Survivin Identifies Keratinocyte Stem Cells and Is Downregulated by Anti-1 Integ - 《stemcells》干细胞学杂志干细胞之家



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作者：Alessandra Marconi, Katiuscia Dallaglio, Roberta Lotti, Cristina Vaschieri, Francesca Truzzi, Fabrizio Fantini, Carlo Pincelli作者单位：Department of Medicine, Institute of Dermatology, University of Modena and Reggio Emilia, Modena, Italy # S7 r. O' @+ d  ~6 X% O3 r

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      【摘要】1 t( `, l8 C* i4 }
   Survivin belongs to the family of inhibitor of apoptosis proteins and is involved in regulation of cell death as well as cell division. Here, we show that wild-type (WT) survivin is expressed in a subpopulation of basal keratinocytes in normal human skin at the cytoplasmic level. WT survivin is highly expressed in keratinocyte stem cells (KSCs), whereas its mRNA level decreases in transit amplifying (TA) cells and disappears in postmitotic (PM) cells. Likewise, WT survivin protein is expressed in KSCs, almost undetectable in TA cells, and absent in PM cells. Real time polymerase chain reaction demonstrates that the putative antiapoptotic isoforms survivin-2B and survivin-Ex3 are expressed at the highest levels in KSCs, whereas they tend to decrease in TA cells and disappear in PM cells. On the contrary, the putative proapoptotic variants of survivin, survivin-3B, and survivin-2 tend to be high in PM and TA cells and are almost absent in KSCs. By confocal microscopy, survivin is predominantly expressed at the nuclear level in KSCs, which proliferate significantly better than TA cells, which, in turn, express mostly cytosolic WT survivin. Blocking &szlig;1 integrin signal downregulates WT survivin mRNA and protein expression and induces apoptosis (anoikis) in KSCs. On the other hand, inhibition of &szlig;1 integrin upregulates mRNA expression of survivin-2. Taken together, these results indicate that survivin identifies human KSCs. Expression of nuclear survivin could reflect the different behavior between KSCs in vitro and in vivo, in terms of proliferation. Finally, survivin could be part of the "niche" protection by preventing anoikis in KSCs.
      【关键词】 Survivin Stem cells Anoikis Keratinocytes
   INTRODUCTION0 ~8 L' h: \+ V2 ~. v* e7 r

The inhibitor of apoptosis proteins (IAPs) are a family of antiapoptotic molecules characterized by one to three BIR (Baculoviral IAP repeats) domains, a COOH-terminal RING finger domain, and a caspase recruitment domain. The IAP family includes X-linked inhibitor-of-apoptosis protein (XIAP), IAP-like protein (ILP2), cIAP1, cIAP2, melanoma inhibitor of apoptosis protein (MLIAP), neuronal apoptosis inhibitory protein (NAIP), Bruce, and Survivin .4 b4 }) B9 j3 a; S& F1 K) }

A population of keratinocyte stem cells (KSCs) governs the renewal of human epidermis. KSCs generate transient amplifying (TA) cells that divide only a small number of times. TA cells produce daughter cells that are irreversibly committed to terminal differentiation (postmitotic .+ d$ c2 \9 U% m+ D; c0 m
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In this study, we present evidence that KSCs express the highest levels of survivin protein, which is barely detectable in TA cells and absent in PM cells. In particular, survivin is predominantly localized in the nucleus of KSCs and in the cytosol of TA cells. On the contrary, the proapoptotic variant survivin-2 is nearly absent in KSCs, whereas it increases in TA cells and is mostly expressed in PM cells. We also show that WT survivin is downregulated, whereas the proapoptotic survivin-2 is upregulated during keratinocyte anoikis.
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MATERIALS AND METHODS' o+ j( z8 ~6 ^9 A$ n- h

Keratinocyte Cultures% ?3 V$ N- U! b; N* |0 [/ g% J; @- n
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Normal human keratinocytes were obtained from foreskin and cultured as described . This allowed us to obtain a population enriched in KSCs, a population consisting of TA cells, and a third population of PM cells. Keratinocytes were then cultured in serum-free medium (KGM; Cambrex Bio Science, Walkersville, MD, http://www.cambrex.com) and used for further experiments.

Anoikis Assay

For blocking adhesion experiments, stem cells were trypsinized and suspended in polypropylene at 37¡ãC with the addition of anti-ß1 integrin-neutralizing antibody (1:250; Immunotech, Marseille, France, http://www.immunotech.cz) or in KBM alone for 0, 2, 4 and 6 hours.( s' a- L* \7 z, n# Z( u

Western Blot Analysis

Cells were washed with phosphate-buffered saline (PBS) and lysed on ice in RIPA buffer, pH 7.5, as described previously , and with anti-ß-actin mouse monoclonal antibody (1:5,000; Sigma-Aldrich) as a control. Then membranes were washed in PBS/Tween 20, incubated with peroxidase-conjugated goat anti-rabbit antibody and anti-mouse antibody (1:3,000; Bio-Rad, Hercules, CA, http://www.bio-rad.com) for 45 minutes at room temperature, washed, and developed using the ECL chemiluminescent detection system (Amersham Biosciences UK Limited, Little Chalfont Buckinghamshire, U.K., http://www.amersham.com).2 y" p8 h# E$ W: t# d- d2 I% D

Real Time Polymerase Chain Reaction7 V" C7 J$ \/ R( ]  g
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Total cellular RNA was extracted from the three populations using TRI Reagent method performed as described by Sigma-Aldrich. To verify the quality of the RNA, we ran an agarose minigel in RNase-free conditions. Quantitative real time polymerase chain reaction (PCR) was performed with an ABI 7500 (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) for WT survivin, survivin-2B, survivin-3B, survivin-Ex3, and survivin-2. As an internal control, housekeeping gene ß-actin mRNA expression was measured in a separated tube. RNase-free water was used as a negative control. We performed three independent experiments, and each one was analyzed in a triplicate PCR. Then, 1 µl of RNA was subjected to retrotranscription and amplification in a 50-µl reaction mixture using the One-Step RT-PCR Master Mix Reagents kit (Applied Biosystems). To distinguish the five splice variants of survivin, the sequences of primers for WT survivin and survivin variants were designed to correspond to exon-exon borders of the complementary strand (Fig. 1). Real time probe sets were designed using the Primer Express program, version 2.0 (Applied Biosystems). The sequence used for primers and probes were designed as above for WT survivin (GenBank accession number BC065497; forward, 5'-CGAGGCTGGCTTCATCCA; probe, 5'-ACCCCATAGAGGAACATA; reverse, 5'-GCAACCGGACGAATGCTTT), survivin-2B (GenBank accession number NM_001012271; forward, 5'-CGGGCACGGTGGCTTA; probe, 5'-ACCAGCACTTTGGGAGG; reverse, 5'-CAACCGGACGAATGCTTTTT), survivin-3B (GenBank accession number AB154416; forward, 5'-GAAAAAATTGGAAGCCAGATTCAG; probe, 5'-CTGGAAGCAAAAGAA; reverse, 5'-ACAGACCCTGGCAAACATCAG), survivin-Ex3 (GenBank accession number NM_001012270; forward, 5'-GCTGGGAGCCAGATGACG; probe, 5' -CCCCATGCAAAGGAAACCAACAATAAGAA; reverse, 5'-TCCGCAGTTTCCTCAAATTCTTT), and survivin-2 (forward, 5'-TGACGACCCCATGTAAGTCTTCT; probe, 5'-TGGCCAGCCTCGAT; reverse, 5'-TTGACAACTCAGTTCAAAACAAAGC). MGB probes were FAM dye-labeled. For ß-actin, we used predeveloped TaqMan Assay Reagents (MGB probe was VIC dye-labeled; Applied Biosystems). Thermal cycling conditions for one-step reverse transcription-PCR were: initial reverse transcription at 48¡ãC for 30 minutes, then DNA polymerase activation at 95¡ãC for 10 minutes, followed by 40 cycles of denaturation at 95¡ãC for 15 seconds, and annealing/extension at 60¡ãC for 1 minute. Data from each sample were compared with stem cells or untreated control or WT survivin expression, as calibrators, using the Sequence Detection Software, version 1.2.3, according to the Relative Quantification Study method (Applied Biosystems). Results were obtained as the mean from three independent experiments. A double-sided Student's t test was performed between samples and calibrator.& r  B& ]3 J' o* n! P
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Figure 1. Real time polymerase chain reaction primer and probe position. Survivin pre-mRNA generates at least five mature mRNA transcripts. Exons are represented with boxes, and the black arrows indicate the stop codon. Primers and probes are designed to correspond to exon-exon borders to distinguish survivin variants. The double-line arrows represent primers; probes are represented by dotted lines. Abbreviations: ex., exon; UTR, untranslated region; WT, wild type.

Confocal Immunofluorescence

Stem and TA cells growing on collagen-coated coverslips for 96 hours were washed in PBS and fixed in situ in formaldehyde (3.6%) for 20 minutes, whereas PM cells were collected, fixed, and cytospun onto slides precoated with 0.01% poly(L-lysine) and air-dried at 48 hours. After a rehydration in PBS, cells were permeabilized by incubation for 10 minutes with 0.5% Triton X-100, for 15 minutes with 0.5% bovine serum albumin and 5% goat serum, and then for 60 minutes at 37¡ãC with the rabbit polyclonal anti-survivin antibody (1 µg/ml) (Abcam), which recognizes both the cytoplasmic and nuclear pool of survivin , and with FBS buffer alone as a control. After a brief washing in PBS, the cells were incubated for 60 minutes with the secondary antibody anti-rabbit tetramethylrhodamine B isothiocyanate (DakoCytomation, Glostrup, Denmark, http://www.dako.com) at 1:50 dilution. Fluorescent specimens were analyzed by a confocal scanning laser microscope (Leica TCS SP2; Leica, Heerbrugg, Switzerland, http://www.leica.com).4 W+ z( W9 |0 E. Q, L9 |: q2 D: j6 j+ x
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Flow Cytometry

After 96 hours, cells were trypsinized and resuspended (1,000,000 cells) in 1 µl of hypotonic fluorochrome solution: 50 µg/ml propidium iodide, containing 0.1% sodium citrate, and 0.5% Triton X-100 (Sigma). After a 15-minute incubation, cells were analyzed using an Epics XL flow cytometer (Coulter Electronics Inc., Hialeah, FL, Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com). This univariate analysis of proliferation index (PI) staining cellular DNA content and deconvolution of the cellular DNA content frequency histograms reveals distribution of cells in three major phases of the cycle (G1 vs. S vs. G2/M) and makes it possible to detect apoptotic cells with fractional DNA content. The PI of cells was calculated by the following formula: PI (%) = () x 100%.4 \4 D& N: x! h+ c( `
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Immunohistochemistry

Five-micron sections cut from formalin-fixed, paraffin-embedded normal skin were deparaffinized in xylene and rehydrated in graded alcohol. The slides were first boiled in citrate buffer, pH 6, for 20 minutes in a standard pressure cooker. Quenching of the endogenous peroxidase activity was obtained by treatment with 6% H2O2 in PBS. Samples were then incubated either with 10 µg/ml anti-survivin rabbit polyclonal antibody (antinuclear and anticytoplasmic survivin; Abcam) or with 200 µg/ml anti-survivin mouse monoclonal antibody (anti-nuclear survivin; Abcam) at 4¡ãC overnight. After three 5-minute washes in PBS, slides were incubated with secondary antibody anti-rabbit biotinylated (DakoCytomation) at 1:300 dilution and anti-mouse biotinylated (DakoCytomation) at 1:200 dilution for 45 minutes at room temperature, followed by incubation with conjugated streptavidin-biotinylated-alkaline phosphatase complex for 30 minutes (BioSPA Division; Societ¨¤ Prodotti Antibiotici S.P.A., Milan, Italy, http://www.spaspa.it/biospa/home.htm) at 1:100 dilution. After three washes of 5 minutes in PBS, the slides were stained with 0.1% New Fuchsin (Sigma-Aldrich). Negative controls were obtained by omitting primary antibody.

RESULTS
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Survivin Is Expressed in a Subpopulation of Normal Basal Keratinocytes% T8 K5 E* U- ]9 ?$ n
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Contradictory results have been reported on the expression of survivin in normal human skin. A number of works indicate that normal human keratinocytes do not express survivin . To clarify survivin expression in normal epidermis, we stained skin section with an antibody recognizing the nuclear as well as the cytoplasmic pool of survivin. Survivin expression was detected only in the basal layer of normal human epidermis. In particular, survivin appeared to be localized only in a subpopulation of basal keratinocytes. Within this population, keratinocytes expressed a cytoplasmic staining (Fig. 2A). No reactivity was observed when the antibody against the nuclear pool of survivin was used (Fig. 2B) or when the primary antibody was omitted (Fig. 2C).6 h& ?* n3 r1 w3 }% _3 g& e

Figure 2. Survivin in normal human skin. Normal human skin was formalin-fixed, paraffin-embedded, and stained with anti-survivin polyclonal antibody able to recognize both nuclear and cytoplasmic survivin (A) and with a monoclonal antibody against the nuclear pool of survivin (B). Control was obtained by omitting the primary antibody (C).7 n& [! }- {( M  j4 F6 [+ f8 d

Expression of Survivin in TA and PM Cells and KSCs

To determine the precise expression of survivin in human epidermis, we isolated three populations of basal keratinocytes, based on their adhesive capacity to type IV collagen, as previously described .4 d* W* }9 B; Q. X, V
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We first investigated whether the keratinocyte subpopulations were characterized by different expression patterns of survivin. Survivin protein was overexpressed in KSCs, whereas it was almost absent in TA cells and not detectable in PM cells (Fig. 3).# y1 j# U% s* ~/ ~- z3 [! R! u% x# X

Figure 3. Expression of survivin in keratinocyte subpopulations. Western blotting was performed on protein extracts from KSCs and TA and PM cells. Nitrocellulose membrane was blotted against polyclonal anti-survivin antibody. A protein of approximate molecular mass, 16.5 kDa, corresponding to the predicted size of survivin, was detected in keratinocyte subpopulations. ß-Actin was used as internal control. Abbreviations: KSC, keratinocyte stem cell; PM, postmitotic; TA, transit amplifying.

Expression of Survivin-Spliced Variants in TA and PM Cells and KSCs

We analyzed the expression of the different survivin isoforms and measured the difference in the three keratinocyte subpopulations by quantitative real time PCR. First, keratinocytes expressed all of the survivin isoforms. In particular, in KSCs, survivin-3B and survivin-2 levels are scarce and significantly lower than WT survivin, survivin-Ex3, and -2B (Fig. 4A). On the other hand, in TA cells, survivin-3B and survivin-2 are significantly more expressed than the other variants (Fig. 4B). In PM cells, survivin-2 and survivin-3B are highest and significantly more expressed than WT survivin, survivin-Ex3, and survivin-2B (Fig. 4C).
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Figure 4. Relative quantification of survivin spliced variants in individual keratinocyte subpopulations. RNA extracts from keratinocyte stem cells (KSCs) and transit amplifying (TA) and postmitotic (PM) cells were amplified by real time polymerase chain reaction to quantify the spliced variants of survivin. Isoforms (Ex3, 2B, 3B, 2) were quantified in the individual keratinocyte subpopulations using WT survivin expression as a calibrator. KSC (A), TA (B), and PM (C). A double-sided Student's t test was performed between samples and calibrator. Abbreviations: Ex3, survivin-Ex3; 2, survivin-2; 2ß, survivin-2ß; 3ß, survivin-3ß; WT, wild type.! E& D9 @3 Z' X) D3 v; D8 }" K
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We next compared the expression pattern of each survivin variant in relation with the three keratinocyte subpopulations. WT survivin mRNA was significantly more expressed in KSCs than in TA cells (Fig. 5A). Unlike the protein level, the difference in WT survivin mRNA expression between KSCs and TA cells is less marked, suggesting possible post-transcriptional regulations. Similarly to WT survivin, survivin-2B was more expressed in KSCs than in TA cells (Fig. 5B). Survivin-Ex3 expression appeared to be similar in KSCs and TA cells, whereas its expression almost disappeared in PM cells (Fig. 5C). Finally, survivin-2 and survivin-3B expression was higher in TA and PM cells compared with KSCs (Fig. 5D, 5E).
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Figure 5. Comparison of individual survivin-spliced variants in keratinocyte subpopulations. RNA extracts of the three keratinocyte subpopulations were amplified by real time PCR to quantify the spliced variants of survivin, using KSCs as a calibrator. WT survivin (A), survivin-2B (B), survivin-Ex3 (C), survivin-2 (D), and survivin-3B (E). A double-sided Student's t test was performed between samples and calibrator. Abbreviations: KSC, keratinocyte stem cell; PM, postmitotic; TA, transit amplifying.. ]' ?6 x3 Z7 U3 _$ ^

Subcellular Localization of Survivin in TA and PM Cells and KSCs# h9 ~7 y- B7 y8 A# H

Survivin exists in distinct subcellular pools, and a ratio of 1:6 for nuclear to cytosolic survivin has been obtained by quantitative fractionation . Using an antibody that recognizes both nuclear and cytosolic pool, by confocal microscopy, survivin was predominantly localized in the nucleus of KSCs (Fig. 6A). On the other hand, TA cells expressed mostly cytosolic survivin (Fig. 6B), whereas no survivin expression was detected in PM cells (Fig. 6C). No staining was observed when the primary antibody was omitted (Fig. 6D). To evaluate whether the nuclear localization correlates with the rate of proliferation, we measured the proliferation index by flow cytometry at the time when subcellular distribution was studied. The rate of proliferation in KSCs was significantly higher than in TA cells (Fig. 6E).( b1 A7 j9 ?- h
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Figure 6. Subcellular localization of survivin in keratinocyte subpopulations. The three subpopulations were fixed and stained in situ with anti-survivin polyclonal antibody 96 hours after plating (A, KSC; B, TA; C, PM). As a negative control, primary antibody was omitted (D). Cells were observed by confocal microscopy. The three subpopulations were trypsinized, stained at 96 hours with propidium iodide, and analyzed by flow cytometry. PI was then calculated, and a double-sided Student's t test was used for comparison of the means (E). KSC versus TA, p 5 R& e6 K6 c/ h0 {7 b& Z
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Survivin Is Downregulated During Anoikis% M+ `6 S8 h2 X! g, W' w, A
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KSCs are protected from anoikis because of the high levels of ß1 integrin . As we present evidence that KSCs express the highest levels of survivin, we wanted to investigate the expression of survivin in keratinocytes undergoing anoikis following anti-ß1 integrin antibody treatment. By real time PCR, WT survivin expression levels significantly decreased in suspended cells and more strikingly in cells treated with anti-ß1 integrin antibody (Fig. 7A). Within 4 hours of suspension, survivin protein levels were slightly decreased, whereas anti-ß1 integrin antibody markedly enhanced this effect. At 6 hours, survivin was hardly detectable in suspended keratinocytes and completely disappeared in anti-ß1 integrin-treated cells (Fig. 7B). To investigate survivin isoform expression in KSCs during anoikis, we evaluated mRNA levels of each isoform in KSCs treated with anti-ß1 integrin. Compared with untreated KSCs, KSCs undergoing anoikis were characterized by a marked upregulation of survivin-2 and survivin-3B expression and by a downregulation of WT survivin, survivin-2B, and survivin-Ex3 expression (Fig. 7C).

Figure 7. Expression of survivin in KSCs undergoing anoikis. KSCs were suspended in polypropylene with or without anti-ß1 integrin antibody and then lysed at different time points. A quantitative relative real time PCR was performed with RNA extracts after 0- and 6-hour treatment, using KSCs at 0 hours as a calibrator for WT survivin (A) and for each isoform (C). A double-sided Student's t test was performed between samples and calibrator. Protein extracts were analyzed by Western blotting with anti-survivin antibody at 0, 2, 4, and 6 hours (B). ß-Actin was used as internal control. Abbreviations: cntrl, control; h/hrs, hour(s).' }4 F1 f4 p% m1 i, y

DISCUSSION" I' Y" g) r) G: P
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In this study, we have definitely shown that survivin is expressed in normal adult human epidermis. This is consistent with the localization of survivin in other normal human epithelia, such as the colonic mucosa and the endometrium . Indeed, we report that isolated KSCs strongly express survivin, whereas differentiated cells do not. This might suggest that survivin contributes to preventing apoptosis and to conferring prolonged survival in the stem population, which drives the renewal process of the epidermis.2 p3 `& s7 \. u# B8 ]

To our knowledge, survivin is the first antiapoptotic protein definitely distinguishing KSCs from TA cells. Other classic apoptotic proteins are differently regulated in the two populations as a function of susceptibility to cell death but are not mutually exclusive . We propose that survivin, in addition to these markers, can be used to identify KSCs.# N! L3 d0 E1 K# Z9 B
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Studying the function of this newly identified marker will shed light on the property of KSCs. In this respect, we have shown that the antiapoptotic WT survivin strongly identifies KSCs. This is in keeping with the proposed function of survivin and with the small degree of susceptibility to apoptosis of this population .8 ~+ _0 T6 @6 n, P( m' i
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To further evaluate the survivin expression and function in keratinocytes, we have studied survivin isoforms in keratinocyte subpopulations. Survivin variants are differently expressed in keratinocyte subpopulations. The high survivin-2B expression in KSCs is at variance with previous literature that claims this isoform is a potential proapoptotic protein .: C3 k5 Q/ Y% z/ t: H* _6 ?4 _2 B
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KSCs are more protected from apoptosis compared with TA cells .

Survivin not only exerts antiapoptotic activities but is also involved in cell-cycle regulation , as well as with cancer cells the high proliferative potential. Indeed, we present evidence that nuclear survivin is mostly restricted to KSCs, which present a high proliferation rate. This is consistent with the concept that nuclear survivin is involved in cell-cycle regulation., T' Y7 a! h  w$ E( r' d8 t( d
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The overexpression of survivin in most tumors renders them resistant to cell death, thus maintaining cancer cell viability . Blocking ß1 integrin completely abolishes survivin in KSCs, suggesting that survivin partially contributes to the integrin survival signal.
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CONCLUSION+ z  p  ?+ Z! B) [( k

Stem cells are critical in tissue homeostasis as well as in cancer development. Moreover, the use of stem cells as a novel therapy for the treatment of several diseases is promising. However, the use of stem cells is hampered by the difficulties in identifying this population in most adult tissues, including epidermis, because of the lack of a specific marker. In addition, the mechanisms underlying stem cell regulation at the epidermal level are not fully understood; in particular, it is still unclear how KSCs lose their stemness and escape the niche protection. There is evidence that KSCs leave this microenviroment not only through differentiation but also via an apoptotic mechanism . Survivin as a molecule that identifies KSCs and their involvement in the protection of these cells from cell death is an important contribution to the understanding of the correlation between KSC phenotype and function. To further clarify the role of survivin in KSC isolation and activity, experiments are now in progress to correlate survivin isoforms expression and function. The discovery of new markers and pathways for the characterization of KSCs is of paramount importance for the clinical use of epidermal cell cultures and possibly for gene therapy.7 w1 j# j6 D; D  s
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DISCLOSURES
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The authors indicate no potential conflicts of interest.& H& J& j$ h; V; }. Z1 ~
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ACKNOWLEDGMENTS* W' V7 z; h( g
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We thank Marco Pignatti for the valuable assistance in the preparation and submission of the manuscript. This work was supported by a grant from Ministry of Instruction, University and Research 2003061751_004 (2003¨C2005). A.M. and K.D. contributed equally to this article./ P! K9 v' }2 q. G% j' x
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