1. p21-Activated Kinase 4 Critically Regulates Melanogenesis via Activation of the CREB/MITF and β-Catenin/MITF Pathways 
Cheong-Yong Yun, Soon-Tae You, Jin-Hwa Kim, Jin H. Chung, Sang-Bae Han, Eun-Young Shin, Eung-Gook Kim 
Journal of Investigative Dermatology 
Volume 135, Issue 5, Pages (May 2015)
DOI: /jid
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2. Figure 1
Expression of p21-activated kinase (PAK) isoforms in the skin and B16 melanoma cells. (a) The skin of HRM-2 mice was co-stained for PAK1, 2, 4, or 6 (green) with SOX10, a melanocyte marker (red). Immunostaining for the pairs PAK3/SOX10 and PAK5/SOX10 was performed using two adjacent slides. White arrows indicate SOX10-positive cells. The nucleus was then stained with 4',6-diamidino-2-phenylindole (DAPI) (blue). Scale bar=5 μm. (b) Immunoblotting was performed to detect the expression of PAK isoforms in B16 melanoma cells. Each exogenous PAK isoform in a myc-tagged form was transfected as a positive control.
Journal of Investigative Dermatology  , DOI: ( /jid )
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3. Figure 2
p21-activated kinase-4 (PAK4) inhibition and knockdown suppress α-melanocyte-stimulating hormone (α-MSH)-stimulated melanogenesis. (a and b) α-MSH stimulated PAK4 phosphorylation concentration (a) and time (b) dependently. (c and d) Cells were pretreated with PF  hours before stimulation with α-MSH for 15 minutes and subjected to immunoblotting (c) and immunofluorescence measurement (d, left). Scale bar=10 μm. pPAK4 fluorescence intensity was quantified (d, right). (e) Cells were treated with PF for the first day and stimulated with α-MSH for 3 days. Melanin content was quantified. Values indicate the mean±SE from three independent experiments. *P<0.01. (f) Left, small interfering RNA (siRNA)-transfected cells were stimulated with α-MSH for 3 days, and melanin content was measured. Values are from three independent experiments. *P<0.01. Right, PAK4 knockdown was monitored by immunoblotting.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
p21-activated kinase-4 (PAK4) inhibition suppresses UVB-induced melanogenesis in vivo. (a) Experimental design. HRM-2 mice were exposed to UVB at the indicated doses and times (arrows). Cream base (vehicle) or cream containing PF was applied every day. Animals were killed 8 hours after the final radiation and subjected to analysis in c. (b) Schematic diagram showing the allocation of the dorsal skin of mice. (c) The skin of HRM-2 mice was co-stained for pPAK4 (green) and SOX10 (red, white arrows). Yellow arrows mark pPAK4- and SOX10-positive cells. Scale bar=10 μm. (d) pPAK4-stained cells in SOX10-positive melanocytes were counted, and the percentage of total SOX10-positive melanocytes was determined (n=60). *P<0.01. (e) Quantification of the pigmentation changes in the skin of HRM-2 mice over time. Skin pigmentation levels are presented as the L value.
Journal of Investigative Dermatology  , DOI: ( /jid )
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5. Figure 4
p21-activated kinase-4 (PAK4) knockdown and inhibition downregulate pCREB, cAMP-response element–binding protein (CREB), microphthalmia-associated transcription factor (MITF), and tyrosinase. (a) B16 melanoma (left) and human epithelial melanocyte (HEM; right) cells were transfected with small interfering RNAs (siRNAs) for 3 days and then stimulated with α-melanocyte-stimulating hormone (α-MSH). Immunoblotting was performed. (b) B16 melanoma cells were transfected with siRNAs, MITF-luc reporter, and Renilla plasmids. Cells were treated with H89 for 2 hours before stimulation with α-MSH for 4 hours. Reporter assay was performed. *P<0.01. (c) Experimental design. Cells were pretreated with PF for 22 hours and harvested depending on the activation or expression time of the indicated proteins after α-MSH stimulation. (d and e) PF pretreated cells were harvested at 15 minutes for pCREB and CREB, 4 hours for MITF, and 2 days for tyrosinase after α-MSH stimulation, respectively, and immunoblotted. (f) Top, HRM-2 skin was processed as described in Figure 3, followed by immunohistochemical staining for CREB, MITF, and tyrosinase (green) and SOX10 (red). White arrows, SOX10-positive cells; yellow arrows, double-positive cells. Scale bar=10 μm. Bottom, CREB-, MITF-, and tyrosinase-stained cells in SOX10-positive cells were quantified (n=60). *P<0.01.
Journal of Investigative Dermatology  , DOI: ( /jid )
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6. Figure 5
p21-activated kinase-4 (PAK4)-β-catenin signaling in α-melanocyte-stimulating hormone (α-MSH)-stimulated melanogenesis. (a) Cells were treated with α-MSH with the indicated doses (top) and for the indicated times (bottom) and then subjected to immunoblotting. (b) Top, cells were pretreated with PF for 6 hours before stimulation with α-MSH for 15 minutes, followed by immunoblotting. Bottom, cells were transfected with small interfering RNAs (siRNAs) for 3 days and then stimulated with α-MSH. (c) Top, cells were immunostained for pβ-cateninS675 (green) and SOX10 (red). Scale bar=10 μm. Bottom, pβ-catenin S675 in SOX10-positive cells was quantified. *P<0.01. (d) Two days after transfection with siRNAs, B16 melanoma (left) and human epithelial melanocyte (HEM; right) cells were treated with MG132 and cycloheximide for 2 hours and then stimulated with α-MSH for 90 minutes. Immunoblotting was performed. (e) Cells were sequentially treated with PF for 6 hours and MG132 and cycloheximide for 2 hours and stimulated with α-MSH for 90 minutes. Immunoblotting was performed.
Journal of Investigative Dermatology  , DOI: ( /jid )
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7. Figure 6
Schematic model for p21-activated kinase-4 (PAK4) signaling in α-melanocyte-stimulating hormone (α-MSH)/UVB-stimulated melanogenesis. α-MSH stimulates PAK4 activation by phosphorylating PAK4 at S474. UVB radiation may activate PAK4 indirectly through the secretion of many paracrine factors, including α-MSH, in neighboring keratinocytes. Activated PAK4 then stimulates the transcription of microphthalmia-associated transcription factor (MITF) via the upregulation or activation of CREB, which in turn induces the expression of melanin synthesis enzymes, such as tyrosinase. In addition, activated PAK4 phosphorylates β-catenin at S675, which promotes the stability of β-catenin and its activity as a transcriptional co-activator. Alternatively, activated PAK4 reduces β-catenin phosphorylation at S33/S37, which leads to ubiquitination-dependent proteolysis. Thus, β-catenin stimulates transcription of tyrosinase in collaboration with MITF in a phosphorylation/dephosphorylation-dependent manner.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions