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Immunocytochemical assessment of cell differentiation of podoplanin-positive osteoblasts into osteocytes in murine bone

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Abstract

In this study, we examined the immunolocalization of podoplanin/E11, CD44, actin filaments, and phosphorylated ezrin in the osteoblasts on the verge of differentiating into osteocytes in murine femora and tibiae. When observing under stimulated emission depletion microscopy, unlike podoplanin-negative osteoblasts, podoplanin-positive osteoblasts showed a rearranged assembly of actin filaments along the cell membranes which resembled that of embedded osteocytes. In the metaphysis, i.e., the bone remodeling site, CD44-bearing osteoclasts were either proximal to or in contact with podoplanin-positive osteoblasts, but the podoplanin-positive osteoblasts also localized CD44 on their own cell surface. These podoplanin-positive osteoblasts, which either possessed CD44 on their cell surface or were close to CD44-bearing osteoclasts, showed phosphorylated ezrin-positivity on the cell membranes. Therefore, the CD44/podoplanin interaction on the cell surface may be involved in the osteoblastic differentiation into osteocytes in the metaphyses, via the mediation of podoplanin-driven ezrin phosphorylation and the subsequent reorganized assembly of actin filaments. Consistently, the protein expression of phosphorylated ezrin was increased after CD44 administration in calvarial culture. Conversely, in modeling sites such as the cortical bones, podoplanin-positive osteoblasts were uniformly localized at certain intervals even without contact with CD44-positive bone marrow cells; furthermore, they also exhibited phosphorylated ezrin immunoreactivity along their cell membranes. Taken together, it seems likely that the CD44/podoplanin interaction is involved in osteoblastic differentiation into osteocytes in the bone remodeling area but not in modeling sites.

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References

  • Amizuka N, Takahashi N, Udagawa N, Suda T, Ozawa H (1997) An ultrastructural study of cell–cell contact between mouse spleen cells and calvaria-derived osteoblastic cells in a co-culture system for osteoclast formation. Acta Histchem Cytochem 30:351–362

    Google Scholar 

  • Amizuka N, Hasegawa T, Oda K, Luiz de Freitas PH, Hoshi K, Li M, Ozawa H (2012) Histology of epiphyseal cartilage calcification and endochondral ossification. Front Biosci 4:2085–2100

    Google Scholar 

  • Barragan-Adjemian C, Nicolella D, Dusevich V, Dallas MR, Eick JD, Bonewald LF (2006) Mechanism by which MLO-A5 late osteoblasts/early osteocytes mineralize in culture: similarities with mineralization of lamellar bone. Calcif Tissue Int 79:340–353

    PubMed  PubMed Central  CAS  Google Scholar 

  • Breiteneder-Geleff S, Matsui K, Soleiman A, Meraner P, Poczewski H, Kalt R, Schaffner G, Kerjaschki D (1997) Podoplanin, novel 43-kd membrane protein of glomerular epithelial cells, is down-regulated in puromycin nephrosis. Am J Pathol 151:1141–1152

    PubMed  PubMed Central  CAS  Google Scholar 

  • Burger EH, Klein-Nulend J (1999) Mechanotransduction in bone-role of the lacuno-canalicular network. FASEB J 13(Suppl):S101-112

    PubMed  CAS  Google Scholar 

  • Burr DB (2002) Targeted and nontargeted remodeling. Bone 30:2–4

    PubMed  CAS  Google Scholar 

  • Costa YF, Tjioe KC, Nonogaki S, Soares FA, Lauris JR, Oliveira DT (2015) Are podoplanin and ezrin involved in the invasion process of the ameloblastomas? Eur J Histochem 59:2451. https://doi.org/10.4081/ejh.2015.2451

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Culty M, Miyake K, Kincade PW, Sikorski E, Butcher EC, Underhill C (1990) The hyaluronate receptor is a member of the CD44 (H-CAM) family of cell surface glycoproteins. J Cell Biol 111:2765–2774

    PubMed  CAS  Google Scholar 

  • Donahue HJ, Li Z, Zhou Z, Yellowley CE (1998) Differentiation of human fetal osteoblastic cells and gap junctional intercellular communication. Am J Physiol Cell Physiol 278:C315-322

    Google Scholar 

  • Farr AG, Berry ML, Kim A, Nelson AJ, Welch MP, Aruffo A (1992) Characterization and cloning of a novel glycoprotein expressed by stromal cells in T-dependent areas of peripheral lymphoid tissues. J Exp Med 176:1477–1482

    PubMed  CAS  Google Scholar 

  • Frost HM (1969) Tetracycline-based histological analysis of bone remodeling. Calcif Tissue Res 3:211–237

    PubMed  CAS  Google Scholar 

  • Hasegawa T, Li M, Hara K, Sasaki M, Tabata C, de Freitas PH, Hongo H, Suzuki R, Kobayashi M, Inoue K, Yamamoto T, Oohata N, Oda K, Akiyama Y, Amizuka N (2011) Morphological assessment of bone mineralization in tibial metaphyses of ascorbic acid-deficient ODS rats. Biomed Res 32:259–269

    PubMed  CAS  Google Scholar 

  • Hasegawa T, Amizuka N, Yamada T, Liu Z, Miyamoto Y, Yamamoto T, Sasaki M, Hongo H, Suzuki R, Freitas PHL, Yamamoto T, Oda K, Li M (2013) Sclerostin is differently immunolocalized in metaphyseal trabecules and cortical bones of mouse tibiae. Biomed Res 34:153–159

    PubMed  CAS  Google Scholar 

  • Hasegawa T, Endo T, Tsuchiya E, Kudo A, Zhao S, Moritani Y, Abe M, Yamamoto T, Hongo H, Tsuboi K, Yoshida T, Nagai T, Khadiza N, Yokoyama A, Luiz de Freitas PH, Li M, Amizuka N (2017) Biological application of focus ion beam-scanning electron microscopy (FIB-SEM) to the imaging of cartilaginous fibrils and osteoblastic cytoplasmic processes. J Oral Biosci 59:55–62

    Google Scholar 

  • Hasegawa T, Yamamoto T, Hongo H, Qiu Z, Abe M, Kanesaki T, Tanaka K, Endo T, Freitas PHL, Li M, Amizuka N (2018) Three-dimensional ultrastructure of osteocytes assessed by focused ion beam-scanning electron microscopy (FIB-SEM). Histochem Cell Biol 149:423–432

    PubMed  CAS  Google Scholar 

  • Hasegawa T, Yamamoto T, Sakai S, Miyamoto Y, Hongo H, Qiu Z, Abe M, Takeda S, Oda K, Freitas PHL, Li M, Endo K, Amizuka N (2019) Histological effects of the combined administration of eldecalcitol and a parathyroid hormone in the metaphyseal trabeculae of ovariectomized rats. J HistochemCytochem 67(3):169–184. https://doi.org/10.1369/0022155418806865

    Article  CAS  Google Scholar 

  • Hazenberg JG, Freeley M, Foran E, Lee TC, Microdamage TD (2006) A cell transducing mechanism based on ruptured osteocyte processes. J Biomech 39:2096–2103

    PubMed  Google Scholar 

  • Hirose S, Li M, Kojima T, de Freitas PH, Ubaidus S, Oda K, Saito C, Amizuka N (2007) A histological assessment on the distribution of the osteocytic lacunar canalicular system using silver staining. J Bone Miner Metab 25:374–382

    PubMed  Google Scholar 

  • Huiskes R, Ruimerman R, van Lenthe GH, Janssen JD (2000) Effects of mechanical forces on maintenance and adaptation of form in trabecular bone. Nature 405:704–706

    PubMed  CAS  Google Scholar 

  • Ikpegbu E, Basta L, Clements DN, Fleming R, Vincent TL, Buttle DJ, Pitsillides AA, Staines KA, Farquharson C (2018) FGF-2 promotes osteocyte differentiation through increased E11/podoplanin expression. J Cell Physiol 233(7):5334–5347. https://doi.org/10.1002/jcp.26345

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Knothe Tate ML, Niederer P, Knothe U (1998) In vivo tracer transport through the lacunocanalicular system of rat bone in an environment devoid of mechanical loading. Bone 22:107–117

    PubMed  CAS  Google Scholar 

  • Knothe Tate ML, Adamson JR, Tami AE, Bauer TW (2004) The osteocyte. Int J Biochem Cell Biol 36:1–8

    PubMed  CAS  Google Scholar 

  • Krishnan H, Ochoa-Alvarez JA, Shen Y, Nevel E, Lakshminarayanan M, Williams MC, Ramirez MI, Miller WT, Goldberg GS (2013) Serines in the intracellular tail of podoplanin (PDPN) regulate cell motility. J Biol Chem 288:12215–12221

    PubMed  PubMed Central  CAS  Google Scholar 

  • Mahtab EA, Vicente-Steijn R, Hahurij ND, Jongbloed MR, Wisse LJ, DeRuiter MC, Uhrin P, Zaujec J, Binder BR, Schalij MJ, Poelmann RE, Gittenberger-de Groot AC (2009) Podoplanin deficient mice show a RhoA-related hypoplasia of the sinus venosus myocardium including the sinoatrial node. Dev Dyn 238:183–193

    PubMed  Google Scholar 

  • Martín-Villar E, Megías D, Castel S, Yurrita MM, Vilaró S, Quintanilla M (2006) Podoplanin binds ERM proteins to activate RhoA and promote epithelial-mesenchymal transition. J Cell Sci 119:4541–4553

    PubMed  Google Scholar 

  • Martín-Villar E, Fernández-Muñoz B, Parsons M, Yurrita MM, Megías D, Pérez-Gómez E, Jones GE, Quintanilla M (2010) Podoplanin associates with CD44 to promote directional cell migration. Mol Biol Cell 21:4387–4399

    PubMed  PubMed Central  Google Scholar 

  • Martín-Villar E, Borda-d’Agua B, Carrasco-Ramirez P, Renart J, Parsons M, Quintanilla M, Jones GE (2015) Podoplanin mediates ECM degradation by squamous carcinoma cells through control of invadopodia stability. Oncogene 34:4531–4544

    PubMed  Google Scholar 

  • Matsui K, Breiteneder-Geleff S, Soleiman A, Kowalski H, Kerjaschki D (1999) Podoplanin, a novel 43-kDa membrane protein, controls the shape of podocytes. Nephrol Dial Transplant 14(Suppl):9–11

    PubMed  CAS  Google Scholar 

  • Mori S, Burr DB (1993) Increased intracortical remodeling following fatigue damage. Bone 14:103–109

    PubMed  CAS  Google Scholar 

  • Nagai T (2019) Immunocytochemical assessment of cell differentiation of podoplanin-positive osteoblasts into osteocytes in murine bone. Preprint at https://eprints.lib.hokudai.ac.jp/dspace/bitstream/2115/77060/1/Tomoya_Nagai.pdf

  • Nefussi JR, Sautier JM, Nicolas V, Forest N (1991) How osteoblasts become osteocytes: a decreasing matrix forming process. J BiolBuccale 19:75–82

    CAS  Google Scholar 

  • Nose K, Saito H, Kuroki T (1990) Isolation of a gene sequence induced later by tumor-promoting 12-O-tettradecanoylphorbor-13-acetate in mouse osteoblastic cell (MC3T3-E1) and expressed constituently in ras transformed cells. Cell Growth Differ 1:511–518

    PubMed  CAS  Google Scholar 

  • Oda K, Amaya Y, Fukushi-Irié M, Kinameri Y, Ohsuye K, Kubota I, Fujimura S, Kobayashi J (1999) A general method for rapid purification of soluble versions of glycosyl phosphatidylinositol-anchored proteins expressed in insect cells: an application for human tissue-nonspecific alkaline phosphatase. J Biochem 126:694–699

    PubMed  CAS  Google Scholar 

  • Palumbo C, Palazzini S, Zaffe D, Marotti G (1990) Osteocyte differentiation in the tibia of a newborn rabbit: an ultrastructural study of the formation of cytoplasmic processes. Acta Anat 137:350–358

    PubMed  CAS  Google Scholar 

  • Ramirez MI, Millien G, Hinds A, Cao Y, Seldin DC, Williams MC (2003) T1alpha, a lung type I cell differentiation gene, is required for normal lung cell proliferation and alveolus formation at birth. Dev Biol 256:61–72

    PubMed  CAS  Google Scholar 

  • Sasaki M, Hongo H, Hasegawa T, Suzuki R, Liu Z, Freitas PHL, Yamada T, Oda K, Yamamoto T, Li M, Totsuka Y, Amizuka N (2012) Morphological aspects of the biological function of the osteocytic lacunar canalicular system and of osteocyte-derived factors. Oral Sci Intern 9:1–8

    Google Scholar 

  • Schacht V, Ramirez MI, Hong YK, Hirakawa S, Feng D, Harvey N, Williams M, Dvorak AM, Dvorak HF, Oliver G, Detmar M (2003) T1alpha/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema. EMBO J 22:3546–3556

    PubMed  PubMed Central  CAS  Google Scholar 

  • Schacht V, Dadras SS, Johnson LA, Jackson DG, Hong YK, Detmar M (2005) Up-regulation of the lymphatic marker podoplanin, a mucin-type transmembrane glycoprotein, in human squamous cell carcinomas and germ cell tumors. Am J Pathol 166:913–921

    PubMed  PubMed Central  CAS  Google Scholar 

  • Shapiro F (1997) Variable conformation of GAP junctions linking bone cells: a transmission electron microscopic study of linear, stacked linear, curvilinear, oval, and annular junctions. Calcif Tissue Int 61:285–293

    PubMed  CAS  Google Scholar 

  • Sprague SM, Popovtzer MM, Dranitzki-Elhalel M, Wald H (1996) Parathyroid hormone-induced calcium efflux from cultured bone is mediated by protein kinase C translocation. Am J Physiol 271:F1139-1146

    PubMed  CAS  Google Scholar 

  • Staines KA, Javaheri B, Hohenstein P, Fleming R, Ikpegbu E, Unger E, Hopkinson M, Buttle DJ, Pitsillides AA, Farquharson C (2017) Hypomorphic conditional deletion of E11/Podoplanin reveals a role in osteocyte dendrite elongation. J Cell Physiol 232:3006–3019

    PubMed  PubMed Central  CAS  Google Scholar 

  • Staines KA, Ikpegbu E, Törnqvist AE, Dillon S, Javaheri B, Amin AK, Clements DN, Buttle DJ, Pitsillides AA, Farquharson C (2019) Conditional deletion of E11/podoplanin in bone protects against load-induced osteoarthritis. BMC MusculoskeletDisord 20(1):344. https://doi.org/10.1186/s12891-019-2731-9

    Article  CAS  Google Scholar 

  • Staines KA, Hopkinson M, Dillon S, Stephen LA, Fleming R, Sophocleous A, Buttle DJ, Pitsillides AA, Farquharson C (2020) Conditional deletion of E11/Podoplanin in bone protects against ovariectomy-induced increases in osteoclast formation and activity. Biosci Rep 40(1):BSR20190329. https://doi.org/10.1042/BSR20190329

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Suzuki K, Fukusumi Y, Yamazaki M, Kaneko H, Tsuruga K, Tanaka H, Ito E, Matsui K, Kawachi H (2015) Alteration in the podoplanin-ezrin-cytoskeleton linkage is an important initiation event of the podocyte injury in puromycin aminonucleoside nephropathy, a mimic of minimal change nephrotic syndrome. Cell Tissue Res 362:201–213

    PubMed  CAS  Google Scholar 

  • Tsuneki M, Yamazaki M, Maruyama S, Cheng J, Saku T (2013) Podoplanin-mediated cell adhesion through extracellular matrix in oral squamous cell carcinoma. Lab Invest 93:921–932

    PubMed  CAS  Google Scholar 

  • Ubaidus S, Li M, Sultana S, de Freitas PH, Oda K, Maeda T, Takagi R, Amizuka N (2009) FGF23 is mainly synthesized by osteocytes in the regularly distributed osteocytic lacunar canalicular system established after physiological bone remodeling. J Electron Microsc (Tokyo) 58:381–392

    CAS  Google Scholar 

  • Vanderbilt JN, Dobbs LG (1998) Characterization of the gene and promoter for RTI40, a differentiation marker of type I alveolar epithelial cells. Am J Respir Cell Mol Biol 19:662–671

    PubMed  CAS  Google Scholar 

  • Wetterwald A, Hoffstetter W, Cecchini MG, Lanske B, Wagner C, Fleisch H, Atkinson M (1997) Characterization and cloning of the E11 antigen, a marker expressed by rat osteoblasts and osteocytes. Bone 18:125–132

    Google Scholar 

  • Williams MC, Cao Y, Hinds A, Rishi AK, Wetterwald A (1996) T1 alpha protein is developmentally regulated and expressed by alveolar type I cells, choroid plexus, and ciliary epithelia of adult rats. Am J Respir Cell Mol Biol 14:577–585

    PubMed  CAS  Google Scholar 

  • Yamamoto T, Hasegawa T, Sasaki M, Hongo H, Tsuboi K, Shimizu T, Ota M, Haraguchi M, Takahata M, Oda K, Luiz de Freitas PH, Takakura A, Takao-Kawabata R, Isogai Y, Amizuka N (2016) Frequency of teriparatide administration affects the histological pattern of bone formation in young adult male mice. Endocrinology 157:2604–2620

    PubMed  CAS  Google Scholar 

  • Zhang K, Barragan-Adjemian C, Ye L, Kotha S, Dallas M, Lu Y, Zhao S, Harris M, Harris SE, Feng JQ, Bonewald LF (2006) E11/gp38 selective expression in osteocytes: regulation by mechanical strain and role in dendrite elongation. Mol Cell Biol 26:4539–4552

    PubMed  PubMed Central  CAS  Google Scholar 

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Acknowledgements

We express our gratitude to the members of the Electron Microscope Laboratory, Research Faculty of Agriculture, Hokkaido University for their technical support for STED microscopic observation.

Funding

This work was supported by the Grants-in Aid for Scientific Research of Japan Society for the Promotion of Science (19K10040 to TH, and 18K19628 to NA).

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TN was the main researcher who contributed to this work, including the preparation of paraffin sections, histochemical analyses, and observation under immune-electron microscopy. TH, TY, HH, MA, and AY performed sample preparations, including fixation and paraffin/epoxy resin-embedding; Yumin and TY performed Western blot analysis. PHLF, ML, AY, and NA participated in the discussion and preparation of the manuscript. TH is the chief of this research project and the corresponding author of this experiment. All the above authors have read and approved the final manuscript.

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Correspondence to Tomoka Hasegawa.

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The preprint of manuscript prior to formal peer review at this journal was posted on the Hokkaido University Collection of Scholarly and Academic Paper (Nagai T. 2019).

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Nagai, T., Hasegawa, T., Yimin et al. Immunocytochemical assessment of cell differentiation of podoplanin-positive osteoblasts into osteocytes in murine bone. Histochem Cell Biol 155, 369–380 (2021). https://doi.org/10.1007/s00418-020-01937-y

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