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Updated: May 1, 2026
Characterization of In Vitro Differentiation of Human Primary Keratinocytes by RNA-Seq Analysis
Published on: May 16, 2020
Multiple pathways are involved in DNA degradation during keratinocyte terminal differentiation
M Yamamoto-Tanaka1, T Makino2, A Motoyama3
11] Shiseido Research Center, 2-2-1 Hayabuchi, Tsuzuki-ku, Yokohama 224-8558, Japan [2] Department of Dermatology, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan.
Abstract:
Loss of the nucleus is a critical step in keratinocyte terminal differentiation. To elucidate the mechanisms involved, we focused on two characteristic events: nuclear translocation of N-terminal fragment of profilaggrin and caspase-14-dependent degradation of the inhibitor of caspase-activated DNase (ICAD). First, we demonstrated that epidermal mesotrypsin liberated a 55-kDa N-terminal fragment of profilaggrin (FLG-N) and FLG-N was translocated into the nucleus. Interestingly, these cells became TUNEL positive. Mutation in the mesotrypsin-susceptible Arg-rich region between FLG-N and the first filaggrin domain abolished these changes. Furthermore, caspase-14 caused limited proteolysis of ICAD, followed by accumulation of caspase-activated DNase (CAD) in TUNEL-positive nuclei. Knockdown of both proteases resulted in a significant increase of remnant nuclei in a skin equivalent model. Immunohistochemical study revealed that both caspase-14 and mesotrypsin were markedly downregulated in parakeratotic areas of lesional skin from patients with atopic dermatitis and psoriasis. Collectively, our results indicate that at least two pathways are involved in the DNA degradation process during keratinocyte terminal differentiation.
Insights
Two pathways involving epidermal mesotrypsin and caspase-14 contribute to nuclear degradation during keratinocyte differentiation. These processes are crucial for skin health and are impaired in conditions like atopic dermatitis and psoriasis.
Area of Science:
- Cell Biology
- Dermatology
- Biochemistry
Background:
- Nuclear loss is essential for keratinocyte terminal differentiation.
- Key events include profilaggrin N-terminal fragment (FLG-N) nuclear translocation and caspase-14-dependent inhibitor of caspase-activated DNase (ICAD) degradation.
Purpose of the Study:
- To investigate the mechanisms of nuclear degradation during keratinocyte terminal differentiation.
- To identify the roles of epidermal mesotrypsin and caspase-14 in this process.
Main Methods:
- Demonstration of FLG-N liberation and nuclear translocation by epidermal mesotrypsin.
- Analysis of caspase-14's role in ICAD proteolysis and subsequent caspase-activated DNase (CAD) activity.
- Utilizing a skin equivalent model with protease knockdown and immunohistochemistry on patient skin samples.
Main Results:
- Epidermal mesotrypsin liberates FLG-N, leading to nuclear translocation and TUNEL positivity.
- Caspase-14 degrades ICAD, releasing CAD into TUNEL-positive nuclei.
- Knockdown of these proteases increases remnant nuclei; both proteases are downregulated in atopic dermatitis and psoriasis skin lesions.
Conclusions:
- At least two distinct pathways, involving epidermal mesotrypsin and caspase-14, mediate DNA degradation during keratinocyte terminal differentiation.
- Dysregulation of these pathways may contribute to the pathogenesis of skin disorders characterized by impaired differentiation.
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