Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents

This Article Abstract PDF (Full Text) PPT slides of all figures Supplemental Material Index Alert me when this article is cited Citation Map Services Email this article Similar articles in this journal Similar articles in PubMed Alert me to new content in the JCB Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Barbaro, V. Articles by De Luca, M. Search for Related Content PubMed PubMed Citation Articles by Barbaro, V. Articles by De Luca, M. Social Bookmarking                      What's this?

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

¹ Epithelial Stem Cell Research Center, The Veneto Eye Bank Foundation, H. SS Giovanni and Paolo, 30100 Venice, Italy
² Department of Biomedical Sciences, University of Modena and Reggio Emilia, 41100 Modena, Italy

Correspondence to Michele De Luca: michele.deluca{at}unimore.it

Human limbal stem cells produce transit amplifying progenitors that migrate centripetally to regenerate the corneal epithelium. Coexpression of CCAAT enhancer binding protein (C/EBP), Bmi1, and Np63 identifies mitotically quiescent limbal stem cells, which generate holoclones in culture. Upon corneal injury, a fraction of these cells switches off C/EBP and Bmi1, proliferates, and differentiates into mature corneal cells. Forced expression of C/EBP inhibits the growth of limbal colonies and increases the cell cycle length of primary limbal cells through the activity of p27^Kip1 and p57^Kip2. These effects are reversible; do not alter the limbal cell proliferative capacity; and are not due to apoptosis, senescence, or differentiation. C/EBP, but not Np63, indefinitely promotes holoclone self-renewal and prevents clonal evolution, suggesting that self-renewal and proliferation are distinct, albeit related, processes in limbal stem cells. C/EBP is recruited to the chromatin of positively (p27^Kip1 and p57^Kip2) and negatively (p16^INK4A and involucrin) regulated gene loci, suggesting a direct role of this transcription factor in determining limbal stem cell identity.

Abbreviations used in this paper: 4OHT, 4-hydroxytamoxifen; C/EBP, CCAAT enhancer binding protein; CFE, colony forming efficiency; ChIP, chromatin immunoprecipitation; ER, estrogen receptor; IRES, internal ribosomal entry site; NGFr, NGF receptor; PGK, phosphoglycerokinase; TA, transit amplifying.

	Introduction

Top Abstract Introduction Results Discussion Materials and methods References

 
Stem 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).

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).

The 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 , ß, 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 Np63ß and Np63 correlate with corneal regeneration and differentiation (Pellegrini et al., 2001; Di Iorio et al., 2005).

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).

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.

	Results

Top Abstract Introduction Results Discussion Materials and methods References

 
Coexpression of C/EBP and Np63 in quiescent human limbal cells
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 Np63ß and Np63, proliferate, and migrate to the central cornea to restore a wounded epithelium (Di Iorio et al., 2005).

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.

View larger version (70K): [in this window] [in a new window] [Download PPT slide]   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 cytoplasm of C/EBP–/Np63+ cells (brackets). Bars, 10 µm.

Expression of C/EBP, Np63, and Bmi1 in limbal stem cells
Immunofluorescence 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 Np63ß 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.

View larger version (60K): [in this window] [in a new window] [Download PPT slide]   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/EBPß, 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 Np63ß and Np63. C/EBPß 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.

Gene 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 hematopoietic 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).

Collectively, 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.

C/EBP regulates the cell cycle of human limbal stem cells
Primary limbal cultures were infected with a lentiviral vector expressing either an epitope-tagged human C/EBP or a control protein (a truncated form of the p75 low-affinity NGF receptor [NGFr]) 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).

View larger version (42K): [in this window] [in a new window] [Download PPT slide]   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 epitope fused to C/EBP is indicated. (B) C/EBP-transduced colonies are indicated by GFP expression (green). (top) 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.

To 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 cytoplasm 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 [top], C, and D). In contrast, C/EBPß was present only in the nucleus, irrespective of the presence of 4OHT (Fig. 4 B [bottom] 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 cytoplasm, 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 cytoplasmic 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.

View larger version (47K): [in this window] [in a new window] [Download PPT slide]   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 cytoplasmic (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/EBPß 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 (top) was found predominantly in the cytoplasm (–4OHT). Nuclear translocation was observed within 12 h from the addition of 1 µM 4OHT to the culture medium (+4OHT; top). Strikingly, in the absence of 4OHT, ER-C/EBP sequestered also the endogeneous C/EBP in the cytoplasm, 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 cytoplasmic extracts (middle). The exposure times of filters in top (ER-C/EBP) and middle (C/EBP) panels were 10 and 75 s, respectively. C/EBPß was present only in nuclear extracts, irrespective of the presence of 4OHT (bottom). (C and D) Cytoplasmic–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/EBPß 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 [bottom] and F [green circles]). In contrast, RRL-ER-G–transduced colonies showed a progressive and linear increase of their size, irrespective of the presence of 4OHT (E [top] and F [black circles]). Values of the size of colonies are in arbitrary units.

View larger version (35K): [in this window] [in a new window] [Download PPT slide]   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.

C/EBP-dependent mitotic quiescence is mediated by p27^Kip1 and p57^Kip2

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-p27^Kip1 or siRNA-p57^Kip2 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 p27^Kip1 and p57^Kip2 mediate C/EBP-induced mitotic quiescence.

C/EBP binds to the p63, involucrin, p27^Kip1, p57^Kip2, and p16^INK4A loci in vivo
To provide evidence for a direct contribution of C/EBP in regulating the expression of Np63, involucrin, p27^Kip1, p57^Kip2, and p16^INK4A, 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 epitope and with control IgGs. Immunoprecipitated chromatin DNA was analyzed by PCR with primers specific for different regions of the p63, involucrin, p27^Kip1, p57^Kip2, and p16^INK4A loci (Fig. 6 A, red arrowheads), containing evolutionarily conserved and/or putative C/EBP binding elements.

View larger version (30K): [in this window] [in a new window] [Download PPT slide]   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.

Chromatin 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 p27^Kip1 loci (Fig. 6 B). These results suggest that the p63, involucrin, p27^Kip1, p57^Kip2, and p16^INK4A loci might be direct targets of C/EBP activity, in some cases in combination with Np63.

C/EBP promotes self-renewal of holoclone-forming cells
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.

Serially 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 Np63ß 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 Np63ß 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 cytoplasm 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).

View larger version (36K): [in this window] [in a new window] [Download PPT slide]   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). ß-Actin (ßact) 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 Np63ß and Np63, whereas C/EBP-transduced cultures expressed high levels of Np63 and low amounts of Np63ß and Np63.

View larger version (28K): [in this window] [in a new window] [Download PPT slide]   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.

	Discussion

Top Abstract Introduction Results Discussion Materials and methods References

 
Exceptional 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/ß-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).

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 haematopoetic and neural stem cells through regulation of the p16^INK4A and p19^ARF 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 p27^Kip1 and p57^Kip2. The growth inhibitory effect of C/EBP is not due to replicative senescence or terminal differentiation, as confirmed by the down-regulation of p16^INK4A 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.

View larger version (28K): [in this window] [in a new window] [Download PPT slide]   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 Np63ß and Np63, which might regulate terminal differentiation and stratification during the regeneration of the damaged corneal epithelium (Di Iorio et al., 2005).

Our data establish an interesting parallel with the hematopoietic system, where quiescence and self-renewal of stem cells have been recently shown to be linked and regulated by p27^Kip1, p57^Kip2, and Mad1 (Scandura et al., 2004; Walkley et al., 2005; Yamazaki et al., 2006). Indeed, loss of p27^Kip1 allows relatively quiescent hematopoietic stem cells to rapidly enter the cell cycle to restore haematopoiesis (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, p27^Kip1, p57^Kip2, and p16^INK4A loci, suggesting a direct role of this transcription factor in determining the genetic program of self-renewing stem cells.

The role of C/EBP described here is intriguing. Indeed, C/EBPs have been mainly related to cellular differentiation. C/EBP, -ß, and - are instrumental in regulating adipogenesis, whereas C/EBP, -, and -ß 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 -ß 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/EBPß 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/EBPß-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 ectopic C/EBPß 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.

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.

	Materials and methods

Top Abstract Introduction Results Discussion Materials and methods References

 
Human specimens, cell culture, and cell cycle analysis
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).

Swiss 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 cytoplasm were classified as holoclones (Di Iorio et al., 2005) and were subjected to immunofluorescence.

For 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).

Immunofluorescence and Western analysis
The following antibodies were used: rabbit anti-C/EBP, anti-Ras^GAP, anti-Rb, and p57^Kip2 purified IgG (Santa Cruz Biotechnology, Inc.); 4A4 pan-p63 mAb (BD Biosciences); p16^INK4A, p21^Waf1/Cip1, and p27^Kip1 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 cytoplasmic protein extraction was performed using the NE-PER Nuclear and Cytoplasmic 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).

Semiquantitative RT-PCR
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. ß-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 ß-actin RT-PCR: p27^Kip1, 5'-AGTGTCTAACGGGAGCCCTA-3' and 3'-GTCCATTCCATGAAGTCAGC-5' (annealing temperature 60°C, 829 bp); p57^Kip2, 5'-CACGATGGAGCGTCTTGTC-3' and 3'-CTTCTCAGGCGCTGATCTCT-5' (annealing temperature 60°C, 699 bp); and ß-actin, 5'-GAGCGCAAGTACTCCGTGT-3' and 3'-ACGAAGGCTCATCATTCAAA-5' (annealing temperature 58°C, 548 bp).

Lentiviral vectors
The 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 epitope–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 epitope 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 encodi