Nucleophagy: A New Look at Past Observations

Accepted Manuscript Nucleophagy: A New Look at Past Observations Han Peng, Robert M. Lavker PII:

S0022-202X(16)31140-X

DOI:

10.1016/j.jid.2016.04.019

Reference:

JID 314

To appear in:

The Journal of Investigative Dermatology

Received Date: 4 April 2016 Revised Date:

22 April 2016

Accepted Date: 23 April 2016

Please cite this article as: Peng H, Lavker RM, Nucleophagy: A New Look at Past Observations, The Journal of Investigative Dermatology (2016), doi: 10.1016/j.jid.2016.04.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Nucleophagy: A New Look at Past Observations Han Peng and Robert M. Lavker

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Departments of Dermatology, Northwestern University, Chicago, IL 60611

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Clinical Relevance •

Inhibition of nucleophagy during keratinocyte differentiation is associated with

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parakeratosis. •

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Corresponding author: Robert M. Lavker, Ph.D., Northwestern University, Feinberg School of Medicine, Department of Dermatology, 303 E. Chicago Ave. Chicago, IL 60611; Tel: 312-503-4315; FAX 312-503-4325; e-mail: [email protected]

Therapies that can induce nucleophagy may be novel therapies for skin diseases

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such as psoriasis.

Studies on the molecular basis of nucleophagy regulation during epidermal

differentiation are needed to identify potential small molecules that promote keratinization.

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Abstract

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Keratinization of the stratum corneum involves a highly choreographed sequence of events in which granular cells lose their nuclei and become desiccated corneocytes. In this issue, Akinduro et al (2016) detail the molecular machinery underlying removal of

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the nucleus (nucleophagy) during the final stages of keratinization. They provide

evidence that nucleophagy is induced when the keratinocytes differentiate and that

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failure in the initiation of nucleophagy is associated with parakeratosis.

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Epidermis functions as a dynamic barrier protecting the organism against a variety of external insults as well as excessive fluid loss. In order to fulfill its protective function, keratinocytes undergo a specific process of differentiation (keratinization) that

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culminates in formation of the stratum corneum. Keratinization begins when the

relatively undifferentiated, keratin filament-filled, proliferating basal cells receive cues to differentiate and seemingly migrate toward the surface. Asymmetric division among the

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proliferating basal cells plays a key role in activating this basal to suprabasal fate switch (Fuchs, 2008; Lopez-Pajares et al., 2013). Furthermore, coordinated activation and

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silencing of specific genes occurs during both the early and late stages of this process(Gdula et al., 2013) and references therein). The appearance of small, membrane-bound granules (lamellar granules) at the apical surface of the lowermost suprabasal (spinous) cells is one of the first morphological indicators that differentiation

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is taking place (Lavker, 1976). An early major differentiation product is filaggrin, which is first observed in the granular cells (Steinert et al., 1981)., and elaboration of the modified cornfield envelope represents one of the last differentiation products(Candi et

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al., 2005; Steinert and Marekov, 1995). Keratin filaments, lamellar granules, filaggrin and the components necessary for the modified corneocyte envelope accumulate in the

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granular cells, and they are subsequently either discharged (lamellar granules) or modified to form the filament/matrix complex, which comprises the bulk of the flattened corneocytes. Another important event is the remodeling and eventual dissolution of the nucleus, which also takes place during keratinization (Gdula et al., 2013). Based on information found in the paper by Akinduro et al. (2016), we are now beginning to

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understand the molecular machinery that underlies the end-stage events in the life of each keratinocyte nucleus (nucleophagy).

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Autophagy The first clue that lytic processes (autophagy) might be involved in the morphological transformation of granular cells into corneocytes comes from a series of ultrastructural

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studies from the early 1970’s (Lavker, 1974; Lavker and Matoltsy, 1970). Using ruminal epithelium (a keratinizing epithelium) as a model, these investigators described the

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events that occur in the intermediate stages, during transition from granular to horny cells. The initial event was an increase in autolysosomes in the granular cells Mitochondrial remnants were observed within the lysosome/autolysosomes (a process now termed mitophagy) in granular cells. This suggested that lytic events triggered the initial transformation stages leading to cornification (Lavker, 1974; Lavker and Maltoltsy,

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1970; Morioka et al., 1999). This increase in autolysosomes preceded disappearance of the metabolic organelles (mitochondria, Golgi, endoplasmic reticulum) and the

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destruction of nuclei. In fact, the nucleus was one of the last structures to undergo degradation, possibly via nucleophagy, although that term was not used at the time

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(Lavker and Matoltsy, 1970). Using multicolor confocal microscopy, image analysis and mathematical modeling have provided a clearer picture of nuclear changes that take place during keratinization(Gdula et al., 2013). As reported in this issue of the JID, Akinduro et al. (2016) employed comprehensive genetic, molecular and cellular biological approaches to address the changes that take place in epidermal nuclei, demonstrating that nucleophagy serves as the means by which nuclei are removed.

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Autophagy is a process of self-cannibalism in which cellular material is engulfed within double-membraned autophagosomes for subsequent digestion (initiation; Fig. 1a). The initial steps in autophagy involve the formation of an isolation membrane (Fig. 1a).

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Genes associated with this event include: unc-51 like autophagy activating kinase 1 (ULK1) complex; phosphatidylinositol-3-kinase (PI3K) complex

(VPS34/Beclin1/ATG14L); and WD repeat domain phosphoinositide interacting 1

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(WIPI1). Activation of these complexes leads to the association of ATG16L1 with the ATG5-ATG12 conjugate. The ATG5-ATG12-ATG16L1 complex then adds

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phosphatidylethanolamine (PE) to the C-terminus of the microtubule associated protein 1 light chain 3 (LC3) protein promoting elongation of the isolation membrane and subsequent autophagosome formation (Fig. 1b). Thus, Beclin1 and the ATG genes are related to the initial formation of the autophagosomes, and LC3 is a well-accepted

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autophagosome marker. At the late stage of autophagy, autophagosomes fuse with lysosomes, resulting in autolysosomes, which function during degradation. Recently, new studies are beginning to detail the steps involved in the end stage of autophagy,

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during which the autolysosomes are recycled to yield lysosomes via autophagic

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lysosome reformation (ALR) (Chen and Yu, 2013). Autophagy-related genes in epidermis To establish the molecular basis by which autophagy occurs in epidermis, Akinduro et al. (2016) examined immunohistologically the detailed expression patterns of several critical genes involved in the initiation of autophagy in epidermis. Among them, LC3, the well-accepted autophagosome marker, was shown to be expressed robustly in the granular layers. Akinduro et al. (2016) also examined LC3 expression in human

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epidermis and observed a similar granular layer distribution. These findings indicate a high degree of autophagy takes place in the granular layer, which is consistent with the reports of excessive numbers of LC3 puncta in the upper layers of GFP-LC3 transgenic

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mouse epidermis as well as in the upper layers of EGFP-LC3-transduced 3D

organotypic raft cultures of normal human epidermal keratinocytes (Moriyama et al., 2014; Rossiter et al., 2013). Additionally, Akinduro et al. (2016) found that autophagy-

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related genes, including LC3, ATG5-ATG12, WIPI1, and ULK1, were upregulated at E16.5, when the granular layer is first observed. These findings suggest that autophagic

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processes play a role in the terminal phases of keratinization, and they support concepts of a major role for nucleophagy in keratinization. Nucleophagy in keratinocyte differentiation

While the morphological evidence for nucleophagy in keratinocyte terminal

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differentiation has been known for many years), our understanding of the molecular components underlying these observations has been scant. Akinduro et al. (2016) observed a dramatic increase in misshaped nuclei within differentiated keratinocytes. At

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morphologically irregular regions of such misshaped nuclei, they detected colocalization of autophagic vesicle markers and nuclear materials. Furthermore, knockdown of

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autophagy-essential genes reduced the number of cells with nucleophagic vesicles in differentiated keratinocytes; with negligible effects on proliferating keratinocytes. Finally, to validate the above observations, Akinduro et al. (2016) found that some of the LC3 puncta in the granular layers were coincident with areas of misshaped nuclei. Collectively, this is strong evidence that nucleophagy plays a key role in nuclear removal during keratinocyte terminal differentiation in vitro and in vivo.

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Autophagy and Psoriasis The links between nucleophagy and human disease are only just emerging.

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Parakeratosis, the retention of nuclei in the stratum corneum, is one of characteristics of psoriasis. Akinduro et al. (2016) showed that LC3, WIPI1 and ULK1 were decreased in the parakeratotic regions of epidermis taken from patients with psoriasis, and they

suggested that a failure in nucleophagy may contribute to its pathogenesis. Several

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studies have suggested that autophagy is impaired in psoriasis. For example, several

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single nucleotide polymorphisms (SNPs) in the ATG16L1 gene, which interacts with ATG5/ATG12 to mediate the conjugation of PE to LC3, were associated with susceptibility to psoriasis (Douroudis et al., 2012). Furthermore, p62, which is degraded during autophagy and is well-accepted as a negative marker for autophagy flux, was reported to be upregulated in the epidermis of psoriatic skin (Lee et al., 2011). Such p62

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accumulation indicates an inhibition of autophagy flux in psoriatic epidermis. Taken together, Akinduro et al. (2016) and others (Douroudis et al., 2012; Lee et al., 2011) have provided evidence that impairment in nucleophagy may contribute to the

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pathogenesis of psoriasis. In support of this idea, UVB therapy (Qiang et al., 2013),

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retinoids (Rajawat et al., 2010), and vitamin D analogs (Hoyer-Hansen et al., 2005), which are used for the treatment of psoriasis, also can induce autophagy. Unanswered nucleophagy questions Most of our knowledge about nucleophagy comes from studies on lower eukaryotes, such as yeast, and the mechanisms of nucleophagy in mammals remain unclear. Akinduro et al. (2016) has demonstraed that WIPI1 and ULK1 are required for nucleophagy during keratinocyte terminal differentiation in vitro. However, to understand

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more fully how nucleophagy is initiated and regulated during terminal differentiation of keratinocytes, additional studies of the molecular mechanisms of nucleophagy are necessary. For example, Akinduro et al. (2016) showed that Beclin1, a key component

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of the PI3K complex, which mediates nucleation at an early stage of autophagy, was increased at day E16.5 in mice, although this protein was not found within the epidermal granular layer. This raises the question of whether Beclin1 is essential for

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autophagy/nucleophagy in the epidermal granular layers. While nuclear changes and nucleophagy have thus far been primarily keratinocyte-centric, this raises the question

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about other cell types such as fibroblasts. A form of nucleophagy has been reported in mouse embryonic fibroblasts carrying lamin mutations as well as in wild-type cells (Park et al., 2009). We anticipate that the present paper by Akinduro et al (2016) will stimulate investigations of nucleophagy in other cellsl that reside in skin. Furthermore, studies on

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autophagy in general have been heavily focused on the initiation stage; whereas, endstage events are just starting to be recognized as important. Akinduro et al (2016) nicely detailed the early events in nucleophagy; however, the events taking place during the

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end stages of nucleophagy remain uncertain. Nonetheless, the present study by Akinduro et al (2016) should stimulate further investigations on the roles of autophagy in

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keratinocyte biology as well as in diseases of abnormal keratinization.

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Figure citation

Figure 1. Autophagy and its initiation. (a) A schematic representation of the stages of

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autophagy. (b) A schematic representation of the initial steps in autophagy involving the formation of an isolation membrane. In this process, unc-51 like autophagy activating kinase 1 (ULK1) complex activates the phosphatidylinositol-3-kinase (PI3K) complex (VPS34/Beclin1/ATG14L). VPS34-derived PI3P recruits double FYVE-containing

protein 1 (DFCP1/ZFYVE1) and WD repeat domain phosphoinositide interacting 1

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(WIPI1) to the outer membrane of autophagosomes, which causes the association of the ATG5/ATG12 conjugate with ATG16L1. The ATG5/ATG12/ATG16L1 complex then

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adds phosphatidylethanolamine group (PE) to the C-terminus of the microtubule associated protein 1 light chain 3 (LC3) protein promoting the elongation of the isolation

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membrane and autophagosome formation.

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