A novel regulatory mechanism underlying asymmetric division of human keratinocytes and the development of skin pigmentation
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Presented by: AKIKO ENOMOTO
Introduction
The mechanisms involved in melanogenesis caused by sunburn and skin inflammation have been clarified previously, and various products have been developed to prevent skin pigmentation. Nevertheless, skin pigmentation develops into a chronic condition with age. Hence, we believe that it is important to not only prevent transient melanogenesis but also improve delayed melanin clearance with age. Therefore, we studied the mechanisms enhancing melanin clearance to develop a more effective treatment for chronic skin pigmentation. In most cases, approaches that promote keratinocyte cell proliferation and differentiation were selected to promote the clearance function. Specifically, because the structure and direction of keratinocyte proliferation in the melanin pigmented-area were different from those in the non-pigmented area, we focused on horizontal keratinocyte division (i.e., asymmetric cell division (ACD)), which plays an important role in maintaining the functionality of the epidermis. In this study, we confirmed that the expression of ACD proteins in melanin pigmented areas was weaker than that in the non-pigmented areas and found a novel regulatory mechanism underlying asymmetric division of human keratinocytes and the development of skin pigmentation. Furthermore, we identified agents that can induce the expression of ACD-related proteins.
Methods
Localization of ACD-related proteins in pigmented skin lesions
Human skin tissue was purchased from BIOPREDIC International and CTI Biotech Visionary Science following all ethical considerations. The tissues were immunostained for the ACD-related proteins, nuclear mitotic apparatus protein (NuMA), G-protein-signalling modulator 2 (GPSM2), and protein inscuteable homologue (INSC). These proteins were selected because NuMA/GPSM2/αi complexes, which are thought to be physically linked by the adapter protein, INSC, are indispensable in ACD.
Effects of ACD-related proteins on pigmentation in skin equivalents
We constructed three-dimensional skin-equivalent models using NUMA1 knockdown (KD) keratinocytes, normal melanocytes, or fibroblasts. Eleven days after air lifting, the skin-equivalent model cells were lysed, and the expression of NuMA and melanogenesis stimulants derived from keratinocytes (i.e. stem cell factor (SCF), endothelin-1 (ET-1), and macrophage migration stimulating factor (MIF)) was determined using western blotting or enzyme-linked immunosorbent assays (ELISAs). In addition, tissues were fixed and stained with Fontana-Masson and immunostained for differentiation markers.
Evaluation of ACD inducers in keratinocytes
Human keratinocytes were pre-treated with cosmetic ingredient-related substances and irradiated with ultraviolet B. Subsequently, the irradiated cells were incubated with fresh samples. After 24 h of incubation, ACD-related mRNA and protein expression was assessed using the reverse transcription-polymerase chain reaction and western blotting, respectively.
Results and discussion
ACD requires establishment of cortical cell polarity and mitotic spindle orientation along the cell polarity axis. The ACD-related proteins, NuMA, GPSM2, and INSC were detected in the epidermis using immunohistochemistry. Co-localisation of these proteins with melanin in the pigmented areas was weaker than that in the non-pigmented areas. We therefore hypothesised that a decrease in ACD-related protein expression induces skin pigmentation. Because NUMA1 KD downregulated GPSM2 expression in keratinocytes, we next examined whether downregulation of ACD-related proteins led to melanin accumulation in the epidermis using reconstituted three-dimensional skin equivalents constructed with NUMA1 KD keratinocytes. The NUMA1 KD skin equivalents became blacker compared with those prepared using control keratinocytes without an increase in the melanogenesis-stimulants, SCF, ET-1, and MIF. Furthermore, melanin accumulation in the basal and lower spinous layers of the epidermis was observed in the NUMA1 KD skin equivalents. Based on the results showing limited localisation of loricrin, a late differentiation marker, and broad localization of keratin 10, an early differentiation marker, in NUMA1 KD skin equivalents, normal keratinisation was inhibited under NUMA1 KD. We thus speculate that NuMA is involved in melanin clearance. Furthermore, our results suggest that ACD-related proteins in keratinocytes are a new potential target for suppressing the development of skin pigmentation. We also found that epigallocatechin gallate present in green tea (Camellia sinensis) extract and ATP in cosmetic materials act as ACD inducers in keratinocytes. Inducers of ACD-related proteins could be expected to suppress the development of skin pigmentation.
Conclusion
Our results indicate that ACD-related proteins in keratinocytes suppress skin pigmentation by inhibiting melanin accumulation in the basal layer of the epidermis, indicating a new mechanism underlying the development of skin pigmentation. We intend to study this mechanism in greater detail to facilitate development of new cosmetic products for treatment of chronic skin pigmentation by enhancing melanin clearance.
The mechanisms involved in melanogenesis caused by sunburn and skin inflammation have been clarified previously, and various products have been developed to prevent skin pigmentation. Nevertheless, skin pigmentation develops into a chronic condition with age. Hence, we believe that it is important to not only prevent transient melanogenesis but also improve delayed melanin clearance with age. Therefore, we studied the mechanisms enhancing melanin clearance to develop a more effective treatment for chronic skin pigmentation. In most cases, approaches that promote keratinocyte cell proliferation and differentiation were selected to promote the clearance function. Specifically, because the structure and direction of keratinocyte proliferation in the melanin pigmented-area were different from those in the non-pigmented area, we focused on horizontal keratinocyte division (i.e., asymmetric cell division (ACD)), which plays an important role in maintaining the functionality of the epidermis. In this study, we confirmed that the expression of ACD proteins in melanin pigmented areas was weaker than that in the non-pigmented areas and found a novel regulatory mechanism underlying asymmetric division of human keratinocytes and the development of skin pigmentation. Furthermore, we identified agents that can induce the expression of ACD-related proteins.
Methods
Localization of ACD-related proteins in pigmented skin lesions
Human skin tissue was purchased from BIOPREDIC International and CTI Biotech Visionary Science following all ethical considerations. The tissues were immunostained for the ACD-related proteins, nuclear mitotic apparatus protein (NuMA), G-protein-signalling modulator 2 (GPSM2), and protein inscuteable homologue (INSC). These proteins were selected because NuMA/GPSM2/αi complexes, which are thought to be physically linked by the adapter protein, INSC, are indispensable in ACD.
Effects of ACD-related proteins on pigmentation in skin equivalents
We constructed three-dimensional skin-equivalent models using NUMA1 knockdown (KD) keratinocytes, normal melanocytes, or fibroblasts. Eleven days after air lifting, the skin-equivalent model cells were lysed, and the expression of NuMA and melanogenesis stimulants derived from keratinocytes (i.e. stem cell factor (SCF), endothelin-1 (ET-1), and macrophage migration stimulating factor (MIF)) was determined using western blotting or enzyme-linked immunosorbent assays (ELISAs). In addition, tissues were fixed and stained with Fontana-Masson and immunostained for differentiation markers.
Evaluation of ACD inducers in keratinocytes
Human keratinocytes were pre-treated with cosmetic ingredient-related substances and irradiated with ultraviolet B. Subsequently, the irradiated cells were incubated with fresh samples. After 24 h of incubation, ACD-related mRNA and protein expression was assessed using the reverse transcription-polymerase chain reaction and western blotting, respectively.
Results and discussion
ACD requires establishment of cortical cell polarity and mitotic spindle orientation along the cell polarity axis. The ACD-related proteins, NuMA, GPSM2, and INSC were detected in the epidermis using immunohistochemistry. Co-localisation of these proteins with melanin in the pigmented areas was weaker than that in the non-pigmented areas. We therefore hypothesised that a decrease in ACD-related protein expression induces skin pigmentation. Because NUMA1 KD downregulated GPSM2 expression in keratinocytes, we next examined whether downregulation of ACD-related proteins led to melanin accumulation in the epidermis using reconstituted three-dimensional skin equivalents constructed with NUMA1 KD keratinocytes. The NUMA1 KD skin equivalents became blacker compared with those prepared using control keratinocytes without an increase in the melanogenesis-stimulants, SCF, ET-1, and MIF. Furthermore, melanin accumulation in the basal and lower spinous layers of the epidermis was observed in the NUMA1 KD skin equivalents. Based on the results showing limited localisation of loricrin, a late differentiation marker, and broad localization of keratin 10, an early differentiation marker, in NUMA1 KD skin equivalents, normal keratinisation was inhibited under NUMA1 KD. We thus speculate that NuMA is involved in melanin clearance. Furthermore, our results suggest that ACD-related proteins in keratinocytes are a new potential target for suppressing the development of skin pigmentation. We also found that epigallocatechin gallate present in green tea (Camellia sinensis) extract and ATP in cosmetic materials act as ACD inducers in keratinocytes. Inducers of ACD-related proteins could be expected to suppress the development of skin pigmentation.
Conclusion
Our results indicate that ACD-related proteins in keratinocytes suppress skin pigmentation by inhibiting melanin accumulation in the basal layer of the epidermis, indicating a new mechanism underlying the development of skin pigmentation. We intend to study this mechanism in greater detail to facilitate development of new cosmetic products for treatment of chronic skin pigmentation by enhancing melanin clearance.