Huirong Li

Microphthalmia-associated transcription factor acts through PEDF to regulate RPE cell migration.

Cells of the retinal pigment epithelium (RPE) play major roles in metabolic functions, maintenance of photoreceptor function, and photoreceptor survival in the retina. They normally form a stable monolayer, but migrate during disease states. Although growth factors produced by the RPE cells primarily control these cellular events, how these factors are regulated in RPE cells remain largely unknown. Here we show that the basic-helix-loop-helix-leucine zipper microphthalmia-associated transcription factor (MITF), which plays central roles in the development and function of a variety of cell types including RPE cells, upregulates the expression of a multifunctional factor PEDF in RPE cells. Consequently, the upregulation of PEDF impairs microtubule assembly and thus inhibits RPE cell migration. Conversely, specific knockdown of PEDF partially rescues the impairment of microtubule assembly and cell migration proceeds in MITF overexpressing stable cells. We conclude that MITF acts through PEDF to inhibit RPE cell migration and to play a significant role in regulating RPE cellular function. We suggest that MITF has a novel and important role in maintaining RPE cells as a stable monolayer and the down-regulation of PEDF that may contribute to retinal degenerative diseases.

Regulation of pigmentation by microRNAs: MITF-dependent microRNA-211 targets TGF-β receptor 2.

There is growing evidence that microRNAs are important regulators of gene expression in a variety of cell types. Using immortalized cell lines and primary neural crest cell explants, we show that microRNA‐211, previously implicated in the regulation of melanoma proliferation and invasiveness, promotes pigmentation in melanoblasts and melanocytes. Expression of this microRNA is regulated by the key melanocyte transcription factor MITF and regulates pigmentation by targeting the TGF‐β receptor 2. Transfection with pre‐miR‐211 precursor molecules in melb‐a and melan‐a cells leads to a decrease in the expression of TGF‐β receptor 2 and reduces the TGF‐β signaling‐mediated downregulation of two melanogenic enzymes, tyrosinase and tyrosinase‐related protein 1. Conversely, downregulation of microRNA‐211 using specific microRNA inhibitors has the opposite effects. It appears, therefore, that microRNA‐211 serves as a negative regulator of TGF‐β signaling which is known to play a important roles in vivo in melanocyte stem cell maintenance and pigmentation.

Dlic1 deficiency impairs ciliogenesis of photoreceptors by destabilizing dynein

Cytoplasmic dynein 1 is fundamentally important for transporting a variety of essential cargoes along microtubules within eukaryotic cells. However, in mammals, few mutants are available for studying the effects of defects in dynein-controlled processes in the context of the whole organism. Here, we deleted mouse Dlic1 gene encoding DLIC1, a subunit of the dynein complex. Dlic1−/− mice are viable, but display severe photoreceptor degeneration. Ablation of Dlic1 results in ectopic accumulation of outer segment (OS) proteins, and impairs OS growth and ciliogenesis of photoreceptors by interfering with Rab11-vesicle trafficking and blocking efficient OS protein transport from Golgi to the basal body. Our studies show that Dlic1 deficiency partially blocks vesicle export from endoplasmic reticulum (ER), but seems not to affect vesicle transport from the ER to Golgi. Further mechanistic study reveals that lack of Dlic1 destabilizes dynein subunits and alters the normal subcellular distribution of dynein in photoreceptors, probably due to the impaired transport function of dynein. Our results demonstrate that Dlic1 plays important roles in ciliogenesis and protein transport to the OS, and is required for photoreceptor development and survival. The Dlic1−/− mice also provide a new mouse model to study human retinal degeneration.

Allele-specific genetic interactions between Mitf and Kit affect melanocyte development.

The tyrosine kinase receptor KIT and the transcription factor MITF, each required for melanocyte development, have been shown to interact functionally both in vitro and in vivo. In vitro, KIT signaling leads to MITF phosphorylation, affecting MITF activity and stability. In vivo, the presence of the Mitf Mi‐wh allele exacerbates the spotting phenotype associated with heterozygosity for Kit mutations. Here, we show that among a series of other Mitf alleles, only the recessive Mitf mi‐bws mimics the effect of Mitf Mi‐wh on Kit. Intriguingly, Mitf mi‐bws is characterized by a splice defect that leads to a reduction of RNAs containing MITF exon 2B which encodes serine‐73, a serine phosphorylated upon KIT signaling. Nevertheless, other Mitf alleles that generally affect Mitf RNA levels, or carry a serine‐73‐to‐alanine mutation that specifically reduces exon 2B‐containing RNAs, do not show similar interactions with Kit in vivo. We conclude that the recessive Mitf mi‐bws is a complex allele that can display a semi‐dominant effect when present in a Kit‐sensitized background. We suggest that human disease variability may equally be due to complex, allele‐specific interactions between different genes.

Microphthalmia-associated transcription factor regulates skin melanoblast migration by repressing the melanoma cell adhesion molecule.

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Microphthalmia-associated transcription factor/T-box factor-2 axis acts through Cyclin D1 to regulate melanocyte proliferation.

Control of cell proliferation is critical for accurate cell differentiation and tissue formation, during development and regeneration. Here, we have analysed the role of microphthalmia‐associated transcription factor MITF and its direct target, T‐box factor TBX2, in regulating proliferation of mammalian neural crest‐derived melanocytes.

Microphthalmia-associated transcription factor regulates the visual cycle genes Rlbp1 and Rdh5 in the retinal pigment epithelium

Regeneration of the visual pigment by cells of the retinal pigment epithelium (RPE) is fundamental to vision. Here we show that the microphthalmia-associated transcription factor, MITF, which plays a central role in the development and function of RPE cells, regulates the expression of two visual cycle genes, Rlbp1 which encodes retinaldehyde binding protein-1 (RLBP1), and Rdh5, which encodes retinol dehydrogenase-5 (RDH5). First, we found that Rlbp1 and Rdh5 are downregulated in optic cups and presumptive RPEs of Mitf-deficient mouse embryos. Second, experimental manipulation of MITF levels in human RPE cells in culture leads to corresponding modulations of the endogenous levels of RLBP1 and RDH5. Third, the retinal degeneration associated with the disruption of the visual cycle in Mitf-deficient mice can be partially corrected both structurally and functionally by an exogenous supply of 9-cis-retinal. We conclude that the expression of Rlbp1 and Rdh5 critically depends on functional Mitf in the RPE and suggest that MITF has an important role in controlling retinoid processing in the RPE.

BMP4 is required for the initial expression of MITF in melanocyte precursor differentiation from embryonic stem cells.

Although the differentiation of melanoblasts to melanocytes is known to depend on many distinct factors, it is still poorly understood which factors lead to the induction of melanoblasts. To determine which factors might induce melanoblasts, we examined a set of candidate factors for their ability to induce expression of MITF, a master regulator of melanoblast development, in an ES cell-based melanocyte differentiation system. It appears that BMP4 is capable of inducing MITF expression in stem cells. In contrast, a number of other factors normally implicated in the development of the melanocyte lineage, including WNT1, WNT3a, SCF, EDN3, IGF1, PDGF, and RA, cannot induce MITF expression. Nevertheless, BMP4 alone does not allow MITF-expressing precursors to become differentiated melanocytes, but the addition of EDN3 further promotes differentiation of the precursors into mature melanocytes. Our results support a model in which BMP4 induces MITF expression in pluripotent stem cells and EDN3 subsequently promotes differentiation of these MITF expressing cells along the melanocyte lineage.

Epilation induces hair and skin pigmentation through an EDN3/EDNRB-dependent regenerative response of melanocyte stem cells

In response to various types of injury, melanocyte stem cells (McSCs) located in the bulge of hair follicles can regenerate mature melanocytes for hair and skin pigmentation. How McSCs respond to injury, however, remains largely unknown. Here we show that after epilation of mice, McSCs regenerate follicular and epidermal melanocytes, resulting in skin and hair hyperpigmentation. We further show that epilation leads to endogenous EDN3 upregulation in the dermal papilla, the secondary hair germ cells, and the epidermis. Genetic and pharmacological disruption of the EDN3 receptor EDNRB in vivo significantly blocks the effect of epilation on follicular and epidermal melanocyte regeneration as well as skin and hair hyperpigmentation. Taken together, these results indicate that epilation induces McSCs activation through EDN3/EDNRB signaling and in turn leads to skin and hair hyperpigmentation. The findings suggest that EDN/EDNRB signaling may serve as a potential therapeutic target to promote repigmentation in hypopigmentation disorders.

DAPL1, a susceptibility locus for age-related macular degeneration, acts as a novel suppressor of cell proliferation in the retinal pigment epithelium

&NA; The retinal pigment epithelium (RPE) forms a monolayer at the back of the vertebrate eye and is fundamental to retinal function and homoeostasis. During early development, RPE cells undergo rapid proliferation, but in the adult, they remain normally nonproliferative throughout life. Nevertheless, under pathological conditions such as in proliferative vitreoretinopathy or after retinal ablation, mature RPE cells can re‐enter the cell cycle and form nodules or multiple cell layers. Here we show that Dapl1, whose human homolog represents a susceptibility locus for age‐related macular degeneration (AMD), is highly up‐regulated in quiescent but not proliferating RPE cells and that experimental overexpression of DAPL1 in proliferating RPE cells inhibits their proliferation. Consistent with this observation, the percent of Ki67‐positive cells is significantly higher in E11.5 Dapl1 knockout mouse embryos compared to age‐matched controls. In adult Dapl1‐/‐ mice, which survive without showing any overt pathology, RPE overgrowth leads to multiple cell layers and/or cellular nodules. The antiproliferative effect of DAPL1 is associated with an increase in CDKN1A protein levels. Reduction of CDKN1A by siRNA in DAPL1‐overexpressing RPE cells in vitro partially restores cell proliferation. Hence, we show that DAPL1 is a novel regulator of RPE cell proliferation that is important for the maintenance of the RPE as a monolayer. The findings suggest that DAPL1 dysregulation may be involved in abnormal RPE‐related proliferative diseases and corresponding retinal dysfunctions in humans.