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BMC veterinary research2010; 6; 16; doi: 10.1186/1746-6148-6-16

Characterization of extracellular matrix macromolecules in primary cultures of equine keratinocytes.

Abstract: Most research to date involving laminins and extracellular matrix protein function in both normal and pathological conditions involves in vitro culture of keratinocytes. Few methods are established to allow for prolonged propagation of keratinocytes from equine tissues, including the hoof lamellae. In this study we modified cell isolation and culture techniques to allow for proliferation and sub-culturing of equine lamellar keratinocytes. Additionally, the production and processing of extracellular matrix molecules by skin and lamellar keratinocytes were studied. Results: Physical and proteolytic tissue separation in combination with media containing a calcium concentration of 0.6 mM in combination with additional media supplements proved optimal for proliferation and subculture of equine lamellar keratinocytes on collagen coated substratum. Immunofluorescence and immunoblotting studies confirmed that equine skin and lamellar keratinocytes produce Ln-332 in vitro and processing of this molecule follows that of other species. As well, matrix components including integrin alpha-6 (alpha 6) and the hemidesmosome proteins, bullous pemphigoid antigen 1 (BP180) bullous pemphigoid antigen 2 (BP230) and plectin are also expressed. Conclusions: Isolation of equine keratinocytes and study of the matrix and adhesion related molecules produced by them provides a valuable tool for future work in the veterinary field.
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Publication Date: 2010-03-15 PubMed ID: 20230631PubMed Central: PMC2847556DOI: 10.1186/1746-6148-6-16Google Scholar: Lookup
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  • Journal Article
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  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research focuses on modifying cell isolation and culture techniques to allow for the growth and development of equine keratinocytes, cells from horse tissues, specifically hoof lamellae. It examines the production and processing of proteins in the extracellular matrix, which is crucial for cell communication and function, in both skin and hoof keratinocytes.

Cell Isolation and Culture Modifications

  • The primary concern of the study was to find an appropriate method for breeding equine keratinocytes, particularly those from the hoof lamella. Traditional methods are limited and do not accommodate long-term propagation of the cells.
  • In order to optimize conditions for the propagation and culture of these cells, this study used a combination of physical and proteolytic separation techniques. These methods involve chemical and physical means to segregate the cells from the tissues.
  • This, coupled with media that had a calcium concentration of 0.6 mM and added media supplements, was found to be the best solution for the growth and sub-culture of equine lamellar keratinocytes on collagen-coated substratum.

Extracellular Matrix Molecule Production

  • The researchers then turned their attention to the extracellular matrix molecules produced by the skin and hoof keratinocytes. The extracellular matrix is essential for cell function, providing structural and biochemical support to surrounding cells.
  • Immunofluorescence and immunoblotting studies, which involve labelling antibodies to see where they bind in the tissue, revealed that both types of keratinocytes produce Ln-332 in vitro. Ln-332 is a type of laminin, which is critical for the structure and function of the matrix.
  • The research showed that the processing of the Ln-332 molecule aligns with that of other species, providing a generalizable outcome.

Expression of Matrix Components

  • Further studies showed that the matrix components integrin alpha-6 (alpha 6), and the hemidesmosome proteins bullous pemphigoid antigen 1 (BP180), bullous pemphigoid antigen 2 (BP230) and plectin are also expressed by equine keratinocytes.
  • These molecules are important for maintaining cell structure and facilitating cell-extracellular matrix adhesion, essential for cellular stability and functionality.

Conclusion

  • Ultimately the study contributes valuable tools and understanding to veterinary research. By establishing a consistently successful isolation and culture method for equine keratinocytes, and demonstrating the extracellular matrix-related molecules they produce, future research in the veterinary field can be expanded.

Cite This Article

APA
Visser MB, Pollitt CC. (2010). Characterization of extracellular matrix macromolecules in primary cultures of equine keratinocytes. BMC Vet Res, 6, 16. https://doi.org/10.1186/1746-6148-6-16

Publication

BMC veterinary research
ISSN: 1746-6148
NlmUniqueID: 101249759
Country: England
Language: English
Volume: 6
Pages: 16

Researcher Affiliations

Visser, Michelle B
  • The Australian Equine Laminitis Research Unit, School of Veterinary Science, University of Queensland, St Lucia, 4072, Australia. michelle.visser@utoronto.ca
Pollitt, Christopher C

    MeSH Terms

    • Animals
    • Cell Culture Techniques / veterinary
    • Cells, Cultured
    • Extracellular Matrix Proteins / metabolism
    • Gene Expression Regulation
    • Hemidesmosomes / metabolism
    • Keratinocytes / cytology
    • Keratinocytes / metabolism

    References

    This article includes 40 references
    1. Aumailley M, Bruckner-Tuderman L, Carter WG, Deutzmann R, Edgar D, Ekblom P, Engel J, Engvall E, Hohenester E, Jones JC, Kleinman HK, Marinkovich MP, Martin GR, Mayer U, Meneguzzi G, Miner JH, Miyazaki K, Patarroyo M, Paulsson M, Quaranta V, Sanes JR, Sasaki T, Sekiguchi K, Sorokin LM, Talts JF, Tryggvason K, Uitto J, Virtanen I, von der Mark K, Wewer UM, Yamada Y, Yurchenco PD. A simplified laminin nomenclature.. Matrix Biol 2005 Aug;24(5):326-32.
      doi: 10.1016/j.matbio.2005.05.006pubmed: 15979864google scholar: lookup
    2. Borradori L, Sonnenberg A. Structure and function of hemidesmosomes: more than simple adhesion complexes.. J Invest Dermatol 1999 Apr;112(4):411-8.
      doi: 10.1046/j.1523-1747.1999.00546.xpubmed: 10201522google scholar: lookup
    3. Litjens SH, de Pereda JM, Sonnenberg A. Current insights into the formation and breakdown of hemidesmosomes.. Trends Cell Biol 2006 Jul;16(7):376-83.
      doi: 10.1016/j.tcb.2006.05.004pubmed: 16757171google scholar: lookup
    4. Spirito F, Charlesworth A, Linder K, Ortonne JP, Baird J, Meneguzzi G. Animal models for skin blistering conditions: absence of laminin 5 causes hereditary junctional mechanobullous disease in the Belgian horse.. J Invest Dermatol 2002 Sep;119(3):684-91.
      doi: 10.1046/j.1523-1747.2002.01852.xpubmed: 12230513google scholar: lookup
    5. Masunaga T. Epidermal basement membrane: its molecular organization and blistering disorders.. Connect Tissue Res 2006;47(2):55-66.
      doi: 10.1080/03008200600584157pubmed: 16754511google scholar: lookup
    6. Powell AM, Sakuma-Oyama Y, Oyama N, Black MM. Collagen XVII/BP180: a collagenous transmembrane protein and component of the dermoepidermal anchoring complex.. Clin Exp Dermatol 2005 Nov;30(6):682-7.
      doi: 10.1111/j.1365-2230.2005.01937.xpubmed: 16197389google scholar: lookup
    7. Tsuruta D, Kobayashi H, Imanishi H, Sugawara K, Ishii M, Jones JC. Laminin-332-integrin interaction: a target for cancer therapy?. Curr Med Chem 2008;15(20):1968-75.
      doi: 10.2174/092986708785132834pmc: PMC2992754pubmed: 18691052google scholar: lookup
    8. Pollitt CC, Daradka M. Equine laminitis basement membrane pathology: loss of type IV collagen, type VII collagen and laminin immunostaining.. Equine Vet J Suppl 1998 Sep;(26):139-44.
      pubmed: 9932105doi: 10.1111/j.2042-3306.1998.tb05133.xgoogle scholar: lookup
    9. French KR, Pollitt CC. Equine laminitis: cleavage of laminin 5 associated with basement membrane dysadhesion.. Equine Vet J 2004 Apr;36(3):242-7.
      doi: 10.2746/0425164044877134pubmed: 15147132google scholar: lookup
    10. French KR, Pollitt CC. Equine laminitis: loss of hemidesmosomes in hoof secondary epidermal lamellae correlates to dose in an oligofructose induction model: an ultrastructural study.. Equine Vet J 2004 Apr;36(3):230-5.
      doi: 10.2746/0425164044877125pubmed: 15147130google scholar: lookup
    11. Rheinwald JG, Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells.. Cell 1975 Nov;6(3):331-43.
      doi: 10.1016/S0092-8674(75)80001-8pubmed: 1052771google scholar: lookup
    12. Fusenig NE, Worst PK. Mouse epidermal cell cultures. II. Isolation, characterization and cultivation of epidermal cells from perinatal mouse skin.. Exp Cell Res 1975 Jul;93(2):443-57.
      doi: 10.1016/0014-4827(75)90471-1pubmed: 808420google scholar: lookup
    13. Freshney R. Culture of Animal Cells: A manual of basic techique. 5. Hoboken. NJ, John Wiley; 2005.
    14. Wunn D, Wardrop KJ, Meyers K, Kramer J, Ragle C. Culture and characterization of equine terminal arch endothelial cells and hoof keratinocytes.. Am J Vet Res 1999 Jan;60(1):128-32.
      pubmed: 9918161
    15. Ekfalck A, Rodriguez-Martinez H, Obel N. Cultivation of tissue from the matrix of the stratum medium of the equine and bovine hoof walls.. Am J Vet Res 1990 Nov;51(11):1852-6.
      pubmed: 2240811
    16. Dahm AM, de Bruin A, Linat A, von Tscharner C, Wyder M, Suter MM. Cultivation and characterisation of primary and subcultured equine keratinocytes.. Equine Vet J 2002 Mar;34(2):114-20.
      doi: 10.2746/042516402776767187pubmed: 11902754google scholar: lookup
    17. Hennings H, Michael D, Cheng C, Steinert P, Holbrook K, Yuspa SH. Calcium regulation of growth and differentiation of mouse epidermal cells in culture.. Cell 1980 Jan;19(1):245-54.
      doi: 10.1016/0092-8674(80)90406-7pubmed: 6153576google scholar: lookup
    18. Tomakidi P, Fusenig NE, Kohl A, Komposch G. Histomorphological and biochemical differentiation capacity in organotypic co-cultures of primary gingival cells.. J Periodontal Res 1997 May;32(4):388-400.
      doi: 10.1111/j.1600-0765.1997.tb00549.xpubmed: 9210093google scholar: lookup
    19. Freshney R. Culture of epithelial cells. New York, Wiley Liss; 2002.
    20. Wille JJ Jr, Pittelkow MR, Shipley GD, Scott RE. Integrated control of growth and differentiation of normal human prokeratinocytes cultured in serum-free medium: clonal analyses, growth kinetics, and cell cycle studies.. J Cell Physiol 1984 Oct;121(1):31-44.
      doi: 10.1002/jcp.1041210106pubmed: 6207187google scholar: lookup
    21. Rheinwald JG, Green H. Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes.. Nature 1977 Feb 3;265(5593):421-4.
      doi: 10.1038/265421a0pubmed: 299924google scholar: lookup
    22. Peehl DM, Ham RG. Clonal growth of human keratinocytes with small amounts of dialyzed serum.. In Vitro 1980 Jun;16(6):526-40.
      doi: 10.1007/BF02626466pubmed: 6156122google scholar: lookup
    23. Wattle O. Cytokeratins of the equine hoof wall, chestnut and skin: bio- and immunohisto-chemistry.. Equine Vet J Suppl 1998 Sep;(26):66-80.
      pubmed: 9932096doi: 10.1111/j.2042-3306.1998.tb05124.xgoogle scholar: lookup
    24. Moll R, Franke WW, Schiller DL, Geiger B, Krepler R. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells.. Cell 1982 Nov;31(1):11-24.
      doi: 10.1016/0092-8674(82)90400-7pubmed: 6186379google scholar: lookup
    25. Biddle D, Spandau DF. Expression of vimentin in cultured human keratinocytes is associated with cell - extracellular matrix junctions.. Arch Dermatol Res 1996 Sep;288(10):621-4.
      doi: 10.1007/BF02505266pubmed: 8919046google scholar: lookup
    26. Champliaud MF, Lunstrum GP, Rousselle P, Nishiyama T, Keene DR, Burgeson RE. Human amnion contains a novel laminin variant, laminin 7, which like laminin 6, covalently associates with laminin 5 to promote stable epithelial-stromal attachment.. J Cell Biol 1996 Mar;132(6):1189-98.
      doi: 10.1083/jcb.132.6.1189pmc: PMC2120759pubmed: 8601594google scholar: lookup
    27. Rousselle P, Keene DR, Ruggiero F, Champliaud MF, Rest M, Burgeson RE. Laminin 5 binds the NC-1 domain of type VII collagen.. J Cell Biol 1997 Aug 11;138(3):719-28.
      doi: 10.1083/jcb.138.3.719pmc: PMC2141627pubmed: 9245798google scholar: lookup
    28. Marinkovich MP, Lunstrum GP, Burgeson RE. The anchoring filament protein kalinin is synthesized and secreted as a high molecular weight precursor.. J Biol Chem 1992 Sep 5;267(25):17900-6.
      pubmed: 1517226
    29. Langhofer M, Hopkinson SB, Jones JC. The matrix secreted by 804G cells contains laminin-related components that participate in hemidesmosome assembly in vitro.. J Cell Sci 1993 Jul;105 ( Pt 3):753-64.
      pubmed: 8408302doi: 10.1242/jcs.105.3.753google scholar: lookup
    30. Becker C, Buttler P, Gräber HG. Influence of anti-CD49f and anti-CD29 monoclonal antibodies on mitotic activity of epithelial cells (HaCaT) and gingival fibroblasts in vitro.. Eur J Oral Sci 2002 Apr;110(2):137-43.
      doi: 10.1034/j.1600-0722.2002.11202.xpubmed: 12013557google scholar: lookup
    31. Hirako Y, Yoshino K, Zillikens D, Owaribe K. Extracellular cleavage of bullous pemphigoid antigen 180/type XVII collagen and its involvement in hemidesmosomal disassembly.. J Biochem 2003 Feb;133(2):197-206.
      doi: 10.1093/jb/mvg024pubmed: 12761182google scholar: lookup
    32. Hieda Y, Nishizawa Y, Uematsu J, Owaribe K. Identification of a new hemidesmosomal protein, HD1: a major, high molecular mass component of isolated hemidesmosomes.. J Cell Biol 1992 Mar;116(6):1497-506.
      doi: 10.1083/jcb.116.6.1497pmc: PMC2289367pubmed: 1541639google scholar: lookup
    33. Gache Y, Chavanas S, Lacour JP, Wiche G, Owaribe K, Meneguzzi G, Ortonne JP. Defective expression of plectin/HD1 in epidermolysis bullosa simplex with muscular dystrophy.. J Clin Invest 1996 May 15;97(10):2289-98.
      doi: 10.1172/JCI118671pmc: PMC507309pubmed: 8636409google scholar: lookup
    34. Riddelle KS, Green KJ, Jones JC. Formation of hemidesmosomes in vitro by a transformed rat bladder cell line.. J Cell Biol 1991 Jan;112(1):159-68.
      doi: 10.1083/jcb.112.1.159pmc: PMC2288810pubmed: 1986003google scholar: lookup
    35. Klatte DH, Jones JC. Purification of the 230-kD bullous pemphigoid antigen (BP230) from bovine tongue mucosa: structural analyses and assessment of BP230 tissue distribution using a new monoclonal antibody.. J Invest Dermatol 1994 Jan;102(1):39-44.
      doi: 10.1111/1523-1747.ep12371728pubmed: 8288909google scholar: lookup
    36. Yang HY, Lieska N, Goldman AE, Goldman RD. A 300,000-mol-wt intermediate filament-associated protein in baby hamster kidney (BHK-21) cells.. J Cell Biol 1985 Feb;100(2):620-31.
      doi: 10.1083/jcb.100.2.620pmc: PMC2113446pubmed: 3881459google scholar: lookup
    37. Pollitt CC, Daradka M. Hoof wall wound repair.. Equine Vet J 2004 Apr;36(3):210-5.
      doi: 10.2746/0425164044877189pubmed: 15147126google scholar: lookup
    38. Rheinwald JG, Beckett MA. Tumorigenic keratinocyte lines requiring anchorage and fibroblast support cultured from human squamous cell carcinomas.. Cancer Res 1981 May;41(5):1657-63.
      pubmed: 7214336
    39. Koshikawa N, Schenk S, Moeckel G, Sharabi A, Miyazaki K, Gardner H, Zent R, Quaranta V. Proteolytic processing of laminin-5 by MT1-MMP in tissues and its effects on epithelial cell morphology.. FASEB J 2004 Feb;18(2):364-6.
      pubmed: 14688206doi: 10.1096/fj.03-0584fjegoogle scholar: lookup
    40. Rousselle P, Lunstrum GP, Keene DR, Burgeson RE. Kalinin: an epithelium-specific basement membrane adhesion molecule that is a component of anchoring filaments.. J Cell Biol 1991 Aug;114(3):567-76.
      doi: 10.1083/jcb.114.3.567pmc: PMC2289097pubmed: 1860885google scholar: lookup

    Citations

    This article has been cited 7 times.
    1. Yang Q, Pinto VMR, Duan W, Paxton EE, Dessauer JH, Ryan W, Lopez MJ. In vitro Characteristics of Heterogeneous Equine Hoof Progenitor Cell Isolates. Front Bioeng Biotechnol 2019;7:155.
      doi: 10.3389/fbioe.2019.00155pubmed: 31355191google scholar: lookup
    2. Baskerville CL, Chockalingham S, Harris PA, Bailey SR. The effect of insulin on equine lamellar basal epithelial cells mediated by the insulin-like growth factor-1 receptor. PeerJ 2018;6:e5945.
      doi: 10.7717/peerj.5945pubmed: 30519508google scholar: lookup
    3. Alkhilaiwi F, Wang L, Zhou D, Raudsepp T, Ghosh S, Paul S, Palechor-Ceron N, Brandt S, Luff J, Liu X, Schlegel R, Yuan H. Long-term expansion of primary equine keratinocytes that maintain the ability to differentiate into stratified epidermis. Stem Cell Res Ther 2018 Jul 4;9(1):181.
      doi: 10.1186/s13287-018-0918-xpubmed: 29973296google scholar: lookup
    4. Reisinger N, Schaumberger S, Nagl V, Hessenberger S, Schatzmayr G. Concentration Dependent Influence of Lipopolysaccharides on Separation of Hoof Explants and Supernatant Lactic Acid Concentration in an Ex Vivo/In Vitro Laminitis Model. PLoS One 2015;10(11):e0143754.
      doi: 10.1371/journal.pone.0143754pubmed: 26599864google scholar: lookup
    5. Leise BS, Watts MR, Roy S, Yilmaz AS, Alder H, Belknap JK. Use of laser capture microdissection for the assessment of equine lamellar basal epithelial cell signalling in the early stages of laminitis. Equine Vet J 2015 Jul;47(4):478-88.
      doi: 10.1111/evj.12283pubmed: 24750316google scholar: lookup
    6. Lan X, Qi D, Ren H, Liu T, Shao H, Zhang J. Chicoric acid ameliorates LPS-induced inflammatory injury in bovine lamellar keratinocytes by modulating the TLR4/MAPK/NF-κB signaling pathway. Sci Rep 2023 Dec 11;13(1):21963.
      doi: 10.1038/s41598-023-49169-zpubmed: 38082032google scholar: lookup
    7. Zhai C, Wu Q, Yang X, Xie Y, Mu J, Lei L, Shi M, Wu W, Li G, Ding J. Quercetin alleviates LPS-induced inflammatory response in dairy cow lamellar keratinocytes through PI3K/Akt/NF-κB signaling pathway. BMC Vet Res 2026 Jan 28;22(1).
      doi: 10.1186/s12917-026-05300-6pubmed: 41606588google scholar: lookup
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