Caspase-14 Protocols

  • Mami Yamamoto-Tanaka
  • Toshihiko Hibino
Part of the Methods in Molecular Biology book series (MIMB, volume 1133)


Unlike other caspase family members, caspase-14 shows restricted expression, being found mostly in epidermis and its appendages. It has been suggested that caspase-14 is not involved in apoptosis or inflammation, but participates in keratinocyte terminal differentiation. Its activation occurs at the corneocyte formation. In previous work, we have purified active caspase-14 from human corneocyte extracts. In addition, we have clarified activation mechanism of caspase-14, where kallikrein-related peptidase 7 (KLK7) generates an intermediate form from procaspase-14 and this form finally converts procaspase-14 to active, mature caspase-14. Here we describe techniques for measurement of caspase-14 activity using synthetic substrate, purification of caspase-14 from corneocyte extract, preparation of constitutively active caspase-14 and specific antibody, quantification of total and active caspase-14 in corneocyte extracts using ELISA, as well as methods for caspase-14 activation and its visualization by immunohistochemistry.

Key words

Caspase-14 Epidermis Keratinocyte Terminal differentiation Corneocyte Kallikrein-related peptidase 


  1. 1.
    Eckhart L, Ban J, Fischer H, Tschachler E (2000) Caspase-14: analysis of gene structure and mRNA expression during keratinocyte differentiation. Biochem Biophys Res Commun 277:655–659PubMedCrossRefGoogle Scholar
  2. 2.
    Van de Craen M, Van Loo G, Pype S, Van Criekinge W, Van den brande I, Molemans F, Fiers W, Declercq W, Vandenabeele P (1998) Identification of a new caspase homologue: caspase-14. Cell Death Differ 5:838–846PubMedCrossRefGoogle Scholar
  3. 3.
    Eckhart L, Declercq W, Ban J, Rendl M, Lengauer B, Mayer C, Lippens S, Vandenabeele P, Tschachler E (2000) Terminal differentiation of human keratinocytes and stratum corneum formation is associated with caspase-14 activation. J Invest Dermatol 115:1148–1151PubMedCrossRefGoogle Scholar
  4. 4.
    Watt FM (1983) Involucrin and other markers of keratinocyte terminal differentiation. J Invest Dermatol 81:100s–103sPubMedCrossRefGoogle Scholar
  5. 5.
    Wertz PW (1997) Integral lipids of hair and stratum corneum. EXS 78:227–237PubMedGoogle Scholar
  6. 6.
    Hitomi K (2005) Transglutaminases in skin epidermis. Eur J Dermatol 15:313–319PubMedGoogle Scholar
  7. 7.
    Yoneda K, Demitsu T, Manabe M, Igarashi J, Kosaka H, Inagaki N, Takahashi H, Kon A, Kakurai M, Kubota Y (2010) Expression of wild-type, but not mutant, loricrin causes programmed cell death in HaCaT keratinocytes. J Dermatol 37:956–964PubMedCrossRefGoogle Scholar
  8. 8.
    Hibino T, Fujita E, Tsuji Y, Nakanishi J, Iwaki H, Katagiri C, Momoi T (2010) Purification and characterization of active caspase-14 from human epidermis and development of the cleavage site-directed antibody. J Cell Biochem 109:487–497PubMedGoogle Scholar
  9. 9.
    Yamamoto M, Kamata Y, Iida T, Fukushima H, Nomura J, Saito M, Tajima M, Okubo Y, Momoi T, Tsuboi R, Hibino T (2011) Quantification of activated and total caspase-14 with newly developed ELISA systems in normal and atopic skin. J Dermatol Sci 61:110–117PubMedCrossRefGoogle Scholar
  10. 10.
    Yamamoto M, Miyai M, Matsumoto Y, Tsuboi R, Hibino T (2012) Kallikrein-related peptidase-7 regulates caspase-14 maturation during keratinocyte terminal differentiation by generating an intermediate form. J Biol Chem 287:32825–32834PubMedCrossRefGoogle Scholar
  11. 11.
    Lamkanfi M, Festjens N, Declercq W, Vanden Berghe T, Vandenabeele P (2007) Caspases in cell survival, proliferation and differentiation. Cell Death Differ 14:44–55PubMedCrossRefGoogle Scholar
  12. 12.
    Xu G, Shi Y (2007) Apoptosis signaling pathways and lymphocyte homeostasis. Cell Res 17:759–771PubMedCrossRefGoogle Scholar
  13. 13.
    Sato Y, Mukai K, Furuya S, Kameya T, Hirohashi S (1992) The AMeX method: a multipurpose tissue-processing and paraffin-embedding method. Extraction of protein and application to immunoblotting. Am J Pathol 140:775–779PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Mami Yamamoto-Tanaka
    • 1
    • 2
  • Toshihiko Hibino
    • 1
  1. 1.Shiseido Research CenterTsuzuki-ku, YokohamaJapan
  2. 2.Department of DermatologyTokyo Medical UniversityTokyoJapan

Personalised recommendations