The osteocalcin gene encodes a 6-kDa polypeptide, which represents one of the most abundant noncollagenous bone proteins, and the present studies establish that osteocalcin mRNA is detected only in bone tissue. An osteocalcin gene was isolated from a rat genomic DNA library, and sequence analysis indicated that the mRNA is represented in a 953-nucleotide segment of DNA consisting of four exons and three introns. A modular organization of the 5' flanking sequences of the gene is reflected by the presence of at least three classes of regulatory elements, which include the following: (i) RNA polymerase H canonical sequences; (U) a series of consensus sequences for hormone receptor binding sites and cyclic nucleotide responsive elements consistent with physiologic expression ofthe osteocalcin gene; and (Uii) a 24-nucleotide sequence in the proximal promoter region with a CAAT motif as a central element. We have designated this highly conserved sequence as an "osteocalcin box" since only 2 nucleotide substitutions are found in the rat and human osteocalcin genes. We have demonstrated two factors regulating osteocalcin gene expression. First, a 200-fold increase occurs in normal fetal calvaria osteoblasts producing a mineralizing matrix, compared to confluent osteoblasts in a nonmineralizing matrix. Second, contained within the 600 nucleotides immediately upstream from the transcription start site are sequences that support a 10-fold stimulated transcription of the gene by 1,25-dihydroxyvitamin D.There has been much interest in the vitamin K-dependent protein of bone, osteocalcin (bone Gla protein), since its discovery over a decade ago (1). A distinguishing feature of this 5.7-kDa protein (46-50 amino acids, depending on the species), and of functional significance, are 3 residues of the calcium binding amino acid, y-carboxyglutamic acid (Gla). Gla residues are posttranslationally synthesized from selected glutamic acid residues by a vitamin K-and C02-requiring enzyme complex (2). They are located at positions 17, 21, and 24 in all species from swordfish to mammals (1). This highly conserved sequence region from residues 20-34 in the central portion of the molecule, which also includes a disulfide loop (Cys-23-Cys-29), accounts for a structural conformation of the protein in the presence of calcium that promotes a tight binding of the protein to hydroxyapatite (1). The appearance of osteocalcin in embryonic bone coincident with mineral deposition (1), its association with the hydroxyapatite component of the matrix (3), its chemoattractant property for cells capable of bone resorption (4), and its modulated synthesis by the calcitrophic hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] (5-7) suggest a role for the protein in bone turnover. Although many properties of the protein have been identified, the precise function of osteocalcin is still unknown.Osteocalcin is synthesized de novo by osteoblasts as a 10,000-kDa precursor (8). While the majority ofthe processed osteocalcin peptide (5.7 kDa) is deposited in b...

Abstract. Basic fibroblast growth factor (bFGF), a potent mitogenic/neurotrophic factor, controls the development and plasticity of many types of neural cells. In adrenal chromaflin cells, the appearance of bFGF protein coincided with the establishment of functional innervation, suggesting induction by trans-synaptic signals. In cultured bovine adrenal medullary cells Western blot analysis revealed 18-, 23-, and 24-kD bFGF isoforms in the cytosolic and nuclear fractions. Stimulation of acetylcholine nicotinic receptors or hormonal angiotensin II receptors or the direct stimulation of adenylate cyclase with forskolin or protein kinase C (PKC) with PMA increased the content of all bFGF isoforms. Increases in the levels of intracellular bFGF did not result in detectable presence of bFGF proteins in culture medium. Instead, bFGF proteins accumulated in the cytoplasm or the nucleus depending on whether PKC or cAMP pathways were activated.The long-term nuclear forskolin-induced accumulation of bFGF was prevented by cycloheximide or by antisense bFGF oligonucleotide and was also accompanied by an increase in bFGF mRNA. We used luciferase reporter plasmids containing the human bFGF promoter to show that the induction of bFGF resulted from transcriptional activation of the bFGF gene and was mediated by regulatory sequences located upstream from its transcription start site. Stimulation of bFGF gene expression by forskolin and PMA was synergistic and was mediated through different promoter regions. The results suggest that stimulation by cAMP and PKC is mediated through novel cis elements. The regulation of bFGF protein content also involves posttranscriptional mechanisms since changes in the levels of individual bFGF isoforms were different depending on whether cells were treated with carbachol or angiotensin II, forskolin, or PMA.The present study indicates that bFGF is an intracrine cytoplasmic-nuclear factor, whose expression is regulated by trans-synaptic and hormonal stimuli and which may act as a direct mediator of genomic responses to afferent stimulation.

Neuronal nicotinic acetylcholine receptors (nAChR) are made from different combinations of subunits encoded by a diverse family of genes. However, the recently cloned a7 gene codes for subunits that can form homooligomeric nAChR complexes when expressed in Xenopus oocytes.Electrophysiological studies reveal that these all-nAChR function as a-bungarotoxin (Bgt)-sensitive, quickly activating/inactivating ion channels with a unique pharmacological profile and an unusually high permeability to calcium ions. Although similar observations have been made in studies of Bgt-sensitive, functional nAChR subtypes that are naturally expressed in neuronal cells, all attempts until now to reconstitute functional a7-nAChR in cell lines have failed. Here we report the successful use of SH-SYSY human neuroblastoma cells, which naturally express low levels of endogenous a7 transcripts, to stably overexpress heterologous rat nAChR a7 transgenes. These transgenes are expressed as the appropriately-sized a7 messages and protein, and stably transfected SH-SYSY cells have over 30-times higher levels of specific Bgt binding sites than do wild-type cells. Whole cell current recordings confhm that transfected cells express functional nAChR that are sensitive to blockade by Bgt and display the typical physiological and pharmacological profiles of a7-nAChR. We conclude that stable, functional expression of a7 transgenes in a mammalian cell line has been achieved for the first time.Key words: Acetylcholine; Nicotine; Nicotinic receptor; a-Bungarotoxin; SH-SYSY cell line lntroauctionNicotinic acetylcholine receptors (nAChR) exist as diverse members of the ligand-gated ion channel (LGIC) superfamily of neurotransmitter receptors [l-3]. Part of the diversity in nAChR is attributable to the existence of at least fifteen different nAChR subunit genes. Proteins encoded by these genes combine in different ways to generate several unique nAChR subtypes. However, roles of nAChR subunits, particularly in the formation and function of neuronal nAChR, are incompletely understood.NeuronaYnicotinic alpha-bungarotoxin binding sites (nBgtS) represent a unique class of nAChR that was first discovered in autonomic and CNS neurons or nervous tissue based on their ability to bind radiolabeled, curaremimetic neurotoxins such as a-bungarotoxin (Bgt) with high affiity and specificity [2,4-g]. Found where nAChR expression was expected, nBgtS also display a clearly nicotinic ligand binding profile, possess many of the physical and chemical properties of other nAChR, and have drug reactivities consistent with roles as LGIC. However, whereas studies of autonomic or CNS neurons that express nBgtS detected functional nAChR responses involved in the mediation of excitatory neurotransmission, those responses were not demonstrated to be sensitive to blockade by curaremimetic neurotoxins (although there were some exceptions; op. cit.). Thus, functional relevance of nBgtS and their identity as LGIC was questioned.Critical breakthroughs in our understanding of nBgtS ...

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