Biomedical subjects

H M Kronenberg

Publications and source records attributed to H M Kronenberg.

At least 73 records · Page 4Linked to original sources

PubMed1992

Interaction of nascent preproparathyroid hormone molecules with microsomal membranes.

To characterize the early steps in the interaction of nascent chains of preproparathyroid hormone (prepro-PTH) with the secretory apparatus, such truncated nascent chains still attached to ribosomes were tested for binding to microsomal membranes and cleavage by signal peptidase. Nascent chains of 114, 97, 88, 81, 70, and 59 residues were tested for their ability to bind tightly to membranes and to undergo signal sequence cleavage. Chains of 81 residues and longer bound tightly to the membranes and were cleaved by signal peptidase. The 88- and 81-residue precursors and their corresponding pro-proteins were less efficiently associated with the membranes than were the 114- and 97-residue precursors and their corresponding pro-proteins. The 70-residue chain bound to the membrane but was not cleaved. When this peptide was subsequently released from the ribosome with puromycin, it was cleaved by signal peptidase. The 59-residue chain bound only slightly to the microsomal membrane and was not cleaved by signal peptidase, even when the nascent peptide was released from the ribosome with puromycin. Thus the critical length for productive binding to microsomal membranes is between 59 and 70 residues; the length required for signal cleavage is between 70 and 81 residues.

Amino Acid Sequence↗

PubMed1992

Regulation of gene transcription and proliferation by parathyroid hormone is blocked in mutant osteoblastic cells resistant to cyclic AMP.

We employed a cyclic AMP-resistant subclone of UMR 106-01 osteoblastic osteosarcoma cells (UMR 4-7) with a regulated, dominant-negative mutation of cyclic AMP-dependent protein kinase (PK-A), to examine the mechanism(s) whereby parathyroid hormone (PTH) regulates growth of these cells. Expression of a transiently transfected CAT reporter gene controlled by the cAMP response element of the rat somatostatin gene ('SST-CAT') was used to monitor PK-A activation in intact cells. Agonist-stimulated SST-CAT expression was specific for agents known to activate adenylate cyclase, required an intact cAMP response element and was specifically blocked following induction of the mutant cAMP-resistant phenotype in UMR 4-7 cells. Inhibition of the proliferation of UMR 106-01 cells by PTH, which is mimicked by forskolin and 8-bromo-cAMP, was blocked completely in mutant cyclic AMP-resistant UMR 4-7 cells. We conclude that control of proliferation in UMR 106-01 cells by PTH involves the cAMP messenger system and requires activation of PK-A.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1992

Assignment of the human cyclin D3 gene (CCND3) to chromosome 6p----q13.

The PRAD1/cyclin D1 gene (CCND1), a member of the D-type cyclin gene family, has been implicated as a protooncogene in parathyroid, lymphoid, and mammary tumors. We cloned and mapped another member of this family, the human cyclin D3 gene (CCND3), to chromosome 6p----q13 using human x rodent hybrids. This assignment raises the hypothesis that cyclin D3 may be involved in the pathogenesis of human neoplasms with abnormalities of chromosome 6.

Blotting, Southern↗

PubMed1992

Somatic gene transfer in the development of an animal model for primary hyperparathyroidism.

The overproduction of hormones is associated with a variety of endocrinological disorders. We have used somatic cell gene transfer of human PTH (hPTH) to develop an animal model of hypercalcemia and osteoclastic skeletal resorption. Recombinant retroviruses were used to transduce a functional hPTH gene into cultured rat fibroblasts. The recombinant-derived preproparathyroid hormone peptide was appropriately processed in this ectopic cell, and intact hPTH (1-84) was secreted at a high level (2-5 ng/10(6) cells/24 h). Transplantation of the PTH-secreting cells into syngeneic rat recipients was associated with the development of hypercalcemia mediated by increasing serum concentrations of hPTH. Thyroparathyroidectomy in these hypercalcemic rats producing hPTH did not result in hypocalcemia and tetany, which was observed in control animals undergoing thyroparathyroidectomy. Chronic overproduction of hPTH (60 days) was associated with severe hypercalcemia, metastatic calcification, and histological changes of osteoclastic resorption of bone. This animal model will be useful in studying the pathophysiology of severe hyperparathyroidism in humans and should help in the evaluation of new medical therapies for hypercalcemia.

3T3 Cells↗

PubMed1992

The carboxy-terminus of parathyroid hormone is essential for hormone processing and secretion.

The biological function of the carboxy-terminal portion of the PTH molecule is unknown. We hypothesized that the carboxy-terminus of PTH may be essential for hormone processing and secretion. To ascertain the potential role of the carboxy-terminus in transport through the secretory pathway, we constructed a series of carboxy-terminally deleted mutants of human preproPTH. PreproPTH and these truncated peptides were expressed in cell-free extracts and in acutely transfected COS 7 cells. Intact preproPTH(1-84) (115 residues) was processed to proPTH(1-84); the PTH was secreted very efficiently. In contrast, translocation across the membrane of the endoplasmic reticulum and signal sequence cleavage were mildly impaired for PTH(1-52) (83 residues), moderately impaired for PTH(1-40) (71 residues), and dramatically impaired for PTH(1-34) (65 residues). Subsequent cleavage of the prosequence and secretion from intact cells were also dramatically impaired for PTH(1-52) and undetectable for PTH(1-40) and PTH(1-34). We conclude that more than 65 residues are needed to cross the endoplasmic reticulum and that more than 83 residues are needed for efficient prosequence cleavage and secretion. Perhaps similar requirements for full transport through the secretory pathway explains why most secreted peptides are synthesized as long propeptides of at least 80 residues.

Animals↗

PubMed1992

Characterization of 1,25-dihydroxyvitamin D3 receptor interactions with target sequences in the rat osteocalcin gene.

The sequences in the rat osteocalcin gene that bind the 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor and mediate its effects on gene transcription contain three copies of a motif homologous to those found in other steroid response elements. To evaluate the relative importance of these motifs and their flanking sequences, functional properties and receptor binding of a series of mutant response elements were analyzed. Mutations in the third motif, including its deletion, have no effect on receptor binding and only modestly detrimental effects on the ability of the upstream motifs to confer 1,25-(OH)2D3 responsiveness in transfected cells. Mutations in the first two motifs have negative effects on both receptor binding and gene activation. Mutagenesis of bases upstream from each of the first two motifs alters receptor binding and gene activation; these bases are, therefore, an integral part of the rat osteocalcin 1,25-(OH)2D3 response element. Two direct hexameric repeats (GGGTGA ATG AGGACA) and not the palindromic motif thus characterize this 1,25-(OH)2D3 response element.

Animals↗

PubMed1991

A G protein-linked receptor for parathyroid hormone and parathyroid hormone-related peptide.

The complementary DNA encoding a 585-amino acid parathyroid hormone-parathyroid hormone-related peptide (PTH-PTHrP) receptor with seven potential membrane-spanning domains was cloned by COS-7 expression using an opossum kidney cell complementary DNA (cDNA) library. The expressed receptor binds PTH and PTHrP with equal affinity, and both ligands equivalently stimulate adenylate cyclase. Striking homology with the calcitonin receptor and lack of homology with other G protein-linked receptors indicate that receptors for these calcium-regulating hormones are related and represent a new family.

Amino Acid Sequence↗

PubMed1991

Negative regulatory elements in the human parathyroid hormone gene.

We have identified and characterized a pair of negative regulatory elements far upstream of the transcription start site of the human parathyroid hormone (hPTH) gene. Transfection of various types of cultured cells with a fusion plasmid containing 4.7 kilobase pairs of the 5'-flanking portion of the hPTH gene linked to the chloramphenicol acetyltransferase (CAT)-coding sequence generated only 10% of the CAT activity of a plasmid containing 684 base pairs of the 5'-flanking region of the hPTH gene. Deletion analyses reveal that there are at least two separate upstream DNA elements in the hPTH gene responsible for the negative regulation. We find that these cultured cells possess nuclear factors which specifically bind to several short DNA sequences within these elements and that these sequences can suppress transcription of the hPTH gene.

3T3 Cells↗

PubMed1991

Rat thymosin beta 4 gene. Intron-containing gene and multiple retroposons.

Thymosin beta 4 (T beta 4), a 43-residue peptide of uncertain function, is widely distributed in most tissues of vertebrates. Southern blot analysis demonstrated that the rat genome contains many closely related T beta 4 sequences. Two distinct T beta 4 sequences were isolated by screening a rat genomic DNA library. Analysis of these clones revealed that they represent T beta 4 retroposons; they lack introns and contain stretches of poly(A) sequence at their 3' ends and direct repeats flanking the cDNA-like sequences. In order to isolate a T beta 4 intron-containing gene, DNA fragments that include T beta 4 introns were generated by polymerase chain reaction, using rat genomic DNA as template. Subsequent Southern blot analysis of rat genomic DNA with intron-specific probes demonstrated the presence of a single T beta 4 intron-containing gene. Upon screening the rat genomic DNA library with an intron-specific probe, two overlapping clones of the T beta 4 intron-containing gene were isolated. Further characterization and sequence analysis showed that the rat T beta 4 intron-containing gene has a 2-kilobase pair-long transcription unit containing two introns. The transcription initiation site and putative regulatory elements of the promoter were identified.

Animals↗

PubMed1991

Mutational analysis of the receptor-activating region of human parathyroid hormone.

The first 4 residues of parathyroid hormone (PTH) are highly conserved in evolution and are important for biological activity. We randomly mutated codons 1-4 of human PTH (hPTH) with degenerate oligonucleotides and, after expression in COS cells, screened the mutants for receptor binding and cAMP-stimulating activity using ROS 17/2.8 cells. This survey identified Glu4 and Val2 as important determinants of receptor binding and activation, respectively. Positions 1 and 3 were more tolerant of substitutions indicating that these sites are less vital to hormone function. Activities of synthetic hPTH(1-34) analogs further demonstrated the importance of positions 2 and 4. The binding affinity of [Ala4,Tyr34] hPTH(1-34)NH2 was 100-fold reduced relative to [Tyr34]hPTH(1-34)NH2 (Kd values = 653 +/- 270 and 4 +/- 1 nM, respectively), and [Arg2, Tyr34]hPTH(1-34)NH2 was a weak partial agonist which bound well to the ROS cell receptor (Kd = 31 +/- 10 nM). The Arg2 analog was nearly as potent as PTH(3-34) as an in vitro PTH antagonist in osteoblast derived cells. However, unlike PTH(3-34), [Arg2]PTH was a full agonist in opossum kidney (OK) cells. These observations suggest that the activation domains of the OK and ROS cell PTH receptors are different. Thus, amino-terminal PTH analogs may be useful as probes for distinguishing properties of PTH receptors.

Amino Acid Sequence↗

PubMed1991

A novel cyclin encoded by a bcl1-linked candidate oncogene.

We have previously identified a candidate oncogene (PRAD1 or D11S287E) on chromosome 11q13 which is clonally rearranged with the parathyroid hormone locus in a subset of benign parathyroid tumours. We now report that a cloned human placental PRAD1 complementary DNA encodes a protein of 295 amino acids with sequence similarities to the cyclins. Cyclins can form a complex with and activate p34cdc2 protein kinase, thereby regulating progress through the cell cycle. PRAD 1 messenger RNA levels vary dramatically across the cell cycle in HeLa cells. Addition of the PRAD1 protein to interphase clam embryo lysates containing inactive p34cdc2 kinase and lacking endogenous cyclins allows it to be isolated using beads bearing p13suc1, a yeast protein that binds cdc2 and related kinases with high affinity and coprecipitates kinase-associated proteins. Addition of PRAD1 also induces phosphorylation of histone H1, a preferred substrate of cdc2. These data suggest that PRAD1 encodes a novel cyclin whose overexpression may play an important part in the development of various tumours with abnormalities in 11q13.

Adenoma↗

PubMed1991

Rearrangement and overexpression of D11S287E, a candidate oncogene on chromosome 11q13 in benign parathyroid tumors.

We report the detailed molecular characterization of a human parathyroid adenoma with a clonal parathyroid hormone gene rearrangement. This rearrangement is similar to one we characterized recently in an independent adenoma. In these two, plus a third partially characterized adenoma, one allele of the PTH gene, on 11p15, is rearranged with DNA from the D11S287 region on 11q13. This region contains a transcribed sequence, D11S287E, distinct from known 11q13 oncogenes, that is expressed in all parathyroid tissues examined, but is overexpressed dramatically in all three tumours with PTH gene-D11S287 rearrangements. These findings suggest that overexpression of D11S287E, perhaps driven by the misplaced PTH gene's regulatory elements, contributed to the development of these benign tumors. D11S287E is a new candidate oncogene with potential importance in parathyroid adenomas and perhaps other tumors with 11q13 abnormalities.

Adenoma↗

PubMed1990

Expression of human parathyroid hormone-(1-84) in Escherichia coli as a factor X-cleavable fusion protein.

Recombinant human parathyroid hormone (hPTH)-(1-84) was obtained from Escherichia coli using a cleavable fusion protein strategy. The fusion protein contains residues 1-138 of human growth hormone as the amino-terminal region and residues 1-84 of hPTH as the carboxyl-terminal region. A 7-residue linker containing the recognition/cleavage sequence of the site-specific blood coagulation protease activated factor X (factor Xa) joins the two regions. Intact hPTH-(1-84) is released from this fusion protein by cleavage in vitro with factor Xa. The fusion protein was produced at a high level and formed inclusion bodies which allowed it to be easily purified by low speed centrifugation, with a yield of approximately 50 mg/liter of culture. After factor Xa cleavage and high performance liquid chromatography purification, highly purified hPTH was obtained, with a final yield of 1.5-3 mg/liter. Physical and biological characterization of the purified hormone demonstrated that it was intact and active hPTH-(1-84).

Animals↗

PubMed1990

DNA sequences in the rat osteocalcin gene that bind the 1,25-dihydroxyvitamin D3 receptor and confer responsiveness to 1,25-dihydroxyvitamin D3.

The 5' flanking region of the rat osteocalcin gene has been shown to confer responsiveness to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] after transfection of fusion genes into ROS 17/2.8 cells. Deletion analysis has demonstrated that there are at least two domains in this 5' flanking region that contribute to 1,25(OH)2D3 responsiveness; however, only the downstream region is able to confer 1,25(OH)2D3 responsiveness to either the native osteocalcin promoter or to a heterologous viral promoter (herpes simplex virus thymidine kinase). The proximal region responsible for 1,25(OH)2D3 induction of the rat osteocalcin gene lies 458 base pairs upstream from the transcription start site of this gene. A 25-base-pair oligonucleotide corresponding to the sequences in this region is able to confer 1,25(OH)2D3 responsiveness to the thymidine kinase promoter in an orientation-independent fashion. This sequence contains three copies of a short sequence that are homologous to "half-sites" of steroid response elements. Gel-retardation assays using porcine intestinal nuclear extract as a rich source of 1,25(OH)2D3 receptor demonstrated retardation in the migration of probes containing the sequence noted above. A monoclonal antibody directed against the 1,25(OH)2D3 receptor caused further retardation in the migration of these protein-DNA complexes. Therefore, the sequences represented in this oligonucleotide encompass the sequences necessary for binding of the 1,25(OH)2D3 receptor to DNA as well as those sequences necessary for 1,25(OH)2D3 to induce osteocalcin gene transcription.

Animals↗

PubMed1990

Mutation of the signal peptide-encoding region of the preproparathyroid hormone gene in familial isolated hypoparathyroidism.

Preproparathyroid hormone (preproPTH) gene mutation has been proposed as a cause of familial isolated hypoparathyroidism (FIH). We cloned the preproPTH alleles of a patient with autosomal dominant FIH and sequenced the coding regions, 5' flanking regions, and splice junctions. The putatively abnormal (based on previous linkage studies) allele differed from the other allele's normal sequence at only one nucleotide. This T to C point mutation changes the codon for position 18 of the 31 amino acid prepro sequence from cysteine to arginine, disrupting the hydrophobic core of the signal sequence. Because the hydrophobic core is required by secreted proteins for efficient translocation across the endoplasmic reticulum, the mutant protein is likely to be inefficiently processed. Indeed, in vitro studies demonstrated dramatically impaired processing of the mutant preproPTH to proPTH. In summary, we observed a point mutation in the signal peptide-encoding region of a preproPTH gene in one FIH kindred and demonstrated a functional defect caused by the mutation. Mutation of the signal sequence constitutes a novel pathophysiologic mechanism in man, and further study may yield important insights both into this form of hormone deficiency and into the role of signal sequences in human physiology.

Amino Acid Sequence↗

PubMed1990

Parathyroid hormone messenger ribonucleic acid in the rat hypothalamus.

Polyadenylated RNA, extracted from rat hypothalami, cross-hybridized with a RNA probe complementary in sequence to rat PTH (rPTH) messenger RNA (mRNA). Amplification of complementary DNA (cDNA) by the polymerase chain reaction also demonstrated the presence of rPTH mRNA in the rat hypothalamus and parathyroid gland. rPTH mRNA was localized by in situ hybridization in the paraventricular and supraoptic nuclei of the rat hypothalamus. These results demonstrate the expression of the PTH gene in the central nervous system of the rat in areas which suggest roles for PTH in neuroendocrine function.

Animals↗

PubMed1990

Thymosin beta 4 is expressed in ROS 17/2.8 osteosarcoma cells in a regulated manner.

The differential expression of mRNAs between the closely related rat osteosarcoma cell lines ROS 17/2.8 and ROS 25/1 was used to identify genes whose expression is associated with the osteoblast phenotype. Thymosin beta 4 cDNA was cloned from an ROS 17/2.8 complimentary DAN library on the basis of its differential hybridization with radiolabeled cDNA prepared from ROS 17/2.8 and ROS 25/1 cells. Northern blot analysis confirmed that thymosin beta 4, hitherto a putative immunodulatory hormone, was indeed differentially expressed. Steady state mRNA levels were severalfold higher in ROS 17/2.8 cells exhibiting an osteoblast-like phenotype, compared with the less osteoblast-like ROS 25/1. Thymosin beta 4 transcripts were also detected in rat UMR 106 osteosarcoma cells and in intact neonatal and fetal rat calvaria. Sequence analysis of the cDNA indicated that thymosin beta 4 transcripts may arise by processing at a more distal polyadenylation signal. Treatment of ROS 17/2.8 cells with dexamethasone increased, while addition of 1,25-dihydroxyvitamin D3 decreased thymosin beta 4 mRNA. The phenotype-dependent expression in the ROS cells and the response to steroid hormone suggest that thymosin beta 4 expression contributes to the osteoblast phenotype.

Amino Acid Sequence↗

PubMed1990

Characterisation of the osteoblastic phenotype by the use of differential hybridization.

To identify genes active in cells of the osteoblast lineage we have begun to characterise the phenotype of the rat osteoblast-like osteosarcoma cell line ROS 17/2.8. We have used the method of differential hybridization to identify cDNA clones encoding mRNA species which are expressed in ROS 17/2.8 cells but not in a non-osteoblast-like osteosarcoma cell line (ROS 25/1). We have identified a number of gene products exhibiting this pattern of expression.

Animals↗