Low levels of glutathione are sufficient for survival of keratinocytes after UV irradiation and for healing of mouse skin wounds

Reduced levels of the cellular antioxidant glutathione are associated with premature skin aging, cancer and impaired wound healing, but the in vivo functions of glutathione in the skin remain largely unknown. Therefore, we analyzed mice lacking the modifier subunit of the glutamate cysteine ligase (Gclm), the enzyme that catalyzes the rate-limiting step of glutathione biosynthesis. Glutathione levels in the skin of these mice were reduced by 70 %. However, neither skin development and homeostasis, nor UVA- or UVB-induced apoptosis in the epidermis were affected. Histomorphometric analysis of excisional wounds did not reveal wound healing abnormalities in young Gclm-deficient mice, while the area of hyperproliferative epithelium as well as keratinocyte proliferation were affected in aged mice. These findings suggest that low levels of glutathione are sufficient for wound repair in young mice, but become rate-limiting upon aging.ISSN:0340-3696ISSN:1432-069

<i>Ko</i>^<i>G</i> mice are viable, but fail to gain weight after weaning.

<p>(A) Scheme of the skin. Keratinocytes of the epidermis and the hair follicles/sebaceous glands, which are affected by the knockout, are shown in blue. Mice heterozygous for the <i>K5Cre</i> transgene and homozygous for the floxed <i>Gclc</i> allele (ho-Gclc^floxed) lack Gclc in keratinocytes (<i>ko</i>^<i>G</i> mice) (B) qRT-PCR of <i>Gclc</i> relative to <i>Rps29</i> using RNA from primary keratinocytes (1°KC; N = 6) or epidermis (3W N = 5; 2M N = 7). (C) Total GSH/GSSG levels (GSH_T) in 1°KC (N = 6/4) and epidermis (3W N = 5; 2M N = 3). (D) Western blot with lysates from epidermis (pooled from 2 mice per genotype), total skin, and liver for Gclc and Gapdh (loading control). (E) Macroscopic appearance of <i>ko</i>^<i>G</i> and <i>ctrl</i> mice at 3W and 2M. (F) Body weight of <i>ctrl</i> and <i>ko</i>^<i>G</i> mice surviving until 2M. Arrow indicates **<i>P</i> ≤ 0.01 for each time point after P23. N = 7/5. (G) Kaplan-Meier survival curves of <i>ko</i>^<i>G</i> versus <i>ctrl</i> mice. N = 14. Significance was analyzed using Log-rank test. *<i>P</i> ≤ 0.05. (H) Hematoxylin/eosin (H&E) staining of stomach sections from mice at 2M. Scale bar: 1 mm. Squares indicate the area shown at higher magnification on the right side. Note the hyperkeratosis in the forestomach of <i>ko</i>^<i>G</i> mice. (I) Picture of the stomach from <i>ko</i>^<i>G</i> and <i>ctrl</i> mice at 2M. Scale bar: 0.5 cm. Scatter plots show the median with interquartile range.</p

Gclc deficiency in keratinocytes causes cell damage through increased levels of ROS and RNS.

<p>(A,B) Representative histograms and quantification of relative median of relative fluorescence units (RFU) of (A) H₂DCF-DA (N = 10/6) and (B) DAF-FM-DA (N = 13/11) assay of primary keratinocytes from <i>ctrl</i> and <i>ko</i>^<i>G</i> mice. (C) Quantification of RFU of H₂DCF-DA assay of primary keratinocytes from <i>ctrl</i>, <i>ko</i>^<i>G</i> and <i>ko</i>^<i>G/N</i> mice. N = 11/4/6. (D) Representative histogram and quantification of a lipid peroxidation assay with the lipid peroxidation sensor C11-BODIPY^581/591 (N = 8/5). (E-G) Immunofluorescence staining of primary keratinocytes and quantification of Ki-67 (E; N = 14/7/7), cleaved caspase-3 (F; N = 16/7/5)) and γH2AX (G; N = 15/11/4) positive cells. Scale bar: 50 μm. (H) Schematic representation of the experimental setup for (I,J). (I) Percentage of primary keratinocytes from individual mice after 24 h, 48 h and 96 h of cultivation in normal medium relative to number of cells at t = 0. N = 6. (J) AlamarBlue cell viability assay after treatment of primary keratinocytes from control or <i>ko</i>^<i>G</i> mice with DMSO (vehicle), Z-VAD, Fer-1, Nec-1 or 3MA in DMSO for 24 h. N = 12/11, analyzed in three independent experiments. Scatter plots show the median with interquartile range. *<i>P</i> ≤ 0.05, **<i>P</i> ≤ 0.01, ***<i>P</i> ≤ 0.001. (K) Electron microscopy of the epidermis at 3W. Left and middle panel: Keratinocytes with signs of ferroptotic cell death, including dysmorphic mitochondria and small mitochondria with increased membrane density and/or membrane accumulation. Right panel: Keratinocyte with signs of necroptotic cell death, including nuclear condensation, vacuolization and presence of autophagosomes. N = nucleus; arrowheads: autophagosomes; arrow: mitochondria. Scale bars: 5 μm (left panel), 700 nm (middle panel), or 3 μm (right panel).</p

Cell damage and apoptosis in the hyperproliferative wound epithelium of Gclc-deficient mice.

<p>(A,B) Immunofluorescence-stained sections of 2-, 3- and 5-day wounds and quantification of (A) cleaved caspase-3 (arrowheads; arrows: positive cells in the adjacent epidermis), and (B) γH2AX positive cells per area HE. White dotted lines show the area of HE in which positive cells were counted. Each picture includes a magnification of the indicated square to show positive cells (if existing). Scale bar: 100 μm. For (A): 2dw N = 6; 3dw N = 6; 5dw N = 10/8. For (B): 2dw N = 4; 3dw N = 7/8; 5dw N = 5. (C) OxyBlot of lysates (pooled from 2 wounds of individual mice) from 3-day wounds. Equal loading was confirmed by Ponceau S staining of the membrane. (D) H&E staining on sections from 3-day wounds. Scale bar: 200 μm. D: dermis Es: eschar, G: granulation tissue, HF: hair follicle, HE: hyperproliferative epithelium. (E-G) Morphometric analysis of (E) percentage wound closure, (F) length HE and (G) area HE of 3-day wounds of <i>ko</i>^<i>G/N</i> and control mice. For (E): N = 6/7; for (F): N = 7/8; for (G): N = 7/8. (H-J) Quantification of (H) Ki-67 (N = 4), (I) cleaved caspase-3 (N = 3/4), and (J) γH2AX (N = 6/5) positive cells per area HE. Scatter plots show the median with interquartile range. *<i>P</i> ≤ 0.05. n.d.: not detectable.</p

Slc7a11 and the thioredoxin/thioredoxin reductase system partially compensate for the loss of Gclc.

<p>(A-C) qRT-PCR of <i>Slc7a11</i> (A), <i>Txn1</i> (B) and <i>Txnrd1</i> (C) relative to <i>Rps29</i> using RNA form the epidermis at 3W (N = 7/8/6/8 for <i>Slc7a11</i>; N = 8/8/7/8 for <i>Txn1</i>, and N = 7/4/7/4 for <i>Txnrd1</i>) and from primary keratinocytes (N = 6). (D) Schematic representation of the experimental setup for (F,G). (E) Schematic representation of the mechanisms of action of the chemical inhibitors used in this study. SSZ inhibits the import of cystine by the xCT transporter, AUR inhibits the catalytic activity of Txnrd, and BSO inhibits the catalytic activity of Gcl. (F) Percentage of primary keratinocytes from individual mice treated with DMSO (vehicle), SSZ or AUR in DMSO for 24 h. N = 6. (G) AlamarBlue cell viability assay after treatment of primary keratinocytes from control mice with DMSO (vehicle), AUR, and/or BSO in DMSO for 24 h. N = 10, analyzed in three independent experiments. Scatter plots show the median with interquartile range. *<i>P</i> ≤ 0.05, **<i>P</i> ≤ 0.01, ***<i>P</i> ≤ 0.001. (H) Schematic representation of the GSH, Txn/Txnrd and Nrf2 antioxidant defense systems and their cross-talk in the epidermis. xCT (Slc7a11), which is stabilized by the CD44 protein, mediates import of cystine and concomitant export of glutamate. Cystine is reduced in the cytoplasm by GSH and most likely by Txn1 or thioredoxin-related protein 14 (Trp14). Oxidized Txn1 or Trp14 are reduced by Txnrd1. Cysteine is used for GSH biosynthesis or incorporated into proteins, including Txn1. Nrf2 is activated upon GSH deficiency and induces the expression of xCT, of enzymes involved in GSH production and recycling (Gclc, gclm, glutathione synthetase (GSS), glutathione reductase (GR)) and of various ROS-detoxifying and antioxidant proteins. Txn/Txnrd, GSH and Nrf2 activities result in a reduction of intracellular ROS and RNS, thereby preventing DNA and protein damage and ultimately cell death.</p

Keratinocyte hyperproliferation and mild inflammation in the skin of <i>ko</i>^<i>G</i> mice.

<p>(A) Scheme of the skin visualizing the region analyzed in (B-E) and the different cell types of the skin that were analyzed in this study. (B) Immunofluorescence staining of skin sections for Ki-67 and quantification of positive cells per length epidermis (3W N = 12; 2M N = 5). Scale bar: 20 μm. (C) Toluidine blue staining of mast cells and quantification of positive cells per area dermis (3W N = 9/10; 2M N = 7/6). Scale bar: 10 μm. (D,E) Immunohistochemistry of the T cell marker CD3 (3W N = 7/7; 2M N = 7) and immunofluorescence for the γδ T cell receptor (3W/2M N = 6). The number of positive cells per length epidermis was determined. Scale bar: 10 μm. White/black dotted lines in (B,D,E) indicate the basement membrane. (F) Schematic representation of the gating strategy used for the detection of epidermal immune cells by flow cytometry. (G-K) Flow cytometry analysis of epidermal cells from mice at 3W or 2M using markers for different types of immune cells and their activation status (3W N = 8/6; 2M N = 7/6). (L-P) qRT-PCR of <i>Tnfα</i>, <i>Tslp</i>, <i>Il6</i>, <i>S100A8</i> and <i>Ptgs2</i> (N = 3–6) relative to <i>Rps29</i> using RNA from the epidermis at 3W and 2M. For <i>Ptgs2</i>, which was hardly detectable in the normal epidermis, epidermis from wounded skin (3 days after wounding) from ctrl and <i>ko</i>^<i>G</i> mice was used as positive control. Scatter plots show the median with interquartile range. *<i>P</i> ≤ 0.05, **<i>P</i> ≤ 0.01, ***<i>P</i> ≤ 0.001.</p

Nrf2 is activated in <i>ko</i>^<i>G</i> mice, but does not compensate for the loss of Gclc.

<p>(A,B) qRT-PCR of <i>Nqo1</i>, <i>Srxn1</i>, <i>Slpi</i> and <i>Sprr2d</i> relative to <i>Rps29</i> using RNA from (A) the epidermis at 3W (N = 5) and from (B) primary keratinocytes (N = 11/9). (C) Scheme of the skin from mice of all four genotypes used for the analysis. Keratinocytes affected by the knockout are shown in the respective color. (D) qRT-PCR of <i>Gclc</i> and <i>Nrf2</i> relative to <i>Rps29</i> using RNA from epidermis at 3W (left panel; N = 8/8/7/8) or primary keratinocytes of control and <i>ko</i>^<i>G/N</i> mice (right panel; N = 8). (E,F) qRT-PCR of <i>Nqo1</i>, <i>Srxn1</i>, <i>Slpi</i>, and <i>Sprr2d</i> relative to <i>Rps29</i> using RNA from the epidermis at 3W (N = 7-8/7-8/5-6/7-8) and of <i>Nqo1</i>, <i>Srxn1</i>, <i>Slpi</i> relative to <i>Rps29</i> using RNA from primary keratinocytes (N = 15/8/7). (G) Macroscopic appearance of mice of all four genotypes at 3W. (H) H&E staining of longitudinal skin sections from mice at 3W and immunofluorescence staining for epidermal differentiation markers. Nuclei were counterstained with Hoechst. Scale bars: 40 μm. (I) Immunofluorescence staining of skin sections for Ki-67 and quantification of positive cells per length epidermis at 3W (N = 22/11/6/8). Scale bar: 20 μm. White dotted lines indicate the basement membrane. (J) TEWL of mice at 3W (N = 25/11/7/8). (K) Body weight of mice at 3W (N = 26/11/5/11). (L) H&E staining of skin from <i>ko</i>^<i>G/N</i> mice at 3W demonstrating malformed hair follicles and cyst formation. The number of abnormal hair follicles per length epidermis is shown in the bar graph (N = 9/9/6/8; n.d. = not detectable). Scale bar: 40 μm. Scatter plots show the median with interquartile range. *<i>P</i> ≤ 0.05, **<i>P</i> ≤ 0.01, ***<i>P</i> ≤ 0.001.</p

Mild wound healing abnormalities in <i>ko</i>^<i>G</i> mice.

<p>(A) Schematic representation of a 3–5 day wound showing the parameters for morphometric analysis: percentage of wound closure = (I+III/I+II+III)*100; length HE = average length I+III; area HE = average area IV+V. D: dermis Es: eschar, G: granulation tissue, HF: hair follicle, HE: hyperproliferative epithelium. (B) Immunofluorescence staining of K14 (green) and Hoechst counterstaining (blue) on sections from 3- and 5-day wounds. Scale bar: 100 μm. (C-E) Morphometric analysis of (C) percentage of wound closure, (D) length HE, and (E) area HE of 2-, 3- and 5-day wounds. For (C): 2dw N = 4/7; 3dw N = 6/5; 5dw N = 9/12. For (D): 2dw N = 5/7; 3dw N = 6/5; 5dw N = 11/14. For (E): 2dw N = 5/7; 3dw N = 6/5; 5dw N = 11/14. (F) Schematic representation of the HE with the region of the cells that were analyzed in (G). (G) Immunofluorescence-stained sections of 2-, 3- and 5-day wounds were used for quantification of Ki-67 positive cells per area HE. White dotted lines show the area of HE in which positive cells were counted. Each picture includes a magnification of the indicated square to show positive cells. Scale bar = 100 μm. 2dw N = 4; 3dw N = 7/8; 5dw N = 5. Scatter plots show the median with interquartile range. **<i>P</i> ≤ 0.01.</p

Progressive skin abnormalities in <i>ko</i>^<i>G</i> mice.

<p>(A) H&E staining of longitudinal skin sections from mice at 3W and 2M and immunofluorescence staining for epidermal differentiation markers. Smaller pictures within the <i>ko</i>^<i>G</i> panel show patches of interfollicular K6. Nuclei were counterstained with Hoechst. Scale bars: 200 μm (H&E) or 40 μm (immunofluorescence). Note the scaling in <i>ko</i>^<i>G</i> mice. The appropriate expression pattern of the differentiation markers and their function in the epidermis are shown schematically in (B). K = Keratin, Flg = Filaggrin, Lor = Loricrin. (C) Thickness of the viable epidermis (all layers except the stratum corneum). 3W (N = 6) and 2M (N = 6/7). (D,E) qRT-PCR of <i>Lor</i> (N = 5/4) and <i>Flg</i> (N = 5) relative to <i>Rps29</i> using RNA from the epidermis at 3W and 2M. (F) Western blot of epidermal lysates at 3W for Lor, Flg and <i>α</i>-Tubulin (loading control). Shown is one representative sample for each genotype (N = 4). (G) Electron microscopy of the <i>stratum corneum</i> at 3W showing reduced adhesiveness of the corneocytes in the epidermis of <i>ko</i>^<i>G</i> mice. Scale bars: 5.5 μm (left panels) or 2.5 μm (right panels). (H) TEWL of mice at 3W (N = 8/9) and 2M (N = 12/8). Scatter plots show the median with interquartile range. *<i>P</i> ≤ 0.05, **<i>P</i> ≤ 0.01.</p