A novel regulatory relationship between RIPK4 and ELF3 in keratinocytes
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.
References (37)
Barrier function of the skin: “la raison d'etre” of the epidermis
J. Invest. Dermatol.
(2003)- et al.
Stem cell heterogeneity and plasticity in epithelia
Cell Stem Cell
(2015) - et al.
RIP4 is an ankyrin repeat-containing kinase essential for keratinocyte differentiation
Curr. Biol.
(2002) - et al.
Mutations in RIPK4 cause the autosomal-recessive form of popliteal pterygium syndrome
Am. J. Hum. Genet.
(2012) - et al.
Exome sequence identifies RIPK4 as the Bartsocas-Papas syndrome locus
Am. J. Hum. Genet.
(2012) - et al.
Protein kinase C-associated kinase (PKK) mediates Bcl10-independent NF-kappa B activation induced by phorbol ester
J. Biol. Chem.
(2002) - et al.
Receptor-interacting protein kinase 4 and interferon regulatory factor 6 function as a signaling axis to regulate keratinocyte differentiation
J. Biol. Chem.
(2014) - et al.
Disease-associated mutations in IRF6 and RIPK4 dysregulate their signalling functions
Cell. Signal.
(2015) - et al.
Interferon regulatory factor 6 promotes differentiation of the periderm by activating expression of Grainyhead-like 3
J. Invest. Dermatol.
(2013) - et al.
RIPK4 activates an IRF6-mediated proinflammatory cytokine response in keratinocytes
Cytokine
(2016)
AP-1 and ets transcription factors regulate the expression of the human SPRR1A keratinocyte terminal differentiation marker
J. Biol. Chem.
Interplay between proximal and distal promoter elements is required for squamous differentiation marker induction in the bronchial epithelium: role for ESE-1, Sp1, and AP-1 proteins
J. Biol. Chem.
Small proline-rich protein 1 is the major component of the cell envelope of normal human oral keratinocytes
FEBS Lett.
Inactivation of the transcription factor Elf3 in mice results in dysmorphogenesis and altered differentiation of intestinal epithelium
Gastroenterology
Identification of ELF3 as an early transcriptional regulator of human urothelium
Dev. Biol.
Ataxia-telangiectasia, mutated (ATM)/nuclear factor kappa light chain enhancer of activated B cells (NFkappaB) signaling controls basal and DNA damage-induced transglutaminase 2 expression
J. Biol. Chem.
Loss of ELF3 immunoexpression is useful for detecting oral squamous cell carcinoma but not for distinguishing between grades of epithelial dysplasia
Ann. Diagn. Pathol.
Epithelial structural proteins of the skin and oral cavity: function in health and disease
Crit. Rev. Oral Biol. Med.
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These authors made equal contributions.
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Current address: Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark.