The transfer of melanin from melanocytes to keratinocytes is a crucial process underlying maintenance of skin pigmentation and photoprotection against UV damage. Here, we present evidence supporting coupled exocytosis of the melanin core, or melanocore, by melanocytes and subsequent endocytosis by keratinocytes as a predominant mechanism of melanin transfer. Electron microscopy analysis of human skin samples revealed three lines of evidence supporting this: (1) the presence of melanocores in the extracellular space; (2) within keratinocytes, melanin was surrounded by a single membrane; and (3) this membrane lacked the melanosomal membrane protein tyrosinase-related protein 1 (TYRP1). Moreover, co-culture of melanocytes and keratinocytes suggests that melanin exocytosis is specifically induced by keratinocytes. Furthermore, depletion of Rab11b, but not Rab27a, caused a marked decrease in both keratinocyte-stimulated melanin exocytosis and transfer to keratinocytes. Thus, we propose that the predominant mechanism of melanin transfer is keratinocyte-induced exocytosis, mediated by Rab11b through remodeling of the melanosome membrane, followed by subsequent endocytosis by keratinocytes.
Choroideremia (CHM), an X-linked degeneration of the retinal pigmented epithelium (RPE), photoreceptors, and choroid, ultimately leads to blindness. It is caused by loss-of-function of the CHM gene product, the Rab escort protein 1 (REP1) that is involved, together with its homologue REP2, in prenylation of Rab GTPases, key regulators of intracellular vesicular traffic. Here, we report the molecular characterization of 20 unrelated Italian families affected by CHM. We identified 19 different mutations, nine of which are new. In most cases, we analyzed the effect of the mutations at the mRNA level. Furthermore, we demonstrated, by in vitro trancription/translation assays, that the mutated mRNAs produced truncated proteins in all cases but one. In fact, we also identified a novel REP1 missense variant (c.1520A>G; p.H507R) associated to CHM. Thus far, only two other CHM-associated missense mutations have been identified, one of which was a splicing alteration. We investigated the impact of the p.H507R amino acid change on REP1 structure and function, thus providing the first experimental demonstration that correlates a missense mutation in CHM with a functional impairment of REP1. Overall, our results indicate that the REP1-Rab geranyl-geranyl transferase interaction and consequently REP1-mediated Rab prenylation is essential for RPE and photoreceptor function.
The NOS (nitric oxide synthase) inhibitor ADMA (asymmetric dimethylarginine) contributes to the pathogenesis of pulmonary hypertension. Reduced levels of the enzymes metabolizing ADMA, dimethylarginine dimethylaminohydrolases (DDAH1 and DDAH2) and increased levels of miR-21 are linked to disease pathology, but the mechanisms are not understood. In the present study we assessed the potential role of miR-21 in the regulation of hypoxia-induced changes in ADMA metabolism in vitro and in vivo. Hypoxia inhibited DDAH1 and DDAH2 expression and increased ADMA levels in cultured human pulmonary endothelial cells. In contrast, in human pulmonary smooth muscle cells, only DDAH2 was reduced whereas ADMA levels remained unchanged. Endothelium-specific down-regulation of DDAH1 by miR-21 in hypoxia induced endothelial dysfunction and was prevented by overexpression of DDAH1 and miR-21 blockade. DDAH1, but not DDAH2, mRNA levels were reduced, whereas miR-21 levels were elevated in lung tissues from patients with pulmonary arterial hypertension and mice with pulmonary hypertension exposed to 2 weeks of hypoxia. Hypoxic mice treated with miR-21 inhibitors and DDAH1 transgenic mice showed elevated lung DDAH1, increased cGMP levels and attenuated pulmonary hypertension. Regulation of DDAH1 by miR-21 plays a role in the development of hypoxia-induced pulmonary hypertension and may be of broader significance in pulmonary hypertension.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.