E2 Polyubiquitin-conjugating Enzyme Ubc13 in Keratinocytes Is Essential for Epidermal Integrity

Koji Sayama,Masahiro Yamamoto,Yuji Shirakata,Yasushi Hanakawa,Satoshi Hirakawa,Xiuju Dai,Mikiko Tohyama,Sho Tokumaru,Myoung-Sook Shin,Hiroaki Sakurai,Shizuo Akira,Koji Hashimoto

doi:10.1074/jbc.m110.106484

Koji Sayama, Masahiro Yamamoto + Show 10 more

Open Access

https://doi.org/10.1074/jbc.m110.106484

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Abstract

The E2 polyubiquitin-conjugating enzyme Ubc13 is a mediator of innate immune reactions. Ubc13 mediates the conjugation of keratin (K)63-linked polyubiquitin chains onto TNF receptor-associated factor 6 and IKKγ during NF-κB activation. In contrast to K48-linked polyubiquitin chains, K63-linked polyubiquitin chains function in nonproteasomal biological processes. Although Ubc13 has been shown to be critical for Toll-like receptor (TLR) and IL-1 receptor signaling, the function of Ubc13 in the epidermis has not been studied. We generated keratinocyte-specific Ubc13-deficient mice (Ubc13(flox/flox)K5-Cre). At birth, the skin of the Ubc13(flox/flox)K5-Cre mice was abnormally shiny and smooth; in addition, the mice did not grow and died by postnatal day 2. Histological analysis showed atrophy of the epidermis with keratinocyte apoptosis. Immunohistochemical analyses revealed reduced proliferation, abnormal differentiation, and apoptosis of keratinocytes in the Ubc13(flox/flox)K5-Cre mouse epidermis. In culture, Ubc13(flox/flox)K5-Cre keratinocyte growth was impaired, and spontaneous cell death occurred. Moreover, the deletion of Ubc13 from cultured Ubc13(flox/flox) keratinocytes by means of an adenoviral vector carrying Cre recombinase also resulted in spontaneous cell death. Therefore, Ubc13 is essential for keratinocyte growth, differentiation, and survival. Analyses of intracellular signaling revealed that the IL-1 and TNF-induced activation of JNK, p38, and NF-κB pathways was impaired in Ubc13(flox/flox)K5-Cre keratinocytes. In conclusion, Ubc13 appears to be essential for epidermal integrity in mice.

Highlights

Kinase 1 (TAK1)3 [3, 4]
Ki67 staining showed decreased keratinocyte proliferation, whereas H&E staining and TUNEL revealed the presence of a small number of apoptotic cells, in the epidermis of each Ubc13flox/flox K5-Cre mouse (Fig. 2B)
Apoptotic cells/basal cells were 0 and 2.9% (p Ͻ 0.01, n ϭ 4) in Ubc13flox/flox mice and Ubc13flox/flox K5-Cre mice, respectively. These results indicate that Ubc13 regulates keratinocyte growth and apoptosis in mice

Summary

Introduction

Kinase 1 (TAK1)3 [3, 4]. The polyubiquitin chains on TNF receptor-associated factor 6 are generated by the E2 ubiquitinconjugating enzyme Ubc13 [3, 5]. Southern and Western blot analyses showed the efficient deletion of Ubc13 in keratinocytes collected from Ubc13flox/flox K5-Cre mice at birth (Fig. 1). Histological analysis revealed atrophy of the epidermis in the Ubc13flox/flox K5-Cre mice (Fig. 2B), indicating that cell growth is impaired by the deletion of Ubc13. Ki67 staining showed decreased keratinocyte proliferation, whereas H&E staining and TUNEL revealed the presence of a small number of apoptotic cells, in the epidermis of each Ubc13flox/flox K5-Cre mouse (Fig. 2B).

Results

Conclusion