Vascular and nervous systems, two major networks in mammalian bodies, show a high degree of anatomical parallelism and functional crosstalk. During development, neurons guide and attract blood vessels, and consequently this parallelism is established. Here, we identified a noncanonical neurovascular interaction in eye development and disease. VEGFR2, a critical endothelial receptor for VEGF, was more abundantly expressed in retinal neurons than in endothelial cells, including endothelial tip cells. Genetic deletion of VEGFR2 in neurons caused misdirected angiogenesis toward neurons, resulting in abnormally increased vascular density around neurons. Further genetic experiments revealed that this misdirected angiogenesis was attributable to an excessive amount of VEGF protein around neurons caused by insufficient engulfment of VEGF by VEGFR2-deficient neurons. Moreover, absence of neuronal VEGFR2 caused misdirected regenerative angiogenesis in ischemic retinopathy. Thus, this study revealed neurovascular crosstalk and unprecedented cellular regulation of VEGF: retinal neurons titrate VEGF to limit neuronal vascularization. PAPERFLICK:

Aldosterone synthase (CYP11B2) is involved in the final steps of aldosterone biosynthesis and expressed exclusively in the adrenal zona glomerulosa cells. Using an electrophoretic mobility shift assay, we demonstrate that COUP-TFI binds to the ؊129/؊114 element (Ad5) of human CYP11B2 promoter. Transient transfection in H295R adrenal cells demonstrated that COUP-TFI enhanced CYP11B2 reporter activity. However, the reporter construct with mutated Ad5 sequences showed reduced basal and COUP-TFI-enhanced activity, suggesting that binding of COUP-TFI to Ad5 is important for CYP11B2 transactivation. To elucidate molecular mechanisms of COUP-TFI-mediated activity, we subsequently screened for COUP-TFI-interacting proteins from a human adrenal cDNA library using a yeast twohybrid system and identified Ubc9 and PIAS1, which have small ubiquitin-related modifier-1 (SUMO-1) conjugase and ligase activities, respectively. The coimmunoprecipitation assays confirmed that COUP-TFI forms a complex with Ubc9 and PIAS1 in mammalian cells. Immunohistochemistry showed that Ubc9 and PIAS1 are markedly expressed in rat adrenal glomerulosa cells. Coexpression of Ubc9 and PIAS1 synergistically enhanced the COUP-TFI-mediated CYP11B2 reporter activity, indicating that both proteins function as coactivators of COUP-TFI. However, sumoylation-defective mutants, Ubc9 (C93S) and PIAS1 (C351S), continued to function as coactivators of COUP-TFI, indicating that sumoylation activity are separable from coactivator ability. In addition, chromatin immunoprecipitation assays demonstrated that ectopically expressed COUP-TFI, Ubc9, and PIAS1 were recruited to an endogenous CYP11B2 promoter. Moreover, reduction of Ubc9 or PIAS1 protein levels by small interfering RNA inhibited the CYP11B2 transactivation by COUP-TFI. Our data support a physiological role of Ubc9 and PIAS1 as transcriptional coactivators in COUP-TFI-mediated CYP11B2 transcription.Aldosterone is exclusively produced in adrenal zona glomerulosa cells due to its unique expression of aldosterone synthase cytochrome P450 (CYP11B2), the enzyme required for the final steps of aldosterone biosynthesis. In aldosterone-producing adrenal cortical adenomas of patients with primary aldosteronism, overexpression of CYP11B2 is demonstrated at the transcriptional level (1, 2). Although the reason for aberrant expression of CYP11B2 in these adenomas is not known, mutations in the CYP11B2 gene do not appear to be the cause (3, 4). We therefore postulated that transcription factors and/or coregulators may play important roles in CYP11B2 overexpression in the tumors.The trans-acting factors that regulate CYP11B2 expression remain poorly defined. The orphan nuclear receptor, steroidogenic factor-1 (SF-1), 1 is shown to play a crucial regulator of most steroid hydroxylase genes, including CYP17 and CYP11B1 (5, 6). However, SF-1 actually represses rather than activates expression of hCYP11B2 (7-9). In addition, other transcription factors that are expressed in the adrenal cortex include the NGFI-B family of or...

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Nuclear receptor coactivators are involved in receptor-mediated transcriptional activation of target genes in a hormone-sensitive manner, and the mechanism of their transactivation has been studied in recent years. The glucocorticoid receptor (GR) interacts with several coactivators, including steroid receptor coactivator-1 (SRC-1) family and CREB-binding protein (CBP). Since coactivators function as transcription amplifiers, subtle changes in expression levels of coactivators in certain cells would markedly intensify receptor-mediated transcriptional activity. The regulation of coactivators by glucocorticoid action, however, has not yet been clarified. In this study, we have shown that one of the coactivators interacting with GR, SRC-1, is downregulated by dexamethasone (DEX) both in vivo and in vitro. In experiments on Sprague-Dawley rats in vivo, the downregulation of SRC-1 was observed in heart, stomach, kidney, liver, and cerebrum, and in experiments on two types of kidney-derived cells in vitro, similar downregulation of SRC-1 was demonstrated in both types of cells. DEX-mediated downregulation of SRC-1 mRNA recovered in 4-8 h, while the downregulation of SRC-1 protein lasted for 12 h and its levels returned to the basal level, 24 h after DEX treatment. Other coactivators examined in this study showed no remarkable changes in expression by DEX treatment, implying that ligand-mediated downregulation of SRC-1 has a pivotal role in the physiology of glucocorticoid action.