Skip to main content
SpringerLink
Log in

Hyaluronan receptors in the human ocular surface: a descriptive and comparative study of RHAMM and CD44 in tissues, cell lines and freshly collected samples

  • Original Paper
  • Published:
Histochemistry and Cell Biology Aims and scope Submit manuscript

Abstract

The purpose of this study was to demonstrate the presence of the receptor for hyaluronan-mediated motility (RHAMM) in human conjunctival epithelium and in two widely used cell lines from human corneal (HCE) and conjunctival (IOBA-NHC) epithelia. We compared the distribution of RHAMM proteins and mRNAs in human ocular surface tissues (corneal, limbal and conjunctival), HCE and IOBA-NHC cell lines, and corneal and conjunctival epithelia primary samples from healthy donors with the previously identified hyaluronan receptor CD44. We also aimed to determine if soluble CD44 (sCD44) was present in human tears, as it could have a role in the interaction of the tear fluid with hyaluronan. Protein expression was evaluated by Western blots and immunofluorescence microscopy. mRNA expression was evaluated by RT-PCR and Q-PCR. sCD44 was analyzed by ELISA in culture supernatants and in human tears. We describe the expression of RHAMM in human healthy conjunctiva and in HCE and IOBA-NHC cells at both protein and mRNA levels, and the presence of sCD44 in human tears. Furthermore, we detected CD44 and sCD44 expression variations in in vitro inflammatory conditions. This study also focused on the necessary caution with which the conclusions extracted from cell lines should be made, and in the great value of using primary samples as often as possible.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  • Abetamann V, Kern HF, Elsasser HP (1996) Differential expression of the hyaluronan receptors CD44 and RHAMM in human pancreatic cancer cells. Clin Cancer Res 2:1607–1618

    PubMed  CAS  Google Scholar 

  • Ahmad S, Kolli S, Li DQ, de Paiva CS, Pryzborski S, Dimmick I, Armstrong L, Figueiredo FC, Lako M (2008) A putative role for RHAMM/HMMR as a negative marker of stem cell-containing population of human limbal epithelial cells. Stem Cells 26:1609–1619

    Article  PubMed  CAS  Google Scholar 

  • Araki-Sasaki K, Ohashi Y, Sasabe T, Hayashi K, Watanabe H, Tano Y, Handa H (1995) An SV40-immortalized human corneal epithelial cell line and its characterization. Invest Ophthalmol Vis Sci 36:614–621

    PubMed  CAS  Google Scholar 

  • Aruffo A, Stamenkovic I, Melnick M, Underhill CB, Seed B (1990) CD44 is the principal cell surface receptor for hyaluronate. Cell 61:1303–1313

    Article  PubMed  CAS  Google Scholar 

  • Assmann V, Jenkinson D, Marshall JF, Hart IR (1999) The intracellular hyaluronan receptor RHAMM/IHABP interacts with microtubules and actin filaments. J Cell Sci 112(Pt 22):3943–3954

    PubMed  CAS  Google Scholar 

  • Budak YU, Akdogan M, Huysal K (2009) Aqueous humor level of sCD44 in patients with degenerative myopia and primary open-angle glaucoma. BMC Res Notes 2:224

    Article  PubMed  Google Scholar 

  • Buganim Y, Rotter V (2008) RHAMM in the complex p53 cell cycle network. Cell Cycle 7:21

    Article  Google Scholar 

  • Cichy J, Puré E (2003) The liberation of CD44. J Cell Biol 161:839–843

    Article  PubMed  CAS  Google Scholar 

  • Contreras-Ruiz L, De la FM, Parraga JE, Lopez-Garcia A, Fernandez I, Seijo B, Sanchez A, Calonge M, Diebold Y (2011) Intracellular trafficking of hyaluronic acid–chitosan oligomer-based nanoparticles in cultured human ocular surface cells. Mol Vis 17:279–290

    PubMed  CAS  Google Scholar 

  • De la Fuente M, Seijo B, Alonso MJ (2008) Novel hyaluronic acid–chitosan nanoparticles for ocular gene therapy. Invest Ophthalmol Vis Sci 49:2016–2024

    Article  PubMed  Google Scholar 

  • De Saint JM, Baudouin C, Di NM, Roman S, Lozato P, Warnet JM, Brignole F (2004) Comparison of morphological and functional characteristics of primary-cultured human conjunctival epithelium and of Wong-Kilbourne derivative of Chang conjunctival cell line. Exp Eye Res 78:257–274

    Article  Google Scholar 

  • Diebold Y, Calonge M, Callejo S, Enriquez-de-Salamanca, Corrales RM, Saez V, Siemasko KF, Stern ME (2003) Characterization of a spontaneously immortalized cell line (IOBA-NHC) from normal human conjunctiva. Invest Ophthalmol Vis Sci 44:4263–4274

    Article  PubMed  Google Scholar 

  • Enriquez-de-Salamanca A, Calder V, Gao J, Galatowicz G, Garcia-Vazquez C, Fernandez I, Stern ME, Diebold Y, Calonge M (2008) Cytokine responses by conjunctival epithelial cells: an in vitro model of ocular inflammation. Cytokine 44:160–167

    Article  PubMed  CAS  Google Scholar 

  • Enriquez-de-Salamanca A, Castellanos E, Stern ME, Fernández I, Carreño E, García-Vázquez C, Herreras JM, Calonge M (2010) Tear cytokine and chemokine analysis and clinical correlations in evaporative-type dry eye disease. Mol Vis 16:862–873

    PubMed  CAS  Google Scholar 

  • Entwistle J, Hall CL, Turley EA (1996) HA receptors: regulators of signalling to the cytoskeleton. J Cell Biochem 61:569–577

    Article  PubMed  CAS  Google Scholar 

  • Franzmann EJ, Reategui EP, Carraway KL, Hamilton KL, Weed DT, Goodwin WJ (2005) Salivary soluble CD444: a potential molecular marker for head and neck cancer. Cancer Epidemiol Biomarkers Prev 14:735–739

    Article  PubMed  CAS  Google Scholar 

  • Fujihara T, Takeuchi T, Saito K, Tsubota K (1997) Flow cytometric analysis of surface antigens on human conjunctival epithelial cells. Ophthalmic Res 29:103–109

    Article  PubMed  CAS  Google Scholar 

  • Gust KM, Hofer MD, Perner SR, Kim R, Chinnaiyan AM, Varambally S, Moller P, Rinnab L, Rubin MA, Greiner J, Schmitt M, Kuefer R, Ringhoffer M (2009) RHAMM (CD168) is overexpressed at the protein level and may constitute an immunogenic antigen in advanced prostate cancer disease. Neoplasia 11:956–963

    PubMed  CAS  Google Scholar 

  • Hamilton SR, Fard SF, Paiwand FF, Tolg C, Veiseh M, Wang C, McCarthy JB, Bissell MJ, Koropatnick J, Turley EA (2007) The hyaluronan receptors CD44 and Rhamm (CD168) form complexes with ERK1, 2 that sustain high basal motility in breast cancer cells. J Biol Chem 282:16667–16680

    Article  PubMed  CAS  Google Scholar 

  • Hardwick C, Hoare K, Owens R, Hohn HP, Hook M, Moore D, Cripps V, Austen L, Nance DM, Turley EA (1992) Molecular cloning of a novel hyaluronan receptor that mediates tumor cell motility. J Cell Biol 117:1343–1350

    Article  PubMed  CAS  Google Scholar 

  • Hornof M, de la FM, Hallikainen M, Tammi RH, Urtti A (2008) Low molecular weight hyaluronan shielding of DNA/PEI polyplexes facilitates CD44 receptor mediated uptake in human corneal epithelial cells. J Gene Med 10:70–80

    Article  PubMed  CAS  Google Scholar 

  • Hughes JL, Lackie PM, Wilson SJ, Church MK, McGill JI (2006) Reduced structural proteins in the conjunctival epithelium in allergic eye disease. Allergy 61:1268–1274

    Article  PubMed  CAS  Google Scholar 

  • Isacke CM, Yarwood H (2002) The hyaluronan receptor, CD44. Int J Biochem Cell Biol 34:718–721

    Article  PubMed  CAS  Google Scholar 

  • Laemmli U (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  • Lerner LE, Schwartz DM, Hwang DG, Howes EL, Stern R (1998) Hyaluronan and CD44 in the human cornea and limbal conjunctiva. Exp Eye Res 67:481–484

    Article  PubMed  CAS  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408

    Article  PubMed  CAS  Google Scholar 

  • Maxwell CA, McCarthy J, Turley E (2008) Cell-surface and mitotic-spindle RHAMM: moonlighting or dual oncogenic functions? J Cell Sci 121:925–932

    Article  PubMed  CAS  Google Scholar 

  • Naor D, Sionov RV, Ish-Shalom D (1997) CD44: structure, function, and association with the malignant process. Adv Cancer Res 71:241–319

    Article  PubMed  CAS  Google Scholar 

  • Nedvetzki S, Gonen E, Assayag N, Reich R, Williams RO, Thurmond RL, Huang JF, Neudecker BA, Wang FS, Turley EA, Naor D (2004) RHAMM, a receptor for hyaluronan-mediated motility, compensates for CD44 in inflamed CD44-knockout mice: a different interpretation of redundancy. Proc Natl Acad Sci USA 101:18081–18086

    Article  PubMed  CAS  Google Scholar 

  • Pilarski LM, Masellis-Smith A, Belch AR, Yang B, Savani RC, Turley EA (1994) RHAMM, a receptor for hyaluronan-mediated motility, on normal human lymphocytes, thymocytes and malignant B cells: a mediator in B cell malignancy? Leuk Lymphoma 14:363–374

    Article  PubMed  CAS  Google Scholar 

  • Sherman L, Sleeman J, Herrlich P, Ponta H (1994) Hyaluronate receptors: key players in growth, differentiation, migration and tumor progression. Curr Opin Cell Biol 6:726–733

    Article  PubMed  CAS  Google Scholar 

  • Slevin M, Krupinski J, Gaffney J, Matou S, West D, Delisser H, Savani RC, Kumar S (2007) Hyaluronan-mediated angiogenesis in vascular disease: uncovering RHAMM and CD44 receptor signaling pathways. Matrix Biol 26:58–68

    Article  PubMed  CAS  Google Scholar 

  • Sneath RJ, Mangham DC (1998) The normal structure and function of CD44 and its role in neoplasia. Mol Pathol 51:191–200

    Article  PubMed  CAS  Google Scholar 

  • Sohr S, Engeland K (2008) RHAMM is differentially expressed in the cell cycle and downregulated by the tumor suppressor p53. Cell Cycle 7:3448–3460

    Article  PubMed  CAS  Google Scholar 

  • Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci 76:4350–4354

    Article  PubMed  CAS  Google Scholar 

  • Turley EA (1982) Purification of a hyaluronate-binding protein fraction that modifies cell social behavior. Biochem Biophys Res Commun 108:1016–1024

    Article  PubMed  CAS  Google Scholar 

  • Turley EA, Austen L, Vandeligt K, Clary C (1991) Hyaluronan and a cell-associated hyaluronan binding protein regulate the locomotion of ras-transformed cells. J Cell Biol 112:1041–1047

    Article  PubMed  CAS  Google Scholar 

  • Turley EA, Noble PW, Bourguignon LY (2002) Signaling properties of hyaluronan receptors. J Biol Chem 277:4589–4592

    Article  PubMed  CAS  Google Scholar 

  • Underhill C (1992) CD44: the hyaluronan receptor. J Cell Sci 103(Pt 2):293–298

    PubMed  CAS  Google Scholar 

  • Yu FX, Guo J, Zhang Q (1998) Expression and distribution of adhesion molecule CD44 in healing corneal epithelia. Invest Ophthalmol Vis Sci 39:710–717

    PubMed  CAS  Google Scholar 

  • Zhu SN, Nolle B, Duncker G (1997) Expression of adhesion molecule CD44 on human corneas. Br J Ophthalmol 81:80–84

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

Authors thank Professor Margarita Calonge, M.D., Ph.D., for critical reading of the manuscript, Ester Carreño, M.D. for technical assistance to obtain tear samples, and Ignacio Amat, M.D. for his help in the immunofluorescence study of HA receptors. The authors also acknowledge the assistance of Britt Bromberg Ph. D. ELS of Xenofile Editing (http://www.xenofileediting.com) for assistance in the final editing and preparation of this manuscript. Financial support was provided by FEDER-CICYT MAT 2010-20452-CO3-01, Spanish Ministry of Science and Education, Regional Grant VA056A08, Networking Research Center on Bioengineering, Biomaterial and Nanomedicine (CIBER-BBN), Ministry of Health, Spain, FPI-Ministry of Science and Innovation and FPU-Ministry of Education Scholarship Programs.

Author information

Authors and Affiliations

  1. Ocular Surface Group, Institute for Applied Ophthalmobiology (IOBA), University of Valladolid, Edificio IOBA, Campus Miguel Delibes, Paseo de Belén 17, 47011, Valladolid, Spain

    Laura García-Posadas, Laura Contreras-Ruiz, Antonio López-García, Sonia Villarón Álvarez, Miguel J. Maldonado & Yolanda Diebold

  2. Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Valladolid, Spain

    Laura García-Posadas, Laura Contreras-Ruiz, Antonio López-García & Yolanda Diebold

Authors
  1. Laura García-Posadas
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Laura Contreras-Ruiz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Antonio López-García
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sonia Villarón Álvarez
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Miguel J. Maldonado
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yolanda Diebold
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yolanda Diebold.

Additional information

L. García-Posadas and L. Contreras-Ruiz contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

García-Posadas, L., Contreras-Ruiz, L., López-García, A. et al. Hyaluronan receptors in the human ocular surface: a descriptive and comparative study of RHAMM and CD44 in tissues, cell lines and freshly collected samples. Histochem Cell Biol 137, 165–176 (2012). https://doi.org/10.1007/s00418-011-0878-z

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00418-011-0878-z

Keywords

Search

Navigation