The Role of Notch Signaling in Genetic Reticular Pigmentary Disorders

Wei-Xue Jia, Ling-Ling Luo, Wen-Rui Li, Yuan-Yuan Zhang, Ping Cheng, Cheng-Rang Li＊

Department of Dermatology, Hospital for Skin Diseases (Institue of Dermatology), Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing, Jiangsu 210042, China.

Abstract

Keywords: genetic, melanocyte, notch signaling, pigmentary disorder

Introduction

Notch signaling is an evolutionarily conserved signal transduction system that controls a broad range of cell differentiation, proliferation and apoptotic, influencing organ formation and morphogenesis.1-3And its dysfunction is implicated in cancer, inflammation, and pigmentary disorder.4-6It is confirmed that Notch signaling is involved in the melanocyte disorders, such as vitiligo and melanoma.In vitiligo, Notch1 signaling is inactivated with consequent loss of epidermal or follicular active melanocytes, and aberrant Notch signaling in vitiliginous white hair and acral and segmental vitiligo may be the cause of treatment resistance.7In melanoma, direct contact of melanoma cells with the remote epidermal layer triggers vertical invasionviaNotch signaling activation.8Moreover, the relationship between Notch signaling and genetic reticular pigmentary disorders becomes more and more evident.POFUT1, POGLUT1,and presenilin enhancer-2(PSENEN)genes are pathogenic genes of Dowling-Degos disease (DDD, also called reticulate pigmented anomaly of the flexures; OMIM 179850, 615696 and 615327).Mutations ofADAM10gene could also lead to one of the genetic reticular pigmentary disorders,reticulate acropigmentation of Kitamura (RAPK; OMIM 615537).Recent years,PSENENand nicastrin(NCSTN)genes are confirmed as the pathogenic genes of familial acne inversa (AI, also called hidradenitis suppurativa;OMIM 142690) associated with pigment abnormalities as well.Products of these genes,POFUT1, POGLUT1,ADAM10, PSENEN,andNCSTN,play vital roles in fucosylation, pyrolysis, and activation of Notch receptors separately.

We searched PubMed by using search strategies(notch [MeSH Major Topic]) AND (pigment [MeSH Major Topic] OR (melanocyte [MeSH Major Topic])from 1997.1 to 2020.12, and summarized the role of Notch signaling in genetic reticular pigmentary disorders in this review.Natural phenotypes of genetic reticular pigmentary disorders provide us an opportunity to explore the underlying mechanism of the Notch signaling pathway in development and function of melanocytes in-depth.

Components and transduction processes of Notch signaling

The gene encoding the Notch receptor was first discovered inDrosophila melanogasterabout a century ago.Orthologs of Notch were identified in other organisms thereafter.And it is proved Notch is highly conserved throughout animal kingdom.3Both Notch receptors and ligands are transmembrane proteins with large extracellular domains that consist of a series of tandem epidermal growth factor (EGF)-like repeats.Therefore, canonical Notch signaling is triggered by the binding of Notch receptors to ligands exposed on neighboring cells.In mammals, there are four different Notch receptors (Notch1-4), and two classes of Notch ligands (Delta 1, 3, 4 and Jagged 1, 2).The maturation of Notch receptors occurs through a series of proteolytic cleavages in the secretory progress.Notch receptor enters the secretory pathway by co-translational insertion into endoplasmic reticulum, and becomes O-fucosylated mediated by O-fucosyltransferase-1 and O-glucosyltransferase-1 encoded byPOFUT1andPOGLUT1,respectively.In Golgi apparatus, the full-length Notch receptor is proteolytically cleaved into two subunits mediated by Furin at site S1.These two subunits form the heterodimeric Notch receptor through calcium-dependent, non-covalent interactions.Binding of ligands to Notch receptors in neighboring cells triggers next two proteolytic cleavage events mediated ADAM-family metal-loproteases and γ-secretase at site S2 and S3, respectively.Two metalloproteases were implicated at site S2 cleavage: ADAM10 (also known as Kuzbanian, Kuz) and tumor-necrosis-factor α-converting enzyme (also known as AD-AM17).The γ-secretase is an enzyme complex that contains presenilin (PSEN),NCSTN, PSENEN and anterior-pharynx-defective-1.The Notch intracellular domain is released from the plasma membrane, and subsequently translocates to the nucleus and forms a complex with the DNA-binding CSL (CBF-1/RBP-Jκ, Suppressor of Hairless/LAG-1)and its coactivator Mastermind-like proteins, to activate various target genes, including members of the Hairy/enhancer of split (Hes) gene and Hairy/enhancer of split related with YRPW gene families.2Recently, evidences show that non-canonical Notch signaling is important in several cellular processes, and associated with cancer and immune dereg-ulations.9The disturbance of endosome-mediated sorting of Notch receptors might result in the triggering of a ligand-independent activation of the receptor during endosomal trafficking.10While the molecular mechanisms and cellular functions of non-canonical Notch cascade need to be elucidated.

Development of melanocytes lineage and melanogenesis

A part of melanoblasts (Mbs), derived from neural crest,colonize epidermal basal layer and differentiate into melanocytes, producing and transferring melanin to adjacent keratinocytes.Another part of Mbs migrate into developing hair follicles and eventually differentiate into two populations: hair matrix melanocytes that colonize in the bulb where they produce melanin that is transferred to hair shaft, and melanocyte stem cells (MSCs) that localize the bulge region at the lower permanent portion of the hair follicle where they could further differentiate into melanocytes and supply melanin to hair.11During the first growth period of postnatal hair, hair matrix melanocytes derived from embryonic Mbs apoptosis occurs.From the anagen phase of second growth period, a part of MSCs migrate to the hair matrix in the bulb, differentiate into mature melanocytes for skin and hair pigmentation.Recently, the secretory portion of eccrine sweat glands in acral volar skin12and the dermis13are also found as the reservoir of melanocytes for skin.

Melanogenesis is a complex biological process responsible for melanin synthesis in melanosomes.Melanin, a multifunctional pigmented biopolymer, plays important roles in determination of skin phenotype and protection from various stimuli, including ultraviolet rays absorbing and scattering, free radicals scavenging, coupled oxidation-reduction reactions, and iron storage.14Tyrosine is catalyzed by a variety of enzyme including tyrosinase,tyrosinase-related protein-1 and tyrosinase-related protein-2, and dopachrome tautomerase through a series of reactions and changes into a polymer ofmelanin, a mixture of pheomelanin and eumelanin.This process is regulated by autocrine or paracrine factors,15involving a variety of signaling pathways, such as Notch, Wnt/β-catenin, kit ligand/Kit, Endothelins/Endothelin receptor type B, transforming growth factor (TGF)-β/TGF-β receptor, α-melanocyte stimulating hormone/Melanocortin 1 receptor.16-17

Genetic reticular pigmentary disorders with responsible genes on notch signaling

Genetic reticular pigmentation disorders seriously affect the patients’ appearance, which causes many patients to mental burden, leading to lower quality of life, social disorder, anxiety, and depression.18Genetic reticular pigmentation disorders include DDD, RAPK, dyschromatosis universalis hereditaria (OMIM 127500), dyschromatosis symmetrica hereditaria (OMIM 127400), and X-linked reticulate pigmentary disorder (OMIM 301220).19

DDD is an autosomal dominant hereditary skin disease,characterized by reticular pigmentation in fold parts, usually in the flexor side such as groin, strands of the medial,armpits, neck, wrist.It usually begins onset of puberty.There have been found four pathogenic gene in DDD: POFUT1,POGLUT1, PSENEN, and KRT5.Three of them, POFUT1,POGLUT1, and PSENEN, are involved in the activation of Notch receptor.POFUT120and POGLUT121encode O-fucosyltransferase-1 and O-gluco-syltransferase-1 respectively.Both of them are essential regulators of Notch signaling activity involving in post-translational modification of Notch receptor.POFUT1 adds O-linked fucose to EGF-like repeats of Notch receptors.Haploinsufficiency of POFUT1 influences the Notch signaling pathway more severely in skin than in hair in humans, and Notch signaling pathway influences the migration of Mbs and melanocyte.20POGLUT1 transfers O-linked glucose from UDP-glucose to serine residues in Notch EGF repeats with the consensus C1-X-S-X-P-C2.22POGLUT1 was essential regulators of Notch activity, which play important role in pigmentation and keratinocyte morphology.21Betzet al.23suggest the haploinsufficiency inKRT5gene induces the dysfunction of melanosome uptake and organelle transport, while the relationship betweenKRT5and Notch signaling remains unknown.

RAPK is an autosomal dominant genetic skin condition,characterized by reticulate, slightly depressed, sharply demarcated pigmented macules mainly affecting the dorsa of hands and feet before puberty and subsequently expand to proximal limb and trunk.Whole-exome sequencing identifiedADAM10mutations as a cause of RAPK.24The ADAM family, membrane-linked metalloprotease, consists of more than 40 members.25ADAM10 cleaves ligand-activated Notch receptor at site S2.In contrast, the rete ridge elongation in RAPK is less prominent than that in DDD and does not show a filiform or antler-like pattern.In addition, the rete ridge elongation in RAPK lesions is seen only in inter follicular epidermis, but not in the follicular infundibulum.Changes in melanocytes and melanosomes seem much more prominent in RAK than in DDD.24The pathogenesis of RAPK and DDD was thought to be associated with Notch signaling pathway,26-27and exact mechanisms calls for further investigation.

AI, also called hidradenitis suppurativa, is a recurrent chronic inflammatory debilitating dermatosis that usually presents with painful inflamed lesions in the apocrine gland-bearing areas of the body after puberty.The gene mutation of subunits of γ-secretase, PSEN, NCSTN,PSENEN, and anterior-pharynx-defective-1, was identified as the pathogenic gene of AI in 2010.28PSENENencodes presenilin enhancer protein-2, andNCSTNencodes NCSTN.They are subunits of γ-secretase complex, involving in the cleavage of Notch receptor at site S3.Recent years, some familial AI patients presented with reticular pigment abnormalities, andPSENENorNCSTNgene of γ-secretase complex in Notch signaling pathway is confirmed as the pathogenic genes.29-32

Overlaps in genetic reticulate pigmentary disorders

Although each disorder above are characterized with unique phenotype, they still overlap greatly.There have been many reports on the coexistence of DDD and RAPK in the literature,33-36and the patients had reticulated hyperpigmentation in the dorsa of hands and feet, axillae and palmoplantar pits.Skin biopsy from hyperpigmented lesion revealed filiform elongation of rete ridges with hyperpigmentation of basilar keratinocytes and clusters of melanocytes at the tip of the rete ridges.Histopathology axillary lesion was also similar except for features of follicular plugging.36Therefore, many scholars suggest that DDD and RAPK are different spectrum manifestations of the same disease.33,36Most treatment attempts for these disorders were unsatisfactory.Adapalene, systemic retinoids, 20% azelaic acid, and Q-switch alexandrite laser have been tried in a few patients with mixed results.36-37Several cases of manifestation of AI-DDD have been described since 1990, and there are several reports of hyperpigmentation in AI patients withPSENENmutations.29-31,38These patients presented with the chronic inflammatory skin disease AI and hyperpigmentation disorder DDD.Recently, a patient with familial AI and concomitant DDD harboring a novel nonsense mutation inNCSTNassociated with a reduced number of critical subunits of γ-secretase was reported.32

DDD, RAPK, and familial AI with pigment abnormalities have similarities and overlaps in clinical manifestations and pathological characteristics: their lesions showed reticular distribution of pigmentation, and their pathology showed dendritic or reticular downward extension of epidermal sudden and melanin increase in basal layer.POFUT1, POGLUT1, ADAM10, PSENEN,andNCSTNgenes are pathogenic genes of DDD, RAPK,and AI with pigment abnormalities separately, which are all vital genes in Notch signaling pathway.According to the similarity in clinic, pathology and pathogenic genes in Notch signaling, Notch signaling play vital roles in pathogenesis of genetic pigmentation disorders.

Notch signaling in melanogenesis

Notch signaling plays a vital role in both development of melanocyte during embryogenesis and maintenance of MSCs to repopulate melanocyte in hair bulb in adulthood in mice.39-40Notch signaling supports survival of immature Mbs by preventing initiation of apoptosisvia Hes1transcription factor.41Conditional knockout of Notch1 and/or Notch2 in melanocytes in mice resulting in a gene dosage-dependent gradual hair graying phenotype after birth, due to the and progressive loss of Mbs and MSCs in the bulge, but does not affect non-follicular melanocytes and pigmentation in the dermis and choroid.42Conditional knockout of RBP-Jκ in melanocyte also led to similar hair graying in mice.42The niche microenvironment plays a crucial role in fate determination for MSCs.11Mbs are strictly regulated by cell-cell interactions with epidermal keratinocytes, and Notch signaling acts as a key component among keratinocyte-melanocyte interactions.

Knockdown ofPOFUT1reduced the expression ofNOTCH1, NOTCH2,HES1, andKRT5in HaCaT cells.20Morpholino knockdown ofpofut1in zebrafish produced a phenotype characteristic of hypopigmentation at 48 hour postfertilization and abnormal melanin distribution at 72 hour postfertilization, replicating the clinical phenotype of DDD.20Therefore, POFUT1 plays a significant and conserved role in melanin synthesis and transport.ADAM10haploinsufficiency was reported to cause freckle-like macules onHairlessHr/HairlessHr, a hairless mouse strain.This mouse strain shows freckle-like pigmentation on the trunk from 5 months of age, and on the glabrous skin ears and feet from 10 months of age, due to the localized clusters of melanocytes.43Interestingly, Adam10 also act as a sheddase for Delta proteins, which serve as Notch ligands.44Shedding of ligand from the cell membrane reduces Notch receptor activation, and researchers hypothesized that decreased Adam10 activity might actually increase Notch signaling.43However, expressing the Notch intracellular domain in melanocytes on a HairlessHr/HairlessHr background did not cause macules to form (C.Van Raamsdonk,personal observation).Using mammalian cell culture studies and knockdown experiments ofpsenenin zebrafish larvae, researchers identifiedPSENENmutations can lead to disturbances in melanocyte migration and differentiation,and the crucial role of Notch signaling in pigmentation and differentiation of the epidermis.30It is demonstrated that zebrafish with insertional mutant of NCSTN presenting depigmentation phenotypes, showed a significant reduction ofNCSTNtranscript possesses mela-nosome maturation defect, Tyrosinase-dependent mitochon-drial swelling, and melanophore cell death.45

Conclusion

From the overlaps of phenotypes of hereditary pigmentation disorders such as DDD, RAPK, and AI with pigment abnormalities, and studies on their pathogenic genesPOFUT1, POGLUT1, ADAM10, PSENEN,andNCSTNin Notch signaling, it is suggested that Notch signaling pathway plays a vital role in melanocyte development and function.Notching signaling is sunlight to clear the fog of the pathogenesis of genetic pigmentary disorders.The limitations of Notch signaling study is that it is not very clearly described in the article about how Notch signaling regulates melanin metabolism.And more work is essential to verify the relationship between Notch signaling and other genetic reticular pigmentary disorders and other depigmentation disorders such as vitiligo.Research on issues of Notch signaling and melanogenesis will be of great help to understand the biological function of melanocyte and the pathogenesis of human genetic pigment abnormalities, and lay foundations for gene therapy and targeted medicine for pigmentary disorders.

Source of funding

This study was funded by the CAMS Innovation Fund for Medical Sciences (No.2016-I2M-1-002) and the Technology Foundation for Selected Overseas Chinese Scholar.