Identification of new signaling pathways that regulate skin pigmentation

The EGR1-STAT3 Transcription Factor Axis Regulates α-MSH-induced Tyrosinase Gene Transcription in Melanocytes

A researcher team led by Professor Soon-young, Shin of Konkuk University has discovered a new mechanism to control skin pigmentation. It was published on website of the Journal of Investigative Dermatology, a dermatological research journal on Jan. 2. This research was carried out as a task of" Support project for advanced researchers" of the Ministry of Science, ICT and Future Planning.

The mechanism of EGR1-STAT3-tyrosinase signaling pathway is a novel one that promotes the biosynthesis of melanin, and found in follow-up studies of the previously reported paper. In 2017, the study began with the first discovery of Agerarin, the ageratum extract. In 2018, a further study was conducted to reveal that Agerarin inhibited the expression of tyrosinease by inhibiting the activation of STAT-3 transcription factor. As a result, its pathway was finally identified. We were very curious about the story contained in the results of subsequent studies. How could these outcomes be achieved?

[Interview] Professor Soon-young, Shin

You first discovered Agerarin in 2017, and I’d like to know the background of the starts of this study.
In 2014, my team conducted a joint research with a team led by Professor Lim, Yoong-ho of Konkuk University, and carried out a research project on ' Development of cosmetic materials based on plant-derived polyphenol glycoside' that was a part of R&D project for' Nutraceutical and Pharmaceutical materials' sponsored by the Rural Development Administration. In the course of the research, we found a new fact that the extract of ' Ageratum'¹, a kind of Compositae, showed skin wrinkle improvement and antioxidant and whitening effects. And we applied it for a patent in cooperation with professor Lim, Yoong-ho. At first, we planned to transfer our patented technology to related companies, however, it was not materialized.

I was disappointed that the results of this functional plant extract would be died out. So, I decided to directly develop cosmeceutical cosmetics². In October 2016, I launched a one-person venture company called ' Agera BioTech' , which allowed me to continue the research.

In the subsequent study at Agera BioTech, I succeeded in separating the effective plant ingredients, which were excellent for skin whitening and moisturizing, from ageratum extract, and found out the molecular structure. Then I named it Agerarin, and applied for domestic and international patents. In October 2018, I created the brand ' Agerin' with the support from the ' Vitalization of women entrepreneurship project' of the Ministry of SMEs and StartupsMSS. Then, the first product group, composed of functional cosmetics package including mask pack, essence oil, and cream products, has been also launched.

What is the main content of this study?

This paper is for the study of the molecular mechanism of skin pigmentation. When exposed to ultraviolet light, α-Melanocyte Stimulating Hormoneα-MSH is secreted from Keratinocytes in the epidermis to stimulate Melanocytes. Melanocyte stimulated by α-MSH promotes the expression of tyrosinase gene, and tyrosinase enzyme oxidizes tyrosine to initiate the melanin biosynthesis process. Melanocyte-derived polymeric melanin moves to keratinocytes. Over time, the newly produced keratinocytes pushed up the melanin-containing keratinocytes to the upper stratum corneum. And finally, the black melanin pigment in keratinocytes made the skin look black.

In the academic world, it has been known that when α-MSH is produced in keratinocytes by ultraviolet stimulation, tyrosinase gene expression is promoted through the cell signaling pathway of Phosphorylating enzyme APKA– CRE binding proteinCREB- Melanin transcription factorMITFin melanocyte. Our research team conducted a study on tyrosinase activity that played a pivotal role in the biosynthesis of melanin pigments. In the meantime, we discovered a new melanin-biosynthesis promoting cell signaling pathway unknown to the existing academic world.

Please describe the mechanism of EGR1-STAT3-Tyrosinase pathway.

EGR1 is a protein about 80 kDa-sized, with 543 amino acids. It is a transcription factor and has a Zinc-finger motif in the form of Cys2-His2 at the c-Terminal. In the case of the EGR1 protein, the expression of EGR1 gene increases in damaged cells when severe damage occurs in the skin cell DNA due to excessive ultraviolet or radiation exposure, which restores damaged DNA or causes apoptosis (inducing cell death) in these cells. This process prevents the cells with damaged DNA (mutant DNA) from proliferating in the body and maintains the homeostasis of the skin cells. Skin keratinocyte, which has been excessively exposed to ultraviolet light, stimulates melanocytes by secreting α -MSH as part of preventing DNA damage. Then EGR1 expression is induced in melanocyte. At this time, EGR1 produced binds to the promoter region of the STAT3 gene, thereby increasing the amount of STAT3 protein. EGR1 produced at this time binds to the promoter region of the STAT3 gene, increasing the amount of STAT3 protein. The increased STAT3 protein in the cytoplasm is activated by JAK1/2Janus kinase 1/2, and the activated STAT3 moves to the nucleus and binds to the tyrosinase gene promoter, thereby increasing the expression of tyrosinase gene. The increased tyrosinase protein oxidizes tyrosine, an amino acid, to initiate the melanin biosynthesis process. Therefore, if EGR1 expression increases in skin cells exposed to ultraviolet light or various stresses, melanin production is promoted, and making the skin color black or increasing pigmentation such as melasma, freckles, and age spots.

There is also a research result showing that ageratum extract can be a moisturizing and whitening ingredient.
Our research team found out the active ingredient that showed moisturizing effects from the ageratum extract, and named it Agerarin. Then we revealed its molecular structure and applied for domestic and international patents. In September 2017, we showed our study result that Agerarin, an ageratum extract, induced expression of circadian molecular bio clock, a gene of the daily periodic molecular biological clock and promoted the physiological activity of skin biorhythm to show skin moisturizing effects³. In July 2018, a study, that showed that Agerarin inhibited the activity of STAT3 transcription factor and melanogenesis caused by ultraviolet exposure to showing skin whitening effects, has been published in the Journal of Dermatological Science. This study proved the fact that the ageratum extract can provide not only moisturizing effects but also whitening function.⁴ And this study shows that transcription factor EGR1 regulates STAT3 gene expression to regulate melanin pigmentation caused by ultraviolet exposure.

In this way, it can be presumed that the ageratum extract Inhibits EGR1 activity and thereby reduce STAT3 gene expression to show whitening function.

How did you start research on EGR1-inhibiting materials?

The regulation of melanin production by EGR1 was discovered accidentally during the study of melanoma, a type of skin cancer.

Melanoma is a malignant skin tumor that occurs in melanocyte, and has a very high rate of cancer growth. This is a malignant tumor with the highest mortality rate among skin cancers, showing an average 5-year survival rate in only 30% of patients because it easily migrates to various other organs such as bones, lungs, and brain along with the lymph nodes. In Koreans, it is known to occur well on extremities such as the palms, soles, fingers, and toes. In Korea, the number of cases of melanoma is increasing.

Our research team has been studying the active function of EGR1 protein- induced metastasis for a long time. One of the projects was to identify the role of EGR1 in lung metastasis of melanoma. In the course of the study, we found that inhibition of EGR1 expression using shRNA⁵ significantly inhibited lung metastasis. At this time, we have also observed that the melanogenesis, itself, was significantly reduced in cancer tissues where EGR1 expression was suppressed.

I repeated the experiment several times, suspecting that ' Is it due to the individual difference of cancer tissues? Is there anything wrong in our experiment?'. However, melanin production was significantly reduced when EGR1 expression was suppressed in all experiments without exception. To investigate whether this phenomenon appeared only in melanoma cancer cells or not, EGR1 expression was suppressed in normal melanocytes. As a result, it was confirmed that if EGR1 expression was inhibited in normal cells, the melanin biosynthesis process was blocked. This phenomenon was thought to be a new molecular mechanism that has never been reported in the academic world. So, I concentrated on the skin whitening effect. It is expected that the suppression of EGR1 expression will not only help whitening effects but also prevent skin cancer.

What are the differences in moisturizing cosmetics based on Flora chrysanthemum extract?
First of all, the moisturizing effect ageratum extracts not only prevent the moisture released from the skin but also promotes the skin physiological activity to show a highly elaborate moisturizing function.

More specifically, the lipid layers such as ceramide and sphingomyelin in the skin epidermis construct a skin barrier and play an important role in maintaining skin moisturization.

The lipids are biosynthesized by a combination of glycerolC3H8O3molecules and fatty acid in epidermal keratinocytes. Since glycerol binds to water molecules and also acts as a natural moisturizing factorNMF that prevents moisture from escaping from the skin, glycerol in the epidermis is very important for the formation of skin barriers and moisturization. Glycerol (glycerin) contained in many cosmetics, forms a thin film on the keratin skin. This has an effect on preventing moisture loss from the skin; however it is hard to expect the role of synthesizing lipids to create skin barrier.

On the other hand, glycerol and water are supplied from the dermis through the transporter protein called ' Aquaporin-3' in the basal cell layer located between epidermis and dermis.

The expression of aquaporin-3 protein is regulated by the circadian molecular biological clock that controls various metabolic and physiological phenomena in our body. ‘Agerarin‘, the ageratum extract promotes the activity of CLOCK gene, a gene of the molecular biological clock active at night, and it increases the amount of aquaporin-3 protein to supply moisture and glycerol from deep skin to epidermis. It also inhibits tyrosinase gene expression, which plays a pivotal role in the biosynthesis of melanin pigments.

In general, our skin damaged during the day is recovered at night by aquaporin-3 that works to fill the damaged skin with moisture. Stimulation, such as stress and excessive UV exposure, disrupts this normal circadian biorhythm to weaken skin regeneration and reduce adequate moisturization and whitening actions during the night. Therefore, ageratum extract- moisturizing cosmetics are based on the principle of activating skin molecular biological clocks to repair damaged skin barriers and promote moisturizing activities by ourselves during the night, rather than artificial water supply. Such a function that is friendly to bio-physiological activity is one of the most attractive advantages of ageratum extract functional cosmetics.

What are the expected results and implications of this study?

In the meantime, most of the functional cosmetics materials for skin whitening have been focused on the development of materials that block cell signaling leading to PKA-CREB-MITF. PKA-CREB-MITF cell signaling pathways play an important role in the melanogenesis process. However, this is also important for other skin physiological functions, so skin pigmentation- inhibiting cosmetic materials that block this pathway can cause various large and small side effects. The implications of this study are a discovery that melanogenesis of the skin is inhibited when the PKA-CREB-MITF cell signaling pathway, that is previously known, is active and the EGR1-STAT3 activation molecular pathway is inhibited. This fact means that the EGR1- STAT3 transcription factor is a cellular pathway necessary in the melanin biosynthesis process, and it is possible to inhibit skin pigmentation without blocking the PKA-CREB-MITF signaling pathway.

Do you have any further study plans based on the signaling pathway you have found?
Based on the newly found EGR1-STAT3 transcription factor, we will discover new natural materials that can control it, and develop a new concept of whitening functional cosmetics. Cosmologically, people may want to avoid melanin that makes the skin dark. However, dermatologically, melanin is a very important physiological substance that prevents DNA damage of the skin cells from strong ultraviolet stimulation.

Unconditional prevention of melanogenesis can cause skin damage and promote skin aging. Whitening means" beautiful white" . However, the beauty we pursue is not the concept of ' Whitening' which simply whitens the face, but the concept of ' Brightening' which makes the skin healthy, bright and glowing. Based on the results of this study, the goal and differentiating points of our research is to create a new healthy and brightening concept of" Skin Brightening functional cosmetics," which effectively prevents abnormal pigmentation such as melasma, freckles and age spots caused by excessive melanogenesis, but maintains the unique protective function of melanin optimally.

I followed your footsteps and was surprised that you' ve been doing research on cancer and inflammation constantly. I wonder how you can constantly show your achievements.
I think it's an ingrained lifestyle habit rather than a secret. I am neither a genius nor an extraordinary person, but I am trying to enjoy the study itself. I still have a lot of questions and I think that there are too many things to learn. I think that life is really mysterious. The more I dig in, the more I know, the more I feel that there are so many things, making me have a sense of awe.

What kind of research do you want to do in the future? What is the specific field of interest?
The more I study in the field of skin science, the more attractive it is. Skin is the largest and most important organ to protect our bodies against harmful environments when our bodies come into direct contact with the external environment. Nevertheless, it seems that the importance of the skin science field has been underestimated in academia and society. There seem to be a lot of unresolved scientific challenges in the field of skin science, just like in other life sciences.

Among the fields, I am especially interested in the field of atopic dermatitis which is a representative incurable disease. The cause of the atopic disease has not been fully identified yet. It involves very complex causes and processes of diseases, the role of various inflammatory cytokines, and the complex immune system.

It is a disease that causes too much pain including extreme itching, especially in children. There is still no cure medicine for this. So, personally, I think that the atopy field is an academically attractive research subject of research, and it is thought to be a global blue ocean field also in industry.

So, I am trying to study the causes and treatment mechanisms of atopic dermatitis more systematically.  Editor Lily Kim

1. Ageratum; Scientific name: Ageratum houstonianum.
2. Functional cosmetics, a combination of Cosmetics and Pharmaceutics, representing pharmacological effects.
3. Published on the Scientific Report in Sep. 2017.
Paper name, Agerarin, identified from Ageratum houstonianum, stimulates circadian CLOCK-mediated aquaporin-3 gene expression in HaCaT keratinocytes. 2017;11;7(1):11175. doi: 10.1038/s41598-017- 11642-x.
4. Published on the Journal of Dermatological Science in 2018.
Paper name, Agerarin inhibits α -MSH–induced TYR gene transcription via STAT3 suppression independent of CREB-MITF pathway. 2018;91:104-112).
5. small hairpin RNA.
It is a technique of molecular biological experiment consequently reduces protein expression by interfering with the translation of mRNA into protein by using a base sequence complementary to mRNA that synthesizes specific proteins