Long-Acting β 2 Adrenergic Receptor Agonist Ameliorates Imiquimod-Induced Psoriasis-Like Skin Lesion by Regulating Keratinocyte Proliferation and Apoptosis


doi: 10.3389/fphar.2022.865715.


eCollection 2022.

Affiliations

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Rui Xu et al.


Front Pharmacol.


.

Abstract

Psoriasis is a chronic inflammatory disease that affects approximately 1%-5% of the population worldwide. Considering frequent relapse, adverse drug reactions, and large costs of treatment, it is urgent to identify new medications for psoriasis. Keratinocytes play an essential role during psoriasis development, and they express high levels of β2-Adrenergic receptor (β2-AR), which increases intracellular cAMP levels when activated. Increased level of cAMP is associated with the inhibition of epidermal cell proliferation. In the present study, we observed the effect of salmeterol, a long-acting β2-AR agonist, on the proliferation and apoptosis of keratinocytes as well as imiquimod-induced psoriasis-like skin lesions in mice. As phosphodiesterase 4 (PDE4) inhibitors increases intracellular cAMP concentration by inhibiting its inactivation, we further explored the synergetic effect of a PDE4 inhibitor and salmeterol on psoriasis-like skin lesions in mice. Our results indicated that salmeterol effectively inhibited the proliferation of HaCaT cells induced by TNF-α and serum, and this effect was accompanied by significantly increased apoptosis and CREB phosphorylation, which were reversed by the PKA inhibitor, H89. Salmeterol ameliorated imiquimod-induced psoriasis-like skin lesions in mice, but salmeterol combined with a PDE4 inhibitor had no synergetic effect in improving skin lesions in mice. Of note, the synergistic effects of anti-proliferation and induction of apoptosis in HaCaT cells appeared by inhibiting ERK signaling. In summary, salmeterol, a long-acting β2-AR agonist, alleviates the severity of psoriasis via inhibiting the proliferation and promoting apoptosis of keratinocytes, partially by activating the cAMP/PKA signaling pathway.


Keywords:

ERK; PDE4 inhibitor; PKA; cAMP; psoriasis; β2 adrenergic receptor agonist.

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

Pharmacological intervention of psoriasis-like dermatitis induced by IMQ in mice. Experimental scheme of psoriasis-like dermatitis induced by IMQ in mice. Sal, Rof, and the combination of Sal and Rof were subcutaneously administered under the skin for 5 days.


FIGURE 2



FIGURE 2

Effect of Salmeterol on cell viability, cell proliferation and apoptosis. (A,B) HaCaT cells were cultured in DMEM containing 10% FBS and treated with Sal at the indicated concentration for 48 h. (A) Cell viability was measured by a CCK8 assay (n = 3). (B) Western blotting analyses of the Bax/Bcl2 ratio (n = 4). (C,D) HaCaT cells were cultured in DMEM containing 10% FBS and then treated with Sal at the indicated concentrations or FSK (10 μM) for 48 h (n = 3). (C) Cell apoptosis was measured by a TUNEL assay (×200, Scale bar = 50 μm). (D) Cell proliferation was measured by EdU incorporation (×400, Scale bar = 50 μm). Data are presented as the mean ± SEM. *p < 0.05 and ***p < 0.001 by one-way ANOVA followed by Tukey’s post-hoc test.


FIGURE 3



FIGURE 3

Salmeterol regulates TNF-α-stimulated cell proliferation and the apoptosis associated with activation of the cAMP/PKA pathway. HaCaT cells were serum starved overnight. During the experiment, HaCaT cells were cultured without serum. HaCaT cells were pretreated with the PKA inhibitor, H89 (1 μM), for 30 min followed by treatment with Sal (100 nM). After 1 h, HaCaT cells were exposed to TNF-α (10 ng/ml) for 48 h (n = 3). (A) Cell viability was measured by a CCK8 assay. (B) Cell proliferation was measured by EdU incorporation (×200, Scale bar = 50 μm). (C) Cell apoptosis was measured by a TUNEL assay (×200, Scale bar = 50 μm). Data are presented as the mean ± SEM. *p < 0.05 and ***p < 0.001 by one-way ANOVA followed by Tukey’s post-hoc test.


FIGURE 4



FIGURE 4

Salmeterol and roflumilast induce PKA/CREB activity. (A,B) HaCaT cells were serum starved overnight. During the experiment, HaCaT cells were cultured without serum. The cells were pretreated with the PKA inhibitor, H89 (1 μM), for 30 min followed by treatment with Sal (100 nM, 1 h) or Rof (100 nM, 30 min). The cells were then treated with TNF-α (10 ng/ml) for 48 h. Representative images and quantification of Western blots for phospho-CREB (pCREB), CREB, and GAPDH (n = 5). (C) PKA activity was measured by a FRET assay. HEK293 cells expressing the nuclear-specific PKA biosensor, NLS-AKAR3, were treated with Sal or Rof at the indicated concentration. The changes in FRET ratio of the PKA biosensor were recorded, and the maximal responses were plotted (n = 11–35). Data are presented as the mean ± SEM. (A,B) **p < 0.01 and ***p < 0.001 by one-way ANOVA followed by Tukey’s post-hoc test. (C) *p < 0.05, **p < 0.01, and ***p < 0.001 by two-way ANOVA followed by Tukey’s multiple comparisons test.


FIGURE 5



FIGURE 5

Effects of salmeterol, roflumilast, and salmeterol combined with roflumilast on psoriasis-like dermatitis induced by IMQ in mice. Psoriasis-like dermatitis was induced by IMQ in mice. Sal, Rof, and the combination of Sal and Rof were subcutaneously administered under the skin for 5 days. (A) Macroscopic presentation of mice on the sixth day after IMQ treatment. (B–E) Quantitative severity assessment on the sixth day after IMQ treatment (n = 5–8). Erythema (B), scales (C), and thickness (D) of the back skin were scored on a scale from 0 to 4. The cumulative score (E) was calculated. (F) Representative H&E staining of cross-sectional slices of the back skin on the sixth day after IMQ treatment. The epidermal thickness of the dorsal skin was measured by five randomly selected fields per section of each mouse. (n = 5–8; ×200, Scale bar = 100 μm). Data are presented as the mean ± SEM. ###p < 0.001 vs. vehicle group; *p < 0.05, **p < 0.01, and ***p < 0.001 vs. IMQ-treated group by one-way ANOVA followed by Tukey’s post-hoc test.


FIGURE 6



FIGURE 6

Effects of salmeterol, roflumilast, and salmeterol combined with roflumilast on cell proliferation and apoptosis in the epidermis of mice with IMQ-induced psoriasis. Psoriasis-like dermatitis was induced by IMQ in mice. Sal, Rof, and the combination of Sal and Rof were subcutaneously administered under the skin for 5 days. (A) Representative TUNEL staining of cross-sectional sections of the back skin of mice on the sixth day (n = 3; ×200, Scale bar = 50 μm). (B) Representative Ki67 staining of cross-sectional sections of the back skin of mice on the sixth day (n = 3; ×200, Scale bar = 50 μm). (C) Expression levels of K17, Bcl2, Bax, pCREB, and CREB in the back skin were detected by Western blotting. GAPDH served as the loading control (n = 4). (D) cAMP levels were detected in skin tissues (n = 4–7). Data are presented as the mean ± SEM. (A–C) *p < 0.05, **p < 0.01, and ***p < 0.001 by one-way ANOVA followed by Tukey’s post-hoc test. (D) *p < 0.05 and **p < 0.01by unpaired t test. ns, not significant.


FIGURE 7



FIGURE 7

ERK activation in the combination treatment mediates the lack of synergisms. (A) Psoriasis-like dermatitis was induced by IMQ in mice. Sal, Rof, and the combination of Sal and Rof were subcutaneously administered under the skin for 5 days. Expression levels of pERK and ERK in the back skin were detected by Western blotting. GAPDH served as the loading control (n = 4). (B–D) HaCaT cells were serum starved overnight. During the experiment, HaCaT cells were cultured without serum. HaCaT cells were pretreated with U0126 (1 μM), for 30 min followed by treatment with Sal (100 nM, 1 h) or Rof (100 nM, 30 min). The cells were then exposed to TNF-α (10 ng/ml) for 48 h (n = 3). (B) Cell viability was measured by a CCK8 assay. (C) Cell proliferation was measured by EdU incorporation (×200, Scale bar = 50 μm). (D) Cell apoptosis was measured by a TUNEL assay (×200, Scale bar = 50 μm). (E) HaCaT cells were serum starved overnight. During the experiment, HaCaT cells were cultured without serum. HaCaT cells were pretreated with U0126 (1 μM), for 30 min followed by treatment with Sal (100 nM, 1 h) or Rof (100 nM, 30 min). The cells were then exposed to TNF-α (10 ng/ml) for 1 h (n = 3). Expression levels of pPDE4D and PDE4D were detected by Western blotting. GAPDH served as the loading control. Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 by one-way ANOVA followed by Tukey’s post-hoc test.

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