Letters to the Editor 213

LETTER TO THE EDITOR

Differentiation-specific expression andlocalization of an autophagosomal mar-ker protein (LC3) in human epidermalkeratinocytes

Striking morphological changes occur during theterminal stages of epidermal differentiation,including the loss of major cellular organelles,

Fig. 1 Western-blot and immunohistochemical analyses ocultured human keratinocytes. (A) The top panel shows thethe arrows) in the whole-keratinocyte extracts obtained fromdifferentiation in the presence of E64d and pepstatin A (10 mg/expression during keratinocyte differentiation. Keratin 10 waweight markers are indicated on the left. (B) The upper (a—ctochemical staining on days 0 and 7 after calcium-induced difshow the pattern of immunohistochemical staining with anti-LCwith FITC (green); (c) and (f) show overlays of (a) and (b), andundifferentiated (day 0) and differentiated (day 7) keratinkeratinization. In the merged images (Merge), the red colorrepresents the fluorescence of anti-keration 10 antibody, andmagnification shows the LC3 distribution in the cytoplasm ofaround the nuclei of the normal human keratinocyte cells (pimmunofluorescence image of differentiated keratinocytes oMacintosh Power PC G4 using the ImageJ software (http://rsb.line square in (a). Note the presence of LC3-positive rings in

aggregation of keratin filaments, and formation ofthe cornified cell envelope. Therefore, cellularhomeostasis of keratinocytes requires a constantbalance to be maintained between biosyntheticand catabolic processes. In regard to the catabolicprocesses, recent studies have revealed that alleukaryotic cells primarily use two distinct mechan-isms for large-scale degradation; one mediated bythe proteasome [1], and the other involving autop-

f LC3 protein during calcium-induced keratinization indetection of the LC3-I and LC3-II proteins (indicated bykeratinocytes on days 0, 3, 5 and 7 after calcium-inducedml each) for 0, 6 and 24 h. Note the increase of LC3-I and IIs also recognized with anti-keratin 10 antibody. Molecular) and lower panels (d—f) show the results of immunohis-ferentiation of keratinocytes. (a) and (d), and (b) and (e)3 IgG tagged with Cy3 (red) and anti-keratin 10 IgG tagged(d) and (e). Bar = 50 mm. Immunofluorescence images ofocytes. Keratin 10 expression is shown as a marker ofrepresents the fluorescence of anti-LC3 IgG, green colorblue color represents the fluorescence of DAPI. (C) Highercultured keratinocytes, which appeared as fine granulesanels C-a and C-b) on day 7 after calcium induction. Ann day 7 stained with anti-LC3 IgG, and deconvolved on ainfo.nih.gov/ij/). (b) A higher magnification of the dottedthe differentiated cells.

214 Letters to the Editor

hagy [2—5], which has the capability of degradingentire cytoplasmic organelles.

Autophagy refers to a major cellular degrada-tion process in which long-lived proteins and orga-nelles are sequestered into autophagosomes(double-membraned vesicular structures) and sub-sequently delivered to lysosomes for degradation.In addition to a role in the routine turnover ofcytoplasmic components, autophagy is alsoinvolved in the development, differentiation andtissue remodeling in various organisms [2—5].Among many proteins known to essential for theautophagic process, LC3, a mammalian homologueof the yeast Apg8p, plays a critical role in autop-hagy. Being synthesized as a soluble cytosolic form(LC3-I), LC3-I is then converted a membrane-boundform (LC3-II) via an autophagy-specific conjugationreaction catalyzed by Atg7 and Atg3. LC3-II is thenrecruited to the autophagosomal membrane.Immunoelectron microscopic analysis has revealedthe existence of LC3-II inside and outside theautophagosomes [6]. It has been demonstrated

Fig. 2 Immunohistochemical localization of LC3 protein inimmunohistological findings from normal human skin obtainlabelled with streptavidin, Alexa 594 conjugate (red) stainingdistribution of LC3 observed almost matched the pattern of soverlay in panel (c). Panel (d) shows the equivalent region ofcornified layer; G, granular layer; S, spinous layer. Scale barhistological labeling with anti-LC3 and haematoxylin and eosinrecognized in the granular layers is significantly decreased in ppanel (a).

that autophagy is transiently upregulated during celldifferentiation and that concomitantly, the cellularlevels of LC3-II are also increased [7]. Based on thisobservation, we attempted to investigate thechanges in the expression levels of LC3-II duringthe differentiation of keratinocytes. We used anti-LC3 antibody, which was raised against full-length ofGST-LC3 expressed in E. coli and subsequently affi-nity-purified on GST-LC3-Sepharose [5].

For gel electrophoresis and immunoblotting,human keratinocytes derived from normal newbornforeskin were maintained in a serum-free standardmedium containing 0.06 mM Ca2+[Epilife-KGM (Kur-abo, Osaka, Japan)]. The keratinocytes (60—70%confluency) in a monolayer were transferred tothe Epilife-KGM medium containing 2 mM Ca2+ andthen incubated for the indicated durations to allowdifferentiation to occur.

Western-blot analysis of protein extracts fromthe human-cultured keratinocytes was performedusing methods described previously [6]. The anti-body showed the same reaction pattern as pre-

normal and psoriatic skin. Panels (a) and (b) show theed using anti-LC3 (green) and anti-ubiquitin antibodiesin paraffin sections of the biopsy specimens. Note that thetaining with the anti-ubiquitin antibody, as shown by thea section stained with haematoxylin and eosin staining. C,= 50 mm. Panels (e) and (f) show the results of immuno-staining in psoriatic skin. Note that the fluorescence of LC3soriatic as skin compared with that in normal human skin in

Letters to the Editor 215

viously described [6,7], and the intensity of theprotein bands at 18 kDa (LC3-I) and 16 kDa (LC3-II) to increase in a time-dependent manner duringcalcium-induced differentiation of the cells(Fig. 1A). It was also noted when autpophagicdegradation of the LC3-II had been inhibited bythe addition of E64d and pepstatin A (lysosomalproteinase inhibitors, 10 mg/ml each) at 6 and24 h before the cells were harvested, LC3-II accu-mulated more abundantly during calcium-inducedkeratinization compared with undifferentiatedstate (day 0), indicating that autophagic turnoverof LC3-II is enhanced during differentiation. Inaddition, we also performed immunofluorescen-cence analysis in the cultured keratinocytes, andfound that a significant increase of LC3-positivepuncta was observed 7 days after the induction ofdifferentiation (Fig. 1B and C), suggesting thatmore autopagosomes accumulate in differentiatedkeratinocytes.

Wealso investigated LC3distribution in thenormalhuman skin (n = 6) by immunohistochemical analysis,and revealed positive staining mainly in the granularlayers, inwhich terminal differentiation starts beforethe loss of the nuclei. No positive stainingwas seen inthe papillary dermis of the skin. Thus, we reasonedthe existence of a possible relationship betweenautophagy and organelle degradation in the granularcells during epidermal differentiation [8].

We further investigated LC3 localization in skinaffected by psoriasis vulgaris (n = 6), a disease char-acterized by the co-existence of accelerated kera-tinization and premature cell death [9,10]. Thedownregulated expression of LC3 found in psoriaticskin might suggest that autophagy in the granularlayer may partly contribute to normal keratinizationand that LC3 might be a useful diagnostic marker foridentifying the status of keratinization in normaland abnormal skin conditions (Fig. 2).

To the best of our knowledge, this is the firstreport to show the existence of LC3, which is essen-tial for the formation of autophagosomes, in theskin. However, the physiological function of thisprotein in the epidermis vis-a-vis its role in epider-mal autophagy is still unknown. Our currentresearch is aimed at obtaining more detailed infor-mation on this subject by immunoelectron micro-scopic analysis using anti-LC3 antibody.

References

[1] Ding WX, Yin XM. Sorting, recognition and activation of themisfolded protein degradation pathways through macroauto-phagy and the proteasome. Autophagy 2008;4:141—50.

[2] Mizushima N, Klionsky DJ. Protein turnover via autophagy:implications for metabolism. Annu Rev Nutr 2007;27:19—40.

[3] Levine B. Unraveling the role of autophagy in cancer. Autop-hagy 2006;2:65—6.

[4] Tanida I, Ueno T, Kominami E. LC3 conjugation system inmammalian autophagy. Int J Biochem Cell Biol 2004;36:2503—18.

[5] Tanida I, Minematsu-Ikeguchi N, Ueno T, Kominami E. Lyso-somal turnover, but not a cellular level, of endogenous LC3 isa marker for autophagy. Autophagy 2005;1:84—91.

[6] Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, NodaT, et al. LC3, a mammalian homologue of yeast Apg8p, islocalized in autophagosome membranes after processing.EMBO J 2000;19:5720—8.

[7] Asanuma K, Tanida I, Shirato I, Ueno T, Takahara H, NishitaniT, et al. MAP-LC3, a promising autophagosomal marker, isprocessed during the differentiation and recovery of podo-cytes from PAN nephrosis. FASEB J 2003;17:1165—7.

[8] Morioka K, Takano-OhmuroH, SameshimaM,Ueno T, KominamiE, Sakuraba H, et al. Extinction of organelles in differentiatingepidermis. Acta Histochem Cytochem 1999;32:465—76.

[9] Griffiths CEM, Camp RDR, Barker JNWN. Psoriasis. In: BurnsT, Breathnach S, Cox N, Griffiths CEM, editors. Rook’s text-book of dermatology. 7th ed., Oxford: Blackwell ScienceLtd.; 2004 . p. 35.1—35.11.

[10] Iizuka H, Takahashi H, Honma M, Ishida-Yamamoto A. Uniquekeratinization process in psoriasis: late differentiation mar-kers are abolished because of the premature cell death. JDermatol 2004;31:271—6.

Kunitaka Haruna*Yasushi Suga

Shigenori MuramatsuKenichi Taneda

Yuki MizunoShigaku Ikeda

Department of Dermatology,Juntendo University School of Medicine,

Tokyo 113-8421, Japan

Takashi UenoEiki Kominami

Isei TanidaDepartment of Biochemistry,

Juntendo University School of Medicine,Tokyo 113-8421, Japan

Isei TanidaKentaro Hanada

Department of Biochemistry and Cell Biology,Laboratory of Biomembranes,

National Institute of Infectious Disease,Tokyo 162-8640, Japan

*Corresponding author. Tel.: +81 3 5802 1089E-mail address: kharuna@med.juntendo.ac.jp

(K. Haruna)

3 April 2008

doi:10.1016/j.jdermsci.2008.07.005