated DISC1 levels.     

Hypoxia inducible factor‐1α‐induced interleukin‐33 expression in intestinal epithelia contributes to mucosal homeostasis in inflammatory bowel disease

Intestinal epithelial cells (IECs), an important barrier to gut microbiota, are subject to low oxygen tension, particularly during intestinal inflammation. Hypoxia inducible factor‐1α (HIF‐1α) is expressed highly in the inflamed mucosa of inflammatory bowel disease (IBD) and functions as a key regulator in maintenance of intestinal homeostasis. However, how IEC‐derived HIF‐1α regulates intestinal immune responses in IBD is still not understood completely. We report here that the expression of HIF‐1α and IL‐33 was increased significantly in the inflamed mucosa of IBD patients as well as mice with colitis induced by dextran sulphate sodium (DSS). The levels of interleukin (IL)?33 were correlated positively with that of HIF‐1α. A HIF‐1α‐interacting element was identified in the promoter region of IL‐33, indicating that HIF‐1α activity regulates IL‐33 expression. Furthermore, tumour necrosis factor (TNF) facilitated the HIF‐1α‐dependent IL‐33 expression in IEC. Our data thus demonstrate that HIF‐1α‐dependent IL‐33 in IEC functions as a regulatory cytokine in inflamed mucosa of IBD, thereby regulating the intestinal inflammation and maintaining mucosal homeostasis.     

The insulin‐PI3K‐Rac1 axis contributes to terminal adipocyte differentiation through regulation of actin cytoskeleton dynamics

Adipocyte differentiation is accompanied by a pronounced change in the actin cytoskeleton characterized by the reorganization of filamentous (F)‐actin stress fibers into cortical F‐actin structures. We previously showed that depolymerization of F‐actin stress fibers induced by inactivation of RhoA–ROCK (Rho‐associated kinase) signaling acts as a trigger for adipocyte differentiation. The relevance and underlying mechanism of the formation of cortical F‐actin structures from depolymerized actin during adipocyte differentiation have remained unclear, however. We have now examined the mechanistic relation between actin dynamics and adipogenic induction. Transient exposure to the actin‐depolymerizing agent latrunculin A (LatA) supported the formation of adipocyte‐associated cortical actin structures and the completion of terminal adipocyte differentiation in the presence of insulin, whereas long‐term exposure to LatA prevented such actin reorganization as well as terminal adipogenesis. Moreover, these effects of insulin were prevented by inhibition of phosphatidylinositol 3‐kinase (PI3K)–Rac1 signaling and the actin‐related protein 2/3 (Arp2/3) complex which is a critical component of the cortical actin networks. Our findings thus suggest that the insulin‐PI3K‐Rac1 axis leads to the formation of adipocyte‐associated cortical actin structures which is essential for the completion of adipocyte differentiation.     

Inhibited expression of hematopoietic progenitor kinase 1 associated with loss of jumonji domain containing 3 promoter binding contributes to autoimmunity in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by T cell overactivation and B cell hyper-stimulation. Hematopoietic progenitor kinase 1 (HPK1, also called MAP4K1) negatively regulates T cell-mediated immune responses. However, the role of HPK1 and the mechanisms that regulate HPK1 expression in SLE remain poorly understood. Using chromatin immunoprecipitation (ChIP) microarray data, we identified markedly increased histone H3 lysine 27 trimethylation (H3K27me3) enrichment at the HPK1 promoter of SLE CD4+ T cells relative to controls, and confirmed this observation using ChIP and real-time PCR experiments. We further found that HPK1 mRNA and protein levels were significantly decreased in CD4+ T cells of patients with SLE, and that this decrease was not caused by exposure to standard SLE medications. Down-regulating HPK1 in healthy CD4+ T cells significantly accelerated T cell proliferation and production of IFNγ and IgG. Consistent with these findings, overexpressing HPK1 in SLE CD4+ T cells caused a significant decrease in T cell reactivity. In addition, we observed a striking decrease in jumonji domain containing 3 (JMJD3) binding, but no marked change in enhancer of zeste homolog 2 (EZH2) binding, at the HPK1 promoter region in SLE CD4+ T cells compared to healthy controls. SiRNA knock down of JMJD3 in healthy CD4+ T cells led to decreased JMJD3 binding and increased H3K27me3 enrichment at the HPK1 promoter region, thus inhibiting the expression of HPK1. Concordantly, plasmid-induced overexpression of JMJD3 in SLE CD4+ T cells led to increased JMJD3 binding, decreased H3K27me3 enrichment, and up-regulated HPK1 expression. Our results show for the first time that inhibited HPK1 expression in SLE CD4+ T cells is associated with loss of JMJD3 binding and increased H3K27me3 enrichment at the HPK1 promoter, contributing to T cell overactivation and B cell overstimulation in SLE. These findings suggest that HPK1 may serve as a novel target for effective SLE therapy.     

miR-126 contributes to Parkinson's disease by dysregulating the insulin-like growth factor/phosphoinositide 3-kinase signaling

Dopamine (DA) neurons in sporadic Parkinson's disease (PD) display dysregulated gene expression networks and signaling pathways that are implicated in PD pathogenesis. Micro (mi)RNAs are regulators of gene expression, which could be involved in neurodegenerative diseases. We determined the miRNA profiles in laser microdissected DA neurons from postmortem sporadic PD patients' brains and age-matched controls. DA neurons had a distinctive miRNA signature and a set of miRNAs was dysregulated in PD. Bioinformatics analysis provided evidence for correlations of miRNAs with signaling pathways relevant to PD, including an association of miR-126 with insulin/IGF-1/PI3K signaling. In DA neuronal cell systems, enhanced expression of miR-126 impaired IGF-1 signaling and increased vulnerability to the neurotoxin 6-OHDA by downregulating factors in IGF-1/PI3K signaling, including its targets p85β, IRS-1, and SPRED1. Blocking of miR-126 function increased IGF-1 trophism and neuroprotection to 6-OHDA. Our data imply that elevated levels of miR-126 may play a functional role in DA neurons and in PD pathogenesis by downregulating IGF-1/PI3K/AKT signaling and that its inhibition could be a mechanism of neuroprotection.     

Aberrant expression of microRNAs in T cells from patients with ankylosing spondylitis contributes to the immunopathogenesis

Ankylosing spondylitis (AS) is a chronic inflammatory disorder characterized by dysregulated T cells. We hypothesized that the aberrant expression of microRNAs (miRNAs) in AS T cells involved in the pathogenesis of AS. The expression profile of 270 miRNAs in T cells from five AS patients and five healthy controls were analysed by real‐time polymerase chain reaction (PCR). Thirteen miRNAs were found potentially differential expression. After validation, we confirmed that miR‐16, miR‐221 and let‐7i were over‐expressed in AS T cells and the expression of miR‐221 and let‐7i were correlated positively with the Bath Ankylosing Spondylitis Radiology Index (BASRI) of lumbar spine in AS patients. The protein molecules regulated by miR‐16, miR‐221 and let‐7i were measured by Western blotting. We found that the protein levels of Toll‐like receptor‐4 (TLR‐4), a target of let‐7i, in T cells from AS patients were decreased. In addition, the mRNA expression of interferon (IFN)‐γ was elevated in AS T cells. Lipopolysaccharide (LPS), a TLR‐4 agonist, inhibited IFN‐γ secretion by anti‐CD3⁺anti‐CD28 antibodies‐stimulated normal T cells but not AS T cells. In the transfection studies, we found the increased expression of let‐7i enhanced IFN‐γ production by anti‐CD3⁺anti‐CD28⁺ lipopolysaccharide (LPS)‐stimulated normal T cells. In contrast, the decreased expression of let‐7i suppressed IFN‐γ production by anti‐CD3⁺anti‐CD28⁺ LPS‐stimulated AS T cells. In conclusion, we found that miR‐16, miR‐221 and let‐7i were over‐expressed in AS T cells, but only miR‐221 and let‐7i were associated with BASRI of lumbar spine. In the functional studies, the increased let‐7i expression facilitated the T helper type 1 (IFN‐γ) immune response in T cells.     

Cloning and embryonic expression patterns of the chicken CELF family.

The CUG-BP and ETR-3-like factor (CELF) protein family has been implicated in the regulation of pre-mRNA alternative splicing, mRNA stability, and translation. Here we discuss the evolution and radiation of the CELF protein subfamilies, and report the cloning of the chicken CELF family members. In this study, we examined the embryonic expression patterns of the CELF family in the chick by in situ hybridization. We found that the tissue specificity reported for CELF proteins in the adult is established early during embryogenesis. Members of one subfamily, CUG-BP1 and ETR-3, are broadly expressed in the early embryo, while members of the second subfamily, CELF4-6, are restricted primarily to the nervous system. Expression patterns of individual CELF genes in several tissues, including the heart, liver, eye, and neural tube, exhibit distinct, yet overlapping, expression patterns. This suggests that different members of the CELF family play distinct functional roles during embryogenesis.     

The expression pattern of nuclear receptors during cerebellar development.

The cerebellum is essential for fine control of movement and posture, and it has been a useful model for studying many aspects of neural development because of its relatively simple anatomy and developmental program. However, the roles of nuclear receptors (NRs) underlying formation of the cerebellum and maintenance of cerebellar functions are still poorly characterized. As a contribution to the Nuclear Receptor Signaling Atlas (NURSA), we employed immunohistochemistry to investigate the expression pattern of 18 NRs in the cerebellum. Ten receptors were demonstrated to be expressed in the postnatal day 21 (P21) cerebellum. Among them, five receptors (COUP-TFI, COUP-TFII, RORalpha, ERbeta, and ERRgamma) were expressed at all stages (embryonic stage, P0, P7, and P21) examined. Interestingly, COUP-TFI and COUP-TFII show differential anterior-posterior expression patterns during cerebellar development. Taken together, our results suggest that members of the nuclear receptor superfamily might play importantly physiological roles in the cerebellum.