GW Bodies: Cytoplasmic Compartments in Normal Human Skin

JM Zee et al. GW Bodies in Skin

GW Bodies: Cytoplasmic Compartments in Normal Human Skin Journal of Investigative Dermatology (2008) 128, 2909–2912; doi:10.1038/jid.2008.154; published online 12 June 2008

microRNAs, degrade mRNAs, interact with stress granules during the stress response, and are differentially regulated during the cell cycle and cell proliferation, GWBs may serve as important regulatory sites for normal, inflammatory, and malignant skin disease. To investigate the presence and distribution patterns of GWBs in normal human skin, 8-mm-thick paraffin-embedded normal skin sections were treated with an antigen retrieval method and stained with mouse mAbs to the recombinant GW182 protein as previously described (Jakymiw et al., 2005). In the epidermis and cutaneous appendages, GWBs were most prominent in the stratum basale and the outer root sheath of the hair follicle (Figure 1a–d). The

a

GWBs visualized in these structures were generally larger and more numerous than in other cutaneous structures. In the epidermis, they were predominantly located in a single row along the basal layer, although transverse sections revealed several layers of keratinocytes containing GWBs in the deep rete ridges. Colocalization studies with a rabbit polyclonal antibody to keratin 15 (ProteinTech Group Inc, Chicago, IL) confirmed the follicular localization of GWBs to bulge progenitor cells (Lyle et al., 1998; Morris et al., 2004; Ohyama et al., 2006) (Figure S1). GW bodies were also visualized in the basaloid cells of the sebaceous glands and the eccrine glands (Figure S2). The germinative matrix of the hair bulb

c

d

b

e

GW182

Control

NHEK

TO THE EDITOR In 2002, unique cytoplasmic compartments were identified and termed GW bodies (GWBs), also known as mammalian processing bodies (P bodies) (Eystathioy et al., 2002; van Dijk et al., 2002; Sheth and Parker, 2003; Eulalio et al., 2007; Jakymiw et al., 2007; Parker and Sheth, 2007). GWBs were named after the GW182 protein, an mRNA-binding protein that resides within these macromolecular foci (Eystathioy et al., 2002). Characterization of this protein revealed numerous GW (glycine–tryptophan) repeats and a canonical RNA recognition motif (Eystathioy et al., 2002). The GW182 protein associates with a specific subset of mRNA, serves as a scaffold or matrix protein, and helps to maintain the integrity of GWBs as knock down of the GW182 protein results in a loss of GWBs (Jakymiw et al., 2005, 2007). GWBs contain a defined subset of mRNA, microRNA, and various proteins involved in mRNA degradation and RNA interference (RNAi) (Eystathioy et al., 2003; Andrei et al., 2005; Jakymiw et al., 2005; Liu et al., 2005a, b; Rehwinkel et al., 2005; Behm-Ansmant et al., 2006; Pauley et al., 2006; Valencia-Sanchez et al., 2006). Intact GWBs are also found to be critical to the RNAi pathway, and microRNA is critical in the formation of GWBs (Jakymiw et al., 2005; Pauley et al., 2006). Lastly, GWBs have been shown to interact with stress granules during cellular stress responses (Kedersha et al., 2005). Current research has focused on improving our understanding of the structure and function of GWBs in a variety of cells and tissues during embryonic development, physiological responses, neoplastic transformation, and aging. As these structures function to localize specific subsets of mRNA and

Figure 1. Cytoplasmic GW bodies are present in the proliferative compartments of normal human skin and in a normal human epidermal keratinocyte (NHEK) cell line. Mouse monoclonal anti-GW182 antibodies (green) were used to carry out indirect immunofluorescence (IIF) studies on normal human skin. GWBs were localized to the stratum basale (a) and the outer root sheath (ORS) of the hair follicle (c). Controls using only secondary antibody are shown for the stratum basale (b) and the ORS of the hair follicle (d). The dotted scale bar and the solid scale bar are representative of the stratum basale and ORS, respectively. Scale bars ¼ 100 mm. (e) NHEKs, stained with the human anti-GW182 serum, 18033 (green), are shown on the left. Arrows indicate select GW bodies. A control, stained with secondary antibody only, is shown on the right. The nuclei were counterstained with 40 ,6-diamidino-2-phenylindole (DAPI, blue). Scale bar ¼ 10 mm.

Abbreviations: NHEK, normal human epidermal keratinocyte; RNAi, RNA interference

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JM Zee et al. GW Bodies in Skin

and the apocrine glands demonstrated weak and sparse staining, respectively (data not shown). Depending on the number and size of GWBs, fluorescence intensity varied and was measured semi-quantitatively (refer to Table 1). Immunohistochemical studies were also performed on serial formalin-fixed, paraffin-embedded skin sections using a mouse mAb to the proliferation marker Ki67 (Dako, Glostrup, Denmark). Within the GWB-containing structures of skin, Ki67-positive cells were noted (Figure S3). Accordingly, GWBs appear more prominent in the proliferative cell populations of normal skin.

To determine whether the GWBs within normal human keratinocytes contain proteins involved in mRNA degradation and RNAi, colocalization studies were performed using antibodies to LSm4, Xrn1, Ge-1 (also known as RCD8 or Hedls), Argonaute-2 (Ago2), and Dicer. Normal human epidermal keratinocytes (NHEK) (Cascade Biologics, Portland, OR) were cultured in EpiLife medium containing human keratinocyte growth supplement (Cascade Biologics). For indirect immunofluorescence studies, the cells were grown at 37 1C with 5% CO2 to B60% confluency on 18 mm glass coverslips. As previously described,

Table 1. The semi-quantitative evaluation of fluorescence intensity for GWBs in normal human skin1 Structure

Fluorescence intensity

Epidermis Stratum basale

+++

Stratum spinosum



Stratum granulosum



Stratum corneum



Eccrine sweat gland Secretory coil Clear cells

++ ++

Dark (intercalated) cells

+

Myoepithelial cells



Duct



Apocrine sweat gland Superficial secretory cells

/+ uneven labeling

Basal cells



Myoepithelial cells



Duct



Sebaceous gland Basal cells

++

Mature sebocytes



Hair follicle Inner epidermal root sheath Matrix

+

Mature



Outer epidermal root sheath2 Basal cell layer Spinous cell layer 1

+++ 

The fluorescence intensities were scored on a 4-point scale:  (negative), + (weak), ++ (moderate), and +++ (strong). The portion below the entry of the sebaceous duct. The external root sheath above the duct entry exhibited a pattern similar to that of the stratum basale.

2

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the colocalization experiments were carried out on NHEKs fixed with 2% paraformaldehyde, permeabilized with 0.5% Triton X-100, and stained using the following antibodies: a human anti-GW182 serum, 18033 (Mitogen Advanced Diagnostics Laboratory, University of Calgary, Calgary, AB, Canada), a mouse anti-Ago2 (a gift from Dr E. Chan, University of Florida), a mouse anti-Dicer, a chicken anti-LSm4 (Abcam, Cambridge, MA), a rabbit antiGe-1 (a gift from Dr D. Bloch, Harvard University), a rabbit anti-Xrn1 (Bethyl Laboratories, Montgomery, TX), and a mouse monoclonal anti-golgin97 (Mitogen Advanced Diagnostics Laboratory, University of Calgary) (Jakymiw et al., 2005; Moser et al., 2007). To quantify the relative frequency of GWBs colocalizing with the mRNA degradation and RNAi-specific markers, Imaris 4.3.1 software (Bitplane Inc, Saint Paul, MN) was used to enumerate both complete and partial overlapping foci. As the staining patterns demonstrated cellular heterogeneity, the calculated percentages of colocalization were obtained from three to six keratinocytes observed over duplicate independent experiments. The GWBs of NHEKs varied in both size and number (Figure 1e). Visualization of the nucleus with 40 ,6-diamidino2-phenylindole (DAPI) confirmed their cytoplasmic localization. A select group of proteins involved in mRNA degradation and RNAi was found to colocalize with keratinocyte GWBs, including Ago2, Dicer, Ge-1, LSm4, and Xrn1 (Figure 2). Approximately 85.17, 18.95, 12.57, and 11.45% of GWBs co-stained with antibodies to Ge-1, Xrn1, LSm4, and Ago2, respectively. To a lesser degree, Dicer colocalized with GW182 at a relative frequency of 2.49% (Table 2). Cells stained with anti-golgin97 (a mAb specific for the Golgi complex protein, golgin97) and secondary antibody alone had colocalization values of 0.19 and 0.80%, respectively, thus demonstrating the specificity of our findings in NHEKs. Our data clearly demonstrate that GWBs contain Ge-1, a protein that enhances mRNA decapping and exonuclease activity within the 50 –30 mRNA degradation pathway (Yu et al., 2005). The lower colocalization frequencies

JM Zee et al. GW Bodies in Skin

I

II

III

IV

Ago2

Dicer

Ge-1

LSm4

Xrn1

10 µm

10 µm

10 µm

10 µm

Figure 2. GW bodies colocalize with mRNA degradation and RNAi-associated proteins in normal human epidermal keratinocytes (NHEK). Using indirect immunofluorescence (IIF) techniques, GWBs were stained with the human anti-GW182 serum 18033 and the secondary AF488 antibody (green; column II). Mouse anti-Ago2 antibody (neat), mouse anti-Dicer antibody (1:100), rabbit anti-Ge-1 antibody (1:400), chicken anti-LSm4 antibody (1:2), and rabbit anti-Xrn1 antibody (1:2) were stained with the secondary AF568 antibody (red; column III). Images were merged and the nuclei were counterstained with DAPI (columns I, IV). Merged images were enlarged in columns II–IV to illustrate the colocalization between GWBs and mRNA degradation/RNAi-associated proteins in NHEKs. Arrows indicate select foci of colocalization.

with Xrn1, LSm4, Ago2, and Dicer suggest that there is microheterogeneity in the compartmentalization of cutaneous GWBs, but that the latter are more involved in mRNA degradation and storage. Alternatively, the association of GWBs and the mRNA degradation and/or RNAi proteins may be

transient and cell cycle-dependent. For instance, in HeLa and HEp-2 cell lines, GWBs have been previously shown to vary in both size and number in relation to cell cycle phase (Yang et al., 2004; Lian et al., 2006). The identification of cytoplasmic GWBs in normal human skin has far-

reaching implications. Their involvement in mRNA storage, regulation, and cell proliferation suggests that GWBs have a functional role in normal human keratinocytes. Further investigations are urgently required to study their significance in skin homeostasis, repair, and disease. www.jidonline.org 2911

JM Zee et al. GW Bodies in Skin

Table 2. Colocalization of GW182 with RNAi and mRNA degradation proteins in normal human epidermal keratinocytes Colocalization with GW182 (%)

Protein Protein function Ago2

An endonuclease that associates with miRNAs and siRNAs within the RISC

11.45

Dicer

A multi-domain RNase III enzyme responsible for cleaving long ds RNA molecules into siRNAs of 19–21 bp and processing premiRNAs into their mature miRNA form

Ge-1

A protein involved in mRNA decapping with exonuclease activity within the 50 –30 mRNA degradation pathway

85.17

LSm4

A protein of a heptameric hLSm complex involved in mRNA splicing and degradation

12.57

Xrn1

A major cytoplasmic 50 –30 exonuclease that degrades mRNAs after they are decapped by Dcp1/Dcp2

18.95

2.49

ds, double-stranded; miRNA, microRNAs; siRNA, small interfering RNAs; RISC, RNA-induced silencing complex.

for eIF4E and eIF4E-transporter in targeting mRNPs to mammalian processing bodies. RNA 11:717–27

CONFLICT OF INTEREST The authors state no conflict of interest.

ACKNOWLEDGMENTS We thank Riley Sullivan and Meifeng Zhang (University of Calgary) for their assistance with the IIF experiments, and Dr Michael Schoel (University of Calgary Microscopy and Imaging Facility) for his technical assistance with deconvolution microscopy. P.R.M. is supported by the Canadian Dermatology Foundation and the Faculty of Medicine (University of Calgary). M.J.F. holds the Arthritis Society Chair (University of Calgary). 1

1

Jennifer M. Zee , Karalee K. Shideler , Theophany Eystathioy2, Andrea K. Bruecks3, Marvin J. Fritzler2 and P. Re´gine Mydlarski1 1

Division of Dermatology, Departments of Medicine & Medical Genetics, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada; 2Division of Rheumatology, Department of Medicine, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada and 3Department of Pathology and Laboratory Medicine, University of Calgary and Calgary Laboratory Services, Calgary, Alberta, Canada E-mail: [email protected] SUPPLEMENTARY MATERIAL Figure S1. GW bodies colocalize with keratin 15 in the follicular bulge progenitor cells. Figure S2. The distribution of GW bodies in normal human skin. Figure S3. GW bodies localize to regions containing highly proliferative Ki67-positive cells.

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