Braun DA et al. (2016), Mutations in nuclear pore genes NUP93, NUP205 a...

XB-ART-52541

Nat Genet 2016 Apr 01;484:457-65. doi: 10.1038/ng.3512.

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Mutations in nuclear pore genes NUP93, NUP205 and XPO5 cause steroid-resistant nephrotic syndrome.

Braun DA , Sadowski CE , Kohl S , Lovric S , Astrinidis SA , Pabst WL , Gee HY , Ashraf S , Lawson JA , Shril S , Airik M , Tan W , Schapiro D , Rao J , Choi WI , Hermle T , Kemper MJ , Pohl M , Ozaltin F , Konrad M , Bogdanovic R , Büscher R , Helmchen U , Serdaroglu E , Lifton RP , Antonin W , Hildebrandt F .

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Nucleoporins are essential components of the nuclear pore complex (NPC). Only a few diseases have been attributed to NPC dysfunction. Steroid-resistant nephrotic syndrome (SRNS), a frequent cause of chronic kidney disease, is caused by dysfunction of glomerular podocytes. Here we identify in eight families with SRNS mutations in NUP93, its interaction partner NUP205 or XPO5 (encoding exportin 5) as hitherto unrecognized monogenic causes of SRNS. NUP93 mutations caused disrupted NPC assembly. NUP93 knockdown reduced the presence of NUP205 in the NPC, and, reciprocally, a NUP205 alteration abrogated NUP93 interaction. We demonstrate that NUP93 and exportin 5 interact with the signaling protein SMAD4 and that NUP93 mutations abrogated interaction with SMAD4. Notably, NUP93 mutations interfered with BMP7-induced SMAD transcriptional reporter activity. We hereby demonstrate that mutations of NUP genes cause a distinct renal disease and identify aberrant SMAD signaling as a new disease mechanism of SRNS, opening a potential new avenue for treatment.

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Species referenced: Xenopus laevis
Genes referenced: bmp7.1 myc nup205 nup93 smad10 xpo5

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	Figure 2. Subcellular localization of NUP93 in podocytes and NUP93 knockdown resulting in reduced podocyte migration, proliferation, and impaired resistance to oxidative stress(a) In BMP7 treated human podocytes the nucleoporin NUP93 (green) and the nuclear transport factor importin7 (red) colocalize to a nuclear rim (arrow heads). (b–c) Neonatal rat kidney sections were stained with antibodies against NUP93 (green) and importin7 (red). Both proteins colocalize in podocyte precursor cells in different stages of glomerular development and other structures of the developing kidney. (c) Magnifications of the capillary loop and renal vesicle stage. (d) In adult rat glomerulus the nuclear exportin XPO5 partially colocalizes with the glomerular slit membrane marker synaptopodin (arrow heads). DAPI (blue) stains DNA. Scale bars are 10 μm in a, d and 25 μm in b, c. (e) Knockdown of NUP93 in human podocytes using two different shRNAs impairs podocyte migration (yellow curve vs. black curve). The decrease in podocyte migration was partially rescued by transfection of mouse Nup93 cDNA (blue curve). (f) Upon knockdown of NUP93 using two different shRNAs, the proliferation rate of human podocytes is severely impaired (yellow curve vs. black curve). Transfection of mouse Nup93 cDNA partially reversed the effect (blue curve). Experiments in e, f were performed in triplicates. Data points represent mean and standard deviation. (g) Addition of sub-lethal concentrations of H2O2 (100 μM, 250 μM, and 500 μM) to human podocyte culture does not induce apoptosis in control cells. In contrast, upon knockdown of NUP93 H2O2 induces apoptotic cell death as shown by increased cleavage of caspase-3.
	Figure 3. NUP93 mutations interfere with nuclear pore complex (NPC) assembly, and a NUP205 mutation affects NUP93-NUP205 interaction(a) Upon overexpression in human podocytes, Myc tagged NUP93 but not the splice site mutation del ex13 or the truncating mutation Lys442Asnfs14, localize to the nuclear envelope (arrow heads). Scale bar 10 μm, see also c. (b) Mutations identified in individuals with SRNS fail to assemble an intact NPC in a depletion-addback assay in X. laevis egg extracts. Upon depletion of NUP93 nuclei fail to assemble properly. Addback of full-length or the mutant constructs Gly591Val or Tyr629Cys restore nuclear envelope and NPC assembly. The constructs of the in-frame deletion of exon 13 (del ex13), the missense mutation Arg388Trp, and the frameshift mutation Lys442Asnfs14 lack this ability. Nuclear membranes are stained with DiIC18 (red, upper row), DNA with DAPI (blue), NPCs with mAB414 (second row, red), and α-Nup93 antibody (third row, green). Scale bar 10 μm, see also d. (c) Quantitation of nuclear localization data from (a) resulting from 50 transfected cells for each condition. (d) Quantitation of depletion-addback assay data from (b) presented as mean and standard deviation resulting from 100 nuclei each from three independent experiments. (e) Upon shRNA knockdown of NUP93 (left panel) immortalized human podocytes show a reduced expression level of NUP205 (right panel). (f) GFP tagged NUP205 precipitates endogenous NUP93 upon overexpression in HEK293 cells. A mutant allele of NUP205 identified in SRNS family A1733 (Phe1995Ser) lacks this interaction.
	Figure 4. NUP93, importin7, and exportin5 (XPO5) interact with SMAD proteins, and NUP93 mutations from individuals with SRNS abrogate the interaction with SMAD4 and importin7 upon coIP in HEK293 cells(a) C-terminally GFP tagged NUP93 precipitates endogenous SMAD4. (b) C-terminally GFP tagged SMAD4 precipitates endogenous NUP93. (c) Upon cooverexpression, GFP tagged NUP93 interacts with Myc tagged SMAD4. Mutations (Lys442Asnfs14, Gly591Val, Tyr629Cys) identified in individuals with SRNS abrogate SMAD4 interaction. (d) Upon BMP7 stimulation C-terminally GFP-tagged NUP93 interacts with phosphorylated/activated SMAD1/5. (e) C-terminally GFP tagged NUP93 precipitates endogenous importin7. (f) C-terminally GFP tagged SMAD4 precipitates endogenous importin7. (g) Upon cooverexpression, GFP tagged NUP93 interacts with Myc tagged importin7. Mutations (Lys442Asnfs14, Gly591Val, Tyr629Cys) identified in individuals with SRNS abrogate importin7 interaction. (h) GFP tagged SMAD4 precipitates endogenous XPO5. Deletion of the SMAD4 nuclear export signal (NES) (aa142-aa149) abrogates the interaction with XPO5. Coimmunoprecipitation experiments shown in a, b, d, e, and f were confirmed using N-terminally GFP-tagged fusion proteins.
	Figure 5. Knockdown of NUP93 interferes with BMP7-induced activation of SMAD signaling(a) Stimulation of human podocytes with recombinant BMP7 (rc-BMP7) (100 ng/μl) induces nuclear accumulation of SMAD4 in control cells (columns 1 vs. 2). Upon knockdown of NUP93 using two different shRNAs (sh1 and sh3) BMP7 fails to induce efficient nuclear translocation of SMAD4 (columns 3, 4). Transfection of full-length wildtype mouse Nup93 cDNA restores this effect (column 5). White dotted lines mark the borders of the nuclear area. Endogenous SMAD4 is stained in red. (b) Transfection of full-length Nup93 restores BMP7-induced nuclear accumulation of SMAD4 in NUP93 knockdown podocytes (column 1). In contrast, cells transfected with clones reflecting all 5 SRNS mutantions do not show efficient nuclear translocation of SMAD4 (columns 2-6). (c) In a luciferase reporter assay, treatment of HEK293 cells with BMP7 increases SMAD reporter activity. Knockdown of NUP93 with two different shRNAs (sh1 and sh3) reduces SMAD reporter activity in response to BMP7 stimulation. Transfection of mouse full-length Nup93 restores a cellular response that is compatible to control cells. In contrast, clones reflecting all 5 SRNS mutations fail to rescue the defects in BMP7-SMAD4 signaling. Data result from three independent experiments each, and are presented as mean and standard deviation. * Indicates statistical significance (p<0.05). (d) Upon stimulation of human podocytes with BMP7, endogenous importin7 (green) accumulates in a nuclear rim pattern (arrow heads). Knockdown of NUP93 by two shRNAs (sh1 and sh3) inhibits this rim formation. Scale bars are 10 μm. Nuclei are stained with DAPI (blue).

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