Elsevier

Cellular Signalling

Volume 28, Issue 12, December 2016, Pages 1916-1922
Cellular Signalling

A novel regulatory relationship between RIPK4 and ELF3 in keratinocytes

https://doi.org/10.1016/j.cellsig.2016.09.006Get rights and content

Highlights

  • RIPK4 signalling regulates ELF3 gene expression in keratinocytes.

  • IRF6 and GRHL3 function downstream of RIPK4 to promote ELF3 gene expression.

  • RIPK4 signalling regulates the expression of SPRR1, a putative ELF3 target gene.

  • TGM1 expression is similarly regulated by RIPK4 signalling.

  • Activation of an IRF6-GRHL3-ELF3 transcriptional pathway by RIPK4 may promote barrier function.

Abstract

Keratinocytes are central to the barrier functions of surface epithelia, such as the gingiva and epidermis. RIPK4 is a key regulator of keratinocyte differentiation; however, the signalling pathways in which it functions remain poorly defined. In this study, we identified a regulatory relationship between RIPK4 and ELF3, an ETS family transcription factor. RIPK4 was shown to be important for the upregulation of ELF3 gene expression by the PKC agonist PMA in both oral and epidermal keratinocytes. RIPK4 promotes keratinocyte differentiation in part by phosphorylating and thereby activating the IRF6 transcription factor. Significantly, silencing of IRF6 inhibited the PMA-inducible expression of ELF3. A role for the GRHL3 transcription factor, a downstream target gene of IRF6, in the regulation of ELF3 expression was similarly demonstrated. ELF3 has previously been shown to regulate the expression of SPPR1A and SPRR1B, small proline-rich proteins that contribute to the cornification of keratinocytes. Consistently, RIPK4 and IRF6 were important for the PMA-inducible expression of SPRR1A and SPRR1B. They were also important for the upregulation of TGM1, a transglutaminase that catalyses the cross-linking of proteins, including small proline-rich proteins, during keratinocyte cornification. RIPK4 was also shown to upregulate the expression of TGM2 independently of IRF6. Collectively, our findings position RIPK4 upstream of a hierarchal IRF6-GRHL3-ELF3 transcription factor pathway in keratinocytes, as well as provide insight into a potential role for RIPK4 in the regulation of keratinocyte cornification.

Introduction

The stratified squamous epithelia of the oral cavity, as well as other surface epithelia (e.g. epidermis), provide protection against mechanical and chemical damage, and biological insults [1], [2]. The epithelia, which are organised into layers of morphologically and biochemically distinct cells, are highly dynamic and maintained through tightly regulated keratinocyte proliferation and differentiation [2], [3]. Tissue renewal, in turn, is initiated by stem cell populations in the basal layer that undergo a limited number of cell divisions before initiating terminal differentiation as they migrate towards the superficial layers. Depending on anatomical location, keratinocytes may also become enucleated, flattened, and cornified [2], [3], [4].

Cornification greatly strengthens the barrier functions of keratinocytes. During the final stages of keratinocyte terminal differentiation, the nucleus and its DNA are degraded and keratin filaments are aggregated into tight bundles by filaggrin to promote the collapsing of the cell into a flattened shape [2], [4]. Concomitantly, the cornified envelope is assembled just under the cell membrane through the cross-linking of various proteins (e.g. involucrin, loricrin, and small proline-rich proteins) by calcium-dependent transglutaminases, for example, transglutaminase-1 (TGM1). Intracellular lipids from lamellar bodies are also deposited in the cell membrane, where they become covalently attached to the cornified envelope as well as extruded from the cell to form intercellular lamellae. Collectively, this results in the replacement of the keratinocyte cell membrane with an insoluble structure that protects the underlying epithelial tissues [2], [4]. Keratinocytes also play active roles in epithelial homeostasis and host defence by producing cytokines that promote inflammation and wound healing in response to injury and infection [5].

Receptor-interacting protein kinase 4 (RIPK4) is an important regulator of keratinocyte differentiation [6]. For instance, the epidermis of Ripk4-deficient mice is disorganised and expanded, and the outermost cornified layers are absent, resulting in defective barrier function [6], [7]. Mutations in RIPK4 cause Bartsocas-Papas syndrome [8], [9], a congenital syndrome that is characterised by severe oral and epidermal abnormalities. At the molecular level, RIPK4 can activate NF-κB [10], [11], [12], [13], a critical regulator of epithelial tissue homeostasis [14]. Significantly, RIPK4 can directly activate Interferon regulatory factor 6 (IRF6) [12]. IRF6 is an important transcriptional regulator of keratinocyte differentiation and promotes the switch from proliferation to differentiation [15], [16]. IRF6 regulates keratinocyte differentiation in part by inducing the expression of the transcription factors Grainyhead-like 3 (GRHL3) and Ovo-like zinc-finger 1 (OVOL1) [12], [17], [18]. Similar to Ripk4-deficient mice, the spinous layer in the epidermis of Irf6-deficient mice is expanded, and the granular and cornified layers appear to be absent [15]. We recently established that RIPK4 also regulates the expression of proinflammatory cytokines by keratinocytes through its activation of IRF6 [19]. Thus, RIPK4 appears to function as a key regulatory nodal point in the maintenance of epithelia homeostasis.

To understand further the role of RIPK4 in keratinocytes, we sought to identify additional target genes of RIPK4 signalling. We show here that RIPK4 signalling regulates the expression of the ETS family transcription factor E74-like factor 3 (ELF3) in human keratinocytes. Specifically, our data suggest that RIPK4 promotes ELF3 gene expression via the IRF6-mediated upregulation of GRHL3. Moreover, this RIPK4-regulated IRF6-GRHL3-ELF3 transcriptional network appears to control the expression of genes (e.g. SPRR1 and TGM1) that directly mediate keratinocyte cornification.

Section snippets

Reagents

Keratinocyte serum-free medium and supplements (human EGF and bovine pituitary extract) (Cat. no. 37010022), GlutaMax-1 (Cat. no. 35050061), Opti-MEM I reduced serum medium (Cat. no. 31985062), Lipofectamine RNAiMAX transfection reagent (Cat. no. 13778150), and the Silencer Select RIPK4 siRNA (Cat. no. 4390824, siRNA ID: s28865) and GRHL3 siRNA (Cat. no. 4392420, siRNA ID: s33754) were from Life Technologies. KGM-Gold BulletKit keratinocyte growth medium (Cat. no. 00192152) and ReagentPack

RIPK4 regulates ELF3 gene expression in human keratinocytes

In addition to regulating the PKC-mediated differentiation of keratinocytes [12], RIPK4 also regulates their expression of proinflammatory cytokines [19]. To understand further the function of RIPK4 in keratinocytes, we sought to identify additional target genes of RIPK4 signalling. To that end, OKF6/TERT-2 human oral keratinocytes (hereafter referred to as OKF6 cells) were transfected with a RIPK4 siRNA (Fig. 1A), and subsequently stimulated with the PKC agonist phorbol 12-myristate 13-acetate

Discussion

Keratinocyte differentiation is central to maintaining the barrier functions of surface epithelia. Depending on anatomical location (e.g. gingiva and epidermis), keratinocytes may also become cornified, which increases their strength and hence barrier functions [2], [3], [4]. Underpinning cornification are transcriptional networks which regulate the expression of structural proteins and enzymes that mediate the formation of the cornified envelope. Herein, we have established a role for RIPK4 in

Conclusions

In summary, we have identified and mechanistically defined a previously unrecognised regulatory relationship between RIPK4 and ELF3 in keratinocytes. Thus, RIPK4 might regulate the barrier functions of surface epithelia, at least in part, through its regulation of a hierarchal IRF6-GRHL3-ELF3 transcription factor pathway.

Acknowledgements

This research was supported by the Australian Government, Department of Industry, Innovation and Science, and National Health and Medical Research Council Project Grant 628769.

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1

These authors made equal contributions.

2

Current address: Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark.

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