Koichi Oba

Establishment and Characterization of a Steroidogenic Human Granulosa-Like Tumor Cell Line, KGN, That Expresses Functional Follicle-Stimulating Hormone Receptor

We established a steroidogenic human ovarian granulosa-like tumor cell line, designated KGN, from a patient with invasive ovarian granulosa cell carcinoma. KGN had a relatively long population doubling time of about 46.4 h and had an abnormal karyotype of 45,XX, 7q-, -22. A steroid analysis of the cultured medium by RIA performed 5 yr after the initiation of culture showed that KGN was able to secrete pregnenolone and progesterone, and both dramatically increased after stimulation with (Bu)(2)cAMP. However, little or no secretion of 17alpha-hydroxylated steroids, dehydroepiandrosterone, androstenedione, or estradiol was observed. The aromatase activity of KGN was relatively high and was further stimulated by (Bu)(2)cAMP or FSH. These findings showed a pattern similar to that of steroidogenesis in human granulosa cells, thus allowing analysis of naturally occurring steroidogenesis in human granulosa cells. Fas-mediated apoptosis of KGN was also observed, which mimicked the physiological regulation of apoptosis in normal human granulosa cells. Based on these findings, this cell line is considered to be a very useful model for understanding the regulation of steroidogenesis, cell growth, and apoptosis in human granulosa cells.

Matrix extracellular phosphoglycoprotein is expressed in causative tumors of oncogenic osteomalacia

Oncogenic osteomalacia (OOM), or tumor-induced osteomalacia, is a rare disease characterized by renal phosphate wasting and osteomalacia. It arises due to the secretion of fibroblast growth factor 23 (FGF-23) from causative tumors. Matrix extracellular phosphoglycoprotein (MEPE) is predominantly expressed in odontoblasts, osteoblasts, and osteocytes. Although the presence of MEPE mRNA has been reported in some OOM tumors, little is known about the prevalence of MEPE expression in OOM tumors. In this study, the expression of MEPE and FGF-23 in OOM tumors was investigated at the transcriptional and translational levels. Eleven causative OOM tumors were analyzed by quantitative real-time reverse transcription-polymerase chain reaction and immunohistochemistry for MEPE and FGF-23 expression. Hemangiopericytomas and giant cell tumors, pathological diagnoses that are common in cases of OOM, were obtained from non-osteomalacic patients and analyzed as controls. The gene expression level of FGF23 and MEPE in OOM tumors was 104- and 105-times higher, respectively, than in non-OOM tumors. Immunohistochemical staining revealed that FGF-23 protein was expressed in all OOM tumors, and MEPE was expressed in 10 out of 11 OOM tumors. Thus, MEPE expression was common in OOM tumors, similar to FGF-23. These results indicate that, in addition to the hypophosphatemic effects of FGF-23, MEPE or the MEPE-derived acidic serine aspartate-rich MEPE-associated motif peptide may contribute to decreased bone mineralization in OOM patients.

The Levels of Somatostatin Receptors in Causative Tumors of Oncogenic Osteomalacia Are Insufficient for Their Agonist to Normalize Serum Phosphate Levels

Oncogenic osteomalacia (OOM) is a rare disease characterized by renal phosphate wasting and osteomalacia and is caused by the secretion of fibroblast growth factor 23 (FGF-23) from causative tumors. Scintigraphy with octreotide, which binds to somatostatin receptors (SSTRs), is a useful way to locate causative tumors in OOM patients. However, the therapeutic effects of octreotide acetate are still controversial. Two OOM patients were administered octreotide acetate intramuscularly. Ten causative OOM tumors, including two resected from the patients participating in the octreotide administration study, were examined for expression of genes encoding SSTRs by quantitative real-time RT-PCR and immunohistochemistry. Octreotide therapy did not improve hypophosphatemia in either case, despite temporal decreases in FGF-23 levels in one patient. The mean expression levels of SSTR1, SSTR3, and SSTR5 were similar in the OOM and non-OOM tumors. Expression of SSTR2 was significantly higher in the OOM tumors than in the non-OOM tumors. Immunohistochemical examinations revealed the presence of SSTR2A, SSTR2B, and SSTR5 in both the OOM and non-OOM tumors. The expression of SSTR genes in OOM tumors contributes to positive imaging using octreotide scintigraphy. However, the levels of SSTRs seem to be insufficient for the octreotide therapy to improve hypophosphatemia. Further studies are needed to clarify the mechanisms by which FGF-23 secretion from OOM tumors is suppressed by octreotide acetate.

Human Ad4BP/SF-1 and its related nuclear receptor

Ad4BP (or SF-1) is an essential transcriptional factor for steroidogenesis as well as for the development of the reproductive axis. We elucidated the structure of the human Ad4BP gene. The spliced variants of Ad4BP gene, ELP1 and ELP2 in mice, are unlikely to be present in humans since the analysis of the human gene revealed an in frame stop codon, 36-bp before the first ATG of Ad4BP. The promoter sequence of human Ad4BP, upstream of non-coding exon 1 was highly conserved, and E-box was also found to be essential for the transcription of human Ad4BP gene. During the process of the human Ad4BP gene cloning, we happened to obtain an Ad4BP-related gene, FTZ-F1beta which also belongs to the nuclear receptor family. We revealed cDNA structures of rat FTZ-F1beta, and found that rat has at least two types of FTZ-F1beta isoforms, which differ only by 21 amino acids length in the A/B domain. The tissue distributions of FTZ-F1beta in rat examined by RT-PCR, was found to be abundant in liver, pancreas, and gastrointestinal tracts. These results suggest that the physiological significance of FTZ-F1beta is different from that of Ad4BP.

Asynchronous Rhythm of Steroidogenic Factor 1 and Period Homolog 2 mRNA Expression in Mouse Y1 Adrenocorticol Tumor Cells

The relationship between the expression of Steroidogenic factor 1 (Sf1) and the circadianrelated gene, period homolog 2 (Per2), in the adrenal cortex is still unknown. We show here that in Y1 adrenocortical tumor cells, expression of steroidogenic-related genes such as P450scc mRNA and Sf1 mRNA were asynchronous with Per2 mRNA. SF1 promoter analyses showed that the E-box element functions in a rhythmic pattern. Rhythmic expression of Upstream factor 1 mRNA, correlated well with Sf1 mRNA expression. We propose that tumorigenesis of adrenocortical lesions cause disruption of synchronous expression of steroidogenic-related and circadian-related genes.

[Phe21]big endothelin‐1(18–34) and [Ala31]big endothelin‐1(18–34) inhibit the human endothelin‐converting enzyme‐1 (ECE‐1) expressed in CHO‐K1 cells in a different fashion

Endothelin‐converting enzyme‐1 (ECE‐1) is one of the most important enzymes to convert big endothelin‐1 (big ET‐1) to ET‐1. To identify the inhibitors of ECE‐1, we examined the effects of variously substituted analogues of big ET‐1 on ECE‐1 activity using solubilized membranes prepared from human ECE‐1‐expressed CHO‐K1 cells. Among the big ET‐1 analogues tested, [Phe21]big ET‐1(18–34) and [Ala31]big ET‐1(18–34) exhibited a significant inhibition of ECE‐1. A kinetic analysis revealed [Phe21]big ET‐1(18–34) to be a competitive inhibitor (K i=20.6 μM) and [Ala31]big ET‐1(18–34) to be a non‐competitive inhibitor (K i=35.6 μM). These results not only support the concept that ECE‐1 recognizes big ET‐1 both at the P1 position and at the C‐terminal region but also revealed that these two regions are recognized by this enzyme in a different manner.