Rnf165/Ark2C enhances BMP-smad signaling to mediate motor axon extension

Little is known about extrinsic signals required for the advancement of motor neuron (MN) axons, which extend over long distances in the periphery to form precise connections with target muscles. Here we present that Rnf165 (Arkadia-like; Arkadia2; Ark2C) is expressed specifically in the nervous system and that its loss in mice causes motor innervation defects that originate during development and lead to wasting and death before weaning. The defects range from severe reduction of motor axon extension as observed in the dorsal forelimb to shortening of presynaptic branches of the phrenic nerve, as observed in the diaphragm. Molecular functional analysis showed that in the context of the spinal cord Ark2C enhances transcriptional responses of the Smad1/5/8 effectors, which are activated (phosphorylated) downstream of Bone Morphogenetic Protein (BMP) signals. Consistent with Ark2C-modulated BMP signaling influencing motor axons, motor pools in the spinal cord were found to harbor phosphorylated Smad1/5/8 (pSmad) and treatment of primary MN with BMP inhibitor diminished axon length. In addition, genetic reduction of BMP-Smad signaling in Ark2C+/− mice caused the emergence of Ark2C−/−-like dorsal forelimb innervation deficits confirming that enhancement of BMP-Smad responses by Ark2C mediates efficient innervation. Together the above data reveal an involvement of BMP-Smad signaling in motor axon advancement

A Novel Function of Apolipoprotein E: Upregulation of ATP-Binding Cassette Transporter A1 Expression

Despite the well known importance of apolipoprotein (Apo) E in cholesterol efflux, the effect of ApoE on the expression of ATP-binding cassette transporter A1 (ABCA1) has never been investigated. The objective of this study was to determine the effect of ApoE on ApoB-carrying lipoprotein-induced expression of ABCA1, a protein that mediates cholesterol efflux. Our data demonstrate that ApoB-carrying lipoproteins obtained from both wild-type and ApoE knockout mice induced ApoAI-mediated cholesterol efflux in mouse macrophages, which was associated with an enhanced ABCA1 promoter activity, and an increased ABCA1 mRNA and protein expression. In addition, these lipoproteins increased the level of phosphorylated specificity protein 1 (Sp1) and the amount of Sp1 bound to the ABCA1 promoter. However, all these inductions were significantly diminished in cells treated with ApoE-free lipoproteins, when compared to those treated with wild-type lipoproteins. Enrichment with human ApoE3 reversed the reduced inducibility of ApoE-free lipoproteins. Moreover, we observed that inhibition of Sp1 DNA-binding by mithramycin A diminished ABCA1 expression and ApoAI-mediated cholesterol efflux induced by ApoB-carrying lipoproteins, and that mutation of the Sp1-binding motif in the ABCA1 promoter region diminished ApoB-carrying lipoprotein-induced ABCA1 promoter activity. Collectively, these data suggest that ApoE associated with ApoB-carrying lipoproteins has an upregulatory role on ABCA1 expression, and that induction of Sp1 phosphorylation is a mechanism by which ApoE upregulates ABCA1 expression

TRPA1- FGFR2 binding event is a regulatory oncogenic driver modulated by miRNA-142-3p

YesRecent evidence suggests that the ion channel TRPA1 is implicated in lung adenocarcinoma (LUAD) where its role and mechanism of action remain unknown. We have previously established that the membrane receptor FGFR2 drives LUAD progression through aberrant protein-protein interactions mediated via its C-terminal proline rich motif. Here, we report that the N-terminal ankyrin repeats of TRPA1 directly bind to the C-terminal proline rich motif of FGFR2 inducing the constitutive activation of the receptor, thereby prompting LUAD progression and metastasis. Furthermore, we show that upon metastasis to the brain, TRPA1 gets depleted, an effect triggered by the transfer of TRPA1-targeting exosomal microRNA (miRNA-142-3p) from brain astrocytes to cancer cells. This downregulation, in turn, inhibits TRPA1-mediated activation of FGFR2 hindering the metastatic process. Our study reveals a direct binding event and characterizes the role of TRPA1 ankyrin repeats in regulating FGFR2-driven oncogenic process; a mechanism that is hindered by miRNA-142-3p.Faculty of Biological Sciences at the University of Leeds, Wellcome Trust Seed Award, Royal Society Research Grant RG150100, MR/K021303/1, Swedish Research Council (2014-3801) and the Medical Faculty at Lund University

Rnf165/Ark2C Enhances BMP-Smad Signaling to Mediate Motor Axon Extension

<div><p>Little is known about extrinsic signals required for the advancement of motor neuron (MN) axons, which extend over long distances in the periphery to form precise connections with target muscles. Here we present that Rnf165 (Arkadia-like; Arkadia2; Ark2C) is expressed specifically in the nervous system and that its loss in mice causes motor innervation defects that originate during development and lead to wasting and death before weaning. The defects range from severe reduction of motor axon extension as observed in the dorsal forelimb to shortening of presynaptic branches of the phrenic nerve, as observed in the diaphragm. Molecular functional analysis showed that in the context of the spinal cord Ark2C enhances transcriptional responses of the Smad1/5/8 effectors, which are activated (phosphorylated) downstream of Bone Morphogenetic Protein (BMP) signals. Consistent with Ark2C-modulated BMP signaling influencing motor axons, motor pools in the spinal cord were found to harbor phosphorylated Smad1/5/8 (pSmad) and treatment of primary MN with BMP inhibitor diminished axon length. In addition, genetic reduction of BMP-Smad signaling in <i>Ark2C</i>^+/− mice caused the emergence of <i>Ark2C</i>^−/−-like dorsal forelimb innervation deficits confirming that enhancement of BMP-Smad responses by Ark2C mediates efficient innervation. Together the above data reveal an involvement of BMP-Smad signaling in motor axon advancement.</p></div

Motor neuron specification is normal in the absence of Ark2C expression.

<p>(A) IF showing Ark2C expression (β-gal, green) and FoxP1 (red) in spinal cord at E13.5; scale bars = 250 µm and 50 µm for the magnified insert. (B) IF showing motor pool marker expression in spinal cord at E13.5. (C) Percentages showing the number of FoxP1-expressing cells (LMC) that also express Isl1 (LMC_m) or high Hb9 (LMC_l) are shown in the histogram; N, number of cells; n.s., not significant; scale bars = 50 µm.</p

Muscles develop normally but atrophy in adult stages in the absence of Ark2C.

<p>(A) Extensor muscles from <i>wt</i> and <i>Ark2C</i>^−/− adult mice. ECRL, extensor carpi radialis longus; ECRB, extensor carpi radialis brevis; EDC, extensor digitorum communis; EDQ, extensor digiti quinti; R, right limb; L, left limb; scale bars = 1 mm. Dot plot showing the ratio of width/length for the muscles from 4 <i>wt</i> and 8 <i>Ark2C</i>^−/− mice. Error bars represent ±SEM; * <i>p</i><0.05; ** <i>p</i><0.01. (B) Whole-mount immunofluorescence (IF) showing developing forelimb extensor muscle at E13.5; yellow arrowhead, extensor carpi and digiti muscles; scale bars = 250 µm. (C) Quantitative RT-PCR showing that <i>Ark2C</i> is not expressed in E12.5 forelimb (<i>n</i> = 3 <i>wt</i> embryos) as compared to spinal cord expression in <i>wt</i> (positive control) and <i>Ark2C</i>^−/− (negative control) embryos. Transcripts measured from exon7–8 including the NRG-TIER and RING domains. Error bars represent ±SD.</p

Ark2C interacts with and ubiquitinates negative regulators via its RING domain.

<p>(A) Immunoprecipitation (IP) in 293T-GArk2C cells after 1 h treatment with dorsomorphin (Inhibitor) or BMP4 in serum-free medium and in the presence of MG132. TL, total lysate; arrow, pSmad1/5/8 specific band. (B) IP in 293T-GArk2C cells showing interactions with mycSmad6 and mycSmad7 but not with mycSmad4. The same membrane was used for IB with a-GFP and leftover signal from mycSmad7 is indicated by *. (C) IP showing the ubiquitination of mycSki in 293T cells transfected as indicated. (D–E) IP showing the ubiquitination of mycSmad6 and mycSmad7 in 293T cells transfected as indicated. The expression of the proteins in the total lysates is shown in (F). Arrowheads indicate specific bands.</p

<i>Ark2C</i>^−/− mice exhibit abnormal forelimb posture and movement, inefficient breathing and feeding.

<p>(A) <i>wt</i> (+/+) and <i>Ark2C</i>^−/− (−/−) mice at postnatal day zero (P0). Black arrowheads indicate the relaxed paw posture. (B) Growth curves of <i>Ark2C</i>^−/− mice and their littermates between P0 and P20. Error bars represent ±SD between individuals. (C) P21 <i>Ark2C</i>^−/− mice and littermate, showing (1) less and (2) more severe phenotypes. Black arrowheads, defective limbs. (D) Blood plasma lactate concentrations from 9 <i>wt</i> and 7 <i>Ark2C</i>^−/− mice at P21 and adult. Error bars represent ±SD of three readings for each individual; * <i>p</i><0.05. (E) Representative footprints from 4 <i>Ark2C</i>^+/+ and 2 <i>Ark2C</i>^−/− adult mice. (F) Histograms showing the percentage of footprints according to how clear the toe prints are. (G) Pawprint trail showing measurements analyzed. (H) Histograms showing results of pawprint analysis from 9 <i>wt</i> and 7 <i>Ark2C</i>^−/− mice. Error bars represent ±SD between individuals; * <i>p</i><0.05; ** <i>p</i><0.01.</p