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IKKα controls formation of the epidermis independently of NF-κB

Abstract

The IKKα and IKKβ catalytic subunits of IκB kinase (IKK) share 51% amino-acid identity and similar biochemical activities: they both phosphorylate IκB proteins at serines that trigger their degradation1,2,3,4. IKKα and IKKβ differ, however, in their physiological functions. IKKβ and the IKKγ/NEMO regulatory subunit are required for activating NF-κB by pro-inflammatory stimuli and preventing apoptosis induced by tumour necrosis factor-α (refs 5,6,7,8,9,10,11). IKKα is dispensable for these functions, but is essential for developing the epidermis and its derivatives12,13,14,15. The mammalian epidermis is composed of the basal, spinous, granular and cornified layers16. Only basal keratinocytes can proliferate and give rise to differentiated derivatives, which on full maturation undergo enucleation to generate the cornified layer. Curiously, keratinocyte-specific inhibition of NF-κB, as in Ikkα-/- mice12,13,14,15, results in epidermal thickening but does not block terminal differentiation17,18. It has been proposed19,20 that the epidermal defect in Ikkα-/- mice may be due to the failed activation of NF-κB. Here we show that the unique function of IKKα in control of keratinocyte differentiation is not exerted through its IκB kinase activity or through NF-κB. Instead, IKKα controls production of a soluble factor that induces keratinocyte differentiation.

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Figure 1: Defective differentiation of cultured Ikkα-/- keratinocytes.
Figure 2: Introduction of IKKα into Ikkα-/- keratinocytes induces their differentiation.
Figure 3: IKKα kinase activity or interaction with IKKγ is not required for induction of keratinocyte differentiation.
Figure 4: Normal differentiation of engrafted Ikkα-/- skin.
Figure 5: IKKα controls production of an autocrine factor that induces keratinocyte differentiation.

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Acknowledgements

We thank K. M. Hodivala-Dilke for advice on keratinocyte isolation; K. Bouic for assistance with adenovirus preparation; D. Brenner and B. Benett for recombinant adenoviruses; M. Delhase for IKKα (EE and 390) mutants; and J. Feramisco for assistance with deconvolution microscopy at the UCSD Cancer Center Core Facility. Y.H., V.B. and T.O. were supported by postdoctoral fellowships from the Arthritis Foundation, Human Frontier Science Program and Japanese Society for Promotion of Science, respectively. Work was supported by grants from the NIH, the Association For International Cancer Research and the CERIES Research Award to M.K., who is the Frank and Else Schilling American Cancer Society Research Professor.

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Correspondence to Michael Karin.

Supplementary information

Figure 1 (jpg 73 KB)

Comparison of differentiation and proliferation markers in skin sections of wt and Ikka-/- mice. Paraffin sections of skin from wt and Ikka-/- (M) 19-day mouse embryos were stained with antibodies to CK5, CK1, and PCNA, CK10, CK6 and filaggrin, as indicated. The top six panels show sections visualized by indirect immunofluorescence. The bottom six panels were visualized by peroxidase staining and hematoxylin was used as counterstaining (200x). BL and SL: basal and suprabasal layers.

Figure 2 (jpg 35 KB)

Hyperproliferation and NF-kB activation in Ikka-deficient keratinoctes. a, Equal numbers of Ikka+/+, Ikka+/- and Ikka-/- keratinocytes were plated at day 0. Cell numbers were determined at the indicated times after culture in normal medium. b, Colony formation by Ikka+/- and Ikka-/- keratinocytes. After fixing in ethanol, colony numbers and sizes were measured using the Image-Pro Plus program. c, Effects of Ca2+ on cell growth. Numbers of Ikka+/+, Ikka+/- and Ikka-/- keratinocytes cultured without or with 0.5 mM Ca2+ (added at day 3). d, IKK and NF-kB activities following treatment of cultured Ikka+/+ and Ikka-/- keratinocytes with TNFa or IL-1a. IKK activity was measured by immunecomplex kinase assay (KA) using IKKg antibody. IKKg recovery was determined by immunoblotting. NF-kB DNA binding activity was measured by a gel mobility shift assay.

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Hu, Y., Baud, V., Oga, T. et al. IKKα controls formation of the epidermis independently of NF-κB. Nature 410, 710–714 (2001). https://doi.org/10.1038/35070605

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