No abstract

Extensive screening of aromatase cDNA was carried out in cDNA libraries from various human tissues. The DNA sequences of all the isolated cDNA clones were identical in the region encoded by exons 2-10 of the aromatase gene. However, tissue-specific sequences, which were classified into four groups, were observed in the 5' portions of the clones corresponding to the region encoded by exon 1. All of them were also found in clones isolated from a human genomic library and mapped between exons 1 and 2 of the human aromatase gene reported previously, suggesting the presence of multiple exons 1 and promoters in the gene. Reverse transcription-PCR analyses of aromatase mRNAs in various tissues revealed that aromatase transcripts are tissue-specifically spliced by alternative use of multiple exons 1, although minor forms of the transcripts were also present in each tissue. Aromatase mRNA is spliced from 10 exons in most tissues, but from 9 exons in the prostate and from 10 or 11 exons in the placenta. This suggests that tissue-specific regulation of the aromatase gene in various tissues may be explained by alternative use of multiple exons 1 flanked with tissue-specific promoters. The alternative use of multiple exons 1 for liver transcripts was found to change developmentally. Furthermore, switch from an adipose-specific exon 1 to another type of exon 1 was observed in aromatase transcripts of adipose tissues of three of five breast cancer patients.

The luminescent jellyfish Aequorea contains a photoprotein, aequorin, which emits light by an intramolecular reaction in the presence of a trace amount of Ca2+. A cDNA library of Aequorea was constructed and clones carrying the cDNA for the Ca2+-dependent photoprotein were isolated by the method of colony hybridization using synthetic oligonucleotide probes. The primary structure of the protein deduced from the nucleotide sequence showed that the protein is composed of 189 amino acid residues and has three F-F hand structures that are characteristic for Ca2+-binding sites. The sequence also suggested that the protein has hydrophobic regions at which the protein may interact with its functional chromophore, coelenterazine.

No abstract

The atheroprotective effects of estrogen are well established and the presence of an estrogen receptor in vascular tissues has recently been reported. Therefore, we investigated the localization of the estrogen-producing enzyme aromatase in vascular tissues to assess the possible contribution of endocrine, paracrine, and autocrine modes of action. Aromatase was found in human vascular smooth muscle cells (SMCs) but not in endothelial cells on in situ hybridization. These observations were further supported by quantitative analysis of aromatase mRNA and the activity in 15 human vascular specimens. Only trace levels of expression were detected in the 3 infants examined, whereas 0.0088 to 0.0806 amol/ microg RNA of aromatase mRNA and 12.9 to 122.3 fmol. h-1. mg-1 protein of the activity were detected in 12 of the adult individuals. The switching of tissue-specific exon 1 of the human aromatase gene was also observed in some cases. Aromatase was found to be expressed only in cultured SMCs and not in cultured endothelial cells of human aorta and pulmonary artery and to be regulated through dexamethasone and the signaling pathways of protein kinase A and C. Study results revealed the localized expression of aromatase in vascular SMCs, which indicated a possible direct action of locally produced estrogen in an autocrine or paracrine manner, with possible cross talk between smooth muscle and endothelial cells.

Transforming growth factor-␤ (TGF-␤) is both abundant in bone and an important regulator of bone metabolism. A T3 C transition at nucleotide 29 in the signal sequence region of the TGF-␤1 gene results in a Leu3 Pro substitution at amino acid position 10. The possible association of this polymorphism with bone mass and the prevalence of osteoporosis has now been investigated in a total of 287 postmenopausal women from two regions (Obu City, Aichi Prefecture, and Sanda City, Hyogo Prefecture) of Japan. A significant association of TGF-␤1 genotype with bone mass was detected in both populations; bone mineral density (BMD) at the lumbar spine was greater in individuals with the CC genotype than in those with the TT or TC genotype. The frequency of vertebral fractures was significantly lower in individuals with the CC genotype than in those with the TC or TT genotypes. For each region, multivariable logistic regression analysis revealed that the frequency of the T allele was significantly higher in subjects with osteoporosis than in controls. Also, the serum concentration of TGF-␤1 in individuals with the CC genotype was significantly higher than that in age-matched subjects with the TC or TT genotype in osteoporotic or osteopenic as well as healthy control groups. These results suggest that the T/C polymorphism of the TGF-␤1 gene is one of the genetic determinants of bone mass and that the T allele is an independent risk factor for the genetic susceptibility to osteoporosis in postmenopausal Japanese women. Thus, analysis of the TGF-␤1 genotype may be useful in the prevention and management of osteoporosis. (J Bone Miner Res 1998;13:1569-1576)

No abstract

] i increase in a dose-dependent manner through the activation of adenyl cyclase. The increases induced by both PTH and hypotonicity were observed primarily in the processes of the osteocytes. In cyclically stretched osteocytes on flexible-bottomed plates, PTH also synergistically elevated the insulin-like growth factor-1 mRNA level. Furthermore, Gd 3؉ and ␣1C antisense significantly inhibited the stretch-induced insulin-like growth factor-1 mRNA elevation. The volume-sensitive calcium influx pathways of osteocytes represent a mechanism by which PTH potentiates mechanical responsiveness, an important aspect of bone formation.Mechanical loading applied to the skeleton has been shown to cause osteotropic influences. On the other hand, prolonged immobilization is one of the important reasons of bone loss. However, the intracellular mechanisms by which bone cells sense mechanical strain are not well known (1).Although osteocytes are the most abundant cells in bone, the role of osteocytes in bone remodeling was not clear until recently. Direct inhibition of osteoclastic activity by osteocytederived protein through osteoclast-osteocyte attachment was demonstrated (2, 3). Furthermore, intermittent mechanical loading within the physiological range enhances IGF-1 1 mRNA expression in osteocytes (4, 5), suggesting that these cells transduce signals induced by mechanical stress to osteoblasts. Although various other mediators such as prostaglandins (6 -8), cyclooxygenase-2 (encoding prostaglandin G/H synthase), (8, 9), endothelial, constitutive nitric oxide synthase (10), or c-fos (8, 11-13) have been suggested, the exact mechanosensing mechanisms in these cells are not clear. Although the involvement of stretch-activated cation channels (SA-Cat) in the reception of mechanical stress has been reported in many other cell types, none has been functionally demonstrated in osteocytes so far.The localization of PTH receptors by in situ hybridization (14), as well as the synergistic effects of mechanical stress and PTH (13, 15), on the other hand, indicated the important role of PTH in regulating the signal transduction of mechanical stress in osteocytes. Stretch-activated cation channels and their activation by PTH were demonstrated in UMR 106 osteoblast-like cells by who suggested either the expression of isoforms of the ␣1C subunit of the voltage-operated Ca 24 channel (VOCC) is required for the activity or that the channel may have homology to the ␣1C subunit of VOCC. Osteocytes differentiate from osteoblasts along with a dramatic elongation of cytoplasmic processes. This morphological change suggests that their levels of sensitivity to mechanical loading and the mechanisms of response could be distinct.In this study, we have identified a stretch-activated Ca 2ϩ entry pathway for the first time in both rat and chicken osteocytes by swelling the cells with hypo-osmotic stress. Also, by using single cell [Ca 2ϩ ] i video-image analysis, we demonstrated that osteocyte processes are furnished with volumesensitive, stretch-act...