Glutaminase inhibitors rejuvenate human skin via clearance of senescent cells: a study using a mouse/human chimeric model

Abstract

Skin aging caused by various endogenous and exogenous factors results in structural and functional changes to skin components. However, the role of senescent cells in skin aging has not been clarified. To elucidate the function of senescent cells in skin aging, we evaluated the effects of the glutaminase inhibitor BPTES (bis-2-(5-phenylacetamido-1, 3, 4-thiadiazol-2-yl)ethyl sulfide) on human senescent dermal fibroblasts and aged human skin. Here, primary human dermal fibroblasts (HDFs) were induced to senescence by long-term passaging, ionizing radiation, and treatment with doxorubicin, an anticancer drug. Cell viability of HDFs was assessed after BPTES treatment. A mouse/human chimeric model was created by subcutaneously transplanting whole skin grafts from aged humans into nude mice. The model was treated intraperitoneally with BPTES or vehicle for 30 days. Skin samples were collected and subjected to reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blotting, and histological analysis. BPTES selectively eliminated senescent dermal fibroblasts regardless of the method used to induce senescence; aged human skin grafts treated with BPTES exhibited increased collagen density, increased cell proliferation in the dermis, and decreased aging-related secretory phenotypes, such as matrix metalloprotease and interleukin. These effects were maintained in the grafts 1 month after termination of the treatment. In conclusion, selective removal of senescent dermal fibroblasts can improve the skin aging phenotype, indicating that BPTES may be an effective novel therapeutic agent for skin aging.

Keywords: aging; glutaminase inhibitor; human skin; senescent cell; therapeutic agent.

Figure 1

Increased aging-related markers in aged HDFs. (A) SA-β-Gal-stained image. Bar = 50 μm. (B) Percentage of SA-β-Gal-positive cells. (C) Percentage of BrdU-FITC-positive cells determined using flow cytometry. (D) Comparison of relative gene expression of p16 and p21. *p < 0.05. (E) Comparison of protein expression of p16 and p21. (F) Immunostaining of p16 and p21. Bar = 50 μm. All results are expressed as mean ± SEM. All experiments were independently repeated in triplicate. NS: non-senescence.

Figure 2

Selective removal of senescent HDFs by treatment with BPTES. After induction of cellular senescence, cells were treated with BPTES at the indicated concentrations for 72 h (n = 4). Cell viability was measured to determine the effect of the senescent cell-removal drugs. Histograms were compared to the DMSO control. The graph shows the mean ± SEM of three independent experiments; *p < 0.05, Mann–Whitney U test; NS: non-senescence.

Figure 3

Removal of SA-β-Gal-positive cells from human skin grafts by BPTES treatment. (A) Skin tissue stained with SA-β-Gal (blue); Bar = 100 μm. (B) Quantification of SA-β-Gal-positive cells in the dermis, excluding the area of the skin appendage. The number of positive cells was counted in high magnification field of view (HPF) (≥5 fields of view per sample). Graphs show the mean ± SEM of three independent experiments. *p < 0.05.

Figure 4

BPTES treatment promotes the removal of skin senescent cells and cell proliferation from human skin grafts. (A) Immunofluorescence staining for p16 or Ki67 in tissue sections of human skin grafts from pre-transplanted aged human skin, treated with control (DMSO) or BPTES. Red: p16, Ki67; Blue: DAPI. The percentage of p16- or Ki67-positive dermal cells at a depth of 100 μm from the epidermal basement layer was counted with DAPI. All results are expressed as mean ± SEM. *p < 0.05. Bar = 100 μm. (B) Relative p16 and p21 mRNA levels were analyzed using RT-qPCR in skin sections of the indicated mice. *p < 0.05. (C) Western blot analysis of p16 and p21. (D) Quantitative analysis of western blot. Band densities normalized against endogenous control GAPDH are shown. *p < 0.05. Graph shows mean ± SEM of three independent experiments.

Figure 5

Effect of senescent cell-depleting agents on SASP expression in aged human skin grafts. Relative mRNA levels of genes associated with SASP in mouse skin measured using qRT-PCR. Data were normalized against mRNA obtained from young human skin; GAPDH was used as endogenous control. All results are expressed as mean ± SEM. *p < 0.05.

Figure 6

Senescent cell elimination drug BPTES improves the skin senescence phenotype of transplanted aged human skin. (A) Evaluation of skin collagen deposition using representative images of MT and H&E staining. (B) Quantification of collagen density in skin sections from the indicated mice. Data represent three or more random sites in each section (n = 3–5). (C) Relative mRNA levels of Col1a1 in the skin from aged mice. *p < 0.05, Mann–Whitney U test; bar = 100 μm.