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Skin chronological aging drives age-related bone loss via secretion of cystatin-A

Nature Aging volume 2, pages 906–922 (2022)Cite this article

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An Author Correction to this article was published on 26 June 2023

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Abstract

Although clinical evidence has indicated an association between skin atrophy and bone loss during aging, their causal relationship and the underlying mechanisms are unknown. Here we show that premature skin aging drives bone loss in mice. We further identify that cystatin-A (Csta), a keratinocyte-enriched secreted factor, mediates the effect of skin on bone. Keratinocyte-derived Csta binds the receptor for activated C-kinase 1 in osteoblast and osteoclast progenitors, thus promoting their proliferation but inhibiting osteoclast differentiation. Csta secretion decreases with skin aging in both mice and humans, thereby causing senile osteoporosis by differentially decreasing the numbers of osteoblasts and osteoclasts. In contrast, topical application of calcipotriol stimulates Csta production in the epidermis and alleviates osteoporosis. These results reveal a mode of endocrine regulation of bone metabolism in the skin, and identify Csta as an epidermally derived hormone linking skin aging to age-related bone loss. Enhancers of skin Csta levels could serve as a potential topical drug for treatment of senile osteoporosis.

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Fig. 1: Premature skin aging drives bone loss in young mice.
Fig. 2: Csta expression and secretion decrease in aged human skin.
Fig. 3: Csta ablation in the epidermis results in low-turnover-osteopenia in mice.
Fig. 4: Csta overexpression in epidermis alleviates bone loss in aged mice.
Fig. 5: Topical calcipotriol alleviates age-related bone loss in mice through increasing Csta production in keratinocytes.
Fig. 6: Csta stimulates osteoblast proliferation via Rack1.
Fig. 7: Csta inhibits osteoclast formation and function via Rack1.
Fig. 8: Csta competes with Src for binding Rack1.

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Data availability

Cytokine array data and mass spectrometry data are provided in Supplementary Data 1 and 2 and Supplementary Information. All other data are provided within the article, Source Data and Supplementary Information or are available from the corresponding author upon reasonable request.

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Acknowledgements

This research was supported by grant nos. 82172507 (B.H.), 81700783 (B.H.) and 81991511 (X.B.) from the National Natural Science Foundation of China, nos. 2019A1515011876 (B.H.) and 2018A030313937 (Z.L.) from Guangdong Natural Science Fund Management Committee and no. 202002030176 (B.H.) from Guangzhou Municipal Science and Technology Bureau. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Author notes

  1. These authors contributed equally: Wenquan Liang, Qingjing Chen, Shasha Cheng.

Authors and Affiliations

  1. Academy of Orthopedics, Guangdong Province, Guangdong Provincial Key Laboratory of Bone and Joint Degeneration Diseases, Department of Orthopedics, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China

    Wenquan Liang, Qingjing Chen, Ruiming Wei, Chenfeng Yao, Zhicong Ouyang, Dawei Kang, Ajuan Chen, Zezheng Liu, Kai Li, Qingchu Li & Bin Huang

  2. Department of Clinical Laboratory, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China

    Shasha Cheng

  3. Department of Orthopedics, The Second People’s Hospital of Panyu District, Guangzhou, China

    Yuejun Li

  4. Department of Cell Biology, School of Basic Medical Science, Southern Medical University, Guangzhou, China

    Xiaochun Bai

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  1. Wenquan Liang
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Contributions

B.H. conceived the ideas for experimental designs and wrote the manuscript. W.L., Q.C. and S.C. conducted the majority of the experiments, analyzed data and helped with manuscript preparation. R.W. and Y.L. maintained mice, collected tissue samples and performed micro-CT analyses. C.Y. and Z.O. conducted cell cultures and immunoblot experiments. D.K., A.C., Z.L. and K.L. performed immunohistochemistry, immunofluorescence and confocal imaging. X.B. and Q.L. developed the concept, supervised the project and critically reviewed the manuscript.

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Correspondence to Bin Huang.

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Extended data

Extended Data Fig. 1 Chemokines and cytokines detected in the serum from young and older male patients with a cytokine antibody array.

a, Scatter plot showing the distribution levels of the 640 cytokines detected in the serum of the patients. Red dots show cytokines up-regulated ≥1.2-fold from older relative to young patients; blue dots show cytokines down-regulated ≥1.2-fold. b, Volcano plot illustrating the magnitude and significance of the differences in cytokine serum concentrations in the patients. Green dots are cytokines with a significantly different serum concentration between groups (True, fold change ≥1.2, p < 0.05), red dots were not different (False).

Extended Data Fig. 2 Skin presents the most abundant Csta expression among tissues in the body.

a, Immunofluorescence staining of Csta in the indicated tissues of adult mice. Dashed lines, basement membrane (skin) or boundary between growth plate and bone marrow (bone). Arrowheads, positively stained cells. BM, bone marrow. Scale bars, 100 μm. Experiments were repeated six times with similar results. b, Real-time PCR analysis of Csta expression levels in the indicated tissues from wild-type mice. Data are presented as mean ± s.d. The number of biological replicates per group is indicated.

Source data

Extended Data Fig. 3 Csta expression and secretion decreases in the skin of aged mice.

a, Immunohistochemical staining of Csta in the skin of young (3 months old) and aged (20 months old) wild-type male mice. Bottom, magnified views of dashed boxed areas. Scale bar, 100 μm. b, c, Protein and mRNA levels of Csta in the mouse skin were analysed by western blotting and qPCR, respectively. d, ELISA analysis of Csta levels in the serum and bone marrow in the mice. e, f, H&E staining in sections of the mouse skin, and quantification of epidermal thickness and cell numbers. Bottom, magnified views of boxed areas (e). Scale bar, 100 μm. g-j, Immunostaining of p16 (cell senescence markers) (g) and Ki-67 (i) (cell proliferation marker) in sections of mouse skin. Arrowheads, positively stained cells. Epidermal cells positively stained by the markers are quantified as their numbers as a percentage of total cells (h, j). Scale bar, 100 μm. k, l, Representative images of micro-CT analyses and quantitative analysis of trabecular and cortical bone microstructure of metaphyseal in the distal femurs from the mice. Scale bar, 500 μm. m, Individual correlations between serum Csta levels and trabecular and cortical bone mass in 21 aged (20-month-old) wild-type mice. Dashed lines, basement membrane. P values were calculatedwith two-tailed Student’s t-test, and all data are presented as mean ± s.d. The number of biological replicates per group and P values are indicated. Experiments were repeated six times (a, e, k), three times (b), nine times (g), and six or seven times (i), with similar results.

Source data

Extended Data Fig. 4 Diminished Csta levels in ovariectomized mice.

a, Fatty body of ovariectomized (OVX) mice relative to their Sham controls. b, Atrophic uterus due to oestrogen deficiency in OVX mice. c, d, Representative images of micro-CT analyses and quantitative analysis of trabecular and cortical bone microstructure of metaphyseal in the distal femurs from the mice. Scale bar, 500 μm. e, f, H&E staining in sections of mice skin, and quantification of epidermal thickness and cell numbers. Scale bar, 100 μm. g, h, Immunohistochemical staining and quantification of the staining intensity of Csta in mice skin in the two groups. Scale bar, 100 μm. i, ELISA analysis of Csta levels in mice serum and bone marrow supernatants. P values were calculated with two-tailed Student’s t-test, and all data are presented as mean ± s.d. The number of biological replicates per group and P values are indicated. Experiments were repeated five times (c) and six times (e, g) with similar results.

Source data

Extended Data Fig. 5 Establishment of epidermis specific and inducible Csta knockout mice.

a, Structure G/Rflox transgenes and schematic diagram of the G/Rflox construct before and after Cre-mediated recombination. G/R consists of a CAG promoter driving a loxP flanked coding sequence of green fluorescent protein (ZsGreen). After Cre-mediated intra-chromosomal recombination, the ZsGreen sequence is excised, thus allowing the promoter to drive expression of the membrane-targeted tandem dimer Tomato (tdTomato). b, G/Rflox transgenic mice were topically treated with AAV2-Cre (G/RAAV2-Cre) or AAV2 (G/RAAV2, control) vector on a patch of dorsal skin once per 7 days for 1 month. The fluorescence of the indicated tissues was observed to monitor AAV2-Cre recombinase activity. AAV2-Cre recombinase was activated in the skin of G/Rflox mice but not in other tissues. Dashed lines, basement membrane; arrowheads, tdTomato-positive regions. Right images are magnified views of the boxed areas (bone). Scale bars, 100 μm. Experiments were repeated five times with similar results.

Extended Data Fig. 6 Csta knockout causes epidermal thinning in mice.

Two-month-old male Cstafl/fl mice were topically treated with AAV2-Cre (CstaAAV2-Cre) or AAV2 (CstaAAV2, control) on shaved patches on dorsal skin once per 7 days for 1 month. The appearance of patches on dorsal skin was photographed (a). b, c, H&E staining in sections of the skin patches, and quantification of epidermal thickness and cell numbers. Bottom, magnified views of boxed areas (b). d, e, Immunostaining of Ki-67 and quantification of Ki-67 positive cells as a percentage of total epidermal cells. Bottom, magnified views of boxed areas (d). f, g, Immunostaining of K10 (a marker for keratinocyte differentiation) and K14 and quantification of K10 positive cells as a percentage of total epidermal cells. Scale bars, 50 μm. P values were calculated with two-tailed Student’s t-test, and all data are presented as mean ± s.d. The number of biological replicates per group and P values are indicated. Experiments were repeated nine times (b), six times (d), and eight or nine (f), with similar results.

Source data

Extended Data Fig. 7 Representative images of FACS isolation of murine osteoclast precursor cells.

Cells were first gated by forward scatter (FSC) and side scatter (SSC) to remove doublets. Osteoclast precursor cells were identified by negative gating for CD11b, and positive gating for CD115 and CD117.

Extended Data Fig. 8 Calcipotriol increases Csta production in keratinocytes.

a-d, Representative images of K10 staining, and quantification of K10 positive cells in dorsal skin from young and older male patients (a, b) and mice (c, d). Right, magnified views of dashed boxed areas (a). Dashed lines, basement membrane. Arrowheads, positively stained cells (c). e-g, Primary mouse keratinocytes were induced to differentiate with 0.2 mM CaCl2 and treated with calcipotriol or left untreated for the indicated times. Csta mRNA, protein, and secretion levels in the cells were analysed by qPCR (e), western blotting (f), and ELISA (g), respectively. h, CSTA-luciferase reporter plasmid or empty vector were transfected into HaCaT cells under low Ca2+ (0.06 mM) conditions. After incubation for 24 h, the transfected cells were incubated with and 10–9 M calcipotriol and 50 µmol/L ceramide C6 (ERK activator) for 12 h, and subjected to detection of ERK phosphorylation and luciferase activity. i, Serum calcium levels and body weights in mice receiving topical calcipotriol or vehicle (Control). j, k, Primary mouse keratinocytes were treated with vehicle (Control) or 10-9 M calcipotriol in the presence of the indicated concentrations of CaCl2 and subjected to detection of cell proliferation rates with EdU assays. Scale bars: 50 μm in a, 20 μm in c, and 200 μm in i. P values were calculated with ANOVA or two-tailed Student’s t-test, and all data are presented as mean ± s.d. The number of biological replicates per group and P values are indicated. Experiments were repeated eight times (a), eight or nine times (c), three times (f, h), and 10 times (j), with similar results.

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Extended Data Fig. 9 Csta promotes proliferation of osteoclast precursors via Rack1.

a, b, CstaAAV2-Cre and CstaAAV2 mice were injected intraperitoneally with EdU. After 4 hours, bone marrow cells were isolated freshly from bilateral femurs and tibias, and subjected to flow cytometric analysis of osteoclast precursor proliferation. Representative flow cytometry plots (a) and the frequencies of proliferative osteoclast precursors (EdU+ CD11bdull) in CD117+- and CD115+-gated bone marrow cells (b) are presented. c-i, Raw264.7 cells stably transfected with Flag-Rack1 or lentiviral vector were treated with recombinant Csta or vehicle. c, d, Representative images and quantification of cell proliferation with EdU assays. Scale bar, 100 μm. e-g, Cells were analysed for cell cycle distribution by flow cytometry (e, f) and for expression and activity of G1 cell cycle regulators (g). h, i, Cell apoptosis, assessed by flow cytometry with Annexin V/PI staining. j, Flow cytometric analysis of osteoclast precursor apoptosis in bone marrow cells freshly isolated from bilateral femurs and tibias of CstaAAV2-Cre and CstaAAV2 mice. Frequencies of Annexin V+ apoptotic cells in CD117+-, CD115+- and CD11bdull- gated bone marrow cells are quantified as apoptosis rates of osteoclast precursors. P values were calculated with ANOVA or two-tailed Student’s t-test, and all data are presented as mean ± s.d. The number of biological replicates per group and P values are indicated. Experiments were repeated three times (a, e, g, h, j), and six times (c), with similar results.

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Extended Data Fig. 10 Csta competes with Src for binding Rack1 in osteoblasts.

MC3T3-E1 cells stably transfected with Flag-Rack1 or lentiviral vector were serum-starved for 5 hours, then treated with recombinant GST-Csta, GST or vehicle (Veh) plus 10%FBS for 1 hour. Proteins in cell lysates were precipitated with anti-Rack1 and subjected to immunoblot analysis with anti-Src and anti-GST (a), or precipitated with anti-Src and analyzed for in vitro protein kinase activity against recombinant Stat3 (b). Experiments were repeated three times (a, b) with similar results.

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Supplementary information

Supplementary Information

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Reporting Summary

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Liang, W., Chen, Q., Cheng, S. et al. Skin chronological aging drives age-related bone loss via secretion of cystatin-A. Nat Aging 2, 906–922 (2022). https://doi.org/10.1038/s43587-022-00285-x

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