Darkening with UVRAG

ABSTRACT

Ultraviolet radiation (UVR)-induced skin pigmentation, afforded by the dark organelles termed melanosomes, accounts for the first-line protection against environmental UVR that increases the risk of developing skin cancers including melanoma. We have recently discovered that UVRAG, originally identified as a BECN1-binding macroautophagy/autophagy protein, appears to have a specialized function in melanosome biogenesis beyond autophagy through its interaction with the biogenesis of lysosome-related organelles complex 1 (BLOC-1). This melanogenic function of UVRAG is controlled by the melanocyte-specific transcription factor MITF as a downstream effector of the α-melanocyte-stimulating hormone (α-MSH)-cAMP signaling in the suntan response, which is compromised in BRAF mutant melanoma. Thus we propose a new mode of UVRAG activity and regulation in melanocyte biology that may affect melanoma predisposition.

KEYWORDS:

• Autophagy
• BLOC-1
• BRAF
• melanosome
• UVRAG

Adaptive skin pigmentation (tanning), the first-line defense against UV radiation, is a fundamental biological process that involves complex crosstalk between keratinocytes and melanocytes. Upon UV exposure, skin keratinocytes secrete α-MSH, which signals melanocytes to ramp up production of UV-blocking melanin in the lysosome-like granule known as melanosomes, a process that is mediated by a complex network of proteins. The pigmented granules are passed on to neighboring keratinocytes to prevent excessive UV penetration and maintain genomic integrity. The melanogenic machinery is not only crucial in cells undergoing toxic UV insult, but also critical for the development of the optic nervous system and retinal function. Recent genomic studies revealed several autophagy-related factors that affect the melanogenic function of melanocytes, suggesting a potential interaction between the autophagy network and cell pigmentation. But the orchestration of the melanogenic process and the autophagy pathway is far from understood, despite the key roles of the 2 processes in cell physiology and pathology. Our recent study shed new light on the matter, identifying an unexpected function of the autophagy-related protein UVRAG (UV radiation resistance associated gene) in melanogenesis and the tanning response.

The initial description of UVRAG focused on its ability to form a complex with BECN1 and the lipid kinase PI3KC3/Vps34, and promote their activity in autophagic membrane remodeling. Later, the story became more complicated when it was discovered that different UVRAG complexes exist and modulate the highly regulated progression of autophagosome formation to lysosomal degradation, as well as other membrane trafficking events that either intersect or converge with autophagic trafficking. Consistent with its autophagic role in maintaining cellular homeostasis, UVRAG at abnormally low levels contribute to the development of diverse types of cancer. Polymorphism in the UVRAG gene is also linked to susceptibility to vitiligo, a pigmentary disorder that is marked by aberrant melanogenesis. However, no study further explored or validated this characteristic, nor is the molecular basis of UVRAG in cell pigmentation known.

To gain further insight into the function of UVRAG in pigment-producing cells, we depleted UVRAG from melanocytes and found not only that the cells become significantly whitened with less melanin production, but also that they are impaired in melanosome integrity, although the expression and activity of the melanogenic enzymes such as TYR, TYRP1, and DCT all remained unaffected [1]. Reassuringly, we could remedy these defects by re-expressing normal UVRAG in these cells. Conversely, when UVRAG is overexpressed, the cells become darkened with an increased number of melanosomes detected in the cytoplasm. A conserved role for UVRAG in melanogenesis and melanocyte differentiation is also observed in fish embryo in vivo. To address the mechanism of this phenomenon, we conducted a yeast two-hybrid screen to assess the spectrum of proteins that interact with UVRAG. Interestingly, a couple of UVRAG partner proteins turned out to be known components of the melanogenic machinery. Particularly intriguing was the interaction between UVRAG and BLOC1S1/BLOS1, which is a key subunit of the BLOC-1 complex involved in melanosomal cargo sorting and delivery – a prerequisite for melanosome pigmentation. We found that UVRAG directly binds to BLOC1S1, and that this interaction is required for the proper localization and function of BLOC-1 during melanosome biogenesis. Removing UVRAG or expressing a mutant form of the protein that cannot bind BLOC1S1 leads to cells defective in the sorting and delivery of melanosomal cargoes to melanosomes, whereas silencing BLOC-1 proteins abolishes the effect of UVRAG in pigmentation. A detailed mechanistic analysis revealed that UVRAG interaction facilitates the assembly of a stable BLOC-1 complex on tubular endosomes during the melanogenic process.

As if the discovery of another function for UVRAG was not surprising enough, we further showed that the role of UVRAG in melanogenesis is independent of its BECN1-associated activity in autophagy. This clearly separates the classical, BECN1-dependent autophagy regulatory function of UVRAG from its role in cell pigmentation. The different roles of UVRAG are relevant for melanocyte adaptation to different environmental cues. For instance, we observed that the melanogenic function of UVRAG as a direct transcriptional target of MITF is particularly required in cutaneous α-MSH-stimulated melanocytes. However, the nutrient-responsive transcription factor TFEB and TFE3, which are more ubiquitously expressed and govern autophagy-lysosome-related gene expression, are dispensable for UVRAG regulation in response to UV tanning. The involvement of autophagy regulators such as UVRAG in melanogenesis and their differential regulation by a MiT/TFE family member suggests that the melanogenic process and the autophagy pathway might be intimately coordinated.

UV-induced melanogenesis is strongly associated with the risk of melanoma. Fair-skinned individuals, especially those who fail to tan after sun exposure, are more likely to develop melanoma than dark-skinned ones. This is even more pertinent in the cases of melanomas with the acquisition of oncogenic BRAF, which are known to be associated with poor UV protection. This is not a purely academic issue, because we found that BRAF mutant melanoma patients have significantly reduced expression of most melanogenic genes including UVRAG compared with those carrying the wild-type genotype. Both the basal levels and α-MSH-induced accrual of melanosomes are compromised in BRAF mutant melanomas as a result of suppressed MITF expression. The link between oncogenic BRAF and dampened melanogenesis is strengthened by the finding that treatment with BRAF inhibitors drastically upregulates MITF and its downstream target UVRAG, leading to increased cell pigmentation and, by extension, UV protection.

Overall, this study clearly established a non-canonical function of a core autophagy regulator in melanogenesis and the suntan response, which also poses interesting questions. It is still unclear how the actions of these various functions of UVRAG and its multiple interactors are integrated in response to different stimuli. It is also of interest as to how melanosomal and autophagosomal vesicle trafficking destined for the specialized lysosome-related organelles is differentially regulated. Future studies on these topics will undoubtedly provide valuable input for melanocyte biology and pathology, and will, hopefully, suggest new approaches to a number of human diseases associated with compromised melanogenesis.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the National Cancer Institute [CA140964].