Background-Deficiencies of iron-sulfur (Fe-S) clusters, metal complexes that control redox state and mitochondrial metabolism, have been linked to pulmonary hypertension (PH), a deadly vascular disease with poorly defined molecular origins. The BolA Family Member 3 (BOLA3) regulates Fe-S biogenesis, and mutations in BOLA3 result in multiple mitochondrial dysfunction syndrome, a fatal disorder associated with PH. The mechanistic role of BOLA3 in PH remains undefined. Methods-In vitro assessment of BOLA3 regulation and gain and loss of function assays were performed in human pulmonary artery endothelial cells (PAECs) using siRNA and lentiviral vectors expressing the mitochondrial isoform of BOLA3. Polymeric nanoparticle 7C1 was utilized for lung endothelial-specific delivery of BOLA3 siRNA oligonucleotides in mice. Overexpression of pulmonary vascular BOLA3 was performed by orotracheal transgene delivery of adenoassociated virus in mouse models of PH. Results-In cultured hypoxic PAECs as well as lung from human Group 1 and 3 PH patients as well as multiple rodent models of PH, endothelial BOLA3 expression was down-regulated, which involved HIF-2α-dependent transcriptional repression via HDAC-mediated histone deacetylation. In vitro gain and loss of function studies demonstrated that BOLA3 regulated Fe-S integrity, thus modulating lipoate-containing 2-oxoacid dehydrogenases with consequent control over glycolysis and mitochondrial respiration. In contexts of siRNA knockdown and naturally occurring human genetic mutation, cellular BOLA3 deficiency down-regulated the glycine cleavage system protein H (GCSH), thus bolstering intracellular glycine content. In the setting of these alterations of oxidative metabolism and glycine levels, BOLA3 deficiency increased endothelial proliferation, survival, and vasoconstriction, while decreasing angiogenic potential. In vivo, pharmacologic knockdown of endothelial BOLA3 and targeted overexpression of BOLA3 in mice demonstrated that BOLA3 deficiency promotes histologic and hemodynamic manifestations of PH. Notably, the therapeutic effects of BOLA3 expression were reversed by exogenous glycine supplementation. Conclusions-BOLA3 acts as a crucial lynchpin connecting Fe-S-dependent oxidative respiration and glycine homeostasis with endothelial metabolic reprogramming critical to PH pathogenesis. These results provide a molecular explanation for the clinical associations linking PH with hyperglycinemic syndromes and mitochondrial disorders. These findings also identify novel metabolic targets, including those involved in epigenetics, iron-sulfur biogenesis, and glycine biology, for diagnostic and therapeutic development. Yu et al.

The neonatal Fc receptor (FcRn) for IgG has been shown to be responsible for IgG transport and to be involved in IgG catabolism. In this study, we show expression of FcRn in normal human epidermal keratinocytes. By RT-PCR, we demonstrate the FcRn alpha-chain mRNA obtained from cultured keratinocytes creating a 457 bp product as confirmed by sequence analysis. Northern blot analysis shows a 1.5 kb transcript. Real-time PCR reveals consistent expression of FcRn alpha-chain mRNA in human keratinocytes from different donors. Anti-FcRn alpha2-extracellular domain and anti-FcRn cytoplasmic tail antibody (Ab) directed against defined antigenic targets were generated and used for immunoblotting and immunoprecipitation revealing protein expression of the 46 kDa FcRn alpha-chain. By immunofluorescence microscopy, we find granular-vesicular staining for FcRn alpha-chain in keratinocytes. Fluorescence-activated cell sorting analysis gives predominantly an intracellular distribution of FcRn in keratinocytes. Biochemically, we demonstrate Fc-dependent binding of human IgG at acidic pH. In normal human epidermis, we find a cytoplasmic vesicular staining of predominantly basal and suprabasal keratinocytes. In summary, we demonstrate expression of a functional FcRn in normal human epidermal keratinocytes. These findings further emphasize the role of keratinocytes as immunomodulating cells in inflammatory and immunologic processes of the skin.

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Retinoids are important modulators for cell growth and differentiation of normal skin. In plasma, retinol is transported coupled to plasma retinol-binding protein. In this study, we investigated gene and protein expression of RPE65, a putative receptor for plasma retinol-binding protein in human epidermal keratinocytes. We performed real-time PCR analysis to evaluate expression of RPE65 mRNA in proliferating and differentiating keratinocytes. Immunoblotting with anti-RPE65 antibody shows distinct reactivity to a 61-kDa protein. Indirect immunofluorescence on normal human epidermis reveals cell surface labeling of keratinocytes. Laser scan microscopy exhibits colocalization of plasma retinol-binding protein and RPE65 on cultured keratinocytes. Internalization experiments with [3H]retinoic acid-retinol-binding protein complex in the presence and absence of excess of retinol-binding protein indicates receptor-dependent uptake of retinoids. We further show isolation of RPE65 protein by affinity chromatography from lysates of keratinocytes using a retinol-binding protein-matrix gel column. In summary, we demonstrate mRNA and protein expression of RPE65 in epidermal keratinocytes. Colocalization of plasma retinol-binding protein with RPE65 and affinity binding suggest a direct interaction of RPE65 with plasma retinol-binding protein in cultured human keratinocytes that might be involved in retinoid uptake of keratinocytes.

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