Sox-4 messenger RNA is expressed in the embryonic growth plate and regulated via the parathyroid hormone/parathyroid hormone-related protein receptor in osteoblast-like cells.

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) exert potent and diverse effects in cells of the osteoblastic and chondrocytic lineages. However, downstream mediators of these effects are characterized inadequately. We identified a complementary DNA (cDNA) clone encoding the 5' end of the transcription factor Sox-4, using a subtracted cDNA library enriched in PTH-stimulated genes from the human osteoblast-like cell line OHS. The SOX-4 gene is a member of a gene family (SOX and SRY) comprising transcription factors that bind to DNA through their high mobility group (HMG)-type binding domain, and previous reports have implicated Sox proteins in various developmental processes. In situ hybridization of fetal and neonatal mouse hindlimbs showed that Sox-4 messenger RNA (mRNA) was expressed most intensely in the zone of mineralizing cartilage where chondrocytes undergo hypertrophy, and by embryonic day 17 (ED17), after the primary ossification center was formed, its expression was detected only in the region of hypertrophic chondrocytes. Sox-4 mRNA was detected in osteoblast-like cells of both human and rodent origin. In OHS cells, physiological concentrations (10(-10)-10(-9) M) of human PTH 1-84 [hPTH(1-84)] and hPT(1-34), but not hPTH(3-84), stimulated Sox-4 mRNA expression in a time-dependent manner, indicating involvement of the PTH/PTHrP receptor. Sox-4 transcripts also were detected in various nonosteoblastic human cell lines and tissues, in a pattern similar to that previously reported in mice. The presence of Sox-4 mRNA in hypertrophic chondrocytes within the mouse epiphyseal growth plate at sites that overlap or are adjacent to target cells for PTH and PTHrP, and its strong up-regulation via activated PTH/PTHrP receptors in OHS cells, makes it a promising candidate for mediating downstream effects of PTH and PTHrP in bone.

The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity.

We describe a hypothalamus-specific mRNA that encodes preprohypocretin, the putative precursor of a pair of peptides that share substantial amino acid identities with the gut hormone secretin. The hypocretin (Hcrt) protein products are restricted to neuronal cell bodies of the dorsal and lateral hypothalamic areas. The fibers of these neurons are widespread throughout the posterior hypothalamus and project to multiple targets in other areas, including brainstem and thalamus. Hcrt immunoreactivity is associated with large granular vesicles at synapses. One of the Hcrt peptides was excitatory when applied to cultured, synaptically coupled hypothalamic neurons, but not hippocampal neurons. These observations suggest that the hypocretins function within the CNS as neurotransmitters.

Overview of the most prevalent hypothalamus-specific mRNAs, as identified by directional tag PCR subtraction.

We applied the directional tag PCR subtractive hybridization method to construct a rat hypothalamic cDNA library from which cerebellar and hippocampal sequences had been depleted, enriching 20-30-fold for sequences expressed selectively in the hypothalamus. We studied a sample of 94 clones selected for enrichment in the subtracted library. These clones corresponded to 43 distinct mRNA species, about half of which were novel. Thirty-eight of these 43 mRNAs (corresponding to 85 of the clones in the sample) exhibited enrichment in the hypothalamus; 23 were highly enriched. In situ hybridization studies revealed that one novel species was restricted to cells in a small bilaterally symmetric area of the paraventricular hypothalamus. Other novel mRNAs showed substantial enrichment in basal diencephalic structures, particularly the hypothalamus, without restriction to single hypothalamic nuclei. The data suggest that the hypothalamus utilizes at least two distinct strategies for employing its selectively expressed proteins. Secretory neuropeptides utilized for intercellular communication are produced by functionally discrete nuclei, while several other proteins are shared by structures that are unrelated in their physiological roles but may share biochemical systems.

High-level production of human parathyroid hormone in Bombyx mori larvae and BmN cells using recombinant baculovirus.

A full-length cDNA encoding human parathyroid hormone (hPTH) containing the prepro region was cloned into Bombyx mori baculovirus under the control of the polyhedrin promoter and polyadenylation sequences. After transfection and generation of the recombinant baculovirus, hPTH production was examined in silkworm larvae and BmN cell cultures. The larvae synthesized and efficiently secreted the correctly processed and authentic hPTH (9.4 kDa) with no sign of internal degradation. In BmN cells, the major secreted form was the correctly sized protein, but small amounts of degraded hPTH could also be detected in the medium by immunoblotting. Unlike the situation in larvae, prepro-hPTH could also be demonstrated intracellularly in BmN cells. The concentration of hPTH in the larval hemolymph was about 70 mg/l, as compared to approx. 55 micrograms/l in the medium per 7.5 x 10(6) cells. Recombinant hPTH (re-hPTH) from the hemolymph was purified by reverse-phase HPLC and subjected to chemical and biological analyses. The authenticity of the purified re-hPTH was confirmed by N-terminal sequencing, amino acid composition and a mass of 9425 Da, close to the theoretical value. The hormone showed high-affinity receptor binding and full biological potency in increasing cellular cAMP.

Isolation and characterization of two biologically active O-glycosylated forms of human parathyroid hormone produced in Saccharomyces cerevisiae. Identification of a new motif for O-glycosylation.

Expression and secretion of human parathyroid hormone in Saccharomyces cerevisiae were achieved by fusing a cDNA encoding the mature human parathyroid hormone (hPTH) to the preproregion of the yeast mating factor alpha. Purified hPTH from yeast-culture medium was found to contain, in addition to the native unglycosylated form, two mannosylated variants with different molecular masses. The three hPTH forms were processed identically, resulting in the same 84 amino acid polypeptides with amino acid sequences identical to the native hormone. In both the O-glycosylated forms that were separated by isocratic reverse-phase HPLC, two mannose-linked residues were localized to Thr79. In addition, the most glycosylated form showed a heterogeneous modification of three, four or five mannosyl residues linked at Ser66. Lysine is N-terminally located to Ser66 and probably stimulates this glycosylation, which introduces a possible new motif for O-glycosylation in yeast. The two glycosylated forms of hPTH had similar biological activity which was identical to the native form of hPTH in a hormone-sensitive adenylate cyclase assay in bone sarcoma cells. Thus, a C-terminal O-glycosylation of hPTH with up to seven mannosyl residues/molecule did not affect the biological activity of the hormone, making possible production of hPTH with potential different pharmacokinetic properties.

Cloning strategies for peptide hormone receptors.

With the advancement in molecular biology in the last decade many receptors have been cloned and the understanding of their mechanism of function has improved proportionally. In addition the concurrent discovery of receptor gene families has been used to design elegant cloning strategies which in turn have facilitated the characterization of new receptors and receptor subtypes. This review covers the most currently used cloning strategies and gives some examples.

Characterization of a K26Q site-directed mutant of human parathyroid hormone expressed in yeast.

Human parathyroid hormone (hPTH) is susceptible to proteolytical cleavage both in humans and when expressed as a secretory product in Escherichia coli (Høgseth, A., Blingsmo, O. R., Saether, O., Gautvik, V. T., Holmgren, E., Hartmanis, M., Josephson, S., Gabrielsen, O. S., Gordeladze, J. O., Alestrøm, P., and Gautvik, K. M. (1990) J. Biol. Chem. 265, 7338-7344) and Saccharomyces cerevisiae (Gabrielsen, O. S., Reppe, S., Saether, O., Blingsmo, O. R., Sletten, K., Gordeladze, J. O., Høgset, A., Gautvik, V. T., Alestrøm, P., Oyen, T. B., and Gautvik, K. M. (1990) Gene (Amst.) 90, 255-262). In the latter system, one major site of cleavage was identified (Arg25-Lys26 decreased Lys27). To produce hPTH resistant to this proteolytic processing, a point mutation changing Lys26 to Gln was introduced, and the modified gene expressed in S. cerevisiae as a fusion protein with the alpha-factor leader sequence. The resulting major form of hPTH secreted to the growth medium was of full length showing that the mutation had eliminated internal processing. Consequently, the yield of the mutant hormone was significantly higher than obtained with the natural peptide. Using improved purification procedures, a significantly higher purity was also obtained. The secreted mutant hPTH-(1-84,Q26) had the correct size, full immunological reactivity with two different hPTH antisera, correct amino acid composition and N-terminal sequence, and correct mass as determined by mass spectrometry. Furthermore, the introduced mutation did not reduce the biological activity of the hormone as judged from its action in three biological assay systems: 1) a hormone-sensitive osteoblast adenylate cyclase assay; 2) an in vivo calcium mobilizing assay in rats; and 3) an in vitro bone resorption assay.

Efficient secretion of human parathyroid hormone by Saccharomyces cerevisiae.

A cDNA encoding mature human parathyroid hormone (hPTH) was expressed in Saccharomyces cerevisiae, after fusion to the prepro region of yeast mating factor alpha (MF alpha). Radioimmunoassay showed high levels of hPTH immunoreactive material in the growth medium (up to 10 micrograms/ml). More than 95% of the immunoreactive material was found extracellularly as multiple forms of hormone peptides. Three internal cleavage sites were identified in the hPTH molecule. The major cleavage site, after a pair of basic amino acids (aa) (Arg25Lys26 decreases Lys27), resembles that recognized by the KEX2 gene product on which the MF alpha expression-secretion system depends. The use of a protease-deficient yeast strain and the addition of high concentrations of aa to the growth medium, however, not only changed the peptide pattern, but also resulted in a significant increase in the yield of intact hPTH (1-84) (more than 20% of the total amount of immunoreactive material). The secreted hPTH (1-84) migrates like a hPTH standard in two different gel-electrophoretic systems, co-elutes with standard hPTH on reverse-phase high-performance liquid chromatography, reacts with two hPTH antibodies raised against different parts of the peptide, has a correct N-terminal aa sequence, and has full biological activity in a hormone-sensitive osteoblast adenylate cyclase assay.

Expression and characterization of a recombinant human parathyroid hormone secreted by Escherichia coli employing the staphylococcal protein A promoter and signal sequence.

Human parathyroid hormone (hPTH) is a peptide hormone consisting of 84 amino acids (hPTH(1-84)). Employing the promoter and signal sequence of Staphylococcus aureus-protein A we have expressed hPTH in Escherichia coli. The expressed proteins are excreted to the growth medium, allowing for rapid and easy purification of the desired products. By amino acid sequence analysis and mass spectrometry, we have shown that the major excreted product is correctly processed human identical hPTH(1-84). The purified recombinant hPTH(1-84) stimulates adenylate cyclase activity in rat osteosarcoma cell membranes to exactly the same extent as synthetic parathyroid hormone standards, indicating that the recombinant product has full biological activity.

Expression of human parathyroid hormone in Escherichia coli.

Human parathyroid hormone (hPTH) is a peptide hormone consisting of 84 amino acids. Using the expression plasmid pKK223-3 with the strong tacpromoter, we have produced a variant of hPTH in E. coli. From the expression plasmid construct the expected product was hPTH with an N-terminal extension of Met-Gly. The peptide was extracted from E. coli cells and purified by high performance liquid chromatography. In two different gel electrophoresis systems including identification by immunoblotting the product behaved exactly as an hPTH standard. N-terminal amino acid sequence analysis of the purified product showed traces of Gly-hPTH. At least 90% of the expressed product was N-terminally blocked, suggesting the presence of N-formyl-methionine. This variant of hPTH did not stimulate adenylate cyclase activity in rat osteosarcoma cell membranes.

Improved diagnosis of primary hyperparathyroidism by defining the inverse relationship between serum immunoreactive parathyroid hormone and calcium.

Serum concentrations of immunoreactive parathyroid hormone (iPTH) measured with a mid-region specific radioimmunoassay and total calcium were correlated in 300 healthy subjects and 158 patients with surgically verified primary hyperparathyroidism (HPT). All the healthy individuals could be separated from the patients by a monoexponential declining curve in which iPTH at concentrations of 0.60 micrograms/l and 0.33 micrograms/l corresponded to calcium concentrations of 2.20 mmol/l and 2.60 mmol/l, respectively. In 22 patients more than one sample was analysed and serum iPTH and calcium were inversely correlated. In contrast, three patients with parathyroid carcinoma showed no reciprocal fluctuations between serum iPTH and calcium. Of 75 patients with hypercalcaemia due to malignant diseases (metastatic mammary carcinoma, bronchial carcinoma, renal carcinoma, myelomatosis), 62 had a normal iPTH/calcium relationship. Two patients with myelomatosis had a temporary elevation of serum iPTH and calcium due to renal impairment. One patient with bronchial carcinoma probably had ectopic production of iPTH. The remaining 10 patients (six mammary carcinomas and four bronchial carcinomas) were found in the pathological iPTH/calcium range. In conclusion, we have demonstrated that an inverse relationship exists between serum iPTH and calcium in patients with non-malignant, primary HPT. Evaluation of iPTH and calcium in the same serum sample gave a correct diagnosis in more than 90% of patients with primary HPT.

Immunoreactive parathyroid hormone and calcitonin in plasma and ultrafiltrate before and after haemodialysis.

The effects of haemodialysis on circulating immunoreactive parathyroid hormone (iPTH) and calcitonin (iCT) were determined in relation to alterations in total (Ca) and ionized (Ca2+) plasma calcium. The hormones were measured in plasma, in dialysate and in ultrafiltrate in eight patients with elevated iPTH concentrations. There was a small, but significant reduction in plasma iPTH during haemodialysis concomitant with an increase in Ca and Ca2+. Only small changes were found in plasma iCT during dialysis. Ultrafiltrate iPTH concentrations were reduced from 46% of plasma values at the beginning of dialysis to 27% of plasma values at the end of 4 h dialysis. Gel chromatography of plasma and ultrafiltrate showed corresponding molecular profiles of intact iPTH and hormone fragments suggesting that none of the PTH peptides were restricted by the dialysis membrane. Clearance of intact iPTH (dialysance) was as expected according to molecular weight. From the PTH clearance we calculated that only 5 to 10 micrograms of intact hormone were removed during dialysis. We therefore conclude that the reduction in plasma iPTH during dialysis is probably related to a relative suppression of PTH secretion rather than due to loss of hormone during dialysis.

Peripheral metabolism of parathyroid hormone in patients with primary hyperparathyroidism as judged by immunological and biological studies.

The present study characterizes the immunological and biological activity of circulating forms of parathyroid hormone (PTH) in patients with primary hyperparathyroidism. In addition, the rate of elimination of intravenously injected 125I-labelled bovine parathyroid hormone (125I-bPTH) was studied in patients with this disease before and after operation. The different molecular forms of serum PTH were characterized by gel chromatography followed by radioimmunoassay employing two antisera with specificities directed against the N-terminal and mid-region part of the peptide, respectively. The major part of immunoreactive PTH (iPTH; on the average above 50%) eluted corresponding to fragments with a molecular size about 7,500 daltons in both radioimmunoassays. Specific immunoreactivity coeluting with the intact hormone represented 9-15%. The biological activity of hyperparathyroid serum after gel chromatography was tested in a hormone-sensitive rat kidney adenylyl cyclase assay system. The basal and PTH-stimulated adenylyl cyclase activity (half-maximal) stimulation at 5 micrograms/l or 0.6 nM) was dependent on Mg2+ and ATP. Maximal responses to PTH, calcitonin, and prostaglandin E2 were 50-200% above basal activity and were obtained in the presence of both GTP and Gpp(NH)p (5 X 10(-4) M). Serum from patients with hyperparathyroidism and PTH extracted from parathyroid tissue stimulated the adenylyl cyclase in a dose-dependent manner, as did the chromatographic fraction representing the intact hormone. Elimination of 125I-bPTH from circulation after intravenous injection to patients with this disease suggested that the hormone, but not its degradation products, were removed more rapidly before than after successful surgery. We conclude that the major part of circulating iPTH in patients with primary hyperparathyroidism is unable to stimulate the rat kidney adenylyl cyclase, and that the biological PTH activity is represented by the intact hormone (15% or less of total iPTH). These patients degrade more rapidly the injected 125I-bPTH and this mechanism introduces a new concept to protect target cells against excessive hormone action.

Characterization of the molecular forms of immunoreactive parathyroid hormone in primary hyperparathyroidism and in hyperparathyroidism due to renal failure.

Radioimmunological determination of immunoreactive parathyroid hormone (iPTH) in sera of patients with primary and secondary hyperparathyroidism was carried out using antisera specific for the amino-terminal and the mid-region part of the hormone. In 58 patients with primary hyperparathyroidism and in 29 patients with the secondary form of the disease due to renal failure, there was a linear correlation between the concentration of serum iPTH determined with the two types of antiserum. In all cases the mid-region-specific antiserum measured the highest iPTH concentrations. The molecular forms of serum iPTH were examined after gel filtration of Sephadex G-75 followed by Bio-Gel P-60 in nine patients. Fractions which represented molecular forms equal to or smaller than intact PTH were collected. Using the mid-region-specific antiserum, the predominant immunoreactivity (50-80%) eluted corresponding to one or several large fragments in both forms of the disease. In contrast, iPTH activity co-eluting with the intact hormone represented 2-8% of the total. Similar elution profiles were observed when the amino-terminal-specific radioimmunoassay was used. However, this antiserum showed that in both disease states relatively higher concentrations of iPTH co-eluted with the intact hormone (about 20%). In conclusion, the higher serum iPTH concentrations measured with mid-region-specific antiserum are mainly due to quantitative differences due to the presence of large and intermediate sized fragments. The appearance of higher concentrations of iPTH in secondary hyperparathyroidism may be due to a reduced degradation and/or filtration by the kidney.