Research ArticleStress responses

The receptor tyrosine kinase HIR-1 coordinates HIF-independent responses to hypoxia and extracellular matrix injury

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Science Signaling  02 Oct 2018:
Vol. 11, Issue 550, eaat0138
DOI: 10.1126/scisignal.aat0138
  • Fig. 1 comt-5p::GFP is up-regulated by hypoxia in an hif-1–independent manner.

    (A) Fluorescent images of WT and hif-1(ia04) mutants carrying the genome-integrated dmaIs1 [comt-5p::GFP] transgene, exposed to normoxia or hypoxia. Representative of >100 animals. Scale bars, 200 μm. (B) Enlarged gray scale images of C. elegans carrying comt-5p::GFP under normoxia and hypoxia. Arrows in normoxia image indicate head neurons with GFP signal. Arrowheads in hypoxia image indicate hypodermal cells with GFP signal. Representative of >100 animals. Scale bars, 40 μm. (C) Schematic diagram of the dmaIs1 transgene. Upstream sequence (2010 bp) of the comt-5 gene was cloned before the GFP-coding sequence, followed by 3′ untranslated region (3′UTR) of the unc-54. The dmaIs1 transgene also carries unc-54p::mCherry as an additional marker of expression. (D) Analysis of the GFP signal by Western blot analysis of 20 randomly picked transgenic animals carrying the dmaIs1 [comt-5p::GFP] transgene. Histone H3 was used as a loading control. Transgenic animals express a cryptic nonspecific protein recognized by the GFP antibody (33 kDa) that does not contribute to fluorescence in living animals. Representative of three independent experiments. (E) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of comt-5 expression in normoxia- or hypoxia-exposed WT and hif-1(ia04) animals. n ≥ 200 total animals of mixed stages for each group analyzed in three independent biological replicates. ***P < 0.001. (F) qRT-PCR analysis of comt-5 expression in animals exposed to hypoxia (1% O2), anoxia (0% O2), or hypoxia-mimicking conditions (CoCl2, NaSH, or KCN). n ≥ 200 total animals of mixed stages for each group analyzed in three independent biological replicates. ***P < 0.001.

  • Fig. 2 HIR-1 is a cell-autonomous inhibitor of comt-5.

    (A) Fluorescent images of transgenic animals carrying the comt-5p::GFP transgene with various hir-1 alleles. Representative of >100 animals. (B) Schematic diagram of hir-1 with indicated LOF alleles isolated from EMS mutagenesis (dma51), CRISPR–Cas9 (CRISPR-associated protein 9) (dma101), and ultraviolet (UV) mutagenesis (tm3911 and tm4098). (C) Schematic map of various hir-1 transgenes. (D) Fluorescent images of transgenic animals expressing comt-5p::GFP (worm 1) with hir-1(dma101) (worm 2) and with hir-1(dma101); hir-1(+) array dmaEx113 (worm 3). Animals carrying extrachromosomal transgene dmaEx113 [dpy-7p::hir-1;myo-2::mCherry] have mCherry fluorescence in the pharynx. Representative of >100 animals. Scale bars, 300 μm. (E) Percentages of animals of indicated genotypes as in (D) with activated comt-5p::GFP. n ≥ 100 total animals of mixed stages for each group with three independent biological replicates. ***P < 0.001.

  • Fig. 3 Deficiency of specific cuticular genes mimics hypoxia-induced comt-5p::GFP activation.

    (A) Fluorescent images of comt-5p::GFP expression in the hypoderm of WT animals or mutants lacking dpy-2, dpy-3, or perl-1. Representative of >100 animals. (B) Schematic gene structures of dpy-2, dpy-3, and perl-1 showing the positions of EMS-derived LOF alleles. (C) Protein domain organization of dumpy 2 (DPY-2), DPY-3, and peroxidase-like 1 (PERL-1), with an asterisk indicating the positions of EMS-derived mutations. The G162R mutation in DPY-3 is in the conserved collagen domain; the Q4R mutation in DPY-2 is in the signal peptide. The missense mutations in the PERL-1 are in the N-terminal peroxidase-like domain. (D) Fluorescent images of animals carrying the col-19::GFP translational reporter exposed to normoxia, hypoxia (24 hours of ≤0.1% O2), or dpy-18(−) and RNAi directed against let-268, perl-1, and dpy-3. Representative of >30 animals. Scale bars, 25 μm. (E) Percentages of animals with COL-19::GFP disorganization with indicated genotypes and conditions. n = 10 1-day-old adults for each group analyzed in three independent biological replicates. ***P < 0.001. (F) Fluorescent images of comt-5p::GFP expression in animals treated with control or RNAi against the indicated genes essential for cuticle integrity. Representative of >100 animals. Scale bars, 200 μm.

  • Fig. 4 HIR-1–mediated regulation of comt-5p::GFP requires NHR-49 and MDT-15.

    (A) Fluorescent images of transgenic animals carrying comt-5p::GFP, and LOF and GOF of the indicated genes. Representative of >100 animals. Scale bars, 200 μm. (B) Western blot analysis of comt-5p::GFP expression in hir-1, hir-1; hif-1, and hir-1; nhr-49 mutants. (C) Schematic gene structures of nhr-49 and mdt-15 showing the position of EMS-derived mutations. (D) Protein sequence alignment of animal proteins orthologous to NHR-49 showing that Gly33 in NHR-49 is fully conserved in orthologs, including human hepatocyte nuclear factor 4 (HNF4). (E) Graph showing penetrance for increased comt-5p::GFP expression during developmental stages for each group. n ≥ 200 total animals of mixed stages for each group analyzed in three independent biological replicates. L1 to L4, larva 1 to larva 4; YA, young adult. (F) Penetrance analysis for increased comt-5p::GFP expression in the indicated strains. n ≥ 200 total adult animals for each group analyzed in three independent biological replicates. ***P < 0.001.

  • Fig. 5 Regulation of HIR-1 by hypoxia and ECM remodeling.

    (A) Fluorescent images of merged comt-5p::GFP and unc-54p::mCherry expression in animals treated with control or indicated drugs or RNAi against let-756. Scale bars, 200 μm. (B) Schematic map of hir-1 transgenes expressing translational reporters. (C) Fluorescent images of transgenic animals carrying the hir-1 translational reporter [rpl-28p::hir-1sig::GFP::hir-1cod; myo-2p::mCherry]. Hypoxia indicates 0% O2 for 24 hours. Arrows indicate GFP::HIR-1 foci. The transgenic marker myo-2p::mCherry expressed in the pharyngeal muscles is also shown. n ≥ 10 total animals for each group analyzed in ≥3 independent biological replicates. Scale bars, 300 μm. (D) Percentages of animals with GFP::HIR-1 foci. n = 10 total adult animals for each group analyzed in three independent biological replicates. ***P < 0.001. (E) SDS–polyacrylamide gel electrophoresis (PAGE) in the presence of the reducing agent β-mercaptoethanol and Western blot analysis of crude extracts of transgenic animals expressing HIR-1::GFP. Hypoxia indicates 0% O2 for 24 hours. The asterisk indicates full-length species of GFP::HIR-1, and the X indicates processed GFP::HIR-1. Representative of three independent experiments. (F) Domain arrangement in HIR-1. (G) SDS-PAGE in the presence of the reducing agent β-mercaptoethanol and Western blot analysis of crude extracts of transgenic animals expressing HIR-1::FLAG. ^, insoluble GFP::HIR-1; *, expected full-length species of HIR-1::FLAG; X, partial fragment of GFP::HIR-1. Hypoxia indicates 0% O2 for 24 hours. Representative of three independent experiments. (H) Quantification of GFP intensity of expressed comt-5p::GFP in the indicated strains. Hypoxia indicates 0.5% O2 for 24 hours. n ≥ 3 total adult animals for each group with three independent biological replicates. ***P < 0.001. a.u., arbitrary units.

  • Fig. 6 hir-1(LOF) is associated with altered ECM homeostasis and defective behavioral recovery after hypoxia.

    (A) Schematic map of dpy-3::Venus transgene (top) and fluorescent images of animals carrying the dpy-3::Venus translational reporter exposed to normoxia, hypoxia (24 hours of ≤0.1% O2), or RNAi against hir-1. Representative of >30 animals. Scale bars, 10 μm. (B) Percentages of animals with disrupted DPY-3::Venus pattern. n = 10 1-day-old adults for each group analyzed in three independent biological replicates. ***P < 0.001. (C) Fluorescent images of animals expressing col-19::GFP in the presence or absence of hir-1 RNAi. Representative of >30 animals. Scale bars, 25 μm. (D) Percentages of animals with COL-19::GFP disorganization. n = 10 1-day-old adults for each group analyzed in three independent biological replicates. ***P < 0.001. (E) WormLab plots showing representative locomotion tracks of WT and hir-1 animals in a 10-min time frame recorded after 24 hours of reoxygenation. Position is detected on the basis of the midpoint of the worm. (F) Displacement of WT and hir-1 mutants in a 10-min time frame recorded before exposure to hypoxia and after 0 to 10 min, 2 hours, and 24 hours of reoxygenation. n = 10 animals for each group analyzed in three independent biological replicates; ***P < 0.001. (G) Schematic model of the proposed HIR-1 pathway. Inactive components are shown in gray, and activated components are shown in black. In WT animals, hypoxia-induced changes in ECM transiently inhibit HIR-1 that permits comt-5 activation and ECM remodeling to maintain ECM homeostasis, whereas hir-1 mutants exhibit constitutive comt-5 activation, maladapted ECM, and decreased locomotor recovery after severe hypoxia.

  • Table 1 Mutants isolated from EMS mutagenesis with constitutive comt-5p::GFP expression.
    StrainAlleleSequenceGeneHomologyLocalizationPenetrance
    DMS99dma11UnknownUnknownUnknownUnknown100% (210)
    DMS176dma51C24G6.2hir-1Receptor tyrosine kinasePlasma membrane82% (134)
    DMS177dma52T14B4.6dpy-2CollagenExtracellular92% (145)
    DMS127dma22EGAP7.1dpy-3CollagenPlasma membrane75% (128)
    DMS696dma236C46A5.4perl-1Animal peroxidaseExtracellular88% (105)

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/550/eaat0138/DC1

    Fig. S1. Genes are differentially regulated by egl-9, hif-1, hypoxia, and hir-1.

    Fig. S2. Expression pattern of hir-1.

    Fig. S3. Hypoxia- and ECM-related genes similarly impair cuticle integrity.

    Fig. S4. Role of NHR-49 in hir-1 signaling.

    Fig. S5. Hypoxia activates comt-5 through a regulatory cascade involving ECM components (LET-756 and DPY-3) and HIR-1.

    Fig. S6. RNA-seq and qRT-PCR analysis of HIR-1–regulated genes.

    Fig. S7. Impaired cuticle integrity in hir-1 mutants under various conditions.

    Table S1. Genes that regulate comt-5p::GFP expression.

    Table S2. Primer sequences.

    Data file S1. RNA-seq analysis of egl-9, hif-1, and hypoxia-regulated genes.

    Data file S2. Gene ontology and KEGG pathway analyses of genes differentially regulated in hir-1 mutants compared with WT animals.

  • The PDF file includes:

    • Fig. S1. Genes are differentially regulated by egl-9, hif-1, hypoxia, and hir-1.
    • Fig. S2. Expression pattern of hir-1.
    • Fig. S3. Hypoxia- and ECM-related genes similarly impair cuticle integrity.
    • Fig. S4. Role of NHR-49 in hir-1 signaling.
    • Fig. S5. Hypoxia activates comt-5 through a regulatory cascade involving ECM components (LET-756 and DPY-3) and HIR-1.
    • Fig. S6. RNA-seq and qRT-PCR analysis of HIR-1–regulated genes.
    • Fig. S7. Impaired cuticle integrity in hir-1 mutants under various conditions.
    • Table S1. Genes that regulate comt-5p::GFP expression.
    • Table S2. Primer sequences.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). RNA-seq analysis of egl-9, hif-1, and hypoxia-regulated genes.
    • Data file S2 (Microsoft Excel format). Gene ontology and KEGG pathway analyses of genes differentially regulated in hir-1 mutants compared with WT animals.

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