doi: 10.3389/fphar.2023.1111798.
eCollection 2023.
Affiliations
Item in Clipboard
Front Pharmacol.
.
Abstract
Introduction: Psoriasis is an inflammatory autoimmune skin disease that is hard to cure and prone to relapse. Currently available global immunosuppressive agents for psoriasis may cause severe side effects, thus it is crucial to identify new therapeutic reagents and druggable signaling pathways for psoriasis. Methods: To check the effects of SOCE inhibitors on psoriasis, we used animal models, biochemical approaches, together with various imaging techniques, including calcium, confocal and FRET imaging. Results and discussion: Store operated calcium (Ca2+) entry (SOCE), mediated by STIM1 and Orai1, is crucial for the function of keratinocytes and immune cells, the two major players in psoriasis. Here we showed that a natural compound celastrol is a novel SOCE inhibitor, and it ameliorated the skin lesion and reduced PASI scores in imiquimod-induced psoriasis-like mice. Celastrol dose- and time-dependently inhibited SOCE in HEK cells and HaCaT cells, a keratinocyte cell line. Mechanistically, celastrol inhibited SOCE via its actions both on STIM1 and Orai1. It inhibited Ca2+ entry through constitutively-active Orai1 mutants independent of STIM1. Rather than blocking the conformational switch and oligomerization of STIM1 during SOCE activation, celastrol diminished the transition from oligomerized STIM1 into aggregates, thus locking STIM1 in a partially active state. As a result, it abolished the functional coupling between STIM1 and Orai1, diminishing SOCE signals. Overall, our findings identified a new SOCE inhibitor celastrol that suppresses psoriasis, suggesting that SOCE pathway may serve as a new druggable target for treating psoriasis.
Keywords:
CRAC channel; Orai1; SOCE; STIM1; calcium; celastrol; psoriasis.
Copyright © 2023 Yuan, Tang, Chen, Zhou, Deng, Han, Zhai, Zhou, Gill, Lu and Wang.
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
Celastrol ameliorated skin symptoms and inhibited epidermal hyperplasia in IMQ-induced psoriasis-like mice. (A) Structure of celastrol (top) and the timeline of various treatments (bottom). BALB/c mice were orally administered with celastrol (CEL-L: 10 mg/kg; CEL-H: 20 mg/kg) or MTX (1 mg/kg) for 10 consecutive days during the topical application of 65 mg IMQ on the dorsal skin. All mice were sacrificed on day 10. (B) Representative images of dorsal skin of mice on day 9. (C) Hematoxylin and eosin (H&E) staining of skin tissue from different groups of mice (magnification × 200, scale bar = 50 μm). (D) Epidermal thickness of mouse dorsal skin (****p < 0.0001 vs. IMQ-induced psoriasis group; n = 8). (E) Severity of psoriasis indicated by the psoriasis area and severity index (PASI).
FIGURE 2
Celastrol attenuated the upregulation of proinflammatory cytokines and NFκB signaling in the skin of psoriatic mouse. The mRNA levels of TNF-α (A), IL-6 (B), IL-17 A (C), IL-23 (D) and p65 (E) in the skin of IMQ-induced psoriasis-like mice determined by RT PCR analysis. (F) Representative WESTERN blotting images of p-IKBα, IKBα, p-NFκB p65 and NFκB p65. (G,H) Quantification of the relative expression of p-IKBα/IKBα and p-NFκB p65/NFκB p65, β-actin expression was used to normalize data. Values were expressed as fold changes relative to control group that was set as 1.0. Data of column graphs are presented as the mean ± SD from three separate experiments.
FIGURE 3
Celastrol dose- and time-dependently inhibited SOCE in HaCaT cells and HEK cells. To empty ER Ca2+ stores, prior to recordings, cells were first treated with 1 μΜ TG in Ca2+ free solution for 10 min. (A) The effect of pre-incubation of celastrol on SOCE and proliferation of HaCaT cells. Left, typical SOCE traces (50 μΜ); right, representative proliferation curves (3 μΜ). Doubling time: control, 18.9 ± 0.2 h; celastrol, 21.3 ± 0.3 h (t-test, ****, p < 0.0001, n = 3). (B) Effects of 10-min pre-incubation of celastrol on SOCE in HEK-GCaMP6m cells. Left, representative traces; right, dose response curves. (C) Action of celastrol on SOCE in HEK STIM1-Orai1 overexpressing cells. Prior to imaging, cells were pretreated 10 min (yellow), 30 min (red) with 50 μΜ celastrol or DMSO (black). Left, Typical traces; right, Statistics (t-test, ****, p < 0.0001, n = 3). (D) Effects of acute application of 50 μΜ celastrol on SOCE in HEK GCaMP6m cells. Effects of 50 μΜ 2-APB were used as a positive control. Left, Typical traces; right, Statistics (t-test, ***, p < 0.0001, n = 3).
FIGURE 4
50 μΜ celastrol disrupted functional couplings between Orai1 and STIM1 but had no effect on Ca2+ influxes mediated by Orai1 and SOAR. (A) Typical confocal images showing the effect of 10-min celastrol on the colocalization between STIM1-YFP (green) and Orai1-CFP (red) in HEK STIM1-Orai1 cells. Top panels, store replete cells. Unlike control cells that show even distribution, 10 min-incubation with celastrol induced the formation of small STIM1 puncta at rest (upper images). Bottom panels, store-depleted cells after 10-min bath with 1 μΜ TG. STIM1 in control cells aggregated into large puncta, while STIM1 showed no-further aggregation in celastrol-treated cells. Scale bar, 10 μm. (B) Celastrol’s effects on FRET signals between STIM1-YFP and Orai1-CFP in HEK STIM1-Orai1 cells. Compared to control, pretreatment with celastrol resulted in a rise in basal FRET signal, and a decrease of the FRET signal after store depletion. Store-emptying ionomycin failed to induce any further increase in celastrol-treated cells. (C,D) Celastrol did not inhibit the constitutive Ca2+ entry mediated in HEK SK cells transiently expressing Orai1-SS, or co-expressing Orai1 and SOAR. Left, Typical traces; right, Statistics. (E) Pre-incubation with celastrol inhibited SOCE in cells co-expressing Orai1 and STIM11-442. Prior to recordings, ER Ca2+ stores were emptied by bathing cells in Ca2+ free solution containing 1 μΜ TG for 10 min. Left, Typical traces; right, Statistics.
FIGURE 5
50 μΜ celastrol inhibited the formation of STIM1 puncta and Ca2+ influxes through constitutively active Orai1 mutants. (A) Representative confocal images showing the effects of celastrol on the store-dependent distribution of YFP-STIM1 or YFP-STIM11-442 transiently expressed in HEK OK cells. Protocols used to induce store depletion by TG were the same as those in Figure 4A. For full-length STIM1 (Left two panels), unlike those in blank control cells (left most panels), celastrol-treatments induced minimal STIM1 punctate at rest and blocked the formation of store-dependent STIM1 puncta (right). As to YFP-STIM11-442 (right two panels), they behave similarly to full length STIM1 in control cells (left), while celastrol completely blocked the formation of STIM11-442 puncta (right). (B) Celastrol’s effects on FRET signals between YFP-STIM1 and CFP-STIM1 transiently co-expressed in HEK SK cells. Compared to control, pretreatment with celastrol resulted in a higher basal STIM1-STIM1 FRET signal, and abolished ionomycin-induced increases in this FRET signal. (C) Celastrol’s effects on FRET signals between STIM11-310-CFP and YFP-SOAR transiently co-expressed in HEK SK cells. Compared to control, pretreatment with celastrol resulted in a significantly lower basal STIM11-310– SOAR FRET signals, and abolished ionomycin-induced increases seen in control cells. (D–F) Celastrol’s effects on Ca2+ influxes mediated by constitutively active Orai1 mutants in GSK cells transiently expressing Orai1-ANSGA (D), Orai1-ANSGA-ΔC (1-265)
(E) or Orai1-ANSGA-ΔN (64-301)
(F). The constitutive Ca2+ influx were greatly inhibited by celastrol. Left, Typical traces; right, Statistics. At least three independent repeats were carried out for each experiments.
References
Grant support
This work was supported by the National Natural Science foundation of China (91954205 for WY, U20A20397 for LC), the Ministry of Science and Technology of China (2019YFA0802104 for WY), Guangdong-Hong Kong-Macau Joint Lab on Chinese Medicine and Immune Disease Research (2020B1212030006 for LC), State Key Laboratory of Dampness Syndrome of Chinese Medicine, The Second Clinical College of Guangzhou University of Chinese Medicine (SZ2021ZZ45 for LC).
Dies ist ein automatisch übersetzter Artikel. Er kann nur einer groben Orientierung dienen. Das Original gibt es hier: psoriasis
