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Vol. 19, Issue 12, 5541-5549, December 2008
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*Institut Jacques Monod, Unité Mixte de Recherche Centre National de la Recherche Scientifique 7592, Universités Paris 6 and Paris 7, 75251 Paris Cedex 5, France; and Institut Gustave Roussy, Genetic Instability and Cancer Unit, Centre National de la Recherche Scientifique Formation de Recherche en Evolution 2939, 94805 Villejuif Cedex, France
Submitted February 15, 2008;
Revised September 2, 2008;
Accepted September 19, 2008
Monitoring Editor: Benjamin Margolis
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,b). The 15 mammalian members known to date can be classified into three groups on the basis of their structure, namely, "proto-type" galectins containing one CRD domain (14 kDa); galectin-3 (30 kDa), which is a unique chimeric molecule containing a single CRD motif combined with a proline-rich N-terminal domain; and tandem-repeat galectins that are composed of two CRDs separated by a short linker sequence (30 kDa) (Hirabayashi and Kasai, 1993). Despite their lack of signal peptide, galectins can be secreted by an endoplasmic reticulum (ER)-Golgi-independent pathway (Lindstedt et al., 1993; Sato et al., 1993). They can be found both intracellularly (cytoplasm and/or nucleus) and extracellularly depending on the cell type, cell cycle stage, and differentiation state. Galectins can be associated with the plasma membrane, but they do not contain transmembrane domain. Consistent with this variable subcellular location, galectins have been implicated in a wide range of cellular processes, including cell–cell interactions, extracellular matrix remodelling, apoptosis, the cell cycle, intracellular trafficking, and splicing (Hughes, 2001; Liu et al., 2002; Hsu and Liu, 2004; Delacour et al., 2006; Elola et al., 2007). Their roles in cancer biology (Lahm et al., 2004; Takenaka et al., 2004; Liu and Rabinovich, 2005; Thijssen et al., 2007), immune response (Sato and Nieminen, 2004; Rabinovich et al., 2007), and morphogenesis (Poirier, 2002) are well documented for the most studied members of the family. In addition, recent evidence indicates that galectin expression and function are highly sensitive to environmental stress resulting from neuroendocrine and immunological stimuli (Blois et al., 2007; Plachta et al., 2007; Toscano et al., 2007), suggesting a critical role of these glycan-binding proteins in maintaining and restoring tissue homeostasis after different insults.
Galectin-7 is a prototype galectin that forms homodimers (Leonidas et al., 1998). Thus, like other prototype members, galectin-7 can act as a bridging molecule between glycoproteins or glycolipids containing similar glycan structures. Galectin-7 was first isolated in two independent differential screens, one screen searching for human epidermal genes responsive to retinoic acid (Magnaldo et al., 1995), and the other screen for genes down-regulated in actively dividing human keratinocytes transformed in vitro (Madsen et al., 1995). Galectin-7 is preferentially expressed in stratified epithelia, including epidermis, cornea, oral cavity, esophagus and anorectal epithelium (Magnaldo et al., 1998) and major changes in its level of expression have been observed in various types of cancer, most notably in skin tumors (Saussez and Kiss, 2006).
Several lines of evidence indirectly suggested that galectin-7 might play a role in epithelial tissue response to environmental stimuli. First, galectin-7 could intervene in the process of wound-healing because it is up-regulated in wounded cornea (Cao et al., 2003) and addition of the recombinant protein accelerates the speed of healing in culture (Cao et al., 2002). Second, galectin-7 has been described as a proapoptotic factor involved in the epidermal response after UVB injury. Hence, following an early report in which galectin-7 was identified as one of the first genes responding to overexpression of the p53 tumor suppressor gene in DLD-1 human colon cancer cells (Polyak et al., 1997), Bernerd et al. (1999) reported 1) an increased galectin-7 expression in the apoptotic sunburn keratinocytes of human skin explants exposed to UVB and 2) the appearance of apoptotic cells in cultures of transfected human keratinocytes overexpressing galectin-7. Finally, ectopic expression of galectin-7 also renders HeLa cells or DLD-1 cells more sensitive to apoptosis induced by a variety of stimuli in addition to UVB (Kuwabara et al., 2002).
To elucidate the role(s) of galectin-7 in vivo, we have generated galectin-7 null mutant mice and focused our analysis on the consequences of this mutation in adult epidermis. The epidermis is a self-renewing stratified epithelium, which provides a protective barrier that serves multiple key functions, including protection from dehydration, infections, and various environmental insults. We present here the first evidence, using null mutant mice, showing that galectin-7 is involved in maintaining epidermal homeostasis in response to major challenges, i.e., UVB irradiation and wounding.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). G418 and gancyclovir doubly resistant clones were screened by Southern blotting. Genomic DNA was digested with HindIII/SpeI and hybridized first with neo probe and then with a specific 5' external probe (Figure 1, A and B). Chimeric males were obtained by injection of a positive clone into C57Bl/6 blastocysts. Heterozygous individuals were intercrossed, and the mutation was maintained on mixed genetic background (50% 129/Sv; 50% C57Bl/6). We also introduced the galectin-7–/– mutation into the C57Bl/6 background by a series of eight backcrosses. Mice were kept in a specific pathogen-free animal house facility and handled respecting the French regulations for animal care.
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Western Blot Analysis
Protein extracts from two anterior foot pads of adult mice were prepared in 400 µl of lysis buffer (Bernot et al., 2004). Fifteen microliters of each preparation was loaded on to a 12% acrylamide gel. Mouse monoclonal anti-β-tubulin antibody (Ab) was first used (1:8000, Amersham N357; GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) and detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse Ab (1:15,000, A9044, Sigma-Aldrich, St. Louis, MO), then rabbit anti-galectin-7 Ab (Magnaldo et al., 1995) was added, followed by HRP-conjugated goat anti-rabbit (1:10,000; Amersham NA934). Signal was revealed using ECL Plus detection system (GE Healthcare RPN2132).
Electron Microscopy
Median back skin samples of 2-mo-old wild-type (wt) and galectin-7–/– mice were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 1 h at room temperature followed by 1% osmium for 1 h at 4°C and contrasted with uranyl acetate before embedding in Epon.
Immunostaining of Skin Sections
Median back skin samples from shaved or depilated mice (UVB experiment) were fixed overnight at 4°C with 4% formaldehyde in phosphate-buffered saline (PBS) before embedding in paraffin (Paraplast). Anterior adult foot pads were embedded in Tissue-Tek OCT (Sakura Finetek, The Netherlands) and immediately snap frozen in liquid N2 before storing at –80°C. All stainings were performed using standard protocols, except for Ki67, which required a preliminary unmasking step at 98°C for 30 min in pH 6.0 solution (S1700; Dako, Trappes, France).
Primary Abs.
Primary antibodies were as follows: anti-galectin-7 rabbit Ab (1:3000; Magnaldo et al., 1995), anti-Ki67 rabbit Ab (1:250, NCL-Ki67p; Novocastra, New Castle, United Kingdom), anti-keratin5 rabbit Ab (1:2500, PR-B160-P; Covance Research Products, Princeton, NJ), anti-keratin10 mouse monoclonal antibody (mAb) (1:2000, C-7284; Sigma-Aldrich), anti-loricrin rabbit Ab (1:200, PRB-145P; BAbCO, Richmond, CA), anti-E-cadherin mouse mAb (1:500, C20820
[GenBank]
; BD Biosciences, San Jose, CA), anti-β-catenin mouse mAb (1:500, C19220
[GenBank]
; BD Biosciences), anti-
6-integrin rat mAb (1:50, MAB1378; Millipore, St-Quentin-en-Yvelines, France), anti-desmoglein mouse mAb (1:10, 03-61002; American Research Products, Belmont, MA), anti-desmocollin1 rabbit Ab (1:2000, NB600-666; Novus Biologicals, Littleton, CO), anti-plakoglobin rabbit Ab (1:25, C2069-60A; U.S. Biological, Swampscott, MA), and anti-cortactin monoclonal Ab (1:200, 05-180; Millipore).
Secondary Abs. Secondary Abs were as follows: Alexa488-conjugated goat anti-rabbit Ab (1:500, A11008 [GenBank] ; Invitrogen, Cergy Pontoise, France), Alexa568-conjugated goat anti-mouse Ab (1:500, A11004 [GenBank] ; Invitrogen); and biotinylated goat anti-rat Ab (1:500, 112-065-003; Jackson ImmunoResearch Laboratories, West Grove, PA) followed by Alexa568-conjugated streptavidin (1:500, S11226 [GenBank] ; Invitrogen).
In Vivo UVB Irradiation
UVB irradiation was done using Philips TL20W/12 fluorescent tubes (LumièreService, Paris, France), with an emission peak at 312 nm (Bernerd et al., 1999). A Kodacel filter (Eastman Kodak, Rochester, NY) blocking wavelengths below 305 nm was added and the irradiance was monitored with a Centra dosimeter (Osram, Berlin, Germany) placed 20 cm away from the source. Back hair of 2-mo-old females (129/Sv; C57Bl/6 background) was removed with depilatory cream under general anesthesia. Five days later, the posterior region of the back was exposed to a single dose of 2000 J/m2 of UVB under general anesthesia. The depilated unirradiated anterior region was used as a control. Mice were killed at different times after UVB irradiation and skin samples (1 cm x 0.5 cm) were taken from the median part of posterior and anterior regions.
Detection of Apoptotic Cells
Apoptotic sunburn cells were identified on paraffin sections stained with hematoxylin and eosin (Kerr et al., 1972). Alternatively, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was carried out using the In Situ Cell Death Detection kit (Roche Diagnostics, Base, Switzerland).
For each animal, the analysis used the entire 1-cm section from two separate slides, spaced 40 sections apart. Results were expressed as the number of apoptotic cells per centimeter of epidermis. This analysis was restricted to the interfollicular regions.
In Vivo Wounding Experiment
Adult mice were anesthetized before a superficial scratch was made along the sagittal axis of tail by using a sterile blood lancet (Assistant, Sondheim, Germany). The animals were killed at 24 or 48 h after injury. Three segments (0.5 cm) of each injured tail were snap-frozen in Tissue-Tek OCT and sectioned (10 µm) for histological analysis and immunostaining. The distance between wound margins was measured after staining with anti-keratin17 rabbit Ab (1:5000; gift from P. Coulombe, Johns Hopkins University, Baltimore, MD). All results are expressed as a mean of three values per individual.
Ex Vivo Wound-healing Assay
Skin explant cultures of 1.5-d-old C57Bl/6 wild-type and mutant mice were performed as described previously (Mazzalupo et al., 2002), except that the biopsies were obtained from ventral skin instead of dorsal skin. Cultures were maintained in medium as described previously (Rheinwald and Green, 1975), fixed on day 7, and stained with anti-keratin17 rabbit Ab (1:5000; gift from P. Coulombe) (Mazzalupo et al., 2002). The surface area of the keratin17-positive outgrowth was quantified. Some explants were treated for 2 h with mitomycin C (10 µg/ml, M0503; Simnga-Aldrich) on day 2, and then they were placed back in normal medium until day 7. For immunofluorescence staining, explants were cultured on glass coverslips for 7 d, fixed in 4% paraformaldehyde for 15 min and then permeabilized with 0.025% saponin in PBS for 20 min. Primary Abs included the following: anti-galectin-7 rabbit Ab (1/3000; Magnaldo et al., 1995), anti-β-tubulin mouse mAb (1:1000), anti-cortactin mouse mAb (1:200, 05-180; Millipore), and anti-lysosome–associated membrane protein (Lamp)-1 mouse mAb (1:50; a gift from H.-P. Hauri, University of Basel, Basel, Switzerland) overnight at 4°C. For β1-integrin staining, the primary goat anti-β1-integrin Ab (1:50, AF2405; R&D Systems Europe, Abingdon, Oxfordshire, United Kingdom) was added in culture medium for 1 h 30 min before fixation. Secondary Abs were as follows: Alexa488-conjugated goat anti-rabbit Ab (1:500, A11008
[GenBank]
: Invitrogen), Alexa568-conjugated goat anti-mouse Ab (1:500, A11004
[GenBank]
; Invitrogen), and Alexa568-conjugated donkey anti-goat Ab (1:500, A11057
[GenBank]
; Invitrogen).
Quantification of Cell Proliferation and Epidermal Thickness
After UVB irradiation, Ki67-positive cells were counted along the entire 1-cm section of epidermis from two separate slides, spaced 40 sections apart. Data are expressed as a number of Ki67-positive cells per centimeter of epidermis. After wounding, Ki67-positive cells were counted over a distance corresponding to one 20x field on either side of the wound.
Epidermal thickness was measured between the basal membrane and the surface of granular cells. Thirty-three measurements were done along the 1-cm section of epidermis. These analyses were restricted to interfollicular regions.
Statistics
The data are expressed as the mean ± SD. The nonparametric Mann–Whitney U-test was used for comparisons (Software StatEL, ad Science, Paris, France).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In wt adult epidermis, galectin-7 is present in both basal and suprabasal (spinous and granular) cells, but its subcellular distribution differs between the two compartments (Figure 1E). In basal cells, staining is uniformly cytoplasmic, whereas in suprabasal cells, there is a strong, irregular, punctiform signal associated with the plasma membranes. No galectin-7 immunoreactivity is detected in null mutant epidermis (Figure 1E).
We looked more carefully at proliferation and differentiation in the galectin-7 null mutants. Using Ki67 as a marker of dividing cells, we found that the mitotic index was similar between wt (115 ± 14 cells/cm) and galectin-7–/– (116 ± 23 cells/cm) epidermis. Consistent with this, the epidermal thickness is comparable between wt tissue (13.9 ± 1.1 µm; N = 3) and galectin-7–/– tissue (13.1 ± 1.3 µm; N = 3).
In addition, the differentiation program does not seem to be affected either in the null mutant epidermis, because the markers for the basal (keratin5), suprabasal (keratin10), and granular (loricrin) layers are all normally expressed (Figure 2A). The distribution of the junction molecules, E-cadherin, β-catenin, 6-integrin, and of the desmosomal components desmoglein, desmocollin-1 and plakoglobin, is also unaffected by the lack of galectin-7 and the intensity of the signals is comparable between wt and galectin-7–/– samples (Figure 2B).
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Apoptotic Response.
In wt animals, the number of apoptotic cells peaked at 12 h post-UVB, with 
60 sunburn cells per centimeter of epidermis. This elevated level of apoptosis was limited to the narrow time frame between 9 and 15 h post-UVB, and it returned to basal levels before 48 h. In galectin-7–/– epidermis, we found twice as many sunburn cells as in wt epidermis at the early time points, i.e., 6 and 9 h post-UVB (p < 0.01) (Figure 4A). This was confirmed by TUNEL assay, which also detected a twofold increase in apoptotic cells in galectin-7–/– (58.9 ± 23 cells/cm) compared with wt (26.6 ± 4.7 cells/cm) epidermis at 6 h. After the 6- to 9-h initial phase, the level stabilized until 15 h post-UVB exposure of mutant mice. Thus, unlike wt epidermis, galectin-7–/– epidermis displayed no distinct narrow peak of apoptosis. Beyond 15 h, we observed no significant difference between the wt and galectin-7–/– reactions. In summary, the apoptotic response is prematurely triggered and lasts for a longer period in the absence of galectin-7.
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), but they occurred in all keratinocytes of the exposed area (Figure 4C). Initially, at 6 h post-UVB, the cytoplasm of all basal and suprabasal cells was positive for galectin-7. At 12 h post-UVB, the time of maximal apoptotic response, some basal cell nuclei displayed intense galectin-7 staining. These are likely to be sunburn cells since their nuclei were picnotic (data not shown), and some of them were clearly detaching from their neighboring cells. At 18 h post-UVB, galectin-7–positive dots were present in the cytoplasm of all epidermal cells, before reappearance of the characteristic punctiform distribution of galectin-7 along the membranes at 24 h post-UVB. In conclusion, there is a clear redistribution of galectin-7 in all cells of the irradiated region of the epidermis. Regenerative Response. We found that the phase of cell proliferation was also severely affected in the mutant mice (Figure 4B). At 24 h post-UVB injury, the number of dividing cells in galectin-7–/– epidermis was already slightly higher than that in wt epidermis. At 48 h, there was an abrupt increase in the number of Ki67-positive cells in galectin-7–/– epidermis, resulting in 2.5-fold excess relative to wt epidermis. This hyperproliferative activity was transient since comparable numbers of Ki67-positive cells were detected at 72 and 96 h in both mutant and wt epidermis. In summary, there is a burst of proliferation taking place in the galectin-7–/– epidermis, whereas during the 24- to 96-h period post-UVB, the number of dividing cells increased moderately and regularly in wt epidermis.
Defective Wound-healing Capacity of Galectin-7–deficient Mice
The extent to which galectin-7 may play a role in posttraumatic skin repair was also assessed after wounding in vivo (Figure 5). We made a superficial scratch along the length of the tail, which damaged the epidermis but left the dermis mostly intact. In these conditions, the wound was closed and restratification was well underway 48 h after injury (Figure 5A). We thus studied the dynamics of the healing process during this time frame. At 24 h, we found that the closure of the wound was less advanced in the galectin-7–/– than in the wt animals (20% difference, p < 0.05) (Figure 5B). At 48 h, closure was completed in both control and experimental groups.
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At 48 h after injury, when the wound is closed and the process of regeneration is taking place, we observed an intense hyperproliferative reaction in the mutant epidermis compared with the wt tissue (Figure 5C). Together with the data presented in Figure 4B, we conclude that galectin-7 plays a key role in the control of keratinocyte proliferation after both types of injury.
To further investigate the role of galectin-7 in keratinocyte migration, we used a recently established ex vivo wound-healing assay. In this assay, newborn circular skin biopsies are placed in culture and keratinocytes move out of the explant, forming a continuous sheet around the original biopsy. This process mimics the epithelialization events taking place during skin repair after wounding in vivo. In addition, the surface of keratinocyte outgrowths gives a quantitative estimate of their epithelialization potential (Mazzalupo et al., 2002). In these conditions, we found that the surface of outgrowths derived from galectin-7–/– explants was reduced, by a factor of 20%, compared with the controls (p < 0.0001) (Figure 6, A). As predicted based on the in vivo results, this difference was due to defects in the migrating capacity of galectin-7–/– keratinocytes. Indeed, when cell division was irreversibly blocked by a treatment with mitomycin C at day 2 of culture, we still observed that mutant outgrowths were 15% smaller than wt outgrowths at day 7 (p < 0.01) (Figure 6A). In addition, we also observed striking differences in the subcellular localization of cortactin at the edge of explants. In wt cells, a characteristic sharp, intense signal was detected along the membrane protrusions whereas, in mutant cells, the signal was diffuse indicating a defect in the cortical domain of lamellopodia (Figure 6B).
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). Thus, galectin-7 is recruited in podosomes, which may account for its role in keratinocyte migration.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
In contrast, we did find that the lack of galectin-7 disturbed the epidermal response to environmental injury. After UVB exposure in vivo, the time course of the apoptotic response was affected in the null mutant epidermis; sunburn cells occurred earlier, the response was less acute, and lasted longer compared with wt tissue. These results are not what might be expected from a mutation in a proapoptotic gene, which galectin-7 was reported to be (Bernerd et al., 1999; Kuwabara et al., 2002). Rather, galectin-7 operates in vivo as a fine tuner, ensuring the completion of a robust and short apoptotic reaction in response to UVB stress. It is worth noting that this information could only have been obtained by examining the kinetics of the apoptotic response over the entire period of 24 h. A single time point, as is commonly reported in the field (Ziegler et al., 1994; Gillardon et al., 1999; Grossman et al., 2001), would have been misleading. For example, at 12 h post-UVB, the reduced number of apoptotic cells in the mutant mice could have been taken as a proof of a proapoptotic role, whereas the opposite conclusion could have been drawn from the results obtained at 9 h post-UVB.
The extent to which galectin-7 may play a role in posttraumatic skin response was also assessed in the context of mechanical injury in vivo. We found that the process of wound closure was less efficient in the absence of galectin-7 (20% wider wound bed in mutant skin compared with wt at 24 h after injury). In an ex vivo assay for wound healing, keratinocyte outgrowth from galectin-7–/– skin explants was also reduced by 20% compared with the wt controls. Because this difference was maintained when proliferation was blocked by mitomycin-C treatment, we conclude that it is primarily due to reduced keratinocyte migration. Consistently, cortactin distribution is severely affected in migrating keratinocytes lacking galectin-7, suggesting that the formation and/or stabilization of actin-based lamellopodia is abnormal. In addition, the coordination of events implicated in wound closure seems to be perturbed in the absence of galectin-7 because cortactin is ectopically expressed in cells located away from the front. How are these effects mediated by galectin-7 remains to be elucidated, but, one clue came from the study of its fine distribution in migrating cells. Hence, we observed that galectin-7 accumulates in podosomes, which are specialized cell–matrix adhesion complexes connecting the ECM to the microfilament network (Spinardi et al., 2004). We propose therefore that galectin-7 might participate in keratinocyte migration by modulating the stability of these highly flexible and dynamic structures. Alternatively, galectin-7 may be secreted through the posodomes, which would be consistent with the recent discovery that these structures are sites of secretion (Linder, 2007). In support of this hypothesis, Cao et al. (2002) found that adding exogenous galectin-7 accelerates the rate of healing of wounded cornea. Once externalized, like other members of this gene family, galectin-7 might function as a matricellular molecule modulating adhesion, for example by regulating interactions between integrins and ECM components and/or by rapidly remodelling local ECM (Hikita et al., 2000; Levy et al., 2001; Elola et al., 2007).
Surprisingly, we also detected a significant increase in keratinocyte proliferation in galectin-7 mutants after both types of injury. This intense proliferation occurred during the regeneration of the damaged tissue, i.e., after completion of the post-UVB apoptotic response and after wound closure. Because the level of cell death is comparable between wt and mutant epidermis after UVB damage, the excess proliferation observed in the mutant is likely to be a primary consequence of the galectin-7 mutation, and not a secondary response to an excessive loss of cells. These results reveal therefore a potential function for galectin-7 in regulating keratinocyte proliferation during tissue repair. Although there is precedent for other galectins having roles in cell growth and cell cycle progression (Lin et al., 2002; Fischer et al., 2005), this is the first report showing the importance of galectin-7 in cell proliferation. The hyperproliferation documented here in the absence of galectin-7 was triggered by only a single dose of UVB, or a mild mechanical injury. It will be interesting to assess the consequences of subjecting galectin-7–/– skin to further stress such as chronic UVB irradiation. Such a regimen might lead to various skin disorders ranging from benign skin hyperplasia to increased incidence of cancer.
In conclusion, these in vivo experiments provide an integrated and dynamic picture of the multiple roles of galectin-7 at the level of the entire tissue. We now have genetic evidence that galectin-7 plays a critical role in the maintenance of epidermal homeostasis by modulating keratinocyte apoptosis and proliferation as well as participating in the process of cell migration. Because galectin-7 is an abundant component of normal keratinocytes, it is possible that it is present as a "sentinelle" molecule, constantly sensing the integrity of the tissue in steady-state conditions while contributing to posttraumatic skin regeneration under environmental stress.
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ACKNOWLEDGMENTS |
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Footnotes |
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These authors contributed equally to this work. 
Address correspondence to: Françoise Poirier (poirier{at}ijm.jussieu.fr).
Abbreviations used: Ab, antibody; ECM, extracellular matrix; CRD, carbohydrate recognition domain; UV, ultraviolet; wt, wild type.
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