Establishment of immortalized human erythroid progenitor cell lines able to produce enucleated red blood cells.

Transfusion of red blood cells (RBCs) is a standard and indispensable therapy in current clinical practice. In vitro production of RBCs offers a potential means to overcome a shortage of transfusable RBCs in some clinical situations and also to provide a source of cells free from possible infection or contamination by microorganisms. Thus, in vitro production of RBCs may become a standard procedure in the future. We previously reported the successful establishment of immortalized mouse erythroid progenitor cell lines that were able to produce mature RBCs very efficiently. Here, we have developed a reliable protocol for establishing immortalized human erythroid progenitor cell lines that are able to produce enucleated RBCs. These immortalized cell lines produce functional hemoglobin and express erythroid-specific markers, and these markers are upregulated following induction of differentiation in vitro. Most importantly, these immortalized cell lines all produce enucleated RBCs after induction of differentiation in vitro, although the efficiency of producing enucleated RBCs remains to be improved further. To the best of our knowledge, this is the first demonstration of the feasibility of using immortalized human erythroid progenitor cell lines as an ex vivo source for production of enucleated RBCs.

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.

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.

[A case of Henoch-Schönlein purpura in a patient on hemodialysis].

Henoch-Schönlein purpura(HSP) is a systemic vasculitis and characterized by the tissue deposition of IgA-containing immune complexes. A 50-year-old man with end stage renal failure due to diabetic nephropathy on maintenance hemodialysis, presented purpura, hematuria, abdominal pain, and joint pain. He also presented a high fever with neutrophilia. Biopsy of skin lesions revealed inflammation of the small vessel accompanied by vascular IgA deposition. Based on the clinical symptoms and skin biopsy, we made the diagnosis of HSP. Oral prednisolone was administered resulting in an improvement of the clinical symptoms. A skin biopsy should be performed for histological and immunofluorescence studies in the case of clinical suspicion of HSP with end stage renal disease on hemodialysis.

[Case of nephrotic syndrome due to AL-type primary amyloidosis associated with renal cell carcinoma].

An 80-year-old man was admitted because of appetite loss, mild proteinuria, and leg edema. A computed tomography examination revealed a tumor in his left kidney, and a left nephrectomy was performed. The tumor was histologically diagnosed as a clear cell type renal cell carcinoma, and hematoxylin eosin staining of the non-tumor region of the resected kidney showed an almost normal morphology. Three months later, he was readmitted because of the development of nephrotic syndrome with a urinary protein excretion of 4.2 g/day, a serum total protein concentration of 5.0 g/dL, a serum albumin concentration of 2.4 g/dL, a serum total cholesterol concentration of 214 mg/dL, and generalized edema. A full examination revealed no evidence of metastasis or recurrence of the renal cell carcinoma or any other malignant tumor. Congo red staining and immunohistochemical staining were performed using the non-tumor region of his resected kidney, and the presence of amyloid deposits in the microvascular walls and glomeruli that did not disappear when treated with potassium permanganate was disclosed. In this manner, the patient was diagnosed as having AL-type primary amyloidosis. Bence-Jones proteinuria and gastric amyloidosis were also observed, but a bone marrow examination showed no signs of multiple myeloma. Previous studies have reported an association between renal cell carcinoma and renal amyloidosis, mainly AA-type secondary amyloidosis. To our knowledge, only two cases of renal cell carcinoma associated with primary amyloidosis have been previously reported. Therefore, the present patient not only represents a rare case of renal cell carcinoma associated with primary amyloidosis, but also reminds us that careful histological examination of the non-tumor region of the resected kidney is needed to evaluate the proteinuria associated with renal cell carcinoma, particularly in elderly patients.

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.

Ubc9 and Protein Inhibitor of Activated STAT 1 Activate Chicken Ovalbumin Upstream Promoter-Transcription Factor I-mediated Human CYP11B2 Gene Transcription.

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 two-hybrid 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.

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.

FHL2, UBC9, and PIAS1 are novel estrogen receptor alpha-interacting proteins.

Estrogen plays important roles in the pathophysiology of atherosclerosis and cardiovascular diseases mediated by estrogen receptor alpha (ERalpha). To elucidate the molecular mechanisms, we screened ERalpha-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 ERalpha in the presence of 17beta-estradiol, but not of tamoxifen or raloxifene in yeast. FHL2 mainly interacted with N-terminal A/B domain of ERalpha but not C-terminal ligand-binding domain. However, overexpression of full-length FHL2 did not affect ERalpha-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 ERalpha with Ubc9 and PIAS1 in yeast. Ubc9 and PIAS1, small ubiquitin-related modifier-1 (SUMO-1) conjugating enzyme and ligase, respectively, markedly interacted with ERalpha in a 17beta-estradiol-dependent manner. These proteins mainly interacted with the DNA-binding and ligand-binding domains of ERalpha. Overexpression of Ubc9 or PIAS1 potentiated ERalpha-mediated transcriptional activities in COS-1 cells in a dose-dependent manner, indicating that both Ubc9 and PIAS1 function as coactivators of ERalpha. In addition, the SUMOylation-defective mutant, Ubc9 (C93S) continued to enhance ERalpha-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 ERalpha exhibit tissue-specific functions utilizing multiple tissue-restricted receptor-interacting proteins.

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 trans-repression 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.

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.