Targeting phosphorylation circuits on CREB and CRTCs as the strategy to prevent acquired skin hyperpigmentation

Background: The cAMP response element-binding protein (CREB) and CREB-regulated transcription coactivators (CRTCs) cooperate in the transcriptional activation of microphthalmia-associated transcription factor subtype M (MITF-M) that is a master regulator in the biogenesis, pigmentation and transfer of melanosomes at epidermal melanocytes. Here, we propose the targeting of phosphorylation circuits on CREB and CRTCs in the expression of MITF-M as the rationale to prevent skin hyperpigmentation by elucidating the inhibitory activity and mechanism of yakuchinone A (Yaku A) on facultative melanogenesis. Methods: We employed human epidermal melanocyte cell, mouse skin, and mouse melanoma cell, and applied Western blotting, reverse transcription-polymerase chain reaction, immunoprecipitation and confocal microscopy to conduct this study. Results: This study suggested that α-melanocyte stimulating hormone (α-MSH)-induced melanogenic programs could switch on the axis of protein kinase A-salt inducible kinases (PKA-SIKs) rather than that of PKA-AMP activated protein kinase (PKA-AMPK) during the dephosphorylation of CRTCs in the expression of MITF-M. SIK inhibitors rather than AMPK inhibitors stimulated melanin production in melanocyte cultures in the absence of extracellular melanogenic stimuli, wherein SIK inhibitors increased the dephosphorylation of CRTCs but bypassed the phosphorylation of CREB for the expression of MITF-M. Treatment with Yaku A prevented ultraviolet B (UV-B)-irradiated skin hyperpigmentation in mice and inhibited melanin production in α-MSH- or SIK inhibitor-activated melanocyte cultures. Mechanistically, Yaku A suppressed the expression of MITF-M via dually targeting the i) cAMP-dependent dissociation of PKA holoenzyme at the upstream from PKA-catalyzed phosphorylation of CREB coupled with PKA-SIKs axis-mediated dephosphorylation of CRTCs in α-MSH-induced melanogenic programs, and ii) nuclear import of CRTCs after SIK inhibitor-induced dephosphorylation of CRTCs. Conclusions: Taken together, the targeting phosphorylation circuits on CREB and CRTCs in the expression of MITF-M could be a suitable strategy to prevent pigmentary disorders in the skin.


Appendix S1. Supplementary methodology: isolation of Yaku A from Alpinia oxyphylla.
General experimental procedures.UV spectra were recorded on a JASCO UV-550 spectrophotometer, and IR spectra were measured on a JASCO FT-IR 4100 spectrometer.NMR spectra were recorded on a Bruker AVANCE 400 MHz, spectrometer using CDCl3 as solvent.

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Nucleotide sequence Amplicon Plant material.The dried fruits of A. oxyphylla were purchased from Kyungdong herbal market in Seoul, Korea, in October 2019.A voucher specimen (CBNU-2019-10-AO) was authenticated by B.Y.H. and deposited at the Herbarium of the College of Pharmacy, Chungbuk National University, Korea.

Figure S1 .
Figure S1.Inhibitory effect of Alpinia oxyphylla extract on melanin production in α-MSHactivated B16F0 cells.The cells were stimulated with α-MSH in the presence of A. oxyphylla extract (A) or arbutin (B) for 72 h. A. oxyphylla extract and arbutin inhibited α-MSH-induced melanin production in the cultures of B16F0 cells.*P < 0.05 vs. α-MSH alone.

Figure S3 .Figure S4 .Figure S5 .Figure S6 .
Figure S3.Effects of Yaku A on CREB phosphorylation and CRTC1 dephosphorylation in UV-B-irradiated co-culture of PAM212 keratinocyte and B16F0 melanoma cells.Coculture with trans-well insert between PAM212 and B16F0 cells was pretreated with Yaku A for 2 h.After stimulation with a single irradiation of UV-B, the co-culture was incubated in the presence of Yaku A for another 30 min.Treatment with Yaku A inhibited UV-B-irradiated phosphorylation of CREB at the S133 residue and the dephosphorylation of CRTC1 at the S151 residue in PAM212 cells (upper) as well as those in B16F0 cells (lower).

Figure S7 .Figure S8 .
Figure S7.Time course study of YKL 06-061-induced CREB phosphorylation in B16F0 cell.The cells were stimulated with YKL 06-061 for indicated time points, and their protein extracts were subjected to Western blot (WB) analysis.YKL 06-061 did not phosphorylate CREB at the S133 residue during 4 h.