Pyruvate Kinase M2 Mediates Glycolysis Contributes to Psoriasis by Promoting Keratinocyte Proliferation


doi: 10.3389/fphar.2021.765790.


eCollection 2021.

Affiliations

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Yun-Zi Liu et al.


Front Pharmacol.


.

Abstract

Psoriasis is characterized by keratinocyte proliferation and immune cell infiltration. M2 isoform of pyruvate kinase (PKM2) was reported to have an important role in cell proliferation, which is a rate-limiting enzyme that regulates the final step of glycolysis. However, how PKM2 regulates cell metabolism and proliferation in psoriatic keratinocytes is still poorly understood. Interestingly, we found that PKM2 was highly expressed in psoriatic epidermis from patients and mouse models. PKM2 overexpression promoted keratinocyte glycolytic metabolism while knockdown inhibited keratinocyte proliferation and glycolysis. Mice lacking PKM2 specifically in keratinocytes, pharmacological inhibition of PKM2 or glycolysis inhibited keratinocyte proliferation and showed obvious remission in an imiquimod-induced psoriatic mouse model. Moreover, the inhibitor of the EGF-receptor blocked EGF-stimulated PKM2 expression and glycolysis in keratinocytes. We identify PKM2 as an upregulated gene in psoriasis. PKM2 is essential in keratinocyte over-proliferation and may represent a therapeutic target for psoriasis.


Keywords:

glycolysis; keratinocyte; psoriasis; pyruvate kinase M2; shikonin.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures


FIGURE 1



FIGURE 1

M2 isoform of pyruvate kinase (PKM2) expression is elevated in human psoriatic skin. (A)
PKM is frequently upregulated in human psoriasis lesional skin. Scatterplot showing PKM mRNA levels from psoriasis lesional skin and their corresponding normal tissues from the GEO datasets. The fold change between mean psoriasis lesional and mean normal skin expression is shown in the bar plot on a log 2-transformed scale. (B) Immunohistochemical staining of serial sections of human skin tissues with antibodies against PKM2 and PKM1. Scale bar: 100 μm. (C) Protein levels of PKM2 and PKM1 in the lesions of psoriasis patients and healthy controls. HT-29 is a colorectal adenocarcinoma cell line; Mouse muscle lysate was included as a control for PKM1 antibody staining; Mouse liver lysate was included as a negative control for PKM1 and PKM2 antibody staining. GAPDH was used as loading control.


FIGURE 2



FIGURE 2

PKM2 expression is elevated in imiquimod (IMQ)-induced psoriatic mouse skin. IMQ or control cream (vaseline) was applied daily to the shaved backs for female BALB/c mice. (A) Phenotypical presentation of mouse back skin after 5 days of control cream (left panel) or IMQ treatment (right panel). (B) The skin thickness was measured on the days indicated. Significant differences are indicated, n = 7 per group (mean ± SEM), p < 0.01. (C) Light microscopy examination of skin sections stained with H&E, or with Abs against the PKM2 and PKM1. Scale bars, 100 μM. H&E, haematoxylin and eosin. (D) Representative immunoblotting of PKM2 from skin samples of control cream or IMQ treated mice on day 5. (E) Serum lactate level is elevated in IMQ treatment mice compared to control cream on day 5 (n = 7, p < 0.001). GAPDH was used as loading control. IMQ, Imiquimod.


FIGURE 3



FIGURE 3

PKM2 play a critical role in the keratinocyte glucose metabolism and proliferation. (A) Immunoblotting of HaCaT cell stably expressing Flag-PKM2. Cells were infected with lentivirus containing the empty vector (GFP) or Flag-PKM2 (MOI = 40). The cells were then selected in puromycin (2 μg/ml) for 1 week. Total cell extracts were immunoblotted with antibodies for PKM1, PKM2, Flag, and GAPDH. (B, C) Lactate production and glucose consumption were measured in HaCaT-GFP and HaCaT-Flag-PKM2 cells. (D) Efficiency of PKM2 shRNA knockdown. Immunoblot analyses indicate ∼70% reduction of PKM2 in PKM2 shRNA-expressing cells compared with control shRNA. (E, F) Lactate production and glucose consumption were measured in HaCaT-control shRNA and HaCaT-PKM2 shRNA cells. (G) Proliferation curves of the PKM2 knockdown and control cells. (H) Immunoblotting of NHEK treated with Flag-PKM2 lentivirus or PKM2 shRNA adenovirus. (I, J) Lactate production and glucose consumption were measured in NHEK. NHEK were transfected with Flag-PKM2 lentiviruses or PKM2 shRNA adenovirus. NHEK: normal human epidermal keratinocytes. Data are means ± SEM (n = 6). GAPDH was used as loading control.


FIGURE 4



FIGURE 4

Effects of shikonin and 2-DG on the keratinocyte glucose metabolism and proliferation. (A) The lactate production in the supernatants of HaCaT cells after stimulated with different concentrations of shikonin or 2-DG for 1 h was determined using a Lactate Assay kit. (B) HaCaT cells were stimulated with different concentrations of shikonin for 1 h and the glucose consumption in the supernatants was analyzed. (C, D) The lactate production and glucose consumption in the supernatants of HaCaT cells after stimulated with shikonin or 2-DG for indicated times were determined. (E) Intracellular ATP levels following treatment with shikonin (10 μM) or 2-DG (10 mM). ATP concentration was quantified using a luciferase based ATP assay. (F) The effects of shikonin and 2-DG on cell proliferation of HaCaT cells were determined by CCK-8 assay. (G) Effects of shikonin and 2-DG on lactate production of NHEK cells. (H) The glucose consumption in NHEK cells following shikonin treatment. The final DMSO concentration was 0.1% DMSO (v/v). 2-DG, 2-deoxy-d-glucose; NHEK, normal human epidermal keratinocytes. Data are means ± SEM (n = 6).


FIGURE 5



FIGURE 5

PKM2 deficiency in keratinocytes reduced cutaneous signs and epidermal thickness on the IMQ-induced psoriasis mouse model. (A) Breeding strategy to develop PKM2flox/flox K14-Cre mouse. (B) PCR showing genotyping of PKM2flox/flox and PKM2flox/flox K14-Cre mice. Band in the upper image indicates the product from PKM2flox/flox, and lower image indicates product from K14-Cre. (C) Representative images of the dorsal skin after IMQ treatment for 5 days. (D) The skin thickness was measured after IMQ treatment for 5 days n = 6 per group. (E) Representative images of skin sections stained with H&E or the indicated antibodies from mice of the indicated genotypes after IMQ treatment for 5 days. Arrows indicate the epidermis. Nuclei were stained with DAPI (blue). Scale bar, 200 µm.


FIGURE 6



FIGURE 6

Therapeutic effect of Shikonin or 2-DG on the IMQ-induced psoriasis mouse model. Topical treatment of IMQ cream (5%) for 5 d on the shaved back skins of BALB/C mice. Vaseline was used as a control. Skin samples were collected before euthanasia. (A) Pictures of the back skin of the mice before euthanasia. (B) The skin thickness was measured on the days indicated. Significant differences are indicated, n = 7 per group (mean ± SEM), p < 0.01. (C) Serum lactate level was measured on day 5 (n = 6). (D) Pathological analysis with H&E staining of the back skin samples of mice from different treatment groups. IMQ, Imiquimod; 2-DG, 2-deoxy-d-glucose.


FIGURE 7



FIGURE 7

EGF induces the PKM2 up-regulation and glycolysis in the keratinocyte. (A) HaCaT cells were pre-treated with or without AG1478 (1 μΜ) for 2 h, and followed incubation of EGF (100 ng/ml) for 24 h. The cell lysates were collected and PKM1, PKM2, ERK, and p-ERK were detected by Western blot analysis. (B) HaCaT cells were treated with EGF (100 ng/ml) for 24 h. Immunofluorescence analyses were performed with the indicated antibodies. Nuclei were stained with Hoechst 33,258 (blue). Bar = 50 μm. (C, D) Cultured HaCaT cells were treated with or without EGF (100 ng/ml) for 24 h in the presence or absence of AG1478 (1 μΜ). Lactate production and glucose consumption were measured in the cell medium. (E) NHEK cells were incubated with EGF (100 ng/ml) for 24 h. The cell lysates were collected and PKM2 were detected by Western blot analysis. (F, G) Lactate production and glucose consumption in NHEK cells following EGF treatment (100 ng/ml) for 24 h. (H) Summary of PKM2-dependent reprogramming of central carbon metabolism and proliferation in psoriatic keratinocyte. GAPDH was used as loading control.

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