Biochimica et Biophysica Acta 1830 (2013) 2427–2434

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Biochimica et Biophysica Acta

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Review

Biochemistry of epidermal stem cells☆

Richard L. Eckert a,b,c,d,e,⁎, Gautam Adhikary a, Sivaprakasam Balasubramanian a, Ellen A. Rorke d,Mohan C. Vemuri f, Shayne E. Boucher f, Jackie R. Bickenbach g, Candace Kerr a

a Department of Biochemistry and Molecular Biology, The University of Maryland School of Medicine, USAb Department of Dermatology, The University of Maryland School of Medicine, USAc Department of Reproductive Biology, The University of Maryland School of Medicine, USAd Department of Microbiology and Immunology, The University of Maryland School of Medicine, USAe The Marlene and Stewart Greenebaum Cancer Center, The University of Maryland School of Medicine, USAf Life Technologies, Inc. Primary and Stem Cell Culture Systems, Frederick, MD, USAg Department of Anatomy and Cell Biology, University of Iowa Carver College of Medicine, USA

Abbreviations: HF, hair follicle; IF, interfollicular;microRNA; SCC, squamous cell carcinoma; KSC, keratino☆ This article is part of a Special Issue entitled Bioche⁎ Corresponding author at: Biochemistry and Molecular

School of Medicine, 108 N. Greene Street, Baltimore, MD3220; fax: +1 410 706 8297.

E-mail address: reckert@umaryland.edu (R.L. Eckert

0304-4165/$ – see front matter © 2012 Elsevier B.V. Alldoi:10.1016/j.bbagen.2012.07.002

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 2 May 2012Accepted 10 July 2012Available online 20 July 2012

Keywords:Stem cellHair follicleInterfollicular stem cellEpidermisKeratinocyte

Background: The epidermis is an important protective barrier that is essential for maintenance of life.Maintaining this barrier requires continuous cell proliferation and differentiation. Moreover, these processesmust be balanced to produce a normal epidermis. The stem cells of the epidermis reside in specific locationsin the basal epidermis, hair follicle and sebaceous glands and these cells are responsible for replenishment ofthis tissue.Scope of review:A great deal of effort has gone into identifying protein epitopes that mark stem cells, in identifyingstem cell niche locations, and in understanding how stem cell populations are related.We discuss these studies asthey apply to understanding normal epidermal homeostasis and skin cancer.Major conclusions: An assortment of stem cell markers have been identified that permit assignment of stem cellsto specific regions of the epidermis, and progress has beenmade in understanding the role of these cells in normal

epidermal homeostasis and in conditions of tissue stress. A key finding is the multiple stem cell populations existin epidermis that give rise to different structures, and that multiple stem cell types may contribute to repair indamaged epidermis.General significance:Understanding epidermal stem cell biology is likely to lead to important therapies for treatingskin diseases and cancer, and will also contribute to our understanding of stem cells in other systems. This articleis part of a Special Issue entitled Biochemistry of Stem Cells.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction — the epidermis

The epidermis provides an important barrier that protects againstenvironmental stress and loss of body fluids. As such it generates anumber of structures that provide key homeostatic functions includ-ing the epidermis, hair follicles, and sebaceous and sweat glands.This dynamic structure renews continually to maintain the epidermalbarrier and to respond to damage. Construction of an appropriate andfunctional barrier requires a balance between keratinocyte prolifera-tion and differentiation [37], in addition to apoptosis when the tissueis stressed [49,50].

During mouse development, the embryonic epidermis begins as asingle layer of cells until embryonic day 12.5 when keratinocytes initiate

SG, sebaceous gland; miRNA,cyte stem cellmistry of Stem Cells.Biology, University of Maryland21201, USA. Tel.: +1 410 706

).

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stratification andmultiple layers are established. As amultilayered tissuethe epidermis displays a number of morphologically distinct zones,including the basal, spinous, granular and cornified layers (Fig. 1) [37].The basal layer contains the undifferentiated cells that possess prolifera-tive capacity. These cells are anchored to the basementmembrane, a bar-rier that separates the epidermis from the underlying dermis. As such,cells of the basal layer are polarized with their basal surface anchoredby integrins to the basement membrane extracellular matrix compo-nents (including laminin and collagen IV secreted by keratinocytes).The integrins include a variety of α- and β-integrin subunits, such asα6 integrin and β4 integrin, putative epidermal stem cell markers,which act as heterodimeric transmembrane receptors [82,125]. In partic-ular, integrins have an important role in organizing hemidesmosomeswhich anchor the basal keratinocytes to the extracellular matrix [120].

At intervals, some of these basal cells lose the ability to proliferate andini\tiate a programof terminal differentiationwhich involvesmaturationas the cells are pushed upwards towards the cell surface. Ultimately, therole of these cells is to form corneocytes, which are dead cells, thatconsist of a covalently crosslinked (cornified) envelope surroundingstabilized keratin bundles [37,94]. In the course of assembly of this

Fig. 1. Structure of the hair follicle and the epidermis. The schematic shows hair follicle structures including the infundibulum, junctional zone, isthmus, sebaceous gland, bulge,bulb and hair shaft. The epidermal layers (basal, spinous, granulate and cornified) are also shown. The interfollicular epidermis is the stratified epidermis located between thehair follicles. The text describes various regions of these structures where specific stem cell populations, identified by specific markers, are located.

Table 1Distribution of stem cell markers in human and murine epidermis.

Marker Location References Species

α6bri/CD71dim IF epidermis [74,128,129] Humanβ1+/MCSP+/Lrg1+ IF epidermis [65,66,71] HumanSurvivin+ IF epidermis [80] HumanLgr6+ IF epidermis [133] MouseCD133 (prominin) IF epidermis [141] HumanCD200+ HF (bulge) [98,99] HumanK15+ HF (bulge) [88] MouseCD200+/CD34−/K15bri HF (bulge) [64] HumanCD200+/CD34−/K15dim HF (bulge) [64] HumanCD34+ HF (bulge) [22] MouseK15 Promoter HF (bulge) [88,118] MouseK19 HF (bulge) [85] Mouseα6dim/MTS24+/Lrig1+ HF (isthmus) [96] MouseSca-1+ HF (infundibulum) [67] MouseLrig1+ HF (isthmus) [65] MouseLgr6+ SG (isthmus) [112] MouseBlimp1+ SG [63] MouseNGFRp75, nestin, Oct-4 MSC (dermis) [76] Mouse

Neural crest (bulge area) [140] MouseCD133+ Cancer stem [100] Humanα6bri/β1dim/CD34bri

α6bri/β1dim/CD34dimCancer stem [106] Mouse

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terminal structure, the nucleus and all other organelles are destroyed[57]. Ultimately, these cells are shed from the skin surface as the termi-nal event in differentiation. This tissue includes three major functionalzones, the proliferative cells of the basal layer (which include thestem cells), partially differentiated cells of the spinous and granularlayers which are viable but non-proliferating, and the terminal differen-tiated dead cells of the cornified layer. Regulation of cell survival is im-portant in each of these zones (Fig. 1). The molecular processes thatcontrol differentiation are complex and involve many gene products[36,38].

Keratinocyte stem cells (KSC) are proliferation units that display lim-ited and slowdivision, can self-renew and are responsible for generatingthe various lineages present in themature tissue [17,18,21,56,127–129].These were initially identified as label-retaining cells [16,19,20]. Currentstudies identify three populations of KSCs in epidermis. These includethe interfollicular (IF) epidermal stem cells in the epidermal basallayer, the hair follicle (HF) stem cells of the bulge and the sebaceousgland (SG) stem cells located immediately above the hair bulge. The IFepidermis is the skin surface located between hair follicles. Under nor-mal conditions these stem cell populations appear to function indepen-dently to produce the IF epidermis, hair follicle and sebaceous gland.However, recent studies suggest that each of these cell populations canfunction to replicate any skin structure [46,73], whichmaybe particular-ly important when the epidermis is stressed.

Specific differences are evident in the microanatomy and compo-sition of the stem cells between mice and humans. These differencesemphasize the importance in distinguishing among animal modelswhen studying epidermal stem cells. For instance, the hair bulge is avery distinct structure in the follicle and is a primary source of epider-mal stem cells in the mouse, while in humans the hair bulge is lessdistinct and IF stem cells appear to be the more abundant stem cell.However, studies of label-retaining cells indicated that human hairfollicle stem cells like those in the mouse can also be found in thebulge-like structure [79,98].

Stem cells, located in the IF epidermis in the basal layer, are an im-portant population of cells involved in maintaining the IF epidermis.

Barrandon and Green characterized differences in proliferative poten-tial of keratinocyte cultures prepared from the human epidermis [11].Three populations of cells were identified and called holo-, para- andmetaclones. The holoclones give rise to the largest colonies in clonalgrowth assays and are rich in markers of the epidermal basal layer, in-cluding β1-integrin [69]. Subsequent studies reveal that these cells arerich in α6-integrin and express low levels of CD71 (transferrin recep-tor) [128,129]. α6-integrinbri/CD71dim cells display many stem cell fea-tures, including quiescence and long-term growth capacity (Table 1)[74,128,129]. These cells are generally detected at the downward tipof the rete ridges. However, another population of cells, that areβ1-integrin+/MCSP (melanoma chondroitin sulfate proteoglycan)+/

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Lrg1+, are present in the upper segment of the rete ridge and theseshare stem cell properties [65,66,71]. Additional markers of these cellshave also been identified, including survivin [80]. The existing informa-tion suggests that IF epidermal stem cells are randomly distributed inthe basal layer and represent 1% to 7% of the basal cell population. The1% level was determined by labeling experiments using human epider-mal organotypic skin cultures [91] and the higher values were deter-mined based on detection of α6-integrinbri/CD71dim human cells[129]. It is interesting that neonatal foreskin includes more stem cells(7%) than skin derived from adults (5%) [128,129]. The localization ofthe murine IF epidermal stem cells is controversial. Evidence suggeststhat Lgr6+ cells, present in the HF, near the infundibulum, serve as astem cell reservoir for the murine IF epidermis [133]. CD133 is a puta-tive marker of the murine dermal papilla [141].

Various other proteins have also been identified that help to distin-guish epidermal stem cells. For instance, connexin43 (Cx43) a gap junc-tion protein is absent in 10% of human basal keratinocytes which alsoexhibited clonal ability, small size and low granularity. Cx43dim cellsalso helped distinguish p63+/ABCG2+/β1-integrin+ label-retainingcells in the human limbal epithelium [31]. Desmoglein3 (Dsg3) is an-other intercellular junction protein that may be a useful marker fordistinguishing epithelial stem cells. High β1-integrin expressinghuman keratinocytes have been shown to express low levels of Dsg3and exhibit greater long-term proliferative capacity in vitro thanintegrin β1hi/Dsg3hi cells. The β1hi/Dsg3lo cells isolated from the adulthuman palm show comparable clonogenic ability to α6hi/CD71lo cells[121,122]. Some evidence suggests that CD146,melanoma cell adhesionmolecule (MCAM), may also distinguish stem cells. For example,CD146lo selection, in conjunction with selection for other markers, in-cluding CD200+, CD24lo, CD34lo, CD71lo, isolates human hair folliclecells with high colony-forming efficiency [98]. Other markers thathave been studied include human EGFRlo (epidermal growth factorreceptor) cells which undergo long-term expansion and produce astratified epidermis in models of skin reconstruction [45]. Low majorhistocompatibility complex, MHC Class I-HLA expression is observedin pluripotent stem cells and also in a subpopulation of basal humankeratinocytes [83].

2. Two models of epidermal stem cell amplification

In addition to holoclones, Barrandon and Green identified otherdividing cells called paraclones, which give rise to abortive coloniesthat differentiate after only limited proliferation and merocloneswhich are intermediate in morphology and proliferative capacity [11].Based on these and other findings, it has been theorized that the IF epi-dermis includes a mixture of proliferating cells consisting of holoclonesand paraclones [11]. The holoclones are thought to correspond to thelabel-retaining stem cell population and the paraclones to the transientamplifying cells. These cells are distinguished based on differences inlabel-retention [28], cell surface marker expression, proliferation fre-quency, and ability to grow as clones in culture [11,11,28,64,96,98,117].In murine epidermis cell populations have been distinguished as epider-mal stem cells which are label-retaining and occasionally give rise to anidentical daughter stem cell (symmetrical division) and a transientamplifying cell (asymmetrical division). Unlike the epidermal stem cell,the transit amplifying cell divides rapidly and, after several rounds ofcell division, undergoes terminal differentiation.

However, fate mapping experiments question the existence oftransient-amplifying cells [34,68]. These studies used inducible geneticlabeling to track progenitor cells in murine tail epidermis for one year.Results showed that the average number of basal layer cells per cloneincreases in a linear fashion with time and does not follow an “epider-mal proliferation unit” pattern which would be expected if transientamplifying cells were present. Since the clones remained cohesive andexpand in size over time, this suggests that only one type of proliferativestem cell exists that undergoes an unlimited number of symmetrical

divisions. If these results can be replicated in areas outside the tailregion, it would suggest a new model for stem cell renewal in theepidermis.

3. Stem cells of the hair follicle

The hair follicle is a structure that varies from the interfollicular epi-dermis in several importantways. First, it projects down into the dermiswhere the cells are exposed to a different environment and, second, thehair follicle undergoes intermittent cycles of growth, regression andquiescence [92]. In each growth cycle, the follicle is generated from aspecific set of stem cells that are resident in the hair bulge. In mouse,the hair follicle bulge is a highly recognizable structure. In humanhair, the bulge is not as readily recognized. The mouse and human sys-tems also differ in other ways. The human hair follicle cycle takes nearlya decade while the murine hair cycle is on the order of weeks [23].

CD200 (cluster of differentiation 200), a type I membrane glyco-protein which contains two immunoglobulin domains, is a reliablemarker of human bulge cells (Table 1), but not of murine bulge cells[98,99]. In contrast, keratin 15 (K15), is a reliable marker in mousebulge cells [88], but is not as specific for human hair bulge stemcells [97]. Distinct bulge-localized K15-positive stem cell subpopula-tions have been identified in human epidermis which are CD200+/CD34−/K15bri (basal) and CD200+/CD34−/K15dim (suprabasal) [64].The CD200+/CD34−/K15bri cells form larger clonal growth colonies[64].

CD34 (cluster of differentiation 34), a single-pass transmembranesialomucin protein, marks bulge cells in murine follicles [22], but isabsent from human bulge tissue [23,61]. In murine hair folliclebulge cells, the CD34+ cells are label-retaining and readily form clon-al colonies [118,119]. Keratin 19 (K19) also stains label-retaining cellsin mice [85]. An extremely useful finding is that the K15 promoteralso marks these cells [88,118].

The region between the bulge and the IF epidermis in murineepidermis also contains putative stem cells. This region, the isthmus(Fig. 1), contains a population of MTS24+/Lrig1+/α6dim cells that arehighly clonogenic [96]. These cells are also marked by an absence ofCD34, K15 and Sca-1. In contrast, Sca-1+ cells are present in theinfundibulum [67] and these cells can repopulate the IF epidermis butnot the HF. Lrig1+ cells have also been identified in the junctional zone(Fig. 1) [65]. Lrig1 probably acts in these cells to maintain quiescenceand in reconstitution assays these cells give rise to all epidermal celllineages [65].

4. Sebaceous gland stem cells

The SG is an important structure in the epidermis. In mouse, stemcells that produce this gland are located in a region of the HF abovethe CD34+/K15+ bulge region. This is evidenced by the presence ofa population of Lgr6+ cells at this location that can produce seba-ceous cells [112]. This population is also multipotent and can replaceHF and IF epidermis and SG structures in mouse [112]. However,these cells are not label-retaining. The Lrg6+ cells are particularly in-teresting in that they can renew the sebaceous gland and sebaceousgland stem cells which are Blimp-1 positive [63]. Lineage-tracking ex-periments indicate that these cells give rise to the entire gland [63].Although its role is not well understood, Blimp-1 is an interestingprotein that must have a negative control role on gland growth, assuppression of Blimp-1 expression results in SG hyperplasia [63].The bulge area also contains melanocyte stem cells and stem cellswith neural crest properties [76,140].

5. Skin cancer stem cells

Non-melanoma skin cancer is the most common cancer in humanpopulations. Basal cell carcinoma (BCC) is frequent and can be highly

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destructive. The second type of skin cancer, squamous cell carcinoma(SCC), is extremely frequent. SCC precancerous stages are well char-acterized with respect to clinical, histological and molecular featuresand metastasis [9,14]. Immunosuppressed patients are two-hundredtimes more likely to get aggressive and metastatic SCC. Skin cancerprogression is linked to ultraviolet (UV) exposure. Early-age UV expo-sure contributes to BCC development, and chronic UV exposure isthought to be responsible for SCC development and progression [9].Moreover, due to the presence of environmental irritants and expo-sure to UV irradiation, skin cancer incidence is increasing [60]. Thus,skin cancer is an important health concern.

The cancer stem cell theory states that tumor stem cells areself-renewing, slow cycling, quiescent cells that are not impacted byanti-cancer agents that kill rapidly growing tumor cells [3]. Moreover,it is thought that a limited number of cancer stem cells give rise tohighly proliferative non-stem cells that comprise the bulk of thetumor. Since cancer stem cells give rise to other tumor cells, eliminat-ing tumor stem cells is thought to be necessary to halt tumor forma-tion [3]. Well-developed mouse models are available to study the roleof epidermal skin cancer stem cells [14,21,51,58,59,109,127].

It is thought that mutations in rarely dividing long-lived stem cellslead to accumulation of genetic changes that overcome cell controland lead to cancer growth. Squamous cell carcinoma progression in-volves mutational inactivation of p53. Ultraviolet light is a commonskin mutagen and DNA changes characteristic of UV exposure areoften observed in skin cancer cells and tumors [25]. As mutated p53suppresses wild-type p53 activity, the cells are rendered more resis-tant to apoptosis [143]. It is clear that these slowly-dividing stemcells accumulate mutations in the 7,12-dimethyl-1,2‐benz[a]anthra-cene (DMBA)/tetradecanoylphorbol-13-acetate (TPA) two-stage can-cer protocol and that these cells then expand during tumor expansion[70,86,87,111]. Thus, stem cells appear to be an important carcinogentarget.

Human SCC is thought to arise from IF stem cells that give rise tosquamous tumors. SCC generally occurs at sun-exposed sites andmanifests UV-associated p53 mutation [26]. This is consistent withthe fact that basal IF epidermis is accessible to UV irradiation. Actinickeratosis is the apparent precursor lesion to SCC [5,9]. However, astrict origin of SCC in the IF epidermis may not hold under specific ex-perimental conditions. For example, tumors arising in the two stagecarcinogenesis model, following sequential treatment of DMBA andTPA, are associated with DMBA-dependent H-ras mutation. Most ofthe tumors arise from stem cells located in the HF bulge [89]. More-over, SCC still develops when the IF epidermis is removed and miceare UV-irradiated [43]. This is presumably due to an impact on HFbulge stem cells. This suggests that various cell populations can giverise to tumors following UV exposure. Moreover, when SCC-relatedmutations (Ras mutation/p53 deletion) are targeted to the bulgestem cells, these cells give rise to SCC [131]. Taken together, thesestudies suggest that stem cells are the likely target of environmentaldamage, and that depending upon the circumstance, SCC can arisefrom various epidermal stem cell populations. Moreover, it is inter-esting that the targeted mutation of transient amplifying cells doesnot produce tumors [131], further supporting the idea that stemcells are the target. Identifying tumor stem cells in human tumorsamples has recently progressed, as Vogel and coworkers showedthat CD133+ cells are enriched for tumor-initiating cells [100]. A dis-tinct spatial distribution of stem cell markers is also observed in mu-rine SCC, as the cancer stem cells are located along the dermal/tumorinterface and are integrin-positive and CD34bri or CD34dim [106].Other protein markers also identify putative cancer stem cells.These markers have been identified in other epithelia, such as mam-mary cancer. For instance, aldehyde dehydrogenase (ALDH) isexpressed in human epidermis [32] and also in normal mammarystem cells [53]. Likewise, CD44 (hyaluronic acid receptor) has alsobeen identified as a marker of mammary stem cells [4] and head

and neck squamous cell cancer stem cells [101]. CD24 is a glycopro-tein and CD24neg expression is present in more primitive, or lessdifferentiated mammary stem cells, and is a marker of post-mitotichuman keratinocytes [15].

Basal cell carcinoma is restricted to areas of the epidermis withhair, and the marker protein profile observed in BCC is consistentwith a hair follicle-origin of the tumors [109]. Moreover, mutationin Ptch1, the Sonic hedgehog receptor, which is involved in hair folli-cle regeneration, is observed [8,75], and the level of Lgr5, a HF stemcell marker, is increased in most BCCs [116]. These findings stronglysuggest that HF stem cells give rise to BCC, although recent resultssuggest that this model may be more complicated than previouslyappreciated [55,123,139].

6. Other stem cells in the epidermis

Non-epithelial stem cells are also found in the epidermis. These in-clude bone marrow‐derived stem cells (BMSCs) which are found inthe epidermis during epidermal regeneration. Several studies suggestthat BMSCs have the ability to derive both dermal and epidermal cellsduring regeneration [10,24,29,35,42]. An alternative explanation forsuch results is the demonstrated ability of BMSCs to fuse with othercells in vitro. However, while ex vivo studies have demonstrated theability of BMSCs to fuse with keratinocytes [12], several studieshave shown that in vivo fusion does not occur [24,62,134] and thatBMSCs differentiate into epidermal cells [135].

7. Signaling proteins in epidermal stem cells

A requirement for stem cell survival is that the quiescent state betightly controlled. This involves a number of signaling cascades, someof which are discussed here. The hair follicle utilizes sonic hedgehog(Shh) and Wnt signaling to control cell function. Wnt/β-catenin sig-naling regulates cell fate. Wnt is a secreted glycoprotein that bindsto Frizzled receptors to trigger a cascade that results in displacementof GSK-3β from the APC/Axin/GSK-3β complex. In the absence of Wntsignaling, β-catenin is phosphorylated and targeted for degradationby the APC/Axin/GSK-3β complex. In the presence of Wnt, Dishev-elled is phosphorylated and activated and recruits GSK-3β from thecomplex. This leads to β-catenin stabilization and Rac1 nuclear trans-location and recruitment of LEF/TCF DNA binding factors to activatetranscription [102]. Wnt signaling, via its impact on TCF3 transcrip-tion factor, is essential for maintenance of HF stem cells [48,84,144],and blocking Wnt action causes cells to differentiate into keratinocytesand sebocytes [78,95]. These findings indicate that Wnt signalingcontrols commitment of HF bulge cells in mouse to maintain the stemstate or permit entry into a differentiation pathway.

Sonic hedgehog (Shh) is a ligand that binds to itsmembrane-spanningreceptor, Patched (Ptc). Under resting conditions, Ptc binds to and inhibitsSmoothened (Smo), a transmembrane protein. The hedgehog signalingcomplex is downstream of Smo. When Shh binds to Ptc, the inhibitorimpact on Smo is relieved and Smo becomes phosphorylated. Anotherprotein called Ci is then released and enters the nucleus to driveexpression of hedgehog target genes. Sonic hedgehog (Shh) is asecond important controller of HF bulge stem cells. Shh is a keygene that is activated in order to organize dermal cells to form thedermal papilla [104]. Inhibition of the Shh pathway during embryonicdevelopment arrests the follicle at the hair bud stage after placodeformation and initial ingrowth [114]. Furthermore, inhibition of Shh inadult skin prevents anagen hair growth [124].

Notch and EGFR signaling are also key pathways in the IF epider-mis [47]. Notch signaling is pronounced in the basal layer of the epi-dermis with high activity in areas featuring stem cells [77]. Notchsignaling also drives keratinocyte differentiation as the cells departthe stem cell niche [77]. In mouse epidermis differentiation andmaintenance of IF epidermis are balanced by the opposing actions

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of Notch and another key signaling molecule, p63 [47,126]. Themitogen-activated protein kinases (MAPK) and epidermal growthfactor receptor (EGFR) are also important. Numerous studies indicatethat EGFR and MAPK signaling are required for normal epidermaldifferentiation, particularly of the IF keratinocyte [2,38–41,52].

8. MicroRNA regulation of epidermal stem cells

MicroRNAs (miRNAs) are small (~19–24 nucleotides) noncodingRNAs that regulate gene expression. One way that miRNAs modulategene expression is by interacting with the complimentary targetmessenger RNA (mRNA) to alter RNA stability or translation. It hasbeen estimated that miRNAs regulate more than one-third of theprotein-coding genome. As a consequence, it is expected that many bio-logical processes are affected. Several groups have demonstrated thatmice with epidermal specific deletion of Dicer, an essential componentof the miRNA regulatory machinery, are born normal but eventuallylose weight and die neonatally without developing a hair coat [6,137].The lack of hair is manifested by underdeveloped and misaligned hairfollicles, which exhibited increased apoptosis, and failure to invaginateinto the dermis [6,137]. This is due to the loss of the follicular stem cellpool, as evidenced by the lack of bulge stem cell markers K15 andCD34 [6]. In contrast, the interfollicular epidermis of these mice was un-affected [6,137]. Epidermal-specific deletion of co-factor Dgcr8 reveals asimilar phenotype, including neonatal lethality, dehydrated skin, a disor-dered dermis, and the presence of apoptotic cells in the HFs [137].

Specific miRNAs are expressed in developing mouse skin and HFs[6,137]. Several miRNAs appear to exert specific functions [1,136]. Forinstance, two independent studies found that miR-203 is enriched inIF epidermis compared to HFs [6,137]. Another study emphasizes theimportance of miRNA regulation in development. For instance,miR-203 is barely detectable in mouse E13.5 skin when epitheliumis still one layer, but after E15.5 and stratification begins miR-203 isamong the most abundant miRNAs. These data suggest that its ex-pression is induced during differentiation and stratification [138].Studies using in situ hybridization of mature skin revealed thatmiR-203 is expressed at high levels only in differentiated cells suchas those in the suprabasal epidermis or the inner root sheath of theHF, but not in progenitor/stem cells such as those in basal epidermis,in the HF bulge, or in the HF matrix [138]. Similar results were alsodemonstrated in zebrafish [132] and human skin [130], indicating aconserved function across species. Furthermore, the expression ofmiR-203 is rapidly upregulated when keratinocytes are induced todifferentiate in vitro by calcium [72,138], by phorbol ester [113] orby vitamin D treatment. Functional analysis of miR-203 with trans-genic mice engineered to over-express miR-203 in the epidermalbasal layer showed that animals frequently died shortly after birth,and histological evaluation revealed a thinner epidermis and deple-tion of K5+ basal cells [138].

In these studies, increased miR-203 expression reduced colonyformation capacity and caused cell cycle exit in vitro [72,93]; yet dem-onstrated limited ability to promote terminal keratinocyte differenti-ation. This suggests that the primary role of miR-203 expression maybe in the differentiation of the transient amplifying cells or to limittheir proliferative lifespan. Interestingly, miR-203 has been shownto target p63 another putative stem cell marker of epidermalkeratinocytes. The transcription factor p63 has been shown, in severalspecies, to be required to maintain the stem cell pool in epidermis. Inmice, its deletion produces a complete loss of stratified epithelia[110]. Studies have shown that the expression of p63 and miR-203is mutually exclusive, and that miR-203 directly represses p63 ex-pression as demonstrated by its ability to facilitate cell cycle arrestduring the transition in cells from the basal layer to suprabasal layer[72,138]. Specifically, one mechanism by which p63 regulates thiscell cycle progression in keratinocytes is through repression ofmiR-34 family members [7], while in cancer, p63 regulates tumor

and metastasis suppression through the transcriptional regulation ofDicer and miR-130b [115].

HF cycling involves stages of expansion, regression and quiescence,and involves dramatic changes in the epidermal and HF architecture[105]. Key regulators of this process are miRNA targets. For example,miR-31 was shown to increase during follicular growth and decreaseduring regression and quiescence [81]. MiR-31 negatively regulatesFgf10, the components of the Wnt and BMP signaling pathways,sclerostin and BAMBI, and Dlx3 transcription factor, as well as keratingenes. Luciferase reporter assays show that it directly targets K16,K17, Dlx3 and Fgf10 [81]. More recently, it is reported thatmiR-125b re-presses skin stem cell differentiation in mouse. In this study, miR-125bexpressionwas high inmouse stemcells and decreased in progeny cells.Moreover, knocking down miR-125b in transgenic mice results inhyper-thickened epidermis, enlarged sebaceous glands, and failure togenerate a hair coat [142], indications that miR-125b alters stem celldifferentiation. Potential targets of miR-125b included Blimp1 and vita-min D receptor (VDR) [142].

9. MicroRNA in melanoma

Several miRNAs have been discovered that regulate the metastatic orproliferative behavior of melanoma cells and, in some cases, their targetormode of action has been identified. For instance,miR-196a suppressionincreases tumor cell migration via an impact on HOX-B7 and HOC-C8[27,90]. MiR-137 downregulation of microphthalmia-associated tran-scription factor (MITF) in melanoma cell lines is associated with melano-ma metastasis [13]. MiR-182 increases migration of melanoma cell linesand their metastatic potential by repressing MITF and FOXO3 expres-sion [108]. MiR-15b upregulation is associated with increased cell prolif-eration and decreased apoptosis, though its targets are unclear [103].Let-7a repression increases the invasive behavior of melanocytes byimpacting β3-integrin [33], while miR-221/222 overexpression increasescell proliferation, migration and invasion via p27 and c-Kit receptor [44].miR-125b is reduced in primarymelanomas that exhibit earlymetastasesbut its target genes are currently unknown [54]. In contrast to these miRswhich facilitate melanomamalignancy, other miRNAs are repressors. Forinstance,miR-193b overexpression represses cell proliferation by increas-ing cyclin D1 in melanoma cell lines [30], while Let-7b inhibits cell cycleprogression and anchorage-independent growth when overexpressed[107].

10. Conclusions

These studies indicate that much progress has been made in un-derstanding the processes that control stem cell quiescence and dif-ferentiation in epidermis. Particular progress has been made usingmouse models where targeted changes in cell populations can be pro-duced using genetic methods. Studies are less advanced in studies ofhuman epidermal stem cells, but progress is accelerating due to theability to grow human keratinocytes as organotypic cultures with ap-propriate fibroblast support. It appears reasonable to assume that tu-mors arising in epidermis are derived from stem cells, localized eitherin the HF or IF epidermis. Moreover, the stem cell niche and locationof the stem cells relative to the mutagenic stimulus profoundly influ-ences cancer development. For example, UV irradiation is not likely toimpact stem cells deep in the HF as compared to the IF epidermalstem cells. This progress bodes well for the application of new knowl-edge to the understanding of stem cell biology and treatment of stemcell-related conditions, including cancer and other skin diseases.

Acknowledgements

Thisworkwas supported by grants to R. Eckert (NIHR01-AR053851,NIH R01-CA131074) and a grant from the State of Maryland Stem CellFund.

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