Galectin-7 in the control of epidermal homeostasis after injury - PubMed (original) (raw)
Galectin-7 in the control of epidermal homeostasis after injury
Gaëlle Gendronneau et al. Mol Biol Cell. 2008 Dec.
Abstract
Galectins, a family of beta-galactoside binding lectins, have recently emerged as novel regulators of tissue homeostasis. Galectin-7 is predominantly expressed in stratified epithelia, especially in epidermis. We report here the generation of galectin-7-deficient mice that are viable and do not display phenotypical abnormalities in skin structure or expression of epidermal markers. However, these mice show unique defects in the maintenance of epidermal homeostasis in response to environmental challenges. First, after UVB irradiation in vivo, the apoptotic response is prematurely triggered and lasts longer in the mutant epidermis. This result contrasts with the proapoptotic role that had been proposed for galectin-7. Second, wound-healing experiments in vivo revealed that galectin-7-deficient mice displayed a reduced reepithelialization potential compared with wild-type littermates. This effect could be attributed to a defect in cell migration. Because galectin-7 is located in the podosomes of keratinocytes migrating out of skin explants in culture, we propose that this glycan-binding protein may directly influence cell/extracellular matrix interactions. Finally, we also detected an unexpected intense hyperproliferative reaction consecutive to both types of stress in galectin-7-deficient mice. Together, these studies provide the first genetic evidence showing that galectin-7 can modulate keratinocyte apoptosis, proliferation, and migration during skin repair.
Figures
Figure 1.
Establishment of galectin-7 null mutant mice. (A) Targeting strategy. Mouse galectin-7 gene comprises five exons (black boxes). The ATG initiation codon is located in exon 2. The 7-kb fragment of 5′ homology contains exon 1, and the 3.8-kb fragment of 3′ homology contains exons 4 and 5 (see Materials and Methods). H, HindIII; B, BamHI; S, SpeI; Bg, BglII; TK, thymidine kinase gene; and Neo, neomycin resistance gene. (B) Southern blot. G418/Gancyclovir double resistant ES cell clones were picked. Genomic DNA was digested with HindIII/SpeI. The wt (14-kb) and targeted (7.5-kb) fragments were detected with a specific 5′ external probe (A). Clone 3 was used in all subsequent experiments. (C) RT-PCR analysis. Skin RNA was prepared from 2-mo-old littermates obtained by crossing heterozygous animals. Galectin-7 mRNA was amplified using the gal7L/gal7R primers (300-bp product). Control amplification using gadph mRNA was done using gapdh5′/gapdh3′ primers, which gave the expected 1193-bp product (see Materials and Methods). (D) Western blot. Fifteen micrograms of protein extracts prepared from foot pads were loaded on 12% acrylamide gel. After blotting, galectin 7 (14-kDa) and β-tubulin (55-kDa) proteins were detected. (E) Galectin-7 distribution in adult epidermis. Paraffin sections from back skin samples of wt and _galectin-7_−/− mice were immunostained with anti-galectin-7 Ab. White lines indicate the dermoepidermal junction. B, basal layer; s, suprabasal layers. We noted autofluorescence in cornified layer. Bar, 10 μm.
Figure 2.
Expression of differentiation and junctional markers in wt and null mutant adult epidermis. Cryosections (5 μm) of anterior foot pads from adult wt and _galectin-7_−/− mice were stained. (A) Differentiation markers: keratin5 (K5) specific for basal cells, keratin10 (K10) specific for suprabasal cells and loricrin (Lor) specific for superficial granular layers. (B) Junctional markers E-cadherin (E-Cad), β-catenin (β-Cat), α6-integrin (α6), desmoglein (Dg), desmocollin1 (Dsc1), and plakoglobin (Pg). White lines indicate the dermoepidermal junction. Bar, 20 μm.
Figure 3.
Ultrastructure of wt and null mutant adult epidermis. The wt (A and C) and _galectin-7_−/− (B and D) epidermises were examined by electron microscopy. (A and B) Comparison of the overall organization of the tissue. There is no detectable difference between wt and mutant tissue. Cellular interactions seem normal. Bar, 3 μm. (C and D) High magnification of the superficial layers. The size of desmosomes does not seem affected in the mutant. The distribution and shape of keratohyalin grains are similar between wt and mutant epidermis. Bar, 1 μm. b, basal layer; sp, spinous layer; and gr, granular layer.
Figure 4.
In vivo UVB irradiation. The wt and _galectin-7_−/− adult female mice were depilated and irradiated with 2000 J/m2 UVB. (A) Apoptotic kinetics. Sections of unirradiated (−UV) or irradiated back skin samples were stained with hematoxylin and eosin, and the number of sunburn cells per centimeter of epidermis was determined. Three to six mice per genotype were used for each time point. Each bar represents the mean value ± SD. Statistical differences between wt and mutant animals are noted *p < 0.01. (B) Proliferative response. Sections of unirradiated (−UV) or irradiated back skin samples were labeled with anti-Ki67 Ab, and the number of positive cells per centimeter of epidermis was determined. Three to six mice per genotype were used for each time point. Each bar represents the mean value ± SD (*p < 0.01) (C) Confocal analysis of galectin-7 distribution. Representative sections of unirradiated (−UV) or irradiated wt back skin samples were stained with anti-galectin-7 Ab. White lines indicate the dermoepidermal junction. Bar, 10 μm.
Figure 5.
In vivo wound-healing experiments after tail injury. Superficial scratches were made along the sagittal axis of the tail of wt and _galectin-7_−/− adult mice. (A) Transverse cryosections of injured wt tails at 24 and 48 h after injury were stained with hematoxylin and eosin. Bar, 75 μm. Arrows indicate the wound margins. (B) Distance between the two wound margins 24 h after injury in wt and _galectin-7_−/− mice. Results were calculated as a mean of three independent measurements per animal. (C) Immunohistochemical detection of Ki67-positive nuclei at the wound site (left). Number of Ki67-positive cells at 24 h and 48 h after wounding (right). Results were calculated as a mean of three independent measurements per animal. (D and E) Double immunostaining with anti-galectin-7 Ab (red) and anti-cortactin Ab (green) of the wound margins, 24 h after injury. Nuclei were detected by Hoechst 33342 staining. Arrows indicate the direction of migration. Note the ectopic cortactin-positive domains (arrowheads) in cells away from the leading edge in _galectin-7_−/− explants. Bar, 10 μm.
Figure 6.
Galectin-7 in reepithelialization process ex vivo. (A) Picture of wt and _galectin-7_−/− newborn skin explants processed for keratin17 immunostaining after 7 d in culture. Dashed lines indicate the leading edge of keratinocyte outgrowths. Bar, 2 mm. Keratinocyte outgrowth area of wt (white bars) and _galectin-7_−/− (black bars) explants were measured either after 7 d in normal culture medium (bars on the left) or when a 2 h mitomycin-C treatment had been applied on the second day. n, number of animals used. (B) Subcellular distribution of cortactin in lamellopodia of migrating keratinocytes in wt and _galectin-7_−/− explants. On day 7, cultures were fixed, permeabilized and stained with anti-cortactin antibody. Nuclei were detected by Hoechst 33342 staining. Bar, 20 μm.
Figure 7.
Confocal analysis of galectin-7 distribution in leading edge keratinocytes. Double immunostainings with anti-galectin-7 Ab combined with anti-β-tubulin, anti-β1-integrin, anti-cortactin, or anti-Lamp1 Abs were performed on wt skin explants fixed after 7 d in culture. Pictures were taken in the focal plane of cell adhesion structures. Bar, 10 μm.
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