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Lysyl Hydroxylase 2 Is Secreted by Tumor Cells and Can Modify Collagen in the Extracellular Space*

  • Author Footnotes
    1 Both authors contributed equally to this work.
    Yulong Chen
    Footnotes
    1 Both authors contributed equally to this work.
    Affiliations
    From the Department of Thoracic/Head and Neck Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas 77030,
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  • Author Footnotes
    1 Both authors contributed equally to this work.
    Houfu Guo
    Footnotes
    1 Both authors contributed equally to this work.
    Affiliations
    From the Department of Thoracic/Head and Neck Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas 77030,
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  • Masahiko Terajima
    Affiliations
    Oral and Craniofacial Health Sciences, School of Dentistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599,
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  • Priyam Banerjee
    Affiliations
    From the Department of Thoracic/Head and Neck Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas 77030,
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  • Xin Liu
    Affiliations
    From the Department of Thoracic/Head and Neck Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas 77030,
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  • Jiang Yu
    Affiliations
    From the Department of Thoracic/Head and Neck Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas 77030,
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  • Amin A. Momin
    Affiliations
    the Department of Clinical Cancer Prevention, University of Texas MD Anderson Cancer Center, Houston, Texas 77030,
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  • Hiroyuki Katayama
    Affiliations
    the Department of Clinical Cancer Prevention, University of Texas MD Anderson Cancer Center, Houston, Texas 77030,
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  • Samir M. Hanash
    Affiliations
    the Department of Clinical Cancer Prevention, University of Texas MD Anderson Cancer Center, Houston, Texas 77030,
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  • Alan R. Burns
    Affiliations
    the College of Optometry, University of Houston, Houston, Texas 77004, and
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  • Gregg B. Fields
    Affiliations
    the Department of Chemistry and Biochemistry, Florida Atlantic University, Jupiter, Florida 33458
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  • Mitsuo Yamauchi
    Correspondence
    To whom correspondence may be addressed.
    Affiliations
    Oral and Craniofacial Health Sciences, School of Dentistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599,
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  • Jonathan M. Kurie
    Correspondence
    To whom correspondence may be addressed:
    Affiliations
    From the Department of Thoracic/Head and Neck Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas 77030,
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants R21AR060978 (NIAMS; to M. Y.) and R01CA105155 (NCI; to J. M. K. and M. Y.). This work was also supported by the Elza A. and Ina S. Freeman Professorship in Lung Cancer (to J. M. K.) and MD Anderson Cancer Center Support Grant CA016672. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
    1 Both authors contributed equally to this work.
Open AccessPublished:November 01, 2016DOI:https://doi.org/10.1074/jbc.M116.759803

      Abstract

      Lysyl hydroxylase 2 (LH2) catalyzes the hydroxylation of lysine residues in the telopeptides of fibrillar collagens, which leads to the formation of stable collagen cross-links. Recently we reported that LH2 enhances the metastatic propensity of lung cancer by increasing the amount of stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs), which generate a stiffer tumor stroma (Chen, Y., et al. (2015) J. Clin. Invest. 125, 125, 1147–1162). It is generally accepted that LH2 modifies procollagen α chains on the endoplasmic reticulum before the formation of triple helical procollagen molecules. Herein, we report that LH2 is also secreted and modifies collagen in the extracellular space. Analyses of lung cancer cell lines demonstrated that LH2 is present in the cell lysates and the conditioned media in a dimeric, active form in both compartments. LH2 co-localized with collagen fibrils in the extracellular space in human lung cancer specimens and in orthotopic lung tumors generated by injection of a LH2-expressing human lung cancer cell line into nude mice. LH2 depletion in MC3T3 osteoblastic cells impaired the formation of HLCCs, resulting in an increase in the unmodified lysine aldehyde-derived collagen cross-link (LCC), and the addition of recombinant LH2 to the media of LH2-deficient MC3T3 cells was sufficient to rescue HLCC formation in the extracellular matrix. The finding that LH2 modifies collagen in the extracellular space challenges the current view that LH2 functions solely on the endoplasmic reticulum and could also have important implications for cancer biology.

      Introduction

      Collagens are the main component of the extracellular matrix and the most abundant protein in mammals (
      • Di Lullo G.A.
      • Sweeney S.M.
      • Korkko J.
      • Ala-Kokko L.
      • San Antonio J.D.
      Mapping the ligand-binding sites and disease-associated mutations on the most abundant protein in the human, type I collagen.
      ). Of the 29 types of collagen identified, fibrillar type I collagen is the most abundant and important in providing tissues and organs with form and mechanical strength. Type I collagen biosynthesis involves a series of post-translational modifications of nascent procollagen α chains on the endoplasmic reticulum; for example, specific lysine (Lys) and proline (Pro) residues undergo hydroxylation, and hydroxylysine (Hyl) residues undergo mono- and di-glycosylation. In the extracellular space, telopeptidyl Lys and Hyl residues on collagen molecules are oxidatively deaminated by lysyl oxidases (LOXs),
      The abbreviations used are: LOX, lysyl oxidase, LH2, lysyl hydroxylase 2, HLCCs, hydroxylysine aldehyde-derived collagen cross-links, deH-HLNL, dehydro-hydroxylysinonorleucine, deH-DHLNL, deH-dihydroxylysinonorlecine, Pyr, pyridinoline, d-Pyr, deoxypyridinoline, deH-HHMD, deH-histidinohydroxymerodesmosine, LCCs, Lys aldehyde-derived cross-links, MC, MC3T3-E1, SILAC, stable isotope labeling by amino acids in cell culture, Q-PCR, quantitative real-time PCR, FT, Fourier transform, (IKG)3, IKGIKGIKG.
      The abbreviations used are: LOX, lysyl oxidase, LH2, lysyl hydroxylase 2, HLCCs, hydroxylysine aldehyde-derived collagen cross-links, deH-HLNL, dehydro-hydroxylysinonorleucine, deH-DHLNL, deH-dihydroxylysinonorlecine, Pyr, pyridinoline, d-Pyr, deoxypyridinoline, deH-HHMD, deH-histidinohydroxymerodesmosine, LCCs, Lys aldehyde-derived cross-links, MC, MC3T3-E1, SILAC, stable isotope labeling by amino acids in cell culture, Q-PCR, quantitative real-time PCR, FT, Fourier transform, (IKG)3, IKGIKGIKG.
      producing the reactive aldehydes Lysald and Hylald, respectively, which initiates a series of condensation reactions to form various covalent intermolecular cross-links involving juxtaposed Lys, Hyl, and histidine (His) residues on the neighboring molecules, resulting in the formation of Hylald-derived collagen cross-links (HLCCs) (
      • Yamauchi M.
      • Sricholpech M.
      Lysine post-translational modifications of collagen.
      ).
      HLCCs are generated through a series of intermediate steps involving multiple enzymatic reactions. For example, HLCCs are produced through the divalent iminium cross-links dehydro-hydroxylysinonorleucine (deH-HLNL) when paired with a juxtaposed Lys residue (i.e. HylaldXLys) on a neighboring molecule, and deH-dihydroxylysinonorlecine (deH-DHLNL) with a Hyl residue (i.e. HylaldXHyl). These are then spontaneously rearranged to form the stable ketoamines by Amadori rearrangement then mature into the formation of the trivalent pyridinium cross-links, pyridinoline (Pyr) (i.e. HylaldXHylaldXHyl) and deoxypyridinoline (d-Pyr) (i.e. HylaldXHylaldXLys). This pathway is predominant in skeletal tissues such as bone and cartilage. The Lysald-derived cross-linking pathway, on the other hand, is predominant in soft connective tissues. Telopeptidyl Lysald can condense with another Lysald residue within the same molecule to form an intramolecular aldol, which then eventually leads to a tetravalent intermolecular cross-link, deH-histidinohydroxymerodesmosine (deH-HHMD) (i.e. LysaldXLysaldXHisXHyl) (
      • Tanzer M.L.
      • Housley T.
      • Berube L.
      • Fairweather R.
      • Franzblau C.
      • Gallop P.M.
      Structure of two histidine-containing crosslinks from collagen.
      ). In skin and cornea, the Lysald-derived cross-linking pathway can also lead to a non-reducible, trivalent cross-link, histidinohydroxylysinonorleucine by involving the divalent, iminium cross-link, deH-HLNL (LysaldXHyl), and a His residue (i.e. LysaldXHylXHis) (
      • Yamauchi M.
      • London R.E.
      • Guenat C.
      • Hashimoto F.
      • Mechanic G.L.
      Structure and formation of a stable histidine-based trifunctional cross-link in skin collagen.
      ).
      Lys hydroxylation is catalyzed by lysyl hydroxylases 1–3 (LH1–3; EC 1.14.11.4) in -X-Lys-Gly- sequences in a reaction that requires Fe2+, 2-oxoglutarate, O2, and ascorbate (
      • Kivirikko K.I.
      • Pihlajaniemi T.
      Collagen hydroxylases and the protein disulfide isomerase subunit of prolyl 4-hydroxylases.
      ). In addition to the -X-Lys-Gly- sequence, -X-Lys-Ala-and -X-Lys-Ser- sequences present in the telopeptides (both N and C termini) of fibrillar collagens can be hydroxylated. It has been reported that LH2 catalyzes Lys hydroxylation in the telopeptides (
      • Uzawa K.
      • Grzesik W.J.
      • Nishiura T.
      • Kuznetsov S.A.
      • Robey P.G.
      • Brenner D.A.
      • Yamauchi M.
      Differential expression of human lysyl hydroxylase genes, lysine hydroxylation, and cross-linking of type I collagen during osteoblastic differentiation in vitro.
      ,
      • Mercer D.K.
      • Nicol P.F.
      • Kimbembe C.
      • Robins S.P.
      Identification, expression, and tissue distribution of the three rat lysyl hydroxylase isoforms.
      • van der Slot A.J.
      • Zuurmond A.M.
      • Bardoel A.F.
      • Wijmenga C.
      • Pruijs H.E.
      • Sillence D.O.
      • Brinckmann J.
      • Abraham D.J.
      • Black C.M.
      • Verzijl N.
      • DeGroot J.
      • Hanemaaijer R.
      • TeKoppele J.M.
      • Huizinga T.W.
      • Bank R.A.
      Identification of PLOD2 as telopeptide lysyl hydroxylase, an important enzyme in fibrosis.
      ) and thereby drives the Hylald-derived collagen cross-linking pathway (
      • Pornprasertsuk S.
      • Duarte W.R.
      • Mochida Y.
      • Yamauchi M.
      Lysyl hydroxylase-2b directs collagen cross-linking pathways in MC3T3-E1 cells.
      ). Altered LH2 expression has a profound impact on the collagen matrix (
      • Pornprasertsuk S.
      • Duarte W.R.
      • Mochida Y.
      • Yamauchi M.
      Overexpression of lysyl hydroxylase-2b leads to defective collagen fibrillogenesis and matrix mineralization.
      ). Although all LH family members (LH1–3) appear to be capable of hydroxylating helical Lys residues, only LH2 modifies the telopeptidyl Lys residues (
      • van der Slot A.J.
      • Zuurmond A.M.
      • Bardoel A.F.
      • Wijmenga C.
      • Pruijs H.E.
      • Sillence D.O.
      • Brinckmann J.
      • Abraham D.J.
      • Black C.M.
      • Verzijl N.
      • DeGroot J.
      • Hanemaaijer R.
      • TeKoppele J.M.
      • Huizinga T.W.
      • Bank R.A.
      Identification of PLOD2 as telopeptide lysyl hydroxylase, an important enzyme in fibrosis.
      ,
      • Takaluoma K.
      • Lantto J.
      • Myllyharju J.
      Lysyl hydroxylase 2 is a specific telopeptide hydroxylase, while all three isoenzymes hydroxylate collagenous sequences.
      ). Inherited skeletal disorders caused by inactivating mutations in the gene that encodes LH2 and a putative LH2 foldase, FKBP10 (
      • Kelley B.P.
      • Malfait F.
      • Bonafe L.
      • Baldridge D.
      • Homan E.
      • Symoens S.
      • Willaert A.
      • Elcioglu N.
      • Van Maldergem L.
      • Verellen-Dumoulin C.
      • Gillerot Y.
      • Napierala D.
      • Krakow D.
      • Beighton P.
      • Superti-Furga A.
      • De Paepe A.
      • Lee B.
      Mutations in FKBP10 cause recessive osteogenesis imperfecta and Bruck syndrome.
      ,
      • Schwarze U.
      • Cundy T.
      • Pyott S.M.
      • Christiansen H.E.
      • Hegde M.R.
      • Bank R.A.
      • Pals G.
      • Ankala A.
      • Conneely K.
      • Seaver L.
      • Yandow S.M.
      • Raney E.
      • Babovic-Vuksanovic D.
      • Stoler J.
      • Ben-Neriah Z.
      • et al.
      Mutations in FKBP10, which result in Bruck syndrome and recessive forms of osteogenesis imperfecta, inhibit the hydroxylation of telopeptide lysines in bone collagen.
      • Shaheen R.
      • Al-Owain M.
      • Faqeih E.
      • Al-Hashmi N.
      • Awaji A.
      • Al-Zayed Z.
      • Alkuraya F.S.
      Mutations in FKBP10 cause both Bruck syndrome and isolated osteogenesis imperfecta in humans.
      ) demonstrate the importance of telopeptidyl Lys hydroxylation in normal collagen biosynthesis and function (
      • Puig-Hervás M.T.
      • Temtamy S.
      • Aglan M.
      • Valencia M.
      • Martínez-Glez V.
      • Ballesta-Martínez M.J.
      • López-González V.
      • Ashour A.M.
      • Amr K.
      • Pulido V.
      • Guillén-Navarro E.
      • Lapunzina P.
      • Caparrós-Martín J.A.
      • Ruiz-Perez V.L.
      Mutations in PLOD2 cause autosomal-recessive connective tissue disorders within the Bruck syndrome: osteogenesis imperfecta phenotypic spectrum.
      ,
      • Ha-Vinh R.
      • Alanay Y.
      • Bank R.A.
      • Campos-Xavier A.B.
      • Zankl A.
      • Superti-Furga A.
      • Bonafé L.
      Phenotypic and molecular characterization of Bruck syndrome (osteogenesis imperfecta with contractures of the large joints) caused by a recessive mutation in PLOD2.
      ).
      It has been reported that LH3, a multifunctional enzyme possessing both LH and glycosyltransferase activities, can be secreted and modifies Lys residues on native proteins and possibly “microfolded” mature collagen molecules in the extracellular space (
      • Salo A.M.
      • Wang C.
      • Sipilä L.
      • Sormunen R.
      • Vapola M.
      • Kervinen P.
      • Ruotsalainen H.
      • Heikkinen J.
      • Myllylä R.
      Lysyl hydroxylase 3 (LH3) modifies proteins in the extracellular space, a novel mechanism for matrix remodeling.
      ). Further studies showed that the glucosyltransferase activity site is required for LH3 to be secreted into the extracellular space (
      • Wang C.
      • Ristiluoma M.M.
      • Salo A.M.
      • Eskelinen S.
      • Myllylä R.
      Lysyl hydroxylase 3 is secreted from cells by two pathways.
      ,
      • Salo A.M.
      • Sipilä L.
      • Sormunen R.
      • Ruotsalainen H.
      • Vainio S.
      • Myllylä R.
      The lysyl hydroxylase isoforms are widely expressed during mouse embryogenesis, but obtain tissue- and cell-specific patterns in the adult.
      ). These findings are the basis of the current paradigm that LH3 is the only LH isoform with an extracellular function. However, whether other LH members are secreted and capable of modifying Lys residues in the extracellular space has not been thoroughly explored.
      In breast cancer, lung cancer, sarcoma, and other tumor types, high LH2 expression increases tumor stiffness, promotes tumor cell invasion and metastasis, and is a predictor of poor clinical outcome (
      • Gilkes D.M.
      • Bajpai S.
      • Wong C.C.
      • Chaturvedi P.
      • Hubbi M.E.
      • Wirtz D.
      • Semenza G.L.
      Procollagen lysyl hydroxylase 2 is essential for hypoxia-induced breast cancer metastasis.
      ,
      • Chen Y.
      • Terajima M.
      • Yang Y.
      • Sun L.
      • Ahn Y.H.
      • Pankova D.
      • Puperi D.S.
      • Watanabe T.
      • Kim M.P.
      • Blackmon S.H.
      • Rodriguez J.
      • Liu H.
      • Behrens C.
      • Wistuba I.I.
      • Minelli R.
      • et al.
      Lysyl hydroxylase 2 induces a collagen cross-link switch in tumor stroma.
      • Eisinger-Mathason T.S.
      • Zhang M.
      • Qiu Q.
      • Skuli N.
      • Nakazawa M.S.
      • Karakasheva T.
      • Mucaj V.
      • Shay J.E.
      • Stangenberg L.
      • Sadri N.
      • Puré E.
      • Yoon S.S.
      • Kirsch D.G.
      • Simon M.C.
      Hypoxia-dependent modification of collagen networks promotes sarcoma metastasis.
      ). In lung cancer, high LH2 expression increases the amount of HLCCs in tumor stroma (
      • Chen Y.
      • Terajima M.
      • Yang Y.
      • Sun L.
      • Ahn Y.H.
      • Pankova D.
      • Puperi D.S.
      • Watanabe T.
      • Kim M.P.
      • Blackmon S.H.
      • Rodriguez J.
      • Liu H.
      • Behrens C.
      • Wistuba I.I.
      • Minelli R.
      • et al.
      Lysyl hydroxylase 2 induces a collagen cross-link switch in tumor stroma.
      ). Moreover, LH2 has been detected in the secretome of breast cancer cells (
      • Blanco M.A.
      • LeRoy G.
      • Khan Z.
      • Alečković M.
      • Zee B.M.
      • Garcia B.A.
      • Kang Y.
      Global secretome analysis identifies novel mediators of bone metastasis.
      ), which raises the possibility that LH2 has an extracellular function. In this study we demonstrate that LH2 is associated with extracellular collagen fibrils in lung cancer tissue specimens and is secreted into the extracellular space in an active dimeric form. Importantly, our data strongly indicate that secreted LH2 is capable of hydroxylating telopeptidyl Lys residues of collagen in the extracellular space, thereby producing stable HLCCs. This implies that extracellular LH2 modifies telopeptidyl Lys residues before their LOX-catalyzed conversion to aldehyde, which challenges the current paradigm that LH2 modifies only intracellular nascent procollagen α chains.

      Author Contributions

      Y. C., H. G., and M. T. conducted the experiments, including generation of stable cell lines, purification of the recombinant proteins, and performance of in vitro hydroxylase activity assays, biochemical and mass spectrometric analyses, and analysis of and interpretation of the data. A. A. M., H. K., and S. M. H. conducted and interpreted the stable isotope labeling of amino acids in cell culture analyses. P. B. performed the immunofluorescent staining and colocalization analysis. A. R. B. conducted the immunogold labeling and transmission electron microscopy experiment. G. B. F., J. Y., and X. L. provided technical assistance. J. M. K., M. Y., and Y. C. conceived the project, interpreted all of the data, and wrote the paper with other co-authors. All authors reviewed the results and approved the final version of the manuscript.

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