Johanna Myllyharju

University of Oulu, Oulu, Oulu, Finland

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Publications (142)

  • [Show abstract] [Hide abstract] ABSTRACT: Erythrocytosis is mainly driven by erythropoietin, which is regulated by hypoxia-inducible factor (HIF). Mutations in HIF prolyl 4-hydroxylase 2 (HIF-P4H-2/PHD2/EGLN1), the major downregulator of HIFα subunits, are found in familiar erythrocytosis, and large-spectrum conditional inactivation of HIF-P4H-2 in mice leads to severe erythrocytosis. Although bone marrow is the primary site for erythropoiesis, spleen remains capable for extramedullary erythropoiesis. We studied HIF-P4H-2 deficient mice ( Hif-p4h-2 gt/gt ) which show slightly induced erythropoiesis upon aging despite non-increased erythropoietin levels, and identified spleen as the site of extramedullary erythropoiesis. Splenic hematopoietic stem cells (HSCs) of these mice exhibited increased BFU-E growth and the mice were protected against anemia. HIF-1α and HIF-2α were stabilized in the spleens, while the Notch ligands and target Jag1, Jag2, Dll1 and Hes1 became downregulated upon aging HIF-2α-dependently. Inhibition of notch signaling in wild-type spleen HSCs phenocopied the increased BFU-E growth. HIFα stabilization can thus mediate non-erythropoietin-driven splenic erythropoiesis via altered Notch signaling.
    Article · Nov 2016 · Molecular and Cellular Biology
  • Nadiya Byts · Jenny jaurilla · Prodeep Poudel · [...] · Johanna Myllyharju
    Poster · Sep 2016
  • [Show abstract] [Hide abstract] ABSTRACT: Age-related macular degeneration (AMD), affecting the retinal pigment epithelium (RPE), is the leading cause of blindness in middle-aged and older people in developed countries. Genetic and environmental risk factors have been identified, but no effective cure exists. Using a mouse model we show that a transmembrane prolyl 4-hydroxylase (P4H-TM), which participates in the oxygen-dependent regulation of the hypoxia-inducible factor (HIF), is a potential novel candidate gene for AMD. We show that P4h-tm had its highest expression levels in the mouse RPE and brain, heart, lung, skeletal muscle and kidney. P4h-tm(-/-) mice were fertile and had a normal life span. Lack of P4h-tm stabilized HIF-1α in cortical neurons under normoxia, while in hypoxia it increased the expression of certain HIF target genes in tissues with high endogenous P4h-tm expression levels more than in wild-type mice. Renal erythropoietin levels increased in P4h-tm(-/-) mice with aging, but the resulting ∼2-fold increase in erythropoietin serum levels did not lead to erythrocytosis. Instead, accumulation of lipid-containing lamellar bodies in renal tubuli was detected in P4h-tm(-/-) mice with aging, resulting in inflammation and fibrosis, and later glomerular sclerosis and albuminuria. Lack of P4h-tm was associated with retinal thinning, rosette-like infoldings and drusen-like structure accumulation in RPE with aging, as is characteristic of AMD. Photoreceptor recycling was compromised, and electroretinograms revealed functional impairment of the cone pathway in adult P4h-tm(-/-) mice and cone and rod deficiency in middle-aged mice. P4H-TM is therefore imperative for normal vision, and potentially a novel candidate for age-induced diseases, such as AMD.
    Article · Jul 2016 · Human Molecular Genetics
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    [Show abstract] [Hide abstract] ABSTRACT: The kidney vasculature is critical for renal function, but its developmental assembly mechanisms remain poorly understood and models for studying its assembly dynamics are limited. Here, we tested whether the embryonic kidney contains endothelial cells (ECs) that are heterogeneous with respect to VEGFR2/Flk1/KDR, CD31/PECAM, and CD146/MCAM markers. Tie1Cre;R26RYFP-based fate mapping with a time-lapse in embryonic kidney organ culture successfully depicted the dynamics of kidney vasculature development and the correlation of the process with the CD31+ EC network. Depletion of Tie1+ or CD31+ ECs from embryonic kidneys, with either Tie1Cre-induced diphtheria toxin susceptibility or cell surface marker–based sorting in a novel dissociation and reaggregation technology, illustrated substantial EC network regeneration. Depletion of the CD146+ cells abolished this EC regeneration. Fate mapping of green fluorescent protein (GFP)-marked CD146+/CD31- cells indicated that they became CD31+ cells, which took part in EC structures with CD31+ wild-type ECs. EC network development depends on VEGF signaling, and VEGF and erythropoietin are expressed in the embryonic kidney even in the absence of any external hypoxic stimulus. Thus, the ex vivo embryonic kidney culture models adopted here provided novel ways for targeting renal EC development and demonstrated that CD146+ cells are critical for kidney vasculature development.
    Full-text available · Article · Apr 2016 · Kidney International
  • [Show abstract] [Hide abstract] ABSTRACT: Objective: Small-molecule hypoxia-inducible factor-prolyl 4-hydroxylase (HIF-P4H) inhibitors are being explored in clinical studies for the treatment of anemia. HIF-P4H-2 (also known as PHD2 or EglN1) inhibition improves glucose and lipid metabolism and protects against obesity and metabolic dysfunction. We studied here whether HIF-P4H-2 inhibition could also protect against atherosclerosis. Approach and results: Atherosclerosis development was studied in low-density lipoprotein (LDL) receptor-deficient mice treated with an oral HIF-P4H inhibitor, FG-4497, and in HIF-P4H-2-hypomorphic/C699Y-LDL receptor-mutant mice, all mice being fed a high-fat diet. FG-4497 administration to LDL receptor-deficient mice reduced the area of atherosclerotic plaques by ≈50% when compared with vehicle-treated controls and also reduced their weight gain, insulin resistance, liver, and white adipose tissue (WAT) weights, adipocyte size, number of inflammation-associated WAT macrophage aggregates and the high-fat diet-induced increases in serum cholesterol levels. The levels of atherosclerosis-protecting circulating autoantibodies against copper-oxidized LDL were increased. The decrease in atherosclerotic plaque areas correlated with the reductions in weight, serum cholesterol levels, and WAT macrophage aggregates and the autoantibody increase. FG-4497 treatment stabilized HIF-1α and HIF-2α and altered the expression of glucose and lipid metabolism and inflammation-associated genes in liver and WAT. The HIF-P4H-2-hypomorphic/C699Y-LDL receptor-mutant mice likewise had a ≈50% reduction in atherosclerotic plaque areas, reduced WAT macrophage aggregate numbers, and increased autoantibodies against oxidized LDL, but did not have reduced serum cholesterol levels. Conclusions: HIF-P4H-2 inhibition may be a novel strategy for protecting against the development of atherosclerosis. The mechanisms involve beneficial modulation of the serum lipid profile and innate immune system and reduced inflammation.
    Article · Feb 2016 · Arteriosclerosis Thrombosis and Vascular Biology
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    [Show abstract] [Hide abstract] ABSTRACT: The Wnts can be considered as candidates for the Congenital Anomaly of Kidney and Urinary Tract, CAKUT diseases since they take part in the control of kidney organogenesis. Of them Wnt5a is expressed in ureteric bud (UB) and its deficiency leads to duplex collecting system (13/90) uni- or bilateral kidney agenesis (10/90), hypoplasia with altered pattern of ureteric tree organization (42/90) and lobularization defects with partly fused ureter trunks (25/90) unlike in controls. The UB had also notably less tips due to Wnt5a deficiency being at E15.5 306 and at E16.5 765 corresponding to 428 and 1022 in control (p<0.02; p<0.03) respectively. These changes due to Wnt5a knock out associated with anomalies in the ultrastructure of the UB daughter epithelial cells. The basement membrane (BM) was malformed so that the BM thickness increased from 46.3 nm to 71.2 nm (p<0.01) at E16.5 in the Wnt5a knock out when compared to control. Expression of a panel of BM components such as laminin and of type IV collagen was also reduced due to the Wnt5a knock out. The P4ha1 gene that encodes a catalytic subunit of collagen prolyl 4-hydroxylase I (C-P4H-I) in collagen synthesis expression and the overall C-P4H enzyme activity were elevated by around 26% due to impairment in Wnt5a function from control. The compound Wnt5a+/-;P4ha1+/- embryos demonstrated Wnt5a-/- related defects, for example local hyperplasia in the UB tree. A R260H WNT5A variant was identified from renal human disease cohort. Functional studies of the consequence of the corresponding mouse variant in comparison to normal ligand reduced Wnt5a-signalling in vitro. Together Wnt5a has a novel function in kidney organogenesis by contributing to patterning of UB derived collecting duct development contributing putatively to congenital disease.
    Full-text available · Article · Jan 2016 · PLoS ONE
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    Data: S1 Fig
    [Show abstract] [Hide abstract] ABSTRACT: Lack of Wnt5a signaling causes severe anomalies in the basement membrane of embryonic kidney. Wild-type (Wt) and Wnt5a-/- kidneys at the E16.5 were sectioned and stained with antibodies against type IV collagen (A, B) and pan-laminin (C,D). Production of type IV collagen (compare B with A, arrows) and laminin (compare D with C, arrows) in the collecting duct are reduced in the Wnt5a-/- mice relative to Wt. Expression of certain genes encoding basement membrane components were analysed by qRT-PCR in the Wnt5a-/- and wild-type (Wt) E16.5 kidneys (E). Expression of the genes Lama2 (laminin α2), Lamb1 (laminin β1), Lamc2 (laminin γ2), Lamc3 (laminin γ3), Col4a1, Col4a2, and Col4a3 (α1–3 chains of collagen IV) was decreased, whereas expression of Lamb2 (laminin β2) was increased in the Wnt5a-/- kidneys relative to Wt. Western blotting studies depict a notable decrease in laminin-111 production in lysates derived from whole kidneys at E16.5 (G). CD, collecting duct, scale bars, A-D 100 μm. (PDF)
    File available · Data · Jan 2016
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    Data: S2 Fig
    [Show abstract] [Hide abstract] ABSTRACT: Identification of a novel human WNT5A variant in a CAKUT patient. Sanger sequencing was performed for the coding region of the WNT5A from DNA samples that were derived from a cohort of 115 CAKUT and 14 Alport syndrome patients respectively. A) The status of the father is unknown, while the mother had been diagnosed to have CAKUT in the past. Sequence traces for the case-parent trio indicated the inheritance for the heterozygous c.779G>A variant in the patient and the father, depicted by arrows. A reference sequencing that represents a consensus of 189 healthy Dutch control individuals serves as controls. B) Alignment of the human WNT5A amino acid sequence with those of several other vertebrates reveals conservation of the amino acid sequence where the variant p. R260H transition was identified. (PDF)
    File available · Data · Jan 2016
  • [Show abstract] [Hide abstract] ABSTRACT: Key messages: HIF-P4H-2 deficiency protects skeletal muscle from ischemia-reperfusion injury. The mechanisms involved are mediated via normoxic HIF-1α and HIF-2α stabilization. HIF-P4H-2 deficiency increases capillary size but not number. HIF-P4H-2 deficiency maintains energy metabolism during ischemia-reperfusion.
    Article · Oct 2015 · Journal of Molecular Medicine
  • Data · Oct 2015
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    [Show abstract] [Hide abstract] ABSTRACT: Overcoming resistance to chemotherapy is a major challenge in colorectal cancer (CRC) treatment, especially since the underlying molecular mechanisms remain unclear. We show that silencing of the prolyl hydroxylase domain protein PHD1, but not PHD2 or PHD3, prevents p53 activation upon chemotherapy in different CRC cell lines, thereby inhibiting DNA repair and favoring cell death. Mechanistically, PHD1 activity reinforces p53 binding to p38α kinase in a hydroxylation-dependent manner. Following p53-p38α interaction and chemotherapeutic damage, p53 can be phosphorylated at serine 15 and thus activated. Active p53 allows nucleotide excision repair by interacting with the DNA helicase XPB, thereby protecting from chemotherapy-induced apoptosis. In accord with this observation, PHD1 knockdown greatly sensitizes CRC to 5-FU in mice. We propose that PHD1 is part of the resistance machinery in CRC, supporting rational drug design of PHD1-specific inhibitors and their use in combination with chemotherapy. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.
    Full-text available · Article · Aug 2015 · EMBO Molecular Medicine
  • Ellinoora Aro · Antti M Salo · Richa Khatri · [...] · Johanna Myllyharju
    [Show abstract] [Hide abstract] ABSTRACT: Collagen prolyl 4-hydroxylases (C-P4Hs I-III) catalyze formation of 4-hydroxyproline (4Hyp) residues required to form triple-helical collagen molecules. Vertebrate C-P4Hs are α2β2 tetramers differing in their catalytic α subunits. C-P4H-I is the major isoenzyme in most cells and inactivation of its catalytic subunit (P4ha1(-/-)) leads to embryonic lethality in mouse, whereas P4ha1(+/-) mice have no abnormalities. To study the role of C-P4H-II, which predominates in chondrocytes, we generated P4ha2(-/-) mice. Surprisingly, they had no apparent phenotypic abnormalities. To assess possible functional complementarity, we established P4ha1(+/-);P4ha2(-/-) mice. They were smaller than their littermates, had moderate chondrodysplasia and developed kyphosis. A transient inner cell death phenotype was detected in their developing growth plates. The columnar arrangement of proliferative chondrocytes was impaired, the amount of 4Hyp and the Tm of collagen II were reduced and the extracellular matrix (ECM) was softer in the growth plates of newborn P4ha1(+/-);P4ha2(-/-) mice. No signs of uncompensated ER stress were detected in the mutant growth plate chondrocytes. Some of these defects were also found in P4ha2(-/-) mice, although in a much milder form. Our data show that C-P4H-I can to a large extent compensate for the lack of C-P4H-II in proper endochondral bone development, but their combined partial and complete inactivation, respectively, leads to biomechanically impaired ECM, moderate chondrodysplasia and kyphosis. Our mouse data suggest that inactivating mutations in human P4HA2 are not likely to lead to skeletal disorders and a simultaneous decrease in P4HA1 function would most probably be required to generate such a disease phenotype. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Article · May 2015 · Journal of Biological Chemistry