Impact of retinoic acid exposure on midfacial shape variation and manifestation of holoprosencephaly in Twsg1 mutant mice

Holoprosencephaly (HPE) is a developmental anomaly characterized by inadequate or absent midline division of the embryonic forebrain and midline facial defects. It is believed that interactions between genes and the environment play a role in the widely variable penetrance and expressivity of HPE, although direct investigation of such effects has been limited. The goal of this study was to examine whether mice carrying a mutation in a gene encoding the bone morphogenetic protein (BMP) antagonist twisted gastrulation (Twsg1), which is associated with a low penetrance of HPE, are sensitized to retinoic acid (RA) teratogenesis. Pregnant Twsg1^+/− dams were treated by gavage with a low dose of all-trans RA (3.75 mg/kg of body weight). Embryos were analyzed between embryonic day (E)9.5 and E11.5 by microscopy and geometric morphometric analysis by micro-computed tomography. P19 embryonal carcinoma cells were used to examine potential mechanisms mediating the combined effects of increased BMP and retinoid signaling. Although only 7% of wild-type embryos exposed to RA showed overt HPE or neural tube defects (NTDs), 100% of Twsg1^−/− mutants exposed to RA manifested severe HPE compared to 17% without RA. Remarkably, up to 30% of Twsg1^+/− mutants also showed HPE (23%) or NTDs (7%). The majority of shape variation among Twsg1^+/− mutants was associated with narrowing of the midface. In P19 cells, RA induced the expression of Bmp2, acted in concert with BMP2 to increase p53 expression, caspase activation and oxidative stress. This study provides direct evidence for modifying effects of the environment in a genetic mouse model carrying a predisposing mutation for HPE in the Twsg1 gene. Further study of the mechanisms underlying these gene-environment interactions in vivo will contribute to better understanding of the pathogenesis of birth defects and present an opportunity to explore potential preventive interventions.KEY WORDS: Twisted gastrulation, Twsg1, Bone morphogenetic protein, Holoprosencephaly, Retinoic acid, Apoptosis, Oxidative stress     

Cell-Autonomous Requirement for Rx Function in the Mammalian Retina and Posterior Pituitary

Rx is a paired-like homeobox gene that is required for vertebrate eye formation. Mice lacking Rx function do not develop eyes or the posterior pituitary. To determine whether Rx is required cell autonomously in these tissues, we generated embryonic chimeras consisting of wild type and Rx−/− cells. We found that in the eye, Rx-deficient cells cannot participate in the formation of the neuroretina, retina pigment epithelium and the distal part of the optic stalk. In addition, in the ventral forebrain, Rx function is required cell autonomously for the formation of the posterior pituitary. Interestingly, Rx−/− and wild type cells segregate before the morphogenesis of these two tissues begins. Our observations suggest that Rx function is not only required for the morphogenesis of the retina and posterior pituitary, but also prior to morphogenesis, for the sorting out of cells to form distinct fields of retinal/pituitary cells.     

Retinoic acid exerts sexually dimorphic effects on muscle energy metabolism and function

The retinol dehydrogenase Rdh10 catalyzes the rate-limiting reaction that converts retinol into retinoic acid (RA), an autacoid that regulates energy balance and reduces adiposity. Skeletal muscle contributes to preventing adiposity, by consuming nearly half the energy of a typical human. We report sexually dimorphic differences in energy metabolism and muscle function in Rdh10+/− mice. Relative to wild-type (WT) controls, Rdh10+/− males fed a high-fat diet decrease reliance on fatty-acid oxidation and experience glucose intolerance and insulin resistance. Running endurance decreases 40%. Rdh10+/− females fed this diet increase fatty acid oxidation and experience neither glucose intolerance nor insulin resistance. Running endurance increases 220%. We therefore assessed RA function in the mixed-fiber type gastrocnemius muscles (GM), which contribute to running, rather than standing, and are similar to human GM. RA levels in Rdh10+/− male GM decrease 38% relative to WT. Rdh10+/− male GM increase expression of Myog and reduce Eif6 mRNAs, which reduce and enhance running endurance, respectively. Cox5A, complex IV activity, and ATP decrease. Increased centralized nuclei reveal existence of muscle malady and/or repair in GM fibers. Comparatively, RA in Rdh10+/− female GM decreases by less than half the male decrease, from a more modest decrease in Rdh10 and an increase in the estrogen-induced retinol dehydrogenase Dhrs9. Myog mRNA decreases. Cox5A, complex IV activity, and ATP increase. Centralized GM nuclei do not increase. We conclude that Rdh10/RA affects whole body energy use and insulin resistance partially through sexual dimorphic effects on skeletal muscle gene expression, structure, and mitochondria activity.     

Change in the developmental fate of the chick optic vesicle from the neural retina to the telencephalon

The forebrain develops into the telencephalon, diencephalon, and optic vesicle (OV). The OV further develops into the optic cup, the inner and outer layers of which develop into the neural retina and retinal pigmented epithelium (RPE), respectively. We studied the change in fate of the OV by using embryonic transplantation and explant culture methods. OVs excised from 10-somite stage chick embryos were freed from surrounding tissues (the surface ectoderm and mesenchyme) and were transplanted back to their original position in host embryos. Expression of neural retina-specific genes, such as Rax and Vsx2 (Chx10), was downregulated in the transplants. Instead, expression of the telencephalon-specific gene Emx1 emerged in the proximal region of the transplants, and in the distal part of the transplants close to the epidermis, expression of an RPE-specific gene Mitf was observed. Explant culture studies showed that when OVs were cultured alone, Rax was continuously expressed regardless of surrounding tissues (mesenchyme and epidermis). When OVs without surrounding tissues were cultured in close contact with the anterior forebrain, Rax expression became downregulated in the explants, and Emx1 expression became upregulated. These findings indicate that chick OVs at stage 10 are bi-potential with respect to their developmental fates, either for the neural retina or for the telencephalon, and that the surrounding tissues have a pivotal role in their actual fates. An in vitro tissue culture model suggests that under the influence of the anterior forebrain and/or its surrounding tissues, the OV changes its fate from the retina to the telencephalon.     

Hypoxia-Inducible Factor-2α Is an Essential Catabolic Regulator of Inflammatory Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a systemic autoimmune disorder that manifests as chronic inflammation and joint tissue destruction. However, the etiology and pathogenesis of RA have not been fully elucidated. Here, we explored the role of the hypoxia-inducible factors (HIFs), HIF-1α (encoded by HIF1A) and HIF-2α (encoded by EPAS1). HIF-2α was markedly up-regulated in the intimal lining of RA synovium, whereas HIF-1α was detected in a few cells in the sublining and deep layer of RA synovium. Overexpression of HIF-2α in joint tissues caused an RA-like phenotype, whereas HIF-1α did not affect joint architecture. Moreover, a HIF-2α deficiency in mice blunted the development of experimental RA. HIF-2α was expressed mainly in fibroblast-like synoviocytes (FLS) of RA synovium and regulated their proliferation, expression of RANKL (receptor activator of nuclear factor–κB ligand) and various catabolic factors, and osteoclastogenic potential. Moreover, HIF-2α–dependent up-regulation of interleukin (IL)-6 in FLS stimulated differentiation of T_H17 cells—crucial effectors of RA pathogenesis. Additionally, in the absence of IL-6 (Il6 ^−/− mice), overexpression of HIF-2α in joint tissues did not cause an RA phenotype. Thus, our results collectively suggest that HIF-2α plays a pivotal role in the pathogenesis of RA by regulating FLS functions, independent of HIF-1α.     

Retinoic acid signaling in perioptic mesenchyme represses Wnt signaling via induction of Pitx2 and Dkk2

Morphogenesis during eye development requires retinoic acid (RA) receptors plus RA-synthesizing enzymes, and loss of RA signaling leads to ocular disorders associated with loss of Pitx2 expression in perioptic mesenchyme. Several Wnt signaling components are expressed in ocular tissues during eye development including Dkk2, encoding an inhibitor of Wnt/β-catenin signaling, which was previously shown to be induced by Pitx2 in the perioptic mesenchyme. Here, we investigated potential cross-talk between RA and Wnt signaling during ocular development. Genetic studies using Raldh1/Raldh3 double null mice deficient for ocular RA synthesis demonstrated that Pitx2 and Dkk2 were both down-regulated in perioptic mesenchyme. Chromatin immunoprecipitation and gel mobility shift studies demonstrated the existence of a DR5 RA response element upstream of Pitx2 that binds all three RA receptors in embryonic eye. Axin2, an endogenous readout of Wnt/β-catenin signaling, was up-regulated in cornea and perioptic mesenchyme of RA deficient embryos. Also, expression of Wnt5a was expanded in perioptic mesenchyme of RA deficient eyes. Our findings demonstrate excessive activation of Wnt signaling in the perioptic mesenchyme of RA deficient mice which may be responsible for abnormal development leading to defective optic cup, cornea, and eyelid morphogenesis.     

Hepatic retinoid stores are required for normal liver regeneration

Preliminary studies of liver regeneration induced by partial hepatectomy (PHE) identified a substantial depletion of hepatic retinoid stores, by greater than 70%, in regenerating livers of wild-type C57Bl/6J mice. To understand this, we compared responses of wild-type and lecithin:retinol acyltransferase (Lrat)-deficient mice, which totally lack hepatic retinoid stores, to PHE. The Lrat-deficient livers showed delayed regeneration in the first 24 h after PHE. At 12 h after PHE, we observed significantly less mRNA expression for growth factors and cytokines implicated in regulating the priming phase of liver regeneration, specifically for Hgf and Tgfα, but not Tgfβ. Compared with wild-type mice, the changes in mRNA levels for p21 and cyclins E1, B1, and A2 mRNAs and for hepatocellular BrdU incorporation and mitoses were delayed (i.e., shifted to later times) in regenerating Lrat^−/− livers. Concentrations of all-trans-retinoic acid were significantly lower in the livers of Lrat^−/− mice following PHE, and this was accompanied by diminished expression of known retinoid-responsive genes. At later times after PHE, the rate of liver weight restoration for Lrat^−/− mice was parallel to that of wild-type mice, although additional biochemical differences were observed. Thus, hepatic retinoid stores are required for maintaining expression of signaling molecules that regulate cell proliferation and differentiation immediately after hepatic injury, accounting for the delayed restoration of liver mass in Lrat^−/− mice.     

HLA-DRB1 Shared Epitope-Dependent DR-DQ Haplotypes Are Associated with Both Anti-CCP–Positive and –Negative Rheumatoid Arthritis in Chinese Han

The association between Human Leukocyte Antigen (HLA) class II and rheumatoid arthritis (RA) has been extensively studied, but few reported DR-DQ haplotype. Here we investigated the association of HLA-DRB1, DQA1, DQB1, and DR-DQ haplotypes with RA susceptibility and with anti-CCP antibodies in 281 RA patients and 297 control in Han population. High-resolution genotyping were performed. The HLA-DRB1 shared epitope (SE)-encoding allele *0405 displayed the most significant RA association (P = 1.35×10⁻⁶). The grouped DRB1 SE alleles showed great association with RA (P = 3.88×10⁻¹³). The DRB1 DRRAA alleles displayed significant protective effects (P = 0.021). The SE-dependent DR-DQ haplotype SE-DQ3/4/5 remained strong association with both anti-CCP -positive (P = 3.71×10⁻¹³) and -negative RA (P = 3.89×10⁻⁵). Our study revealed that SE alleles and its haplotypes SE-DQ3/4/5 were highly associated with RA susceptibility in Han population. The SE-DQ3/4/5 haplotypes were associated with both anti-CCP positive RA and -negative RA.     

LincRNA‐EPS impairs host antiviral immunity by antagonizing viral RNA–PKR interaction

LincRNA‐EPS is an important regulator in inflammation. However, the role of lincRNA‐EPS in the host response against viral infection is unexplored. Here, we show that lincRNA‐EPS is downregulated in macrophages infected with different viruses including VSV, SeV, and HSV‐1. Overexpression of lincRNA‐EPS facilitates viral infection, while deficiency of lincRNA‐EPS protects the host against viral infection in vitro and in vivo. LincRNA‐EPS ^−/− macrophages show elevated expression of antiviral interferon‐stimulated genes (ISGs) such as Mx1, Oas2, and Ifit2 at both basal and inducible levels. However, IFN‐β, the key upstream inducer of these ISGs, is downregulated in lincRNA‐EPS ^−/− macrophages compared with control cells. RNA pulldown and mass spectrometry results indicate that lincRNA‐EPS binds to PKR and antagonizes the viral RNA–PKR interaction. PKR activates STAT1 and induces antiviral ISGs independent of IFN‐I induction. LincRNA‐EPS inhibits PKR‐STAT1‐ISGs signaling and thus facilitates viral infection. Our study outlines an alternative antiviral pathway, with downregulation of lincRNA‐EPS promoting the induction of PKR‐STAT1‐dependent ISGs, and reveals a potential therapeutic target for viral infectious diseases.     

Brain-expressed X-linked 2 Is Pivotal for Hyperactive Mechanistic Target of Rapamycin (mTOR)-mediated Tumorigenesis

Frequent alteration of upstream proto-oncogenes and tumor suppressor genes activates mechanistic target of rapamycin (mTOR) and causes cancer. However, the downstream effectors of mTOR remain largely elusive. Here we report that brain-expressed X-linked 2 (BEX2) is a novel downstream effector of mTOR. Elevated BEX2 in Tsc2^−/− mouse embryonic fibroblasts, Pten^−/− mouse embryonic fibroblasts, Tsc2-deficient rat uterine leiomyoma cells, and brains of neuronal specific Tsc1 knock-out mice were abolished by mTOR inhibitor rapamycin. Furthermore, BEX2 was also increased in the liver of a hepatic specific Pten knock-out mouse and the kidneys of Tsc2 heterozygous deletion mice, and a patient with tuberous sclerosis complex (TSC). mTOR up-regulation of BEX2 was mediated in parallel by both STAT3 and NF-κB. BEX2 was involved in mTOR up-regulation of VEGF production and angiogenesis. Depletion of BEX2 blunted the tumorigenesis of cells with activated mTOR. Therefore, enhanced STAT3/NF-κB-BEX2-VEGF signaling pathway contributes to hyperactive mTOR-induced tumorigenesis. BEX2 may be targeted for the treatment of the cancers with aberrantly activated mTOR signaling pathway.     

Impairment of Visual Function and Retinal ER Stress Activation in Wfs1-Deficient Mice

Wolfram syndrome is an early onset genetic disease (1/180,000) featuring diabetes mellitus and optic neuropathy, associated to mutations in the WFS1 gene. Wfs1−/− mouse model shows pancreatic beta cell atrophy, but its visual performance has not been investigated, prompting us to study its visual function and histopathology of the retina and optic nerve. Electroretinogram and visual evoked potentials (VEPs) were performed in Wfs1^−/− and Wfs1^+/+ mice at 3, 6, 9 and 12 months of age. Fundi were pictured with Micron III apparatus. Retinal ganglion cell (RGC) abundance was determined from Brn3a immunolabeling of retinal sections. RGC axonal loss was quantified by electron microscopy in transversal optic nerve sections. Endoplasmic reticulum stress was assessed using immunoglobulin binding protein (BiP), protein disulfide isomerase (PDI) and inositol-requiring enzyme 1 alpha (Ire1α) markers. Electroretinograms amplitudes were slightly reduced and latencies increased with time in Wfs1^−/− mice. Similarly, VEPs showed decreased N+P amplitudes and increased N-wave latency. Analysis of unfolded protein response signaling revealed an activation of endoplasmic reticulum stress in Wfs1 ^−/− mutant mouse retinas. Altogether, progressive VEPs alterations with minimal neuronal cell loss suggest functional alteration of the action potential in the Wfs1 ^−/− optic pathways.