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Double-Stranded RNA Sensing Determines Epithelial Cell Identity

  • Chung-Ping Liao
    Affiliations
    Department of Dermatology, University of Texas Southwestern Medical Center, Dallas, Texas, USA
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  • Edem Tchegnon
    Affiliations
    Department of Dermatology, University of Texas Southwestern Medical Center, Dallas, Texas, USA

    Integrative Molecular and Biomedical Sciences Program, University of Texas Southwestern Medical Center, Dallas, Texas, USA
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  • Lu Q. Le
    Correspondence
    Correspondence: Lu Q. Le, Associate Professor of Dermatology, Hamon Center for Regenerative Science and Medicine, Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9069, USA.
    Affiliations
    Department of Dermatology, University of Texas Southwestern Medical Center, Dallas, Texas, USA

    Integrative Molecular and Biomedical Sciences Program, University of Texas Southwestern Medical Center, Dallas, Texas, USA

    Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA

    Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas, USA
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      Skin epithelium is constituted by heterogeneous keratinocytes in different body areas. In this issue, Zhou et al. used transcriptome analysis to investigate the site-specific epidermal cell identity on volar skin. Keratin 9 is highly enriched in volar keratinocytes, and its expression is dependent on low expression of the double-stranded RNA receptor DDX58, suggesting that double-stranded RNA sensing could allow a potential approach to modulate skin thickness and durability.
      • The ability of dsRNA to engender stem cell features in keratinocytes could be used in cell-based therapy for wound healing and skin regeneration.
      • Engineering of stress- and pressure-resistant volar-like skin by modulating the dsRNA-DDX58-STAT1 axis could be useful for the skin areas that are more susceptible to trauma, such as amputation stumps and decubitus ulcers.
      • The availability of dsRNA analogs could provide a potential therapeutic approach for hyperkeratotic skin associated with palmoplantar keratoderma or lichen simplex chronicus, where KRT9 is highly expressed.

       Introduction

      Skin is the outermost protective organ of the body. During development, its structure is initially formed by a single layer of unspecified epithelial cells soon after gastrulation (
      • Blanpain C.
      • Fuchs E.
      Epidermal stem cells of the skin.
      ). These cells differentiate into multiple layers of the epidermis (Figure 1a) and skin appendages, including hair follicles, sebaceous glands, and sweat glands. Despite continuous cell turnover, these structures maintain their unique identities throughout life. Although the exact molecular mechanisms controlling their fates are not known, interactions between epithelia (intrinsic factors) and mesenchyme (extrinsic factors) are critical to maintain their identities. Even within the non-adnexal epidermis, there are site-specific differences in structures and functions. For example, the skin epithelium of the palms and soles has unique histological and functional features that are distinct from those in other body areas. Volar skin is glabrous (hairless) and is more durable because of a thicker layer of stratum corneum. Although the distinctive nature of volar epithelium has been well appreciated, the underlying biological mechanism regulating its fate and identity remains unclear.
      Figure thumbnail gr1
      Figure 1Regulation of KRT9 expression in volar keratinocytes. (a) KRT9 is a specific marker of suprabasal epithelial cells in volar skin. (b) KRT9 expression is decreasing in volar keratinocytes over time with passages, concomitant with the induction of DDX58, STAT1, and KRT7. (c) Treatment of poly(I:C) reduces KRT9 expression; in contrast, KRT9 is induced by DDX58 or STAT1 siRNA. dsRNA, double-stranded RNA; KRT, keratin; P, passage; poly(I:C), polyinosinic:polycytidylic acid.

       Keratin 9 expression in volar epithelium

      • Zhou R.
      • Wang G.
      • Kim D.
      • Kim S.
      • Islam N.
      • Chen R.
      • et al.
      dsRNA sensing induces loss of cell identity.
      aimed to define possible factors involved in maintaining cell identities in palmoplantar skin by comparing differences in gene expression between keratinocytes from volar versus nonvolar skin. Principal component analysis showed a unique transcriptome profile of volar epithelium. One of the main distinguishing characteristics of volar keratinocytes is the elevated expression of keratin (KRT) 9. This is consistent with previous tissue-staining studies showing the unique expression of KRT9 in the suprabasal layers of volar epithelium. (
      • Kim D.
      • Hossain M.Z.
      • Nieves A.
      • Gu L.
      • Ratliff T.S.
      • Mi Oh S.
      • et al.
      To control site-specific skin gene expression, autocrine mimics paracrine canonical Wnt signaling and is activated ectopically in skin disease.
      ,
      • Knapp A.C.
      • Franke W.W.
      • Heid H.
      • Hatzfeld M.
      • Jorcano J.L.
      • Moll R.
      Cytokeratin no. 9, an epidermal type I keratin characteristic of a special program of keratinocyte differentiation displaying body site specificity.
      ,
      • Schweizer J.
      • Baust I.
      • Winter H.
      Identification of murine type I keratin 9 (73 kDa) and its immunolocalization in neonatal and adult mouse foot sole epidermis.
      ) (Figure 1a). The high KRT9 expression in volar keratinocytes is linked to hypomethylation of the KRT9 promoter, although the epigenetic factor mediating this demethylation is not yet defined.

       Fibroblasts regulate KRT9 expression in keratinocytes

      The expression of KRT9 in volar keratinocytes has been previously shown to be sustained by volar fibroblasts in a co-culture setting. In addition, volar fibroblasts have the ability to induce KRT9 expression in nonvolar keratinocytes (
      • Yamaguchi Y.
      • Itami S.
      • Tarutani M.
      • Hosokawa K.
      • Miura H.
      • Yoshikawa K.
      Regulation of keratin 9 in nonpalmoplantar keratinocytes by palmoplantar fibroblasts through epithelial-mesenchymal interactions.
      ). These findings highlight the participation of critical non–cell-autonomous signaling in skin development.
      • Zhou R.
      • Wang G.
      • Kim D.
      • Kim S.
      • Islam N.
      • Chen R.
      • et al.
      dsRNA sensing induces loss of cell identity.
      also observed that volar keratinocytes are able to maintain KRT9 expression in a solo culture setting, suggesting that the adaptation of volar identity of these cells occurs both intrinsically and in a non–cell-autonomous manner. Although volar keratinocytes independently express KRT9, this ability is lost over time with multiple cell passages, concomitant with an increase of KRT7, a primitive epithelial keratin. Evidently, a reversal in the expression level of these proteins in volar keratinocytes accompanies their conversion into simple epithelial cells. This observation raises the question of whether the conversion of cell identity may result from the lack of mesenchymal (fibroblast) interaction. An understanding of the cellular mechanism regulating KRT9 expression could provide insights on how volar epithelia maintain their identity.

       Double-stranded RNA sensing suppresses site-specific KRT9 expression

      In an attempt to delineate the intrinsic signals controlling KRT9 expression,
      • Zhou R.
      • Wang G.
      • Kim D.
      • Kim S.
      • Islam N.
      • Chen R.
      • et al.
      dsRNA sensing induces loss of cell identity.
      evaluated gene expression profile differences between cells isolated at early (high KRT9 level) and late passages (low KRT9 level). Decreasing KRT9 expression is associated with the induction of a group of genes involved in innate immune activation and double-stranded RNA (dsRNA) sensing, including OAS1, OAS2, OAS3, OASL, RNASEL, DDX58, and STAT1 (Figure 1b). This observation suggests that dsRNA sensing could play a role in the regulation of KRT9 expression. To test this hypothesis, the authors treated early-passaged volar keratinocytes with polyinosinic:polycytidylic acid (poly[I:C]) (a synthetic analogue of dsRNA), and they found that poly(I:C) suppresses KRT9 expression. They also treated late-passaged volar keratinocytes with small interfering RNA (siRNA) against STAT1 and DDX58 (an RNA helicase) and found that both siRNAs could induce KRT9 expression (Figure 1c). Furthermore, skin from DDX58-knockout mice are refractory to poly(I:C)-mediated KRT9 repression. Taken together, this evidence confirms the role of dsRNA sensing in site-specific suppression of KRT9 expression in volar keratinocytes. Although DDX58 knockdown can also reduce the expression of OAS1, OAS2, and RNASEL, knockdown of other dsRNA-sensing molecules, including OAS1/2/3, TLR3 (dsRNA receptor), MAVS (DDX58 downstream target), and IRF9 (STAT1/2 binding protein), has no effect on KRT9 expression. These data suggest that KRT9 expression in volar keratinocytes is regulated by noncanonical dsRNA sensing signaling through the DDX58-STAT1 axis.

       dsRNA sensing determines epithelial cellular identity

      KRT7 is a primitive keratin expressed in simple epithelia lining internal organs (
      • Sandilands A.
      • Smith F.J.
      • Lunny D.P.
      • Campbell L.E.
      • Davidson K.M.
      • MacCallum S.F.
      • et al.
      Generation and characterisation of keratin 7 (K7) knockout mice.
      ). The loss of KRT9 expression in late culture passages is coupled with the up-regulation of KRT7. In addition, knockdown of DDX58 also decreases the expression of KRT7. These results further confirm the role of dsRNA sensing in determining epithelial tissue and cell identity. Activation of RNA helicase DDX58 drives epithelial cells toward KRT7+ primitive epithelium; in contrast, maintenance of site-specific KRT9+ volar epithelium requires low DDX58 expression.
      One of the main questions emerging from these surprising results relates to the source of dsRNA, because this generally results from a viral infection.
      • Zhou R.
      • Wang G.
      • Kim D.
      • Kim S.
      • Islam N.
      • Chen R.
      • et al.
      dsRNA sensing induces loss of cell identity.
      tried to address this question by staining endogenous U1 small nuclear RNA, which can serve as a ligand of TLR3 for dsRNA sensing in the context of noninfectious conditions (
      • Bernard J.J.
      • Cowing-Zitron C.
      • Nakatsuji T.
      • Muehleisen B.
      • Muto J.
      • Borkowski A.W.
      • et al.
      Ultraviolet radiation damages self noncoding RNA and is detected by TLR3.
      ). However, they did not detect a change of U1 RNA in volar keratinocytes associated with loss of KRT9 expression during culture. In a recent study,
      • Dhir A.
      • Dhir S.
      • Borowski L.S.
      • Jimenez L.
      • Teitell M.
      • Rotig A.
      • et al.
      Mitochondrial double-stranded RNA triggers antiviral signalling in humans.
      showed the ability of mitochondria to generate dsRNA, which can escape into the cytoplasm and produce an antiviral immune response that includes up-regulation of DDX58. This raises the possibility of mitochondrial dsRNA-mediated cell identity specification, which can be examined by comparing mitochondrial RNA degradation pathways in volar and nonvolar keratinocytes, as well as in tissues before and after skin wounding.

       Conclusions

      • Zhou R.
      • Wang G.
      • Kim D.
      • Kim S.
      • Islam N.
      • Chen R.
      • et al.
      dsRNA sensing induces loss of cell identity.
      found a unique, and perhaps an unexpected, mechanism for controlling epithelial cell identity. The involvement of dsRNA sensing that is normally part of innate immune signaling represents an interesting and novel discovery. Despite the critical role of mesenchymal fibroblasts in epithelial positional identity, this study highlights the ability of volar keratinocytes to retain their identity in vitro in a cell-autonomous manner. Moreover, the results also identify a platform to explore how positional identity and cell fate can be modulated in vivo. With the advent of gene- and cell-based therapy for many skin diseases, this study has important clinical implications for future human trials.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      CPL is a recipient of the Career Development Award from the Dermatology Foundation. LQL holds a Career Award for Medical Scientists from the Burroughs Wellcome Fund and the Thomas L. Shield, MD Professorship in Dermatology and is supported by funding from the National Cancer Institute of the National Institutes of Health and the US Department of Defense .

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      Linked Article

      • dsRNA Sensing Induces Loss of Cell Identity
        Journal of Investigative DermatologyVol. 139Issue 1
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          How cell and tissue identity persist despite constant cell turnover is an important biologic question with cell therapy implications. Although many mechanisms exist, we investigated the controls for site-specific gene expression in skin, given its diverse structures and functions. For example, the transcriptome of in vivo palmoplantar (i.e., volar) epidermis is globally unique, including Keratin 9 (KRT9). Although volar fibroblasts have the capacity to induce KRT9 in nonvolar keratinocytes, we show here that volar keratinocytes continue to express KRT9 in in vitro solo cultures.
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