Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Jul 1;22(13):7145.
doi: 10.3390/ijms22137145.

Anoctamin1 Induces Hyperproliferation of HaCaT Keratinocytes and Triggers Imiquimod-Induced Psoriasis-Like Skin Injury in Mice

Mi Ran Choi  1 , Hae Dong Kim  2 , Sinyoung Cho  2 , Seong Ho Jeon  2 , Dong Hyun Kim  3 , Jungwon Wee  4 , Young Duk Yang  2
Affiliations

Anoctamin1 Induces Hyperproliferation of HaCaT Keratinocytes and Triggers Imiquimod-Induced Psoriasis-Like Skin Injury in Mice

Mi Ran Choi et al. Int J Mol Sci. .

Abstract

Psoriasis, a long-lasting and multifactorial skin disease, is related to comorbidities such as metabolic disease, depression, and psoriatic arthritis. Psoriasis occurs due to a variety of factors including keratinocyte hyperproliferation, inflammation, and abnormal differentiation. Proinflammatory cytokines upregulated by increased activation of keratinocytes and immune cells in the skin trigger progression of psoriasis. This study aimed to investigate the effects of anoctamin1 (ANO1) on psoriasis development in vitro and in vivo. We analyzed the proliferation of HaCaT keratinocytes and ANO1-related ERK and AKT signaling pathways after ANO1 inhibitor (T16Ainh-A01 and Ani9) treatment and knock-down of ANO1. Furthermore, after applying imiquimod (IMQ) cream or coapplying IMQ cream and T16Ainh-A01 on mouse ears, we not only observed psoriatic symptoms, including ear thickening, but also quantified the effects of treatment on ERK and AKT signaling-involved proteins and proinflammatory cytokines. Inhibition of ANO1 attenuated the proliferation of HaCaT cells and induced reduction of pERK1/2. Coapplication of IMQ and T16Ainh-A01 on ears of mice reduced not only symptoms of IMQ-induced psoriasis such as thickening and erythema, but also expression of ANO1 and pERK1/2 compared to that of application of IMQ alone. In addition, the expression levels of IL-17A, IL-17F, IL-22, IL-23, IL-6, IL-1β, and TNF-α increased after applying IMQ and were significantly reduced by coapplying IMQ and T16Ainh-A01. These results aid in understanding the underlying mechanisms of ANO1 in epidermal layer keratinocyte hyperproliferation and suggest the potential of ANO1 as a _target to treat psoriasis.

Keywords: ERK pathway; anoctamin 1; imiquimod; keratinocyte; psoriasis.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Expression of ANO1 in the human keratinocyte HaCaT cell line and psoriatic tissue. (a) The expression of ANO1 mRNA and protein levels in HaCaT cells. (b) Expression of ANO1 protein in HaCaT cells using immunocytochemistry. Scale bar = 50 µm. (c) The expression of ANO1 protein in psoriatic skin tissue was increased compared to that of healthy skin tissue. Scale bar = 50 µm (insert: 20 µm). (d) Expression of ANO1 in the skin of normal and psoriasis from immunohistochemical analysis (ANO1 intensity of ten cells per unit area was measured using ZEN 3.1). To compare the differences of ANO1 expression between normal and psoriasis skin tissues, Student’s t-test was used. *: significantly different from normal skin (*** p < 0.001).
Figure 2
Figure 2
The effects of ANO1 inhibition on HaCaT cells. (a) Proliferation of cells exposed to 30 µM T16Ainh-A01 for 3 days. (b) Proliferation of cells exposed to 10 µM Ani9 for 3 days. (c) Proliferation of cells following ANO1 knock-down for 3 days. After knock-down of ANO1 using siRNA _targeting ANO1 and in negative controls (mock and si-NC), proliferation rates of HaCaT cells were measured for 3 days. (d) Immunoblots of ANO1 and ERK and AKT signaling pathway-related proteins after ANO1 knock-down for 2 days. (e) Densitometric analysis for the relative intensity of ANO1 and ERK and AKT signaling pathway-related proteins. Treatments with the two blockers (T16Ainh-A01 and Ani9) and ANO1 knock-down were performed as four independent replicates to guarantee reliable results. To compare the differences between control (vehicle or Mock [si-NC]) and blocker-treated or ANO1 knock-down cells, two-way ANOVA and Tukey’s post-hoc tests were used. *: significantly different from vehicle in (a,b), from Mock and si-NC (c), and from Mock (e) (* p < 0.05, ** p < 0.01, and *** p < 0.001). #: significantly different from si-NC (e) (# p < 0.05 and ## p < 0.01).
Figure 3
Figure 3
Inhibition of ANO1 channel activity by resveratrol, curcumin, xanthohumol, and isoxanthohumol. The activity of ANO1 was measured in Fischer rat thyroid (FRT) cells stably co-expressing human ANO1 and the yellow fluorescent protein (YFP). After the cells were exposed to positive controls (NPPB and T16Ainh-A01) or one of the four compounds for 20 min, they were treated with 140 mM NaI solution containing 100 µM ATP to activate ANO1 channel activity, continuously measured every 0.2 s for 6 s. (a) Fluorescence intensity of cells exposed to vehicle, positive controls, resveratrol, and curcumin. (b,d) Relative fluorescence intensity obtained from tested cells at 6 s. (c) Fluorescence intensity of cells exposed to vehicle, positive controls, xanthohumol, and isoxanthohumol. To compare the differences among vehicle, positive controls, resveratrol, curcumin, xanthohumol, and isoxanthohumol, two-way ANOVA and Tukey’s post-hoc tests were used. *: significantly different from vehicle (* p < 0.05, ** p < 0.01, and *** p < 0.001).
Figure 4
Figure 4
Alleviation of psoriasis-like symptoms in mouse ears by inhibiting ANO1. To induce psoriasis-like skin injury, three mouse groups (IMQ [n = 7], IMQ + 30 µM A01 [n = 7], and IMQ + 100 µM A01 [n = 7]) received a daily dose of 25 mg of 5% IMQ cream on their right ears for 7 consecutive days; IMQ + 30 µM A01 and IMQ + 100 µM A01 groups additionally received 15 µL of 30 µM and 100 µM A01, respectively, at 30 min before and after IMQ treatment. The control group received a similar dose of Vaseline cream on their right ears. (a) Changes in right ear thickness caused by IMQ and A01. (b–d) Thickness, erythema, and scaling of right ears were scored daily. (e) The cumulative score (thickening plus erythema plus scaling) was described. Symbols per day depict mean ± SEM of seven mice ears per group. The significant differences among groups obtained from statistical analysis are presented in Figure S2. (f) Phenotypic representation of the right ears in mice treated with IMQ or IMQ + 100 µM A01 for 7 days. Imiquimod, IMQ; T16Ainh-A01, A01.
Figure 5
Figure 5
Changes in histopathological structure and ANO1-related proteins in mouse ears showing psoriasis-like symptoms caused by inhibiting ANO1. The right ears of the mice in all four groups (control, IMQ, IMQ + 30 µM A01, and IMQ + 100 µM A01 groups) were obtained on the eighth day after psoriasis-like skin injury and inhibition of ANO1. (a) H&E staining and ANO1 immunoreaction in the right ears of control, IMQ, and IMQ + 100 µM A01 groups. Scale bar = 500 µm. (b) The quantification of acanthosis in the right ears of control (n = 3), IMQ (n = 3), and IMQ + 100 µM A01 (n = 3) groups. (c) The signal intensity of ANO1 per ten cells in the epidermis of right ears of control (n = 3), IMQ (n = 3), and IMQ + 100 µM A01 (n = 3) groups. Significant differences of acanthosis (b) or ANO1 intensity (c) among control, IMQ, and IMQ + 100 µM A01 groups were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test. *: significantly different from IMQ group (** p < 0.01 and *** p < 0.001). (d) The expression levels of ERK and AKT proteins. The phosphorylation of ERK was increased by IMQ cream but was decreased by A01 in a dose-dependent manner. Imiquimod, IMQ; T16Ainh-A01, A01.
Figure 6
Figure 6
Changes in ANO1 and immune response-related cytokine mRNA levels in mouse ears showing psoriasis-like symptoms caused by inhibiting ANO1. Two mouse groups (IMQ [n = 7] and IMQ + A01 [n = 7]) received a daily dose of 25 mg of 5% IMQ cream on right ears for 7 consecutive days, and the IMQ + A01 group additionally received 15 µL of 100 µM A01 at 30 min before and after IMQ treatment. The control group received a similar dose of Vaseline cream on their right ears. After extracting total RNA from the right ears of the mice (7 animals in each group), obtained on the eighth-day post-treatment onset, _target genes were analyzed using RT-PCR.; their expression levels were measured and represented as densitometric graphs. To compare the differences among groups, one-way ANOVA and Tukey’s post-hoc tests were used. *: significantly different between two groups (* p < 0.05, ** p < 0.01, and *** p < 0.001). Imiquimod, IMQ; T16Ainh-A01, A01.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Langley R.G., Krueger G.G., Griffiths C.E. Psoriasis: Epidemiology, clinical features, and quality of life. Ann. Rheum. Dis. 2005;64:ii18–ii23. doi: 10.1136/ard.2004.033217. - DOI - PMC - PubMed
    1. Koo J., Marangell L.B., Nakamura M., Armstrong A., Jeon C., Bhutani T., Wu J.J. Depression and suicidality in psoriasis: Review of the literature including the cytokine theory of depression. J. Eur. Acad. Dermatol. Venereol. 2017;31:1999–2009. doi: 10.1111/jdv.14460. - DOI - PubMed
    1. Griffiths C.E., Barker J.N. Pathogenesis and clinical features of psoriasis. Lancet. 2007;370:263–271. doi: 10.1016/S0140-6736(07)61128-3. - DOI - PubMed
    1. Michalek I.M., Loring B., John S.M. A systematic review of worldwide epidemiology of psoriasis. J. Eur. Acad. Dermatol. Venereol. 2017;31:205–212. doi: 10.1111/jdv.13854. - DOI - PubMed
    1. Parisi R., Symmons D.P., Griffiths C.E., Ashcroft D.M. Global epidemiology of psoriasis: A systematic review of incidence and prevalence. J. Investig. Dermatol. 2013;133:377–385. doi: 10.1038/jid.2012.339. - DOI - PubMed

MeSH terms

  NODES
todo 1
twitter 2