Gene expression profiles of skin from cyclin dependent kinases 5-knockdown mice.

OBJECTIVE: This study aimed to identify genes regulated by cyclin dependent kinases 5 (CDK5) that participate in hair pigmentation in mice. METHODS: The mRNA expression profiles of skin samples from CDK5-knockdown mice were constructed using high-throughput RNA sequencing and compared with those of wild-type mice. RESULTS: In total, 8,002 known genes were differentially expressed between CDK5-knockdown and wild-type mice. Of these, 3,658 were upregulated and 4,344 were downregulated in the skin of CDK5-knockdown mice. An additional 318 previously unknown genes were also differentially expressed, with 171 downregulated and 147 upregulated genes in the skin of CDK5-knockdown mice. Of the known genes expressed in mouse skin, 80 were associated with hair color, with 61 showing lower expression and 19 exhibiting higher expression in skin of CDK5-knockdown mice. Importantly, the expression of the tyrosinase-related protein 1 (TYRP1) and the calcium signaling pathway were also found to be regulated by CDK5, suggesting that pigmentation is regulated by CDK5 via the calcium signaling pathway and TYRP1. CONCLUSION: The transcriptome profiles obtained from the skin of CDK5-knockdown mice compared to wild-type mice provide a valuable resource to help understand the mechanism by which CDK5 regulates melanogenesis in mice and other animals.

Synaptotagmin-4 promotes dendrite extension and melanogenesis in alpaca melanocytes by regulating Ca2+ influx via TRPM1 channels.

Synaptotagmin-4 (SYT4) is a membrane protein that regulates membrane traffic in neurons in a calcium-dependent or calcium-independent manner. In melanocytes, the intracellular free calcium ion (Ca2+ ) may be important for dendrite growth and melanogenesis. Mammalian melanocytes originating from neural crest cells produce melanins. Therefore, we predicted that SYT4 might play a role in melanogenesis and the dendrite morphology of melanocytes. To investigate whether SYT4 is involved in melanocyte physiology, SYT4 was overexpressed in alpaca melanocytes and B16-F10 cells. The results showed that SYT4 overexpression resulted in a phenotype consistent with melanogenesis and dendrite extension. At the molecular level, SYT4 interacted with extracellular regulated MAP kinase (ERK) to decrease p-ERK activity, which negatively regulated CREB expression. Furthermore, cyclic AMP-responsive element-binding protein (CREB) was upregulated and caused the downregulation of the expression of melanogenic regulatory proteins, including microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), tyrosinase-related protein-1 (TYRP1), dopachrome tautomerase (DCT), and transient receptor potential melastatin 1 (TRPM1). Intracellular free Ca2+ promoted the upregulation of calcium/calmodulin dependent protein kinase IV (CAMK4) expression, which phosphorylated CREB (p-CREB). In turn, p-CREB participated in the transcription of MITF. These results demonstrated that SYT4 promoted melanogenesis through dendrite extension and tyrosinase activity, during which the regulation of Ca2+ influx via the TRPM1 channel was a key factor. SIGNIFICANCE OF THE STUDY: Intracellular Ca2+ is important for the function and survival of melanocytes and melanoma cells. SYT4 stimulated melanogenesis through calcium. These results provide evidence that SYT4 regulates Ca2+ influx through TRPM1 to cause melanogenesis and axonal elongation in alpaca melanocytes and further suggesting that the growth and metastasis of melanoma is controlled by the inhibited expression of SYT4 in melanoma cells.

Characterization and expression of soluble guanylate cyclase in skins and melanocytes of sheep.

The study reported the characterization of soluble guanylate cyclase (sGC) with the size of CDS of 1860bp, encoding a protein of 620 amino acids and containing several conserved functional domains including HNOB, HNOBA, and CHD. Quantitative real time PCR analysis of sGC showed that the expression of sGC mRNA is higher (∼5 fold) in white sheep skin relative to black sheep skin with significant difference (P<0.01). Using a rabbit polyclonal anti-sGC antibody, an immune reactive band corresponding to sheep sGC protein was detected in the skin samples by Western blotting analysis, and the expression of sGC protein was significantly higher in white sheep skin compared to black sheep skin (P<0.01). Immunohistochemical analysis revealed that sGC protein was localized in cytoplasm and intercellular substance of upper hair papilla in hair follicles of white sheep skin, but the protein was localized in cytoplasm and intercellular substance of lower hair bulb and outer root sheath cells in hair follicles of black sheep skin. The immunocytochemical analysis revealed that sGC was expressed in melanocytes in vitro of sheep skin. Over expression of sGC in melanocytes resulted in decreased expression of key melanogenic genes including microphthalmia transcription factor (MITF), tyrosinase (TYR), tyrosinase related protein 1(TYRTP1), and tyrosinase related protein 2(TYRP2) both at mRNA and protein level. Moreover, the melanocytes was capable of producing cGMP and cAMP. The observed differential expression and localization of sGC in sheep skins and melanocytes and the capability of producing cGMP and cAMP, which suggested a potential role for this gene in hair color regulation.