C/EBP regulates cell cycle and self-renewal of human limbal stem cells

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作者：Vanessa Barbaro1, Anna Testa2, Enzo Di Iorio1, Fulvio Mavilio2, Graziella Pellegrini1,2, and Michele De Luca1,2作者单位：Epithelial Stem Cell Research Center, The Veneto Eye Bank Foundation, H. SS Giovanni and Paolo, 30100 Venice, Italy
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          【关键词】 cell
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Introduction
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# g# Q6 K- D- ?# N7 eStem cells have the unique capacity to self-renew and generate committed, transit amplifying (TA) progenitors that differentiate into the cell lineages of the tissue of origin (Niemann and Watt, 2002; Fuchs et al., 2004; Cotsarelis, 2006; Blanpain et al., 2007). The most important function of TA cells is to increase the number of differentiated progeny produced by each stem cell division, thus enabling stem cells to divide infrequently, at least under normal tissue homeostasis. The cornea provides an ideal experimental system for studying stem cells of human stratified epithelia (Lavker and Sun, 2003). Human corneal stem cells are segregated in the basal layer of the limbus, which is the vascularized zone encircling the cornea and separating it from the bulbar conjunctiva. The corneal epithelium lies on the avascular Bowman's membrane and is formed by TA keratinocytes that migrate millimeters away from their parental limbal stem cells (Schermer et al., 1986; Cotsarelis et al., 1989; Lehrer et al., 1998; Pellegrini et al., 1999a).
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Clonal analysis of squamous human epithelia, including the cornea, has identified three types of clonogenic keratinocytes, giving rise to holoclones, meroclones, and paraclones in culture (Barrandon and Green, 1987; Pellegrini et al., 1999a). Holoclone-forming cells have all the hallmarks of stem cells, including self-renewing capacity (Rochat et al., 1994; Claudinot et al., 2005), telomerase activity (Dellambra et al., 2000), and an impressive proliferative potential—a single holoclone can generate the entire epidermis of a human being (Rochat et al., 1994). Holoclone-forming cells generate all the epithelial lineages of the tissue of origin (Pellegrini et al., 1999a; Oshima et al., 2001; Blanpain et al., 2004; Claudinot et al., 2005), permanently restore massive epithelial defects (Gallico et al., 1984; Romagnoli et al., 1990; Pellegrini et al., 1997, 1999b; Ronfard et al., 2000), and can be retrieved from human epidermis regenerated from cultured keratinocytes years after grafting (De Luca et al., 2006). We have recently shown that a defined number of genetically corrected stem cells regenerate a normal epidermis in patients with genetic skin adhesion disorders (Mavilio et al., 2006). The paraclone is generated by a TA cell, whereas the meroclone has an intermediate clonal capacity and is a reservoir of TA cells (Barrandon and Green, 1987; Pellegrini et al., 1999a).
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/ _) H6 |9 p# R+ `  HThe p63 gene produces full-length (TAp63) and N-terminally truncated (Np63) transcripts initiated by different promoters. Each transcript is alternatively spliced to encode three different p63 isoforms, designated , ?, and (Yang et al., 1998). The p63 gene products are essential for the morphogenesis and the regenerative proliferation of stratified epithelia (Mills et al., 1999; Yang et al., 1999). In particular, Np63 sustains the proliferative potential of basal epidermal keratinocytes (Parsa et al., 1999; Koster et al., 2004; McKeon, 2004; Nguyen et al., 2006). In the human corneal epithelium, high levels of Np63 identify limbal stem cells both in vivo and in vitro, whereas Np63? and Np63 correlate with corneal regeneration and differentiation (Pellegrini et al., 2001; Di Iorio et al., 2005).8 ~6 U, p% y- S& m: P3 g* d
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In mammary gland epithelial cells, the CCAAT enhancer binding protein (C/EBP) transcription factor regulates cell cycle by inducing a G0/G1arrest. This effect is specific for epithelial cells and for the G0/G1 phase, as C/EBP expression does not increase in other types of G0/G1-arrested cells or in mammary cells arrested at other stages of the cell cycle (O'Rourke et al., 1999; Hutt et al., 2000). C/EBP is a member of a highly conserved family of leucine zipper transcription factors expressed in a variety of tissues and cell types and involved in the control of cellular proliferation and differentiation, metabolism, and inflammation (Ramji and Foka, 2002; Johnson, 2005). At least six members of the family have been isolated and characterized (C/EBP–C/EBP), with further diversity produced by the generation of different polypeptides by differential use of translational initiation sites, and extensive protein–protein interactions within the family and with other types of transcription factors (Ramji and Foka, 2002; Johnson, 2005).
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In this paper, we show that C/EBP and Np63 are coexpressed by human limbal stem cells in vivo and in vitro and that the expression of C/EBP is restricted to a subset of mitotically quiescent Np63 /Bmi1  cells. Forced expression of a constitutive C/EBP or of a tamoxifen-inducible estrogen receptor (ER)–C/EBP fusion protein in human primary limbal keratinocytes shows that C/EBP is instrumental in regulating self-renewal and cell cycle length of limbal stem cells.
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Results
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Coexpression of C/EBP and Np63 in quiescent human limbal cells, k: B- T8 K8 Y( h7 L5 H3 R
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Experiments were performed on four uninjured and five wounded corneas, referred to as resting and activated cornea, respectively (Di Iorio et al., 2005). We have previously shown that Np63 is expressed by 10% of resting limbal basal cells endowed with stem cell properties and that activated Np63  limbal cells contain Np63? and Np63, proliferate, and migrate to the central cornea to restore a wounded epithelium (Di Iorio et al., 2005).
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Immunofluorescence analysis on resting limbal sections revealed that C/EBP and Np63 were coexpressed in the same patches of basal cells (Fig. 1 A, left). Both transcription factors were undetectable in suprabasal cell layers (Fig. 1 A) and in the entire corneal epithelium (not depicted). Limbal cell nuclei were stained with DAPI to estimate the proportion of C/EBP /Np63  cells in the basal layer. 1 mm of resting limbal epithelium contained a mean of 15 C/EBP /Np63  cells, equivalent to 10% of the basal layer. Upon corneal wounding and limbal activation, Np63 appeared in many basal and some suprabasal limbal cells, whereas C/EBP remained confined to 10% of the basal layer (Fig. 1 A, middle). Of note, C/EBP  limbal cells invariably coexpressed Np63 (Fig. 1 A). In activated limbus, patches of C/EBP /Np63  basal cells flanked by C/EBP–/Np63  cells were commonly observed (Fig. 1 A, right), whereas neither resting nor activated central corneal epithelium expressed C/EBP (not depicted). C/EBP /Np63  resting limbal cells did not express Ki-67, a proliferation-associated nuclear antigen present throughout the cell cycle but absent in G0/G1-arrested cells (not depicted). In activated limbus, proliferating Ki-67  limbal cells expressed Np63, but not C/EBP, whereas C/EBP  cells contained Np63 but not Ki-67 (Fig. 1 B). Thus, C/EBP and Np63 are coexpressed by quiescent limbal basal cells, whereas Np63, but not C/EBP, is expressed in proliferating limbal cells.
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- Z1 I/ n! Y" A. R  E+ I Figure 1. Expression of Np63, C/EBP, p27Kip1, and p57Kip2 in the human limbus. (A) Double immunofluorescence of sections of resting (left) and activated (middle and right) limbus stained with C/EBP-specific (green) and p63-specific (red) purified IgG. Yellow in the merge frames indicates cells stained with both antibodies. Np63 and C/EBP were coexpressed by a discrete number of basal cells of a resting limbus (left). In the activated limbus, many basal and some suprabasal cells expressed Np63, whereas C/EBP was contained only in 10% of Np63  basal cells (middle). Patches of C/EBP /Np63  basal cells (right; arrowheads) were flanked by cells expressing Np63 but not C/EBP (right; brackets). (B) Sections of activated limbus were also stained with mAbs against the proliferation-associated nuclear antigen Ki-67 (blue). Proliferating Ki-67  limbal cells (blue arrows) expressed Np63 (red arrows) but not C/EBP, whereas C/EBP  cells (green arrows) contained Np63 (yellow arrows) but not Ki-67. (C) Serial limbal sections stained with C/EBP-, p63-, p27Kip1-, and p57Kip2-specific IgG (as indicated in frames) showed that C/EBP-positive cells (green arrows) contained Np63 (red arrows) and nuclear p27Kip1 (yellow arrows) and p57Kip2 (orange arrows), whereas the two Cdk inhibitors were expressed only in the cylasm of C/EBP–/Np63  cells (brackets). Bars, 10 μm.4 M+ f& |- \7 H. H0 _
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The cyclin/Cdk inhibitors p27Kip1 and p57Kip2 negatively regulate G1 progression. Nuclear levels of p27Kip1 are high in quiescent cells (Sherr and Roberts, 1999). Mitogenic and/or oncogenic signals activate different kinases that phosphorylate p27Kip1 on serine and tyrosine residues, promoting its export from the nucleus and cylasmic proteolysis, thereby leading to cell proliferation (Rodier et al., 2001; Chu et al., 2007; Grimmler et al., 2007; Kaldis, 2007). Of note, p57Kip2, which inhibits cyclin D–Cdk4/6 complexes (Yamazaki et al., 2006), is highly expressed in mouse epidermal stem, but not TA, cells (Dunnwald et al., 2003). Immunofluorescence analysis on limbal sections revealed that C/EBP, p27Kip1, and p57Kip2 were coexpressed in the nucleus of the same patches of basal cells (Fig. 1 C, arrowheads). Such cells also expressed Np63 (Fig. 1 C, arrowheads). C/EBP  cells were flanked by C/EBP–/Np63  cells containing cylasmic, but not nuclear, p27Kip1 and p57Kip2 (Fig. 1 C, brackets). Finally, p27Kip1 and p57Kip2 were never detected in a fully activated limbus or in the corneal epithelium (not depicted). These data are consistent with the notion that p27Kip1 and p57Kip2 are localized in the nucleus of quiescent cells, appear in the cylasm at the G1–S transition, and are not expressed by actively proliferating cells (Rodier et al., 2001), and confirm that C/EBP is expressed only by quiescent limbal basal cells.7 K9 y! V+ `# X# _# A

9 ^# F" C$ K' kExpression of C/EBP, Np63, and Bmi1 in limbal stem cells3 \9 E& y# H, ]3 u$ u. Z

+ u# G/ |+ @5 _. }5 j1 l2 a1 uImmunofluorescence analysis showed that Np63 is abundantly and uniformly expressed in holoclones (Fig. 2 A), is expressed in a subset of meroclone cells, and is not expressed in paraclones (Di Iorio et al., 2005). Western analysis showed that clonal evolution, i.e., the transition from holoclones to paraclones, is accompained by a progressive disappearance of Np63 and a relative enrichment in Np63? and Np63 (Fig. 2 B). Strikingly, C/EBP expression was detected exclusively in holoclones (Fig. 2 B) and confined to a subpopulation of Np63  cells (Fig. 2 A). C/EBP /Np63  cells were not proliferating, as shown by the mutually exclusive expression of C/EBP and Ki-67 (Fig. 2 C). Of note, although Ki-67 and C/EBP were never expressed in the same cell (Fig. 2 C), large areas of the colony were formed by nonproliferating yet C/EBP-negative cells (Fig. 2 C, dots), suggesting that the expression of C/EBP was not merely related to the proliferative status of the limbal cell./ R+ Y5 p+ a" S$ h$ R) R% ^$ T6 H% Y

7 U0 ?- A/ @7 _  S Figure 2. Expression of Np63, C/EBP, and Bmi1 in limbal clones and resting limbus. (A) Holoclone type colonies were isolated as described in Materials and methods. Double immunofluorescence was performed on PFA-fixed holoclones with p63-specific (red) and C/EBP-specific (green) purified IgG. Yellow in the merge frames indicates cells stained with both antibodies. C/EBP was contained in a subpopulation of Np63  cells. Bars, 50 μm. (B) Clonal analysis of subconfluent primary limbal cultures (Pellegrini et al., 2001). Cell extracts were prepared from cultures generated by holoclones, meroclones, and paraclones, run on SDS-polyacrylamide gels, and immunostained with 4A4 (pan-p63) and anti-Bmi1 mAbs and with anti-C/EBP, anti-C/EBP?, and anti-GAPDH purified IgG. C/EBP and Bmi1 were detected exclusively in holoclones. Clonal evolution was characterized by a progressive disappearance of Np63 and an enrichment in Np63? and Np63. C/EBP? was uniformly expressed in all clonal types. (C) Selected holoclones were double stained with an anti–Ki-67 mAb (blue) and an anti-C/EBP IgG (green). The expression of C/EBP and Ki-67 was mutually exclusive. The dotted areas outline nonproliferating cells that do not express C/EBP. Bar, 50 μm. (D) Double immunofluorescence of sections of resting limbus stained with anti-C/EBP IgG (green) and anti-Bmi1 mAb (violet). The two transcription factors were coexpressed by a defined number of basal limbal cells. Palisades of Vogt are indicated (left) and shown at higher magnification (right). Bars: (left) 100 μm; (right) 10 μm.3 Y' y- G! q+ T! C8 R
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C/EBP and -? are the most commonly expressed and thoroughly studied isoforms of the C/EBP family (Ramji and Foka, 2002). In particular, C/EBP and -? are known to positively regulate the program of squamous differentiation in the epidermis (Oh and Smart, 1998; Zhu et al., 1999). Accordingly, we found that C/EBP and -? were contained in the suprabasal layers of both human limbal and corneal epithelium (unpublished data). Of note, however, although C/EBP? was expressed in all limbal clonal types (Fig. 2 B), we could not detect C/EBP in cultured limbal colonies.9 W! o* H4 P  _  I3 J7 V/ v# q

) [1 Q3 j* s/ x+ S1 PGene profiling experiments have led to the identification of genes that are commonly expressed in adult stem cells. Among these genes, Bmi1, a member of the polycomb group of transcription factors, plays a crucial role in the renewal of hemaoietic and neural stem cells (Lessard and Sauvageau, 2003; Molofsky et al., 2003, 2005; Park et al., 2003) and is expressed in clonogenic, multipotent, and self-renewing murine hair follicle stem cells (Claudinot et al., 2005). Immunofluorescence performed on resting limbal sections revealed that C/EBP and Bmi1 were coexpressed by the same limbal basal cells (Fig. 2 D). In particular, the basal layer of palisades of Vogt, where limbal stem cells are thought to be concentrated, was formed by C/EBP /Bmi1  cells (Fig. 2 D). Accordingly, Western blot analysis showed that Bmi1 was expressed in holoclones but not in meroclones and paraclones (Fig. 2 B).
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, {' T1 Q2 T' v( s6 FCollectively, these data indicate that C/EBP, Np63, and Bmi1 colocalize in limbal stem cells of the resting corneal epithelium in vivo and in limbal holoclone-forming cells in vitro and that expression of C/EBP is restricted to a subset of Np63  cells that are mitotically quiescent both in vivo and in vitro./ A$ Z& [: I  M- {' H( X

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Primary limbal cultures were infected with a lentiviral vector expressing either an epie-tagged human C/EBP or a control protein (a truncated form of the p75 low-affinity NGF receptor ) under the control of a constitutive phosphoglycerokinase (PGK) promoter. Both vectors expressed GFP under the control of an internal ribosomal entry site (IRES) element (Fig. 3 A, RRL--G and RRL-N-G). Transduction efficiency on clonogenic cells was 90%, as calculated by GFP expression. After 2 d of cultivation, the size of untransduced colonies increased nearly threefold (Fig. 3 B, red circles), whereas the size of C/EBP /GFP  colonies increased only slightly (Fig. 3 B, yellow circles). Control cells reached confluency 5 d after plating (Fig. 3 B). In contrast, a 5-d culture of C/EBP /GFP  cells showed well-defined colonies composed of small, tightly packed cells (Fig. 3 B).  f7 C' m" K7 M# t0 j* H

6 e; x1 O. n6 t2 K. O' y Figure 3. Forced expression of C/EBP in human limbal keratinocytes. (A) Schematic map of the RRL--G and RRL-N-G lentiviral vectors (proviral form), expressing C/EBP or NGFr under the control of a constitutive, human PGK promoter. The vectors carry an inactivating deletion in the U3 region of the LTR. Splice donor (SD) and acceptor (SA) sites, Rev-responsive element (RRE), central poly-purine tract (cPPT), and the woodchuck hepatitis posttranscriptional regulatory element (WPRE) are indicated. In both vectors, EGFP is expressed under the control of an IRES. The Flag epie fused to C/EBP is indicated. (B) C/EBP-transduced colonies are indicated by GFP expression (green). () The size of an untransduced colony (red circle) increased nearly threefold after 2 d of culture, whereas the size of a C/EBP-transduced colony contained in the same dish (yellow circle) did not increase considerably. (bottom) After 5 d of culture, untransduced cells reached confluency (left), whereas C/EBP-transduced colonies were well separated and composed of small tightly packed cells (right). Yellow asterisks indicate 3T3 feeder cells, which are not visible in untransduced cultures. Bars, 50 μm. (C) Cell cycle analysis of NGFr-transduced (control; gray bars) and C/EBP-transduced (green bars) limbal cells after 5 d of cultivation. Approximately 55, 35, and 10% of control cells were in the G1, S, and G2–M phases of the cell cycle, respectively, whereas most of the C/EBP-transduced cells were in the G1 phase. Apoptotic cells were virtually undetectable in both cases. Error bars indicate SD. (D) Western blot analysis of the expression of C/EBP, p16, involucrin, p27Kip1, p57Kip2, p21Waf1/Cip1, and Rb in cell extracts from C/EBP- and NGFr-transduced limbal keratinocytes.
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  d( M' i# V7 S. t7 OReplicative senescence and differentiation of keratinocytes are associated with increased levels of p16INK4A and involucrin, which indicate irreversible exit from the cell cycle and onset of terminal differentiation, respectively (Dellambra et al., 2000). C/EBP-transduced cells contained threefold less p16INK4A and involucrin than NGFr-transduced cells (Fig. 3 D). C/EBP-transduced cells contained four- and threefold more p27Kip1 and p57Kip2 than control cells, respectively (Fig. 3 D). A cell cycle profile revealed that 55, 35, and 10% of the control cells were in the G1, S, and G2–M phases, respectively (Fig. 3 C). In sharp contrast, most of the C/EBP-transduced cells were in the G1 phase of the cell cycle (Fig. 3 C). The amount of apoptotic cells was negligible in both C/EBP-transduced and control cells (Fig. 3 C). Finally, C/EBP-dependent growth inhibition was associated with neither increase of p21Waf1/Cip1 or pRb expression (Fig. 3 D) nor activation of the p53 checkpoint pathway (not depicted). These data indicate that the growth inhibitory effect of C/EBP was not due to replicative senescence, terminal differentiation, or apoptosis.+ P! }) O- V1 A) p& d. x1 f

7 W" i2 p' i9 K" MTo investigate whether the growth inhibitory effect of C/EBP was reversible, we transduced primary limbal cells with a lentiviral vector expressing an N-terminal fusion between C/EBP and a modified, 4-hydroxytamoxifen (4OHT)–inducible ligand binding domain of the human ER (Littlewood et al., 1995; Fig. 4 A, RRL-ER-G). In mock-transduced (RRL-ER-G) cells, C/EBP was found predominantly in the nucleus (Fig. 4 B, ER, middle). In the absence of 4OHT, the ER-C/EBP chimeric protein was found in the cylasm of transduced limbal cells, and nuclear translocation was observed within 12 h from the addition of 1 μM 4OHT to the culture medium (Fig. 4, B , C, and D). In contrast, C/EBP? was present only in the nucleus, irrespective of the presence of 4OHT (Fig. 4 B and Fig. D). Members of the C/EBP family are known to form homo- and heterodimers. In the absence of 4OHT, ER-C/EBP sequestered also the endogeneous C/EBP in the cylasm, as indicated by the absence of nuclear immunofluorescent staining (Fig. 4 D, –4OHT), the absence of endogeneous C/EBP in nuclear extracts, and the presence of C/EBP in the corresponding cylasmic extracts (Fig. 4 B, middle, –4OHT) of RRL-ER-G–transduced cells. Colonies of cells transduced with the control vector showed a progressive and linear increase in their size, irrespective of the presence of 4OHT (Fig. 4, E and F). In sharp contrast, the growth of ER-C/EBP /GFP  colonies was strictly dependent on the localization of the ER-C/EBP chimera (Fig. 4, E and F): (1) addition of 4OHT at day 1 considerably slowed the growth of transduced colonies; (2) removal of 4OHT at day 4 was promptly followed by a linear increase of the size of GFP  colonies; and (3) readdition of 4OHT at day 6 again induced a growth arrest. Of note, untransduced, NGFr- and ER-transduced primary limbal cells duplicated every 17–19 h, whereas C/EBP-transduced cells showed a doubling time of 41 h (Fig. 5 E, green). These data show that C/EBP lengthened the limbal cell cycle by forcing cells into the G1 phase without altering their capacity for multiplication.! J: n, u6 z3 t* j! r8 \
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Figure 4. Expression of 4OHT-inducible C/EBP in limbal keratinocytes. (A) Schematic map of the RRL-ER-G and RRL-ER-G lentiviral vectors (proviral form), expressing an N-terminal fusion of C/EBP to a modified ER ligand binding domain, or the ER domain only, under the control of a PGK promoter. In both vectors, EGFP is expressed under the control of an IRES. SD, splice donor site; SA, splice acceptor site; RRE, Rev-responsive element; cPPT, central poly-purine tract; WPRE, woodchuck hepatitis posttranscriptional regulatory element. (B) Nuclear (N) and cylasmic (C) extracts were prepared from RRL-ER-G–transduced (ER) and RRL-ER-G–transduced cells, either treated ( 4OHT) or untreated (–4OHT) with 1 μm 4OHT, run on SDS-polyacrylamide gels, and immunostained with anti-C/EBP and anti-C/EBP? purified IgG. In mock-transduced cells, C/EBP was found exclusively in the nucleus (ER; middle). In the absence of 4OHT, the ER-C/EBP chimeric protein () was found predominantly in the cylasm (–4OHT). Nuclear translocation was observed within 12 h from the addition of 1 μM 4OHT to the culture medium ( 4OHT; ). Strikingly, in the absence of 4OHT, ER-C/EBP sequestered also the endogeneous C/EBP in the cylasm, as indicated by the absence of any C/EBP species in nuclear extracts of –4OHT cells and the presence of C/EBP in the corresponding cylasmic extracts (middle). The exposure times of filters in (ER-C/EBP) and middle (C/EBP) panels were 10 and 75 s, respectively. C/EBP? was present only in nuclear extracts, irrespective of the presence of 4OHT (bottom). (C and D) Cylasmic–nuclear translocation of the ER-C/EBP fusion protein in response to 4OHT treatment of RRL-ER-G-transduced limbal keratinocytes, stained with an anti-C/EBP antibody (green). Staining of C/EBP? is shown for comparison (pink). Note the absence of C/EBP in nuclei of untreated cells. Bars, 20 μm. (E and F) Reversible growth inhibitory effect of C/EBP. In the presence of 4OHT (1–4 d of culture), the size of ER-C/EBP–transduced colonies did not increase considerably; removal of 4OHT at day 4 was followed by a linear increase of the size of GFP  colonies, whereas readdition of 4OHT at day 6 again induced a growth arrest (E and F ). In contrast, RRL-ER-G–transduced colonies showed a progressive and linear increase of their size, irrespective of the presence of 4OHT (E and F ). Values of the size of colonies are in arbitrary units.
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Figure 5. Role of p27Kip1 and p57Kip2 in C/EBP-induced mitotic quiescence. (A and B) Semiquantitative RT-PCR analysis was performed on limbal cells transduced with either control ER (ER), constitutive NGFr (NGFr), constitutive C/EBP (C/EBP), or inducible C/EBP in the absence (–4OHT) or in the presence ( 4OHT) of tamoxifen, using p27Kip1- and p57Kip2-specific primers. A 5–10-fold increase of both p27Kip1 (yellow bars) and p57Kip2 (orange bars) transcripts was observed only in the presence of exogeneous nuclear C/EBP. Error bars indicate SD. (C) C/EBP-transduced cells were then transfected with siRNA-p27Kip1 (left) and siRNA-p57Kip2 (right), with an efficiency of 84 ± 2 and 77 ± 3%, respectively. Bars, 20 μm. (D) Cell extracts were prepared from C/EBP-transduced cells transfected with siRNA-p27Kip1 (left) or siRNA-p57Kip2 (right), run on SDS-polyacrylamide gels, and immunostained with the indicated purified IgG. Note that siRNA-p27Kip1 and siRNA-p57Kip2 determined a strong decrease of the expression of p27Kip1 and p57Kip2, respectively, but not of C/EBP and GAPDH. (E) Cell doubling time was calculated. Untransduced (black) and C/EBP-transduced cells, untransfected (green) or transfected with siRNA-p27Kip1 (yellow), siRNA-p57Kip2 (orange), or the combination of the two siRNA molecules (yellow   orange) showed a doubling time of 18, 41, 23.5, 23, and 18.5 h, respectively.
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  R) `. A! b; E& k6 DC/EBP-dependent mitotic quiescence is mediated by p27Kip1 and p57Kip2
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Semiquantitative RT-PCR was performed on control and C/EBP-transduced cells using p27Kip1- and p57Kip2-specific primers. As shown in Fig. 5 (A and B), we observed a 5–10-fold increase of both p27Kip1 and p57Kip2 transcripts in RRL--G (C/EBP)–transduced limbal cells and RRL-ER-G–transduced cells treated with 4OHT ( 4OHT), as compared with RRL-N-G (NGFr)–, RRL-ER-G (ER)–, and RRL-ER- G–transduced cells not treated with 4OHT (–4OHT). To prove the role of p27Kip1 and p57Kip2 in mediating the effect of C/EBP on keratinocyte cell cycle, C/EBP-transduced limbal cells were transfected with siRNAs specifically targeted to the p27Kip1 and p57Kip2 mRNAs. Transfection efficiency was 84 ± 2 and 77 ± 3%, respectively (Fig. 5 C). Western blot analysis showed that siRNA-p27Kip1 and siRNA-p57Kip2 caused a strong decrease of the expression of p27Kip1 and p57Kip2, but not of C/EBP and a control protein (Fig. 5 D).% U) b: X1 G( h; n) |

- G. K6 d2 f# r$ T1 C/ @# O3 |Untransduced and C/EBP-transduced cells showed a doubling time of 18 and 41 h, respectively (Fig. 5 E). C/EBP-transduced cells transfected with either siRNA-p27Kip1 or siRNA-p57Kip2 showed a doubling time of 23.5 and 23 h, respectively. Of note, C/EBP-transduced cells transfected with both siRNAs simultaneously, showed a doubling time of 18.5 h, a value undistinguishable from that of untransduced control cells (Fig. 5 E). These data show that p27Kip1 and p57Kip2 mediate C/EBP-induced mitotic quiescence.2 k+ R- f" P4 M2 p8 n1 M

9 p9 K! ~; i4 H2 FC/EBP binds to the p63, involucrin, p27Kip1, p57Kip2, and p16INK4A loci in vivo! a8 G7 I8 f. X
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To provide evidence for a direct contribution of C/EBP in regulating the expression of Np63, involucrin, p27Kip1, p57Kip2, and p16INK4A, we analyzed recruitment of C/EBP to these loci by a chromatin immunoprecipitation (ChIP) assay on cultured limbal keratinocytes three and five passages after transduction with either RRL--G or the control, RRL-N-G vector. Protein–DNA complexes were immunoprecipitated with antibodies specific for C/EBP or the Flag epie and with control IgGs. Immunoprecipitated chromatin DNA was analyzed by PCR with primers specific for different regions of the p63, involucrin, p27Kip1, p57Kip2, and p16INK4A loci (Fig. 6 A, red arrowheads), containing evolutionarily conserved and/or putative C/EBP binding elements.
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Figure 6. Recruitment of C/EBP and p63 on selected gene targets in primary limbal keratinocytes in vivo. (A) Schematic representation of the genomic target loci. Positions of amplified regions (red arrowheads) are indicated with respect to transcription start sites (arrows). INV, involucrin. (B) Chromatin from C/EBP- or NGFr-transduced (control) limbal keratinocytes was cross-linked at the third and fifth passage in culture and immunoprecipitated without antibody (–) or with anti-CEBP, anti-Flag, anti-p63 (4A4), and anti-p63 antibodies, and control IgGs, and analyzed by PCR using primers specific for the genomic regions indicated in A. Amplified fragments were run on an agarose gel and stained with ethidium bromide. The last lane on each gel corresponds to the input sample.
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In C/EBP-transduced cells, vector-derived (Flag-tagged) C/EBP was found associated to the p63 locus in intron 3 (at position  147873 to  148041) and in an evolutionarily conserved, keratinocyte-specific enhancer in intron 5 ( 202579 to  202761; Antonini et al., 2006). Primers designed to amplify other sequences from the p63 locus detected the correct fragment only in the input samples (Fig. 6, A and B). Binding of Flag-tagged C/EBP was also observed to a region upstream of the involucrin promoter (–421 to –119) containing a C/EBP responsive element previously characterized in keratinocytes (Agarwal et al., 1999; Balasubramanian and Eckert, 2004) and upstream of the p27Kip1 (–227 to  14), p57Kip2 (–622 to –398), and p16INK4A (–1020 to –871) loci (Fig. 6 B). Binding to all these sites was observed specifically in C/EBP-transduced cells and was more pronounced at the fifth than at the third passage (Fig. 6 B). The signals obtained with the anti-Flag antibody were always weaker than those obtained with the anti-C/EBP antibody, probably reflecting a lower immunoprecipitation efficiency. In control, NFGr-transduced cells, a weak but specific signal was observed at the p63, involucrin, and p27Kip1 loci in chromatin immunoprecipitated with the anti- C/EBP but not the anti-Flag antibody. Binding was observed at the third but not at the fifth passage (Fig. 6 B), most likely as a result of the presence of endogenous C/EBP activity in a subset of early passage cells, which is lost in later passages.
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2 B7 x" i& }; u1 h: oChromatin from the same cells was also immunoprecipitated with antibodies specific for all isoforms or only the isoforms of p63. Binding of Np63 was observed in the intron 5 enhancer of the p63 locus (Antonini et al., 2006) in both C/EBP-transduced and control cells. Binding was more pronounced in C/EBP  than in control cells, reflecting either an increased recruitment of Np63 to the enhancer or simply the increased proportion of cells expressing Np63 in these cultures. Interestingly, Np63 and C/EBP appear to bind the same regions in the p63 and p27Kip1 loci (Fig. 6 B). These results suggest that the p63, involucrin, p27Kip1, p57Kip2, and p16INK4A loci might be direct targets of C/EBP activity, in some cases in combination with Np63.
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C/EBP promotes self-renewal of holoclone-forming cells
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Clonogenic ability and proliferative potential are distinct properties of epithelial cells. Keratinocyte stem cells are endowed with high clonogenic and high proliferative capacity, and self-renewal occurs when both properties are maintained. Conversely, TA cells are clonogenic but have a limited capacity for multiplication.: m  O) |& E5 z- e" g

; b3 [9 `2 d  b) T. K: z8 NSerially cultivated, untransduced, or NFGr-transduced limbal cells showed a progressive decrease of their clonogenic capacity (Fig. 7, A and C) and ceased to proliferate after 60–75 d (or 9–11 passages) in culture (Fig. 7 B). Replicative senescence occurs because of clonal evolution, as indicated by the progressive increase of aborted, paraclone-type colonies (Fig. 7 D) and by the replacement of Np63 with Np63? and Np63 expression (Fig. 2 B and Fig. 7, E and F). In sharp contrast, both clonogenic ability (Fig. 7, A and C) and proliferative capacity (Fig. 7 B) of C/EBP-transduced cells were maintained indefinitely. This effect was due to the capacity of enforced C/EBP expression to promote self-renewal and halt clonal evolution in holoclones, as indicated by the following evidence: (1) serially cultivated C/EBP-transduced cells showed no increase in the number of paraclones (Fig. 7 D) or replacement of Np63 with Np63? and Np63 (Fig. 7, E and F); (2) statistical analysis of cell size (Di Iorio et al., 2006), a major marker of clonogenic stem cells (Barrandon and Green, 1985), showed that C/EBP-transduced cells were nearly 10-fold smaller than control cells (325.93 vs. 3,035.25 μm3); (3) clonal analysis revealed that the percentage of holoclone-forming cells decreased and eventually set to zero in serially cultivated control cells but remained constant in C/EBP-transduced cells (10–15% of inoculated cells); and (4) ER- C/EBP was able to fully sequester also endogeneous C/EBP in the cylasm of limbal cells in the absence of 4OHT (Fig. 4 B). Such cells ceased to express Np63 (not depicted) and underwent replicative senescence in only two passages as compared with 9–11 passages of control untransduced cells (Fig. 7 B).
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Figure 7. C/EBP halts clonal evolution and promotes self-renewal of human limbal keratinocytes. (A) Clonogenic capacity of untransduced and NGFr- and C/EBP-transduced limbal cells was evaluated at the cell passages indicated by numbers. NA (not available) indicates cultures that reached replicative senescence. (B–D) Number of cumulative population doublings (B), CFE values (number of colonies/inoculated cells ratio; C), and aborted colonies values (aborted colonies/total colonies ratio; D) are indicated. Untransduced and NGFr- and C/EBP-transduced cells are indicated by red, black, and green circles, respectively. Note that (1) control cells underwent senescence, whereas C/EBP-transduced cells proliferated indefinitely, and (2) control cells showed a progressive decrease of their CFE and a progressive increase of the percentage of aborted colonies, whereas both the number of clonogenic cells and aborted colonies remained constant during serial cultivation of C/EBP-transduced cells. (E) Semiquantitative RT-PCR analysis was performed using primers specific for each of the p63 N isoforms (Di Iorio et al., 2005) on C/EBP- and NGFr-transduced cells at different passages (indicated by numbers). ?-Actin (?act) has been used as a control. (F) Western blot analysis of cell extracts prepared from the same cultures used for RT-PCR. Upon serial cultivation, control cells showed a progressive disappearance of Np63 and enrichment of Np63? and Np63, whereas C/EBP-transduced cultures expressed high levels of Np63 and low amounts of Np63? and Np63.
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To investigate whether C/EBP was able to rescue TA cells from their terminal fate, we transduced different single cell–derived clones. As expected, holoclone, meroclone, and paraclone type clones displayed a progressive decrease in clonogenicity and Np63 content (Fig. 8). Forced expression of C/EBP was able to sustain the self-renewal of holoclones and meroclones and, hence, of clones still containing Np63  cells, but not that of Np63– paraclones (Fig. 8). All cells in transduced holoclones and meroclones expressed Np63 (not depicted), further suggesting that C/EBP is able to foster self-renewal only of Np63  cells and to maintain expression of Np63 in such cells.
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Figure 8. C/EBP promotes self-renewal of holoclones but not paraclones. Immunofluorescence analysis was performed on clone-derived cytospins using anti-Np63 and anti-C/EBP purified IgG. Representative clonal types are shown, and their Np63 and C/EBP content is expressed in arbitrary values. Immediately after their isolation (p0), daughter cells from each clone were transduced with lentiviral vectors expressing C/EBP (RRL--G), NGFr (RRL-N-G), or Np63 (RRL-N-G). CFEs performed at selected passages (p) are shown. Enforced C/EBP expression sustained self-renewal of holoclones and meroclones indefinitely (CFEs at p10 are shown), but not that of paraclones, which ceased proliferation after p2. Enforced Np63 and NGFr expression were unable to foster self-renewal, irrespective of the clonal type. SD, splice donor site; SA, splice acceptor site; RRE, Rev-responsive element; cPPT, central poly-purine tract; WPRE, woodchuck hepatitis posttranscriptional regulatory element.1 m( p& s2 i% [6 B' H- j: d' Q/ d
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These data prompted us to investigate whether forced expression of Np63 was sufficient to sustain limbal cell self-renewal. Primary limbal cultures and single cell–derived clones were infected with a lentiviral vector expressing the Np63 isoform (Fig. 8, RRL-N-G). Np63-transduced holoclones underwent regular clonal evolution and ceased to proliferate after 11 passages, a value identical to control untransduced cells (Figs. 7 and 8). Np63 was therefore unable to sustain limbal stem cell self-renewal both in primary cultures (unpublished data) and in clones. Finally, simultaneous infection with lentiviral vectors expressing C/EBP and Np63 was unable to rescue clonogenic ability and self-renewal in paraclones, suggesting that loss of self-renewal is an irreversible process, at least in limbal keratinocytes.
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Discussion" j. b$ H# n5 q$ o2 |6 ?, p- U

6 ^' m/ n! r" Q; n% p5 rExceptional progress has been made in understanding the molecular mechanisms regulating keratinocyte stem cells. The role of transcription factors, such as p63, tcf3, CCAAT displacement protein, and GATA-3, and of adhesion and signaling molecules, such as integrins, Wnt/?-catenin, c-Myc, Notch, hedgehog, Sgk3, and bone morphogenic proteins, in controlling hair follicle and epidermal development and stem cell fate has been highlighted (Niemann and Watt, 2002; Fuchs et al., 2004; Cotsarelis, 2006; Blanpain et al., 2007). Molecular phenotyping of some of the keratinocyte stem cell niches helped explain how stem cells interact with the microenvironment to maintain their properties (Morris et al., 2004; Tumbar et al., 2004). Little is known, however, on the regulation of perhaps the most important property of epithelial stem cells, that is, their capacity to self-renew. It has been shown that the Rho guanosine triphosphatase Rac1 sustains murine epidermal stem cell renewal and human epidermal stem cell clonogenicity by negatively regulating MYC (Benitah et al., 2005). However, differences exist between different lining epithelia and among animal species. For instance, Rac1 stimulates differentiation and not self-renewal in the intestinal epithelium (Stappenbeck and Gordon, 2000), whereas the CD34 antigen identifies murine but not human hair follicle stem cells (Cotsarelis, 2006).
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We took advantage of the availability of human corneas to carry out genetic manipulation experiments on primary, clonogenic limbal stem cells and show that C/EBP plays a key role in regulating their cell cycle and self-renewal properties. Our findings are graphically summarized in Fig. 9. According to this model, a defined number of mitotically quiescent limbal stem cells coexpress Bmi1, Np63, and C/EBP under normal homeostasis. Coexpression of Bmi1, Np63, and C/EBP therefore identifies limbal holoclones and is part of the genetic program maintaining stem cell identity. Bmi1 fosters self-renewal of haemaoetic and neural stem cells through regulation of the p16INK4A and p19ARF pathways (Lessard and Sauvageau, 2003; Molofsky et al., 2003, 2005; Park et al., 2003; Walkley et al., 2005) and might play a similar role also in limbal stem cells. Np63 sustains the proliferative potential of stem cells in several stratified epithelia, including the cornea (Parsa et al., 1999; Pellegrini et al., 2001; Koster et al., 2004; McKeon, 2004; Di Iorio et al., 2005; Nguyen et al., 2006). We show here that C/EBP regulates mitotic quiescence of limbal keratinocytes by forcing cells in the G0/G1 phase of the cell cycle. Even under culture conditions specifically designed to promote keratinocyte proliferation, forced C/EBP expression greatly increases the cell cycle length through activation of the cell cycle inhibitors p27Kip1 and p57Kip2. The growth inhibitory effect of C/EBP is not due to replicative senescence or terminal differentiation, as confirmed by the down-regulation of p16INK4A and involucrin. Perhaps more important, C/EBP promotes the self-renewal of Np63  limbal stem cells, as suggested by the block of clonal evolution and the indefinite maintenance of the number of holoclones during serial cultivation of C/EBP-transduced limbal keratinocytes.
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Figure 9. Schematic description of a model for human corneal regeneration. Under normal homeostasis, quiescent stem cells localized in the basal layer of the limbus (Fig. 1 and Fig. 2 D) coexpress C/EBP and Bmi1 (blue), which are responsible for mitotic quiescence and self-renewal properties, and Np63 (red), which sustains the stem cell proliferative potential (Parsa et al., 1999; Yang et al., 1999; Pellegrini et al., 2001; Koster et al., 2004; McKeon, 2004; Di Iorio et al., 2005; Nguyen et al., 2006). These cells give rise to holoclones in culture (Fig. 2, A and C). Under stress conditions, such as those induced by a corneal damage, a fraction of such stem cells switches off C/EBP (and Bmi1) but maintains Np63 (red). Activated Np63  stem cells actively proliferate and migrate to the central cornea to restore and regenerate the corneal epithelium (Fig. 2 A; Di Iorio et al., 2005). Activated stem cells, however, lose their self-renewal properties, enter into the TA compartment, and progressively lose Np63 expression. TA cells switch on Np63? and Np63, which might regulate terminal differentiation and stratification during the regeneration of the damaged corneal epithelium (Di Iorio et al., 2005).
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Stem cells are capable of shifting from a homeostatic state of relative quiescence to rapid proliferation under specific conditions (activation). In the ocular surface, this shift occurs upon central corneal wounding (Lehrer et al., 1998; Di Iorio et al., 2005). This explains the apparently opposing actions of C/EBP and Np63. On one hand, C/EBP induces mitotic quiescence (through a positive regulation of p27Kip1 and p57Kip2) and self-renewal of limbal stem cells; on the other, it preserves their proliferative potential (essential for stem cell–dependent tissue regeneration) through a positive regulation of Np63. In this way, when some limbal stem cells are released from C/EBP-dependent mitotic constraints, as in a corneal damage, they can unchain their remarkable p63-dependent proliferative capacity, multiply, and migrate to repair a corneal wound. This process is, however, irreversible and leads to limbal stem cell terminal differentiation (Fig. 9). Our data therefore strengthen the notion that proliferation and self-renewal capabilities are two related, albeit distinct, processes. At least in human limbal stem cells, proliferation potential relies on the expression of Np63, whereas self-renewal requires also C/EBP. Similarly, Bmi1 is essential for the self-renewal of neural stem cells but does not influence the proliferative capacity of their committed progeny (Molofsky et al., 2003). The notion that Np63 induces the expression of growth factor receptors and adhesion molecules regulating survival and motility of epithelial cells (Carroll et al., 2006) is consistent with our proposed model.* U6 D3 C7 p' V

; x, f- Z$ n4 e/ _% [1 g* POur data establish an interesting parallel with the hemaoietic system, where quiescence and self-renewal of stem cells have been recently shown to be linked and regulated by p27Kip1, p57Kip2, and Mad1 (Scandura et al., 2004; Walkley et al., 2005; Yamazaki et al., 2006). Indeed, loss of p27Kip1 allows relatively quiescent hemaoietic stem cells to rapidly enter the cell cycle to restore haemaoiesis (Walkley et al., 2005). Finally, we show that C/EBP is directly associated in vivo, alone or in combination with Np63, to chromatin-surrounding promoters or regulatory elements of the p63, p27Kip1, p57Kip2, and p16INK4A loci, suggesting a direct role of this transcription factor in determining the genetic program of self-renewing stem cells.
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* S5 G) ~" U; S" A4 s% f" Z) w- tThe role of C/EBP described here is intriguing. Indeed, C/EBPs have been mainly related to cellular differentiation. C/EBP, -?, and - are instrumental in regulating adipogenesis, whereas C/EBP, -, and -? orchestrate myeloid differentiation into mature neutrophils, atypical neutrophils, and macrophages (Rosen et al., 2000; Ramji and Foka, 2002), and C/EBP regulates learning and long-term memory in the central nervous system (Sterneck et al., 1998; Taubenfeld et al., 2001). The importance of the C/EBP family in cellular differentiation also extends to other cell types, including hepatocytes, ovarian luteal cells, intestinal epithelial cells, and epidermal keratinocytes. For instance, it has been shown that C/EBP and -? induces cell cycle exit in normal keratinocytes and positively regulates the program of squamous differentiation in the epidermis (Oh and Smart, 1998; Zhu et al., 1999). However, C/EBP? promotes keratinocyte proliferation and skin tumor formation in the presence of oncogenic Ras or in response to carcinogens (Zhu et al., 2002; Sterneck et al., 2006) and fosters hepatocyte proliferation during liver regeneration after partial hepatectomy (Greenbaum et al., 1998). Mammary epithelial cells from C/EBP?-deficient mice have a proliferation defect that leads to impaired ductal morphogenesis and a failure to lactate (Robinson et al., 1998; Seagroves et al., 1998), and ecic C/EBP? expression in human mammary epithelial cells induces hyperproliferation and a partially transformed phenotype (Bundy and Sealy, 2003). Finally, C/EBP induces late differentiation events in epidermal keratinocytes (Smith et al., 2004) and is indeed detected in the subrabasal layers of the human epidermis (unpublished data). Therefore, the biological effects of C/EBPs appear to be highly species and cell context specific, suggesting that role that C/EBP exerts in the human corneal epithelium might not necessarily be observed in other squamous epthelia.
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The mechanisms controlling C/EBP expression and function in the limbus, as well as the downstream mediators of C/EBP activity in controlling stem cell quiescence and self-renewal, remain to be determined. The expression of the C/EBPs has been found to change markedly during several physiological and pathophysiological conditions through the action of extracellular signals. C/EBPs are subject to extensive species- and tissue-specific posttranscriptional regulation and phosphorylation-mediated changes in DNA binding activity and nuclear localization (Ramji and Foka, 2002). Furthermore, the different C/EBP proteins are able to form heterodimers in all intrafamilial combinations and to associate with other factors (Ramji and Foka, 2002). A combination of biochemical, cellular, and genetic experiments is necessary to acquire a more comprehensive description of upstream regulators and downstream targets of C/EBP and to elucidate the networks of protein interactions and regulatory pathways that control its activity in human limbal stem cells.& U2 S) s- s4 l# v) V/ s
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Materials and methods
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Human specimens, cell culture, and cell cycle analysis
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1 C$ y1 q2 O$ c  A: {Corneas taken from organ donors and considered unsuitable for transplantation (solely because of hepatitis seropositivity of the donor) were examined with a slit lamp immediately before retrieval and classified as resting corneas, which did not show epithelial defect, dehydration, edema, or inflammation, or activated corneas, which had central corneal epithelial defects and/or abrasions, usually as a result of incomplete closure of the eyelids after death. Resting and activated corneas were taken 3.93 ± 0.69 and 6.79 ± 2.9 h from death, respectively. Corneas were provided by D. Ponzin and A. Ruzza (The Veneto Eye Bank Foundation, Venice, Italy).
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# f# }7 x3 U! u" o; \- H+ ZSwiss mouse 3T3-J2 cells (a gift from H. Green, Harvard Medical School, Boston, MA) were grown in DME supplemented with 10% calf serum. Keratinocytes were cultivated on a feeder layer of lethally irradiated 3T3-J2 cells, and colony forming efficiency (CFE) assays and calculation of the number of cell generations and population doublings were performed as described previously (Pellegrini et al., 1999a; Dellambra et al., 2000). Clonal analysis was performed from subconfluent primary cultures as described previously (Pellegrini et al., 1999a, 2001). In brief, single cells were inoculated onto multiwell plates containing a feeder layer of 3T3 cells. Clones were identified after 7 d of culture under an inverted microscope and transferred to replicate dishes. One dish (1/4 of the clone) was fixed 9–12 d later and stained with rhodamine B for clonal type classification (Barrandon and Green, 1987; Pellegrini et al., 1999a). The second dish was used for further experiments and analyses. In selected experiments, 100 limbal cells were plated in 100-cm dishes and cultured for 1 wk. Colonies were then examined under a microscope (Axiovert 200 M; Carl Zeiss MicroImaging, Inc.): large round colonies with smooth and regular borders and formed entirely by small cells with scarce cylasm were classified as holoclones (Di Iorio et al., 2005) and were subjected to immunofluorescence.% @8 i+ {" P) U3 ?2 S5 G* ^2 l

% Y% c& r! i  q* AFor cell cycle analysis, subconfluent keratinocyte cultures were trypsinized and fixed in 70% ethanol at 4°C. Samples (106 cells) were rehydrated in PBS/1% FCS at room temperature for 10 min and stained with 20 μg/ml propidium iodine for 30 min at 4°C. Flow cytometry was performed using a LSR II FACScan (Becton Dickinson).
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Immunofluorescence and Western analysis  _. h4 k2 t1 {1 c3 o, g

; ?5 \% V3 k1 |/ B/ Y. i) X4 bThe following antibodies were used: rabbit anti-C/EBP, anti-RasGAP, anti-Rb, and p57Kip2 purified IgG (Santa Cruz Biotechnology, Inc.); 4A4 pan-p63 mAb (BD Biosciences); p16INK4A, p21Waf1/Cip1, and p27Kip1 mAbs (Exalpha Biologicals, Inc.); involucrin and Ki67 mAbs (Novocastra); Bmi1 mAb (Upstate Biotechnology); rabbit anti-p63 unconjugated and FITC-conjugated purified IgG raised against a synthetic peptide (NH2-DFNFDMDARRNKQQRIKEEGE-COOH) comprising the C terminus post-SAM domain of p63 (Primm; Di Iorio et al., 2005). Secondary rhodamine- or FITC-labeled antibodies were obtained from Santa Cruz Biotechnology, Inc. For immunofluorescence analysis, keratinocyte colonies were fixed (3% paraformaldehyde/2% sucrose in PBS, pH 7.6), permeabilized (0.5% Triton X-100 in PBS), and coated with 0.5% BSA/PBS for 1 h at RT. Paraformaldehyde-fixed corneal samples were embedded in OCT, frozen, and sectioned. Immunofluorescence was performed on fixed colonies and 5–7-μm corneal sections as described previously (Di Iorio et al., 2005). Confocal analyses were done with a confocal analyzer (LSM510-META; Carl Zeiss MicroImaging, Inc.). Multitrack analysis was used for image acquisition. For immunoblots, mass or clonal cultures were extracted on ice with RIPA buffer (0.15 mM NaCl/0.05 mM Tris/HCl, pH 7.5/1% Triton X-100/1% sodium deoxycholate/0.1% SDS). Nuclear and cylasmic protein extraction was performed using the NE-PER Nuclear and Cylasmic Extraction kit (Pierce Chemical Co.) following conditions supplied by the manufacturer. Equal amounts of samples were electrophoresed on 7.5% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride filters (Immobilon-P; Millipore). Immunoreactions were performed as described previously (Pellegrini et al., 2001) using antibodies at a 1:500 dilution. Immobilon bound antibodies were detected by chemiluminescence with ECL (GE Healthcare).
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1 g5 _0 k0 V1 x3 B# ZSemiquantitative RT-PCR
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Total RNA was extracted from keratinocyte cultures, purified with RNase Micro kit (QIAGEN), and quantified by spectrophotometry. RT-PCR was performed using the One Step RT-PCR kit (QIAGEN). cDNAs were synthesized from 0.5–2 μg of total RNA, and PCR reactions were performed using 20, 24, 28, 32, 36, and 40 cycles. ?-Actin was used for normalization. Ethidium bromide–stained agarose gels were visualized with an Image Station 440 CF (Kodak). Quantification was performed using 1D 3.5 software (Kodak). Primer sequences for p63 isoforms and annealing temperatures were as described previously (Di Iorio et al., 2005). The following primers and annealing temperatures were used for p27, p57, and ?-actin RT-PCR: p27Kip1, 5'-AGTGTCTAACGGGAGCCCTA-3' and 3'-GTCCATTCCATGAAGTCAGC-5' (annealing temperature 60°C, 829 bp); p57Kip2, 5'-CACGATGGAGCGTCTTGTC-3' and 3'-CTTCTCAGGCGCTGATCTCT-5' (annealing temperature 60°C, 699 bp); and ?-actin, 5'-GAGCGCAAGTACTCCGTGT-3' and 3'-ACGAAGGCTCATCATTCAAA-5' (annealing temperature 58°C, 548 bp).
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* _4 y' ~2 R7 tLentiviral vectors: n1 t) N# i* w" B

7 O, |* f, m% n7 }% IThe human C/EBP cDNA was cloned by RT-PCR from total RNA extracted from the THP1 cell line using specifically designed primers containing EcoR1 recognition sequences. To generate a N-terminal Flag epie–tagged C/EBP sequence, the EcoR1 C/EBP cDNA fragment was subcloned into the pFlagCMV-2 plasmid (Sigma-Aldrich), after inserting an EcoRV restriction site at position 913 by PCR (for all primers, see Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200703003/DC1). To generate a Flag-tagged ER-C/EBP fusion sequence, a MfeI–EcoRI PCR–amplified fragment containing the modified ligand binding domain of the human ER (Littlewood et al., 1995) was fused to the C terminus of the Flag epie before inserting the C/EBP cDNA. The Flag-C/EBP, Flag-ER- C/EBP, and Flag-ER cassettes were extracted as EcoRV fragments and cloned downstream the human PGK promoter and upstream an IRES-EGFP cassette into the blunted SmaI–BamHI sites of the pRRL.ppt.PGK.IRES.GFP.WPRE lentiviral vector (Urbinati et al., 2005), to obtain the RRL--G, RRL-ER-G, and RRL-ER-G vectors. The control RRL-N-G vector was generated by cloning an NcoI–EcoRV fragment encoding a truncated form of the low-affinity, p75 NGFr (NGFr; Bonini et al., 1997) into the same vector backbone. The RRL-N-G vector was obtained by inserting the cDNA of Np63 isoform (Yang et al., 1998) into the same vector backbone.1 g* R1 I) V; c; [2 s: q- F6 S
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Lentiviral stocks pseudotyped with the vesicular stomatitis G protein (VSV-G) were prepared by transient cotransfection of 293T cells using a three-plasmid system (the transfer vector and the helper plasmids pCMVR8.74, encoding Gag, Pol, Tat, and Rev, and pMD.G, encoding VSV-G), as previously described (Dull et al., 1998). Viral titers were determined by transduction of HeLa cells with serial dilution of the vector stocks and ranged from 107 to 108 TU/ml. Transduction efficiency was evaluated by scoring GFP and/or NGFr transgene expression by flow cytometry.
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Transduction of limbal keratinocytes
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Subconfluent primary or clonal limbal cultures were trypsinized. 2 x 104 cells were resuspended in 1 ml of culture medium containing 8 mg/ml polybrene and transduced with lentiviral vector stocks at a MOI of 25, overnight at 37°C. Gene transfer efficiency was assessed 4 d after transduction by scoring GFP  cells by confocal fluorescence microscopy (LSM510-META; Carl Zeiss MicroImaging, Inc.). In selected experiments (Fig. 4), 1 μM 4OHT was added every 12 h for 3 d to RRL-ER-G–transduced cultures. 4OHT was then removed from the culture medium for 2 d and readded until control cultures reached confluency." ]* i7 T0 A9 Y9 }1 n

7 i! e6 F2 X1 B2 @- WTransfection of siRNA-p27 and siRNA-p57; W2 n& ?. b5 W5 Q& L+ l/ h

5 l' B- \" B6 h# kThese experiments were performed using the siRNA-p27 and siRNA-p57 RNAi Human/Mouse Starter kit (QIAGEN). The siRNA duplexes were designed using the HiPerformance Design Algorithm licensed from Novartis AG, integrated with a stringent in-house homology analysis tool. Double-stranded RNAs were synthesized by QIAGEN. Primary cultured human limbal epithelial cells previously transduced with a lentiviral vector carrying the C/EBP cDNA (RRL--G) were plated into 24-well plates at 4 x 104 cells/cm2. 48 h later, cells were transfected using fluorescently labeled p27Kip1 (Alexa Fluor) and p57Kip2 (Cy3) siRNAs at a final concentration of 67 nM (siRNA to Hiperfect reagent ratio 1:6) and incubated under normal growth conditions (37°C and 5% CO2). Transfection efficiency was determined 24 h after siRNA addition through laser-scanning confocal microscope (LSM510-META) analysis. Nonsilencing siRNAs (Alexa Fluor) were used as negative controls at the same conditions of transfection (67 nM; 1:6 ratio) and cell density.
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ChIP assay
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ChIP assays were performed essentially as described previously (Testa et al., 2005). Chromatin was prepared from 107 limbal keratinocytes at the third and the fifth passage after transduction with either the RRL--G or the RRL-N-G vector. Nuclear extracts were sonicated to obtain DNA fragments ranging from 400 to 800 bp in length. The equivalent of 5 x 105 cells was immunoprecipitated with rabbit anti-CEBP (Santa Cruz Biotechnology, Inc.), mouse anti-Flag (Sigma-Aldrich), mouse anti-p63 (4A4 pan63; BD Biosciences), and rabbit anti-p63 antibodies. Immunoprecipitations with mouse and rabbit IgGs (BD Biosciences) were included as controls. Immunoprecipitated DNA was analyzed by PCR with primers spanning regions containing known or putative CEBP and p53/p63 binding motifs within the genomic loci of p63 (from position  70720 to  70951,  147873 to  148041,  151191 to  151408,  202579 to  202761, and  234830 to  235057 from the transcription start site), involucrin (–421 to –119), p27Kip1 (–227 to  14), p57Kip2 (–622 to –398), and p16INK4A (–1020 to –871). Specific primers are listed in Table S1.
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, S3 N; O1 H2 _6 n% AOnline supplemental material! D$ \* j& c5 o3 S# S0 t
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Table S1 gives the primers used in this study. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200703003/DC1.
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Acknowledgments9 v' Y# V1 E' r

" C; N' _  J$ M! K* dThis work was supported by grants from the Ministry of Health, the Istituto Superiore di Sanità, and the European Commission, VI Framework Program, Cornea Engineering (grant NMP2-CT-2003-504017) and Skintherapy (grant LSHB-CT-2005-512073).
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科研人

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sky蓝

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