Secreted Frizzled-related Protein5Abrogates Wnt3a-Induced Melanogenesis Of The Hair Follicle

Melanocytes are derived from the neural crest and gradually migrateto the epidermis in developmental process, finally formed a hair folliclemelanocyte stem cells. Melanocytes are specialized melanin producingcells, and are responsible for skin, hair, and eye pigmentation in vertebrateorganisms. Recent studies indicate that the differentiation of melanocytestem cells is regulated by numerous signaling pathways, especially theWNT signaling pathway.Activity of Wnt signaling pathway is regulated bysecreted inhibitors, including secreted frizzled-related protein (sFRP). ThesFRP family has an amino-terminal cysteine-rich domain (CRD) that hashigh homology with the ligand-binding domain of frizzled Wnt receptor.Secreted frizzled related protein5(sFRP5) has been demonstrated to be aWnt antagonist; however, its effects on melanocyte have not yet beenreported. Using various in vitro assays, we show that sFRP5suppress thedifferentiation of melanocytes and the development of melanin formationthrough Wnt3a inhibition. sFRP5interfered with melanocyte functions byantagonizing the canonical Wnt/β-catenin signaling pathway. In vivo assays we demonstrated a visible melanin reduction on the skin of Dct-LacZtransgenic mice after sFRP5treatment. This study results are as follows:1.SFRP5express during the hair cycleIn Dct-LacZ transgenic mice, X-gal staining was used to labelLacZ-positive melanocytes. The endogenous expression of SFRP5wasdetermined in dorsal skin hair follicles in different stages of the hair cycle.In the anagen stage (P7), SFRP5was expressed mainly in the ORS of thebulge region as well as between the bulge region and hair matrix. However,the expression level of SFRP5was reduced in the hair matrix. In thecatagen (P18) and telogen (P21) stages, there was no obvious SFRP5protein detected in the hair follicles. Consistent with these data, SFRP5mRNA was most strongly expressed in anagen, decreased in catagen, andlowest in telogen.2. In Vitro StudiesTo test the effect of sFRP5on the melanocytes, we employed a mousemelanocyte cell line, Imc23，which is low degree of differentiation and donot synthesize melanin in normal culture conditions, as an in vitro cellmodel. IMC23cells were infected with an Ad-GFP or administration ofAd-Wnt3a or co-administration of Ad-Wnt3a+Ad-sFRP5.We investigatedthe effect of sFRP5on melanogenesis using a tyrosinase activity assay.Tyrosinase activity was significantly reduced after SFRP5treatment.Further Masson-Fontanas Silver staining assay found that sFRP5 administration induced a significant reduction in numbers of melaningranules and a reduction of melanin content. These results provideevidence that sFRP5reduce the amount of melanin in melanocytes.Tofurther confirm the role of sFRP5in melanocyte differentiation, wemeasured the levels of differentiation-related RNA and proteins (TRP1,tyrosinase,Mitf) and GAPDH in Imc23cells.The data indicate that sFRP5is able to restrain the role of Wnt3a which induced the rise of theseenzymes expression,thereby inhibiting the formation of melanin.3. SFRP5inhibited the Wnt/β-catenin pathwaywe examined the impact of sFRP5on the Wnt/β-catenin pathwayusing IMC23. RT-PCR analysis revealed a reduction in β-catenin RNAexpression after treatment with sFRP5and Luciferase assays indicated thatendogenous β-catenin/Tcf transcriptional activity was decreased in thesFRP5treatment group. Further there was a diminution in β-catenin proteinexpression detected by Western blot analysis.Thus, our data demonstratethat SFRP5is able to repress melanocyte differentiation by blocking theWnt/β-catenin pathway.4. Investigation of sFRP5Effects in an in Vivo EnvironmentTo examine the effect of sFRP5on the melanocytes in hair follicle,wetreated the Dct-LacZ transgenic mice at the telogen stage with intradermalinjections of Ad-GFP or Ad-Wnt3a or Ad-Wnt3a+Ad-SFRP5,which closeto the head,middle, and tail of the dorsal skin respectively. Following the injections(8d or14d), A significant melanin reduction was observed in thearea of sFRP5application on the Dct-LacZ transgenic mice. We stainedsections of the dorsal skin with X-gal and Eosin-hematoxylin. After sFRP5treatment, the group showed a significant decrease in the amount ofLacZ-positive melanocytes. We also found that the number of pigmentationwas a significant reduction.In conclusion, there are no published data demonstrating theinvolvement of sFRP5in melanin formation to date.We report ourinvestigations on the role of sFRP5on melanocyte using a variety of invitro assays and vivo models. The data demonstrate sFRP5to be a potentmelanogenesis inhibitor that warrants further investigation as a therapeuticagent in the control of Pigment-producing disorders.