Parathyroid Secretory Protein Research Articles

Scaffold-free parathyroid tissue engineering using tonsil-derived mesenchymal stem cells

To restore damaged parathyroid function, parathyroid tissue engineering is the best option. Previously, we reported that differentiated tonsil-derived mesenchymal stem cells (dTMSC) restore in vivo parathyroid function, but only if they are embedded in a scaffold. Because of the limited biocompatibility of Matrigel, however, here we developed a more clinically applicable, scaffold-free parathyroid regeneration system. Scaffold-free dTMSC spheroids were engineered in concave microwell plates made of polydimethylsiloxane in control culture medium for the first 7days and differentiation medium (containing activin A and sonic hedgehog) for next 7days. The size of dTMSC spheroids showed a gradual and significant decrease up to day 5, whereafter it decreased much less. Cells in dTMSC spheroids were highly viable (>80%). They expressed high levels of intact parathyroid hormone (iPTH), the parathyroid secretory protein 1, and cell adhesion molecule, N-cadherin. Furthermore, dTMSC spheroids-implanted parathyroidectomized (PTX) rats revealed higher survival rates (50%) over a 3-month period with physiological levels of both serum iPTH (57.7–128.2pg/mL) and ionized calcium (0.70–1.15mmol/L), compared with PTX rats treated with either vehicle or undifferentiated TMSC spheroids. This is the first report of a scaffold-free, human stem cell-based parathyroid tissue engineering and represents a more clinically feasible strategy for hypoparathyroidism treatment than those requiring scaffolds. Statement of SignificanceHerein, we have for the first time developed a scaffold-free parathyroid tissue spheroids using differentiated tonsil-derived mesenchymal stem cells (dTMSC) to restore in vivo parathyroid cell functions. This new strategy is effective, even for long periods (3months), and is thus likely to be more feasible in clinic for hypoparathyroidism treatment. Development of TMSC spheroids may also provide a convenient and efficient scaffold-free platform for researchers investigating conditions involving abnormal calcium homeostasis, such as osteoporosis.

MicroRNA response of inhalation exposure to hexanal in lung tissues from Fischer 344 rats.

In previous studies, we have investigated the relationships between environmental chemicals and health risk based on omics analysis and identified significant biomarkers. Our current findings indicate that hexanal may be an important toxicant of the pulmonary system in epigenetic insights. MicroRNA (miRNA) is an important indicator of biomedical risk assessment and target identification. Hexanal is highly detectable in the exhaled breath of patients with chronic obstructive pulmonary disease (COPD) and chronic inflammatory lung disease. In this study, we aimed to identify hexanal-characterized miRNA-mRNA correlations involved in lung toxicity. Microarray analysis identified 56 miRNAs that commonly changed their expression more than 1.3-fold in three doses (600, 1000, and 1500 ppm) within hexanal-exposed Fischer 344 rats by inhalation, and 226 genes were predicted to be target genes of miRNAs through TargetScan analysis. By integrating analyses of miRNA and mRNA expression profiles, we identified one anti-correlated target gene (Chga; chromogranin A; parathyroid secretory protein 1). Comparative toxicogenomics database (CTD) analysis of this gene showed that Chga is involved with several disease categories such as cancer, respiratory tract disease, nervous system disease, and cardiovascular disease. Further research is necessary to elucidate the mechanisms of hexanal-responsive toxicologic pathways at the molecular level. This study concludes that our integrated approach to miRNA and mRNA enables us to identify molecular events in disease development induced by hexanal in an in vivo rat model. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1909-1921, 2016.

1,25-Dihydroxyvitamin D3Has Opposite Effects on the Expression of Parathyroid Secretory Protein and Parathyroid Hormone Genes

Previous studies have shown that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] decreases levels of mRNA for prepro-PTH as well as PTH secretion after chronic exposure (24-48 h) of parathyroid cells in tissue culture. We have now extended these studies to determine the effects of the vitamin D3 metabolite on parathyroid secretory protein (PSP) gene expression. Primary cultures of bovine parathyroid cells were incubated with 10(-8) M 1,25-(OH)2D3 for periods of time ranging from 24-72 h. As observed in earlier experiments, prepro-PTH mRNA decreased to less than 50% of the control value after 72 h. In marked contrast, PSP mRNA showed a 2.5-fold increase by 24 h and greater than 7-fold stimulation by 72 h. In the same studies, PTH secretion was suppressed (to 60% of control), while PSP secretion was increased by 40% over control values. Exposure to high (2.5 mM) or low (0.5 mM) calcium had no effect on PSP mRNA, even though low calcium stimulated the secretion of PSP while high calcium suppressed secretion. These studies showed that 1,25-(OH)2D3 has opposite effects on the gene expression of PSP and PTH in bovine parathyroid cells in tissue culture.

Inhibitors of cellular proteolysis cause increased secretion from parathyroid cells

The secretion but not the biosynthesis of parathyroid hormone and secretory protein 1 is strongly, negatively regulated by extracellular [Ca2+]. In order to examine the hypothesis that decreased degradation might directly cause increased secretion, we tested the effects of agents that suppress cellular degradation via different mechanisms: 3-methyladenine, chloroquine, and 1-deoxynojirimycin. When secretion was inhibited by high [Ca2+], all agents caused increased secretion, but at low [Ca2+] only deoxynojirimycin was effective. Results derived from the use of two radioimmunoassays for parathyroid hormone suggested that the increased secretion was due to reduced destruction of secretory vesicles by lysosomes, and to reduced proteolysis of their contents by secretory vesicle proteases.

Calcium-binding proteins in the parathyroid gland: Detailed studies of parathyroid secretory protein

Abstract In order to identify calcium (Ca2+)-binding proteins in the parathyroid gland, we used electrophoretic blots of proteins separated by a two-dimensional nondenaturing/denaturing gel system and incubated them with 45Ca2+. Parathyroid secretory protein (PSP) and proteins with approximate molecular weights of 98,000, 88,000, 58,000, and 30,000 were noted to bind Ca2+ in cytosolic fractions from bovine parathyroid, adrenal, and pituitary glands. However, differences in the binding affinity and capacity of the various proteins were observed. PSP displayed a low affinity and high binding capacity for Ca2+. In the presence of 5 mM MgCl2 and 60 mM KCl, native PSP (immobilized on nitrocellulose filters) bound 7.5 mol of Ca2+/mol of protein monomer with an apparent Kd of 1.1 mM. Immunoblotting identified the association of PSP with parathyroid cell membranes in a Ca2+-dependent manner. This property, together with its heat stability, distinguished PSP from other cytosolic Ca2+-binding proteins which were identified. There was also evidence for a Ca2+-dependent protein-protein interaction (aggregation) of PSP present in a Nonidet P-40 extract of cell membranes. The high Ca2+ binding capacity of PSP and its Ca2+-dependent membrane association may be features that make PSP a potentially important protein in secretory cells.

Isolation and purification of bovine parathyroid secretory protein

Isolation and purification of bovine parathyroid secretory protein

Suppression of parathyroid hormone secretion by aluminum

The effect of aluminum on parathyroid hormone secretion was examined using collagenase-dispersed bovine parathyroid cells. An increase in the medium aluminum concentration over the range of 0.5 to 2.0 mM, in low calcium medium, progressively inhibited the secretion of radioimmuno-assayable hormone. At 2.0 mM aluminum hormone secretion was inhibited by 68% while high medium calcium, without aluminum, maximally inhibited parathyroid hormone secretion only 39%. Individually, 2.0 mM aluminum or 2.0 mM calcium inhibited isoproterenol-stimulated hormone secretion by 43%. Either metal suppressed basal and isoproterenol-stimulated cyclic AMP levels of the parathyroid cells. That the inhibitory effect of aluminum on parathyroid hormone secretion was not due to an irreversible toxic effect was demonstrated by a restoration of normal secretion when cells were returned to 0.5 mM calcium medium without aluminum. The incorporation of [3H]leucine into total cell protein, parathyroid secretory protein, proparathyroid hormone, or parathyroid hormone was not affected by aluminum. The secretion of radiolabeled protein was, however, inhibited by aluminum. These results suggest that aluminum does not affect protein biosynthesis of the parathyroid cell or the conversion of proparathyroid hormone to parathyroid hormone. Aluminum appears to directly affect the secretion of protein from dispersed parathyroid cells.

Phosphorylation of parathyroid secretory protein.

The phosphorylation of proteins released into the medium of bovine parathyroid gland slices or isolated cells incubated with 32Pi has been investigated. The primary protein phosphorylated had a Mr of 68,000 and coeluted with newly synthesized parathyroid secretory protein (PSP) on Bio-Gel chromatography and on polyacrylamide gel electrophoresis. Isoelectric focusing of double-labeled samples ([35S]methionine and 32Pi) revealed comigration of the two radioactive markers at a pH of 4.6, which was similar to that of purified PSP. Phosphorylation of the Mr 68,000 protein was also demonstrated in cell homogenates incubated with [gamma-32P]ATP; the Mr 68,000 protein was the predominant labeled protein. Increasing quantities of calcium, with and without added EGTA, caused a progressive decrease in phosphorylation of the protein. These studies demonstrate that PSP is readily phosphorylated in parathyroid cells, that the degree of phosphorylation is inversely proportional to calcium concentration, and that PSP is the major phosphorylated protein released from the gland. The relationship of phosphorylation to the potential physiologic importance of PSP remains to be determined.

Calcium-regulated biosynthesis of the parathyroid secretory protein, proparathyroid hormone, and parathyroid hormone in dispersed bovine parathyroid cells.

Dispersed bovine parathyroid cells were incubated in vitro with 3H-labeled amino acids for 30--120 min. Analysis of cell extracts by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis revealed a calcium-regulated incorporation of 3H-labeled amino acids into three major peaks, one with a molecular weight (Mr) of 70,000 and two peaks in the 10,000 Mr region, and a minor 5,000 Mr peak. Furthermore, [3H]mannose was incorporated into the 70,000 Mr peak, which corresponds to the parathyroid secretory protein (PSP) recognized as a glycoprotein. The two peaks in the 10,000 Mr region had the electrophoretic mobility of [3H]bovine proparathyroid hormone (ProPTH) and [3H]bovine PTH (1--84), respectively. Immunoreactive PTH was only detected in a peak comigrating with [3H]PTH-(1--84). The nonimmunoreactive 5,000 Mr peptide was not further identified. The incorporation of radioactive amino acids into PSP, ProPTH, PTH-(1--84), and the 5000-daltons peptide was inversely related to the calcium concentration in the incubation medium. The incorporation of radioactive mannose into PSP was also greater at low extracellular calcium concentrations. Our findings provide evidence for an inverse relationship between the extracellular calcium concentration and the formation of PSP, ProPTH, PTH-(1--84), and a yet to be identified 5000-dalton peptide in dispersed bovine parathyroid cells.

Extraction, purification, and partial characterization of bovine parathyroid secretory protein.

Parathyroid secretory protein (PSP) was purified from extracts of bovine parathyroid glands using radioactive PSP as a marker during purification. The labeled preparation was obtained by gel filtration of medium resulting from incubation of parathyroid glands with radioactive amino acids for 4 h. Alkaline extraction, ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, and gel filtration over Bio-Gel A 1.5 M and A 5 M resulted in a homogeneous product. Purified PSP monomer had a Mr = 67,000, was highly acidic on gel electrophoresis, and contained over 35% glutamic and aspartic acid residues. Approximately 18% of the protein consisted of carbohydrate residues. The homogeneity of the final product was established by polyacrylamide and sodium dodecyl sulfate (SDS) gel electrophoresis and the identification of a single NH2-terminal leucine. A radioimmunoassay for PSP was developed to identify the protein in tissue extracts and in incubation medium. Purification of PSP should be useful in defining further its biochemistry, biologic function, and control of secretion.

Identification and cell-free translation of mRNA coding for a precursor of parathyroid secretory protein.

Identification and cell-free translation of mRNA coding for a precursor of parathyroid secretory protein.

Subcellular Distributions of Parathyroid Hormone, Hormonal Precursors, and Parathyroid Secretory Protein*

Distributions of parathyroid hormone (PTH), proparathyroid hormone (ProPTH), preproparathyroid hormone (PreProPTH), and parathyroid secretory protein (PSP) were analyzed in subcellular fractions prepared from homogenates of bovine parathyroid glands. Slices of bovine parathyroid glands were incubated with radiolabeled amino acids for 3--30 min to selectively label newly synthesized proteins. Subcellular fractions were prepared from homogenates of the gland slices by differential centrifugation. Newly synthesized labeled hormonal polypeptides in the fractions were analyzed by electrophoresis on polyacrylamide gels, and total amounts of PTH and ProPTH (previously formed and newly synthesized) were determined by immunoassay. Ninety percent of total immunoreactive, 70--80% of newly synthesized PTH, ProPTH, and PreProPTH, and 50% of PSP were found in sedimentable particulate fractions. The low speed (800 X g) pellet, which consisted predominantly of cell debris and nuclei with adherent remnants of cytoplasm, contained 30--50% of the ProPTH and PTH. The intermediate speed (10,000 X g) pellet, which contained granules, was relatively enriched in PTH. Most particulate-associated hormone could be solubilized by treatment with deoxycholate (DOC) 98% and 97% of radiolabeled and 93% and 83% of immunoreactive ProPTH and PTH, respectively, in particulates sedimenting at 10,000 and 105,000 X g were rendered DOC-soluble. Approximately 50% of the PTH and ProPTH in the particulates resisted digestion by combined trypsin and chymotrypsin, whereas PreProPTH was completely susceptible to proteolysis. Up to 50% of the radiolabeled PTH and ProPTH added exogenously to parathyroid gland slices before homogenization became associated with the particulate fractions, and 70--80% or radiolabeled PreProPTH added to the subcellular fractions readily associated with the sedimentable material. The results indicate that in homogenates of parathyroid glands, PTH, ProPTH, PreProPTH, and PSP are associated with particulate structures. Furthermore, up to 50% of the association of ProPTH, PTH, and PSP with particulate fractions seems to be nonsepcific and occurs during the disruption of the tissues. The remaining 50% or more of hormonal protein is presumably sequestered within membrane-limited structures, such as microsomal vesicles. The complete susceptibility in particulate fractions of newly synthesized PreProPTH, but not of ProPTH, to limited proteolysis indicates that the two precursors are located in different subcellular compartments and suggests that PreProPTH is converted to ProPTH before its entry into the intracisternal space of the endoplasmic reticulum. Alternatively, the PreProPTH identified in parathyroid gland slices may represent polypeptide chains synthesized in the cell sol on polyribosomes that are not attached to endoplasmic reticulum but are adsorbed nonspecifically to the particulate fraction of the cell during the process of tissue homogenization.

The effects of calcium and magnesium on the secretion of parathormone and parathyroid secretory protein by isolated porcine parathyroid cells.

The preparation of dispersed parathyroid cells by collagenase digestion of porcine parathyroid glands, essentially as outlined by Brown et al. (Endocrinology 99: 1582, 1976), is described. The cells secrete parathormone linearly for at least 4 h of incubation and rapidly respond in inverse fashion to changes in the medium calcium and magnesium concentrations over the range 0.5-3.0 mM. In terms of inhibition of secretion, either ion was more effective in the presence of a minimum concentration of the other, indicating that calcium and magnesium affect separate cellular sites. Parathormone was identified both by immunoassay of the whole incubation medium and by its separation by polyacrylamide gels and carboxymethylcellulose chromatography. When the cells were incubated with radioactive amino acids and both the medium and cells were subsequently analyzed on gels, we found that parathyroid secretory protein as well as parathormone and some immunoactive fragments were present. Analysis of the radioactive protein contained in the cells at high and low calcium concentrations revealed that calcium decreased the formation of the secretory protein by approximately 40% without appreciably affecting the formation of proparathormone or parathormone. The secretion of both parathyroid secretory protein and parathormone were inversely proportional to the concentrations of medium calcium or magnesium. The secretion of the latter, however, was more sensitive (95% inhibition) than parathormone (40-60% inhibition) to changes in medium divalent cations. These results suggest that the synthesis, intracellular processing, or secretion of parathormone and parathyroid secretory protein utilize independent calcium- and magnesium-regulated pathways.

The Intracellular Pathway for Parathormone Biosynthesis and Secretion

The initial translation product of parathormone messenger RNA--preproparathormone--is larger than parathormone. Two amino terminal peptide segments of the peptide chain are removed sequentially to form the 84-amino acid hormone. The first cleavage, the removal of a 25-amino acid extension from preproparathormone, occurs in the rough endoplasmic reticulum, and results in the formation of proparathormone. This peptide moves via an energy-dependent mechanism to the Golgi region of the cell where a specific converting enzyme cleaves a basic hexapeptide segment yielding parathormone itself. A part of the newly formed hormone then is enclosed within prosecretory vesicles, transported to the cellular membrane and secreted. Another portion is stored in mature secretory vesicles and is subject to subsequent secretion. The moment to moment control of parathormone secretion by calcium resides at the plasma membrane, but a tightly coupled response at the level of intracellular hormone degradation is also necessary in order to control intracellular hormone levels in the face of rapid changes in secretory rate. Three major secretory products are released from the parathyroid under the control of extracellular calcium: (1) parathormone, (2) a large protein--"parathyroid secretory protein"--whose function is unknown, and (3) peptide fragments of parathormone. Secretion of hormonal fragments adds to the population of parathormone immunoreactivity in the blood. These fragments appear to be similar if not identical to those formed by peripheral metabolism of parathormone in the liver and kidney.

The secretion of parathormone and glycosylated proteins by parathyroid cells in culture

The secretion of radioactive peptides by dispersed porcine parathyroid cells incubated with [ 3H]- or [ 14C]amino acids, [ 3H]glucosamine and [ 3H]mannose was analyzed. After incubation, the culture medium contained radioactive parathormone, as expected, and two radioactive glycopeptides: SP I and SP II. SP I appears to be identical with parathyroid secretory protein , heretofore not recognized as a glycoprotein. SP II has not been previously identified. SP I, but not SP II or parathormone, was adsorbed by Concanavalin A possibly reflecting a high mannose content of this molecule. Raising the concentration of calcium in the medium suppressed the secretion of radioactive parathormone and SP I in a similar fashion but did not affect the secretion of SP II. Our results suggest that SP I may play a fundamental role in parathyroid synthetic or secretory processes.