Kazuyuki Yanai

Molecular Variation of the Human Angiotensinogen Core Promoter Element Located between the TATA Box and Transcription Initiation Site Affects Its Transcriptional Activity

Recent genetic studies indicate that several molecular variants discovered in angiotensinogen (AG), the precursor of vasoactive octapeptide angiotensin II, could potentially be responsible for inherited predisposition to human blood pressure variation. We have previously shown that a ubiquitously expressed nuclear factor, AGCF1, bound to AGCE1 (AG core promoterelement 1 including the core nucleotides,CTCGTG, CTC-type) located between the TATA box and transcription initiation site (positions −25 to −1) is an authentic regulator of human AG transcription. In the present study, we showed that AGCF1 has biologically and immunologically similar properties to those of a helix-loop-helix nuclear factor USF1 and examined the effects of two other naturally occurring molecular variants (ATCGTG, ATC-type and ATTGTG, ATT-type) found in the AGCE1 position on the human AG transcriptional activity. Competitive gel-shift and transfection experiments demonstrated that the transcriptional activity for the CTC- and ATC-type promoters was 2.5 times higher than that for the ATT-type through the alteration of AGCF1-binding affinity. These results suggest the possible involvement of USF1 as a component in AGCF1 formation and the potential importance of AGCE1 variation in blood pressure regulation through human AG expression.

Induction of hydroxyapatite resorptive activity in bone marrow cell populations resistant to bafilomycin A1 by a factor with restricted expression to bone and brain, neurochondrin

Bone, one of the favored sites for tumor metastasis, is a dynamic organ undergoing formation and resorption. We found bone metastasis with osteolytic lesion in the bone marrow of the femur by injecting BW5147 T-lymphoma cells into the tail vein of AKR mice. To understand this bone destruction, we constructed a cDNA library from BW5147 with a cloning vector that allowed in vitro synthesis of mRNAs, and then identified a particular cDNA clone by adding the conditioned medium from Xenopus oocytes following injection of the mRNA synthesized in vitro to primary bone marrow heterogeneous cell populations on hydroxyapatite thin films. By means of this method, we isolated a factor with 16% leucine residues, termed neurochondrin, that induces hydroxyapatite resorptive activity in bone marrow cells resistant to bafilomycin A1, an inhibitor of macrophage- and osteoclast-mediated resorption. Expression of the gene was localized to chondrocyte, osteoblast, and osteocyte in the bone and to the hippocampus and Purkinje cell layer of cerebellum in the brain. This may provide insights into the molecular mechanisms underlying bone resorption with potential implications for the activation of cells other than macrophages and osteoclasts in bone marrow cells.

Molecular cloning and expression of human neurochondrin-1 and -2.

Human neurochondrins have been cloned from a brain cDNA library. The human neurochondrin-1 and -2 predict leucine-rich (15.8 and 15.9%) proteins of 729 and 712 amino acid residues, with molecular weights of 78.9 and 77.2 kDa, respectively. The deduced amino acid sequence indicates 98% identity among human, mouse and rat species. Northern analysis indicates that about 4 kb human neurochondrin mRNAs are abundant in the fetal and the adult brain.

Differential action of AGCF2 upon cell type-dependent expression of human angiotensinogen gene.

To investigate the regulatory mechanisms of human angiotensinogen (ANG) gene expression in the brain, we analyzed the 1.3‐kb promoter by transfection studies and gel shift assays. The region from −106 to +44 was sufficient for promoter activity in glioblastoma cells, and multiple nuclear factors including AGCF2 (human N ore promoter binding actor ) bound within this 150‐bp region. The mutations within AGCF2‐binding elements decreased the transcriptional activity in glioblastoma cells but rather increased it in hepatoma cells. These results indicate that AGCF2 has a differential function between these cells and contributes to the glia‐dependent angiotensinogen promoter activity.

Identification of two distinct Sp1- and RBF-1-like nuclear factors that bind to the upstream region of the human angiotensinogen promoter

Angiotensionogen, the protein precursor of angiotensin II that is a crucial regulator of blood pressure and electrolyte balance, is constitutively produced by the liver. In the present study, we identified two nuclear factors that are possibly involved in maintaining the constitutive promoter activity of the human angiotensinogen gene. The 32 bp DNA region between −344 and −313 located in the 1.3 kb angiotensinogen upstream region (−1222 to +44) partially contributed to the maintenance of the efficient promoter activity in HepG2 cells. This segment was able to form the complexes with HepG2 nuclear extracts, which could be dissociated by competing recognition sequences that contain those of either Sp1 or RBF-1. Anin vivo competition experiment demonstrated that the parental promoter activity is reduced about 65% by an RBF-1 competitor more effectively than by an Sp1 competitor. These results suggested that Sp1- and RBF-1-like factors play roles in maintaining the constitutively active angiotensinogen promoter.

Identification of the mouse neurochondrin promoter region and the responsible region for cell type specific gene regulation

Neurochondrin is a cytoplasmic protein possibly involved in neurite outgrowth and chondrocyte differentiation. In the present study, we have identified 202 bp of the mouse neurochondrin minimal promoter sequences encompassing the transcriptional initiation site, and both of the activating and repressing regions in the first exon. These two regulatory regions in the first exon had a cell type dependent effect on the identified minimal promoter. In the regulatory region, the duplication of potential binding sites for GATA family transcriptional factors was observed. Prospective binding sites for sex determining region Y and c-Ets1 were also found in the minimal promoter region. These factors could be potential regulators for the mouse neurochondrin gene.