Sakiko Kobayashi

Neurons Limit Angiogenesis by Titrating VEGF in Retina

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:

Coactivation of the N-terminal Transactivation of Mineralocorticoid Receptor by Ubc9

Molecular mechanisms underlying mineralocorticoid receptor (MR)-mediated gene expression are not fully understood. Various transcription factors are post-translationally modified by small ubiquitin-related modifier-1 (SUMO-1). We investigated the role of the SUMO-1-conjugating enzyme Ubc9 in MR transactivation. Yeast two-hybrid, GST-pulldown, and coimmunoprecipitation assays showed that Ubc9 interacted with N-terminal MR-(1-670). Endogenous Ubc9 is associated with stably expressing MR in 293-MR cells. Transient transfection assays in COS-1 cells showed that Ubc9 increased MR transactivation of reporter constructs containing MRE, ENaC, or MMTV promoter in a hormone-sensitive manner. Moreover, reduction of Ubc9 protein levels by small interfering RNA attenuated hormonal activation of a reporter construct as well as an endogenous target gene by MR. A sumoylation-inactive mutant Ubc9(C93S) similarly interacted with MR and potentiated aldosterone-dependent MR transactivation. An MR mutant in which four lysine residues within sumoylation motifs were mutated into arginine (K89R/K399R/K494R/K953R) failed to be sumoylated, but Ubc9 similarly enhanced transactivation by the mutant MR, indicating that sumoylation activity is dispensable for coactivation capacity of Ubc9. Coexpression of Ubc9 and steroid receptor coactivator-1 (SRC-1) synergistically enhanced MR-mediated transactivation in transient transfection assays. Indeed, chromatin immunoprecipitation assays demonstrated that endogenous MR, Ubc9, and SRC-1 were recruited to an endogenous ENaC gene promoter in a largely aldosterone-dependent manner. Coimmunoprecipitation assays showed a complex of MR, Ubc9, and SRC-1 in mammalian cells, and the endogenous proteins were colocalized in the nuclei of the mouse collecting duct cells. These findings support a physiological role of Ubc9 as a transcriptional MR coactivator, beyond the known SUMO E2-conjugating enzyme.

Expression and regulation of nuclear receptor coactivators in glucocorticoid action

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.

FHL2, UBC9, and PIAS1 are Novel Estrogen Receptor α‐Interacting Proteins

Estrogen plays important roles in the pathophysiology of atherosclerosis and cardiovascular diseases mediated by estrogen receptor α (ERα). To elucidate the molecular mechanisms, we screened ERα‐interacting proteins from a human heart cDNA library using a yeast two‐hybrid system, and identified the four and a half of LIM‐only protein 2 (FHL2). FHL2 interacted with ERα in the presence of 17β‐estradiol, but not of tamoxifen or raloxifene in yeast. FHL2 mainly interacted with N‐terminal A/B domain of ERα but not C‐terminal ligand‐binding domain. However, overexpression of full‐length FHL2 did not affect ERα‐dependent transcriptional activities of a reporter containing 3 copies of estrogen response element in COS‐1 cells. Since tissue distribution of FHL2 was highly restricted to the heart, the function of FHL2 may be observed in a cell type‐ or promoter‐specific manner. We have also detected strong interactions of ERα with Ubc9 and PIAS1 in yeast. Ubc9 and PIAS1, small ubiquitin‐related modifier‐1 (SUMO‐1) conjugating enzyme and ligase, respectively, markedly interacted with ERα in a 17β‐estradiol‐dependent manner. These proteins mainly interacted with the DNA‐binding and ligand‐binding domains of ERα. Overexpression of Ubc9 or PIAS1 potentiated ERα‐mediated transcriptional activities in COS‐1 cells in a dose‐dependent manner, indicating that both Ubc9 and PIAS1 function as coactivators of ERα. In addition, the SUMOylation‐defective mutant, Ubc9 (C93S) continued to enhance ERα‐dependent transcriptional activities. These findings suggest that coactivator abilities and SUMOylation capacities of Ubc9 and PIAS1 are separable and distinct. The present studies indicate that ERα exhibit tissue‐specific functions utilizing multiple tissue‐restricted receptor‐interacting proteins.

Regulation of differential COUP-TF-coregulator interactions in adrenal cortical steroidogenesis.

Hyperfunctioning adrenocortical adenomas produce excessive amounts of various corticosteroids due to dysregulated expression of steroidogenic enzymes. Since no genetic mutations in steroidogenic enzyme genes have been identified as yet, the dysregulated expression at the transcription level may be crucial. Chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) and steroidogenic factor-1 (SF-1) play key roles in the transcriptional regulation of steroidogenic P450 genes. Transfection studies showed that SF-1 activated and COUP-TFs repressed the transcription of bovine CYP17 gene promoter from the CRS2 element in a mutually exclusive manner in Y-1 cells. The results indicate that COUP-TFs negatively regulate the transcriptional activity of SF-1, a steroidogenic cell-specific activator of various steroidogenic P450 genes. Expression of both COUP-TFI and COUP-TFII was significantly decreased in the cortisol-producing adenomas, in which CYP17 was drastically overexpressed, indicating that decreased expression of COUP-TFs play a key role in overexpression of CYP17 in this type of tumors. We then screened for COUP-TFI-interacting proteins from a cortisol-producing adenoma cDNA library using a yeast two-hybrid system and identified a novel RING finger-containing protein which can function as a coregulator for COUP-TFI. Notably, COUP-TFI activated rather than repressed several target genes including the human CYP11B2 gene promoter, the results of which were opposite to those of the CYP17 promoter. The bifunctional activities of COUP-TFI may be derived from the promoter context and our newly identified COUP-TFI coregulator.

NF-YC Functions as a Corepressor of Agonist-bound Mineralocorticoid Receptor

The role of aldosterone has been implicated in the metabolic syndrome and cardiovascular diseases. The biological actions of aldosterone are mediated through mineralocorticoid receptor (MR). Nuclear receptor-mediated gene expression is regulated by dynamic and coordinated recruitment of coactivators and corepressors. To identify new coregulators of the MR, full-length MR was used as bait in yeast two-hybrid screening. We isolated NF-YC, one of the subunits of heterotrimeric transcription factor NF-Y. Specific interaction between MR and NF-YC was confirmed by yeast two-hybrid, mammalian two-hybrid, coimmunoprecipitation assays, and fluorescence subcellular imaging. Transient transfection experiments in COS-7 cells demonstrated that NF-YC repressed MR transactivation in a hormone-sensitive manner. Moreover, reduction of NF-YC protein levels by small interfering RNA potentiated hormonal activation of endogenous target genes in stably MR-expressing cells, indicating that NF-YC functions as an agonist-dependent MR corepressor. The corepressor function of NF-YC is selective for MR, because overexpression of NF-YC did not affect transcriptional activity mediated by androgen, progesterone, or glucocorticoid receptors. Chromatin immunoprecipitation experiments showed that endogenous MR and steroid receptor coactivator-1 were recruited to an endogenous ENaC gene promoter in a largely aldosterone-dependent manner, and endogenous NF-YC was sequentially recruited to the same element. Immunohistochemistry showed that endogenous MR and NF-YC were colocalized within the mouse kidney. Although aldosterone induces interaction of the N and C termini of MR, NF-YC inhibited the N/C interaction. These findings indicate that NF-YC functions as a new corepressor of agonist-bound MR via alteration of aldosterone-induced MR conformation.

Proteasome‐Mediated Mineralocorticoid Receptor Degradation Attenuates Transcriptional Response to Aldosterone

The ubiquitin‐proteasome pathway regulates the turnover of many nuclear hormone receptors, such as the estrogen receptor. For estrogen receptor, proteasome inhibition decreases ligand‐mediated transcription. We provide evidence that the mineralocorticoid receptor (MR) is degraded by the ubiquitin‐proteasome pathway in a ligand‐dependent manner and that proteasomal inhibition results in increased accumulation of the MR with enhancement of transcriptional response to aldosterone. Examination of the primary sequence of human and rat MR has identified two candidate PEST degradation motifs. Mutation of lysine 715 and/or 367 within this PEST element failed to prevent degradation of MR protein or transcriptional activity mediated by aldosterone, indicating that other lysine residues are targeted by proteasomal degradation of MR. These findings demonstrate a coupling between MR up‐regulation and transcriptional hyperactivity.

Risk Factors Associated with Persistent Postoperative Hypertension in Cushing's Syndrome

The aim of the present study was to assess the long-term results of adrenalectomy and to evaluate potential risk factors for the persistence or recurrence of hypertension. Forty-five patients with Cushings syndrome caused by benign cortisol-producing adrenocortical adenomas were evaluated before and for a period of 1 year after surgical cure. When the patients were classified into two groups according to whether their preoperative BP was more (HBP group) or less (NBP group) than 140/90 mmHg, the BP level was found to be continuously higher in the HBP group than in the NBP group during the year after surgery. This finding suggests that the preoperative BP level in Cushings syndrome may be a determinant factor for persistent hypertension after surgery (P<0.05). In addition, a correlation was found between postoperative BP level and duration of hypertension (P<0.05), but no relationships were found between postoperative BP levels and other factors, including age, BMI, tumor size, serum cortisol, aldosterone, potassium, total cholesterol, or glucose levels. The above findings indicate that intensive control of preoperative BP to maintain it below 140/90 mmHg with antihypertensive medication is a very important means of improving prognosis for postoperative BP. Immediate diagnosis and surgical treatment to reduce the duration of hypertension are also crucial for the long-term BP prognosis.

Coactivation of SF-1-Mediated Transcription of Steroidogenic Enzymes by Ubc9 and PIAS1

Steroidogenic factor-1 (SF-1) is a nuclear orphan receptor, which is essential for adrenal development and regulation of steroidogenic enzyme expression. SF-1 is posttranslationally modified by small ubiquitin-related modifier-1 (SUMO-1), thus mostly resulting in attenuation of transcription. We investigated the role of sumoylation enzymes, Ubc9 and protein inhibitors of activated STAT1 (PIAS1), in SF-1-mediated transcription of steroidogenic enzyme genes in the adrenal cortex. Coimmunoprecipitation assays showed that both Ubc9 and PIAS1 interacted with SF-1. Transient transfection assays in adrenocortical H295R cells showed Ubc9 and PIAS1 potentiated SF-1-mediated transactivation of reporter constructs containing human CYP17, CYP11A1, and CYP11B1 but not CYP11B2 promoters. Reduction of endogenous Ubc9 and PIAS1 by introducing corresponding small interfering RNA significantly reduced endogenous CYP17, CYP11A1, and CYP11B1 mRNA levels, indicating that they normally function as coactivators of SF-1. Wild type and sumoylation-inactive mutants of Ubc9 and PIAS1 can similarly enhance the SF-1-mediated transactivation of the CYP17 gene, indicating that the coactivation potency of Ubc9 and PIAS1 is independent of sumoylation activity. Chromatin immunoprecipitation assays demonstrated that SF-1, Ubc9, and PIAS1 were recruited to an endogenous CYP17 gene promoter in the context of chromatin in vivo. Immunohistochemistry and Western blotting showed that SF-1, Ubc9, and PIAS1 were expressed in the nuclei of the human adrenal cortex. In cortisol-producing adenomas, the expression pattern of SF-1 and Ubc9 were markedly increased, whereas that of PIAS1 was decreased compared with adjacent normal adrenals. These results showed the physiological roles of Ubc9 and PIAS1 as SF-1 coactivators beyond sumoylation enzymes in adrenocortical steroidogenesis and suggested their possible pathophysiological roles in human cortisol-producing adenomas.

COUP-TF and transcriptional co-regulators in adrenal steroidogenesis

Chicken ovalbumin upstream promoter‐transcription factors (COUP‐TFs) and steroidogenic factor‐1 (SF‐1) play key roles in the transcriptional regulation of steroidogenic P450 genes. Transfection studies showed that SF‐1 activated bovine CYP17 promoter activity, whereas COUP‐TFs repressed it from the CRS2 element in a mutually exclusive manner in mouse adrenocortical Y‐1 cells. COUP‐TFI and SF‐1 competitively bind to the Ad5 element of the human CYP11B2 gene promoter. Unexpectedly, overexpression of COUP‐TFI increased the CYP11B2 promoter activity, whereas overexpression of SF‐1 repressed it in human adrenocortical H295R cells. In cortisol‐producing adrenal cortical adenomas, down‐regulation of nuclear receptors, including COUP‐TFs was found. We therefore screened for COUP‐TFI‐interacting proteins using a yeast two‐hybrid system and have identified Ubc9 and PIAS1, SUMO‐1 conjugating enzyme and ligase, respectively. Coexpression of Ubc9 and PIAS1 synergistically enhanced COUP‐TFI‐mediated transrepression of CYP17 gene as well as transactivation of CYP11B2 gene. The SUMOylation‐defective mutants of these proteins continued to function as co‐regulators of COUP‐TFI. These findings indicate that Ubc9 and PIAS1 can function as transcriptional co‐regulators of COUP‐TFI to modulate adrenal cortical steroidogenesis in a SUMOylation‐independent manner.