Biomedical subjects

J D Zajac

Publications and source records attributed to J D Zajac.

7 recordsLinked to original sources

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

Multiple endocrine neoplasia syndrome--type 2b. Case report and review.

The multiple endocrine neoplasia syndromes are an association of tumours of 2 or more endocrine glands. Multiple endocrine neoplasia type 2b (MEN 2b) patients develop medullary thyroid carcinoma and pheochromocytomas as well as unique physical characteristics. Most commonly, MEN2b is inherited with an autosomal dominant pattern although sporadic cases are not uncommon. If untreated the disease may be lethal. The facial, oral and ocular characteristics are reliable markers of the disease. These patients give a history most commonly of slipped capital femoral epiphysis, hypertension and life-long diarrhoea and/or constipation. MEN2b is most commonly characterised by nodules on the anterior aspect of the tongue, thickened lips with nodules, thickened upper eyelids, broadened nasal bridge, thickened corneal nerves and dilated, symmetrical, pedunculated nodules on the cheek mucosa. The patient described has most of these characteristics. Radiographic features of the jaws which have not been previously described are reported. These include a markedly enlarged and bifurcated inferior alveolar canal and shortened roots of the lower incisor teeth. Due to the lethality of the disease, patients who present with the above physical characteristics must be further investigated to exclude MEN2b.

Adrenal Gland Neoplasms↗

PubMed1992

An intra-thyroidal branchial cyst: a case report.

Branchial cleft cysts were previously thought not to occur in the thyroid but have recently been described in two patients with Hashimoto's disease. This case report describes a patient with a branchial cleft cyst in an otherwise normal thyroid gland and could provide further evidence that thyroidal follicular cells are derived from the branchial clefts as well as from the primitive gut.

Adult↗

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

Cyclic adenosine 3',5'-monophosphate (cAMP)-dependent and cAMP-independent regulation of parathyroid hormone receptors on UMR 106-01 osteoblastic osteosarcoma cells.

The osteoblast-like cells, UMR 106-01, express PTH receptors that are coupled to adenylate cyclase. Recently, we reported the isolation of a UMR 106-01 subclone, UMR 4-7, that is stably transfected with a Zn(++)-inducible mutant of the regulatory subunit of protein kinase A. Incubation of UMR 4-7 cells with Zn++ renders the cells unresponsive to cAMP agonists. This subclone, therefore, seemed particularly suitable for studies of PTH receptor regulation. In UMR 106-01 cells, PTH receptors are strikingly down-regulated by pretreatment with 8-Br-cAMP or 3-isobutyl-1-methylxanthine for 2 days. In UMR 4-7 cells, this effect is totally prevented by prior and concurrent treatment with Zn++. Zn++ addition to UMR 106 cells does not modify these responses. Treatment with the PTH agonist [Nle8,18,Tyr34]bovine PTH(1-34)NH2 [(NlePTH(1-34)] also markedly down-regulates PTH receptors in UMR 106 cells, but this effect is only partially inhibited in Zn(++)-induced UMR 4-7 cells. At high doses, the PTH antagonist, [Nle8,18,Tyr34]bovine PTH(3-34)NH2 [NlePTH(3-34)] also (partially) reduces PTH receptor availability. Receptor regulation by NlePTH(3-34) is not blocked in the cAMP-resistant cells, however. Coincubation of submaximal doses of NlePTH(1-34) (1 nM) with NlePTH(3-34) (1 microM) reduces receptor availability more than when the cells are exposed to either ligand alone. This decrease is only partially inhibited in Zn(++)-induced UMR 4-7 cells. In contrast to its additive effect on receptor regulation, NlePTH(3-34) efficiently competes for binding to the PTH receptor in UMR 106-01 cells and antagonizes the stimulatory effects of NlePTH(1-34) on both intracellular cAMP accumulation and gene expression driven by a transiently transfected synthetic cAMP-responsive enhancer. In conclusion, homologous down-regulation of PTH receptors is mediated by activation of both cAMP-dependent (via protein kinase A) and cAMP-independent pathways. PTH activates both pathways, whereas the effect of NlePTH(3-34) appears to be exclusively cAMP-independent. These results give new insights into mechanisms of PTH receptor regulation.

1-Methyl-3-isobutylxanthine↗

PubMed1989

Production of parathyroid hormone-related protein by a rat parathyroid cell line.

Parathyroid hormone-related protein (PTHrP), the peptide associated with humoral hypercalcemia of malignancy, has been identified in fetal and adult parathyroid glands. We here report a sub-clone of a rat parathyroid cell line which secretes a single peptide species corresponding in size to PTHrP(1-84). Biological activity of the secretion product was blocked by a specific antiserum against PTHrP, but not by parathyroid hormone (PTH) antiserum. Secretion of PTHrP by these cells was regulated by extracellular calcium in the physiological range. A single messenger RNA species for PTHrP was identified, though PTH mRNA could not be shown in these cells. Hybrid CAT genes containing 700-1000 bp of 5'-flanking DNA from the human PTH or PTHrP genes were transfected into these cells, and the PTHrP gene was expressed at 10-fold higher levels than the PTH gene. These cells thus provide a valuable model system for investigation expression of PTHrP in a non-transformed cell line.

Adenylyl Cyclases↗

PubMed1989

Inhibition of parathyroid hormone responsiveness in clonal osteoblastic cells expressing a mutant form of 3',5'-cyclic adenosine monophosphate-dependent protein kinase.

PTH activates multiple acute intracellular signals within responsive target cells, but the importance of cAMP vs. other second messenger signals in mediating different biological responses to PTH is not known. To address these questions, we developed a genetic approach to block activation of the cAMP-dependent protein kinase (PK-A) in PTH-responsive cell lines. Clonal rat osteosarcoma cells (UMR 106-01) were stably transfected with REV-I, a plasmid that directs synthesis of a mutant cAMP-resistant form of the type I regulatory subunit of PK-A. In the transfected bone cells, most of the catalytic subunits of PK-A were associated with the mutant regulatory subunit, and activation of PK-A by cAMP was correspondingly inhibited. We have characterized one such mutant (UMR 4-7) that expressed large amounts of mutant mRNA and exhibited inducible blockade of PK-A via the REV-1 metallothionein promoter. In the absence of metallothionein induction, these cells exhibited nearly normal PTH responsiveness, but after REV-1 induction by Zn2+, they were resistant to PTH-induced activation of PK-A and regulation of membrane phospholipid synthesis by both PTH and cAMP analogs. The mutant UMR 4-7 cell provides a model system in which the consequences of cAMP production by PTH or other agonists that activate adenylate cyclase in osteoblasts may be specifically inhibited by brief exposure to Zn2+. Such mutant cell lines will facilitate further investigation of the linkage between early signalling events and subsequent biological responses in the action of PTH and other agonists on target cells in bone.

Animals↗