Introduction: Solar lentigo (hereafter called age spots) is a major symptom of photoaging. So far, pleiotropic melanocytic factors from keratinocytes, melanocytes, and dermal layers are involved in age spots. Through UVB exposure, keratinocytes secrete factors such as a-MSH, Endothelin-1 and IL1a, thereby stimulating melanogenesis and inflammation. Our previous studies have shown that in age spots these inflammatory factors further have effects on the destruction of the basement membrane and the abnormal formation of microvascular structures. Therefore, keratinocytes are the origin to create the melanocytic environment of age spots. In this environment, keratinocytes fail to differentiate. Although keratinocyte differentiation is mediated by a cell-cell contact factor E-cadherin, its involvement in age spots remains unknown. Furthermore, while intensive studies have uncovered various factors responsible for age spot formation, how those factors are reciprocally related or integrated has yet to be addressed. Thus, to improve age spots, an integrated solution targeting the origin of the age spot is needed. To find the origin of the age spot and the integrated solution, we focused on a critical regulator of keratinocyte differentiation, E-cadherin, revealing a novel mechanism and solution for improving age spots.
Methods: Immunofluorescent staining of E-cadherin with skin tissues containing age spots was carried out. To analyze the effects of downregulating E-cadherin expression in cultured keratinocytes, specific knockdown of E-cadherin (E-cad-KD) with siRNAs were performed, along with control-siRNA treatments. These siRNA-treated keratinocytes were either cultured alone or co-cultured with melanocytes. To identify secreted factors from keratinocytes, the supernatants of either E-cad-KD or control keratinocytes were analyzed by mass spectrometry. At the same time, to check the melanogenesis factors, melanocytes were cultured in the supernatant-conditioned medium and analyzed for quantitative PCR (qPCR) and immunohistochemistry. For behavioral analyses of melanocytes, fluorescence-labeled melanocytes co-cultured with E-cad-KD keratinocytes were observed by live-imaging. Furthermore, live imaging of the uptake of melanin by either E-cad-KD or the control keratinocytes were performed to analyze the speed of melanin uptake. For the ingredient screening, keratinocytes treated with ingredients were analyzed with qPCR to check the level of E-cadherin expression.
Results: In age spots, keratinocyte E-cadherin in both basal and suprabasal layers were considerably downregulated as compared with non-lesion areas. To test whether E-cadherin downregulation affects melanogenesis and melanin uptake, the knockdown of E-cadherin in keratinocytes by siRNAs were performed. E-cadherin downregulation promoted the secretion of several melanocytic factors, sFRP1, ET-1, and TNFa at about two times higher than the control. In fact, melanocytes treated with Ecad-KD-conditioned medium significantly increased the expression of melanogenesis factors such as MITF, Tyrosinase and TRP1, thereby promoting melanin synthesis. In addition, live imaging of melanocytes cocultured with either the control or Ecad-KD keratinocytes showed that, in the presence of E-cad-KD keratinocytes, melanocytes sustained their migration with less dendrite outgrowth and retraction as compared with the control. Furthermore, Ecad-KD keratinocytes remarkably promoted the melanin uptake at about 2-fold faster than in control keratinocytes. Therefore, these results indicated that the downregulation of E-cadherin expression in keratinocytes triggers the secretion of melanocytic factors, stimulating melanogenesis by affecting melanocyte dynamics and melanin uptake. Finally, to provide a solution to prevent a melanocytic environment by E-cadherin upregulation, we screened ingredients in keratinocytes. As a result, Rosa Multiflora Fruit Extract significantly increased E-cadherin expression.
Discussion/Conclusion: E-cadherin is a key factor for cell adhesion, which is critical for cell invasion and the integrity of tissue homeostasis. The same is true in age spots, in which E-cadherin is not only a key factor for keratinocyte differentiation but also crucial for controlling the homeostasis of a melanocytic environment in the epidermis. Our new finding showed that downregulating E-cadherin triggers various downstream melanocytic processes, secretion of melanocytic factors, melanogenesis, melanocyte dynamics, and melanin uptake. In other words, E-cadherin is a “fort” for keratinocytes that prevents the epidermis from triggering melanocytic factors while forming a melanocytic environment. Our finding opens a new possibility for improving age spot that will approach the core of the age spot, E-cadherin. As a first solution, we showed that Rosa Multiflora Fruit Extract increases E-cadherin expression in keratinocytes. The cosmetic market has been seeking an all-in-one solution to improve age spots. Thus, strengthening cell-cell contact by increasing E-cadherin could be such a solution, creating an integrated solution that covers downstream melanocytic environments.