BoneKEy-Osteovision | Commentary
Fine-wiring the parathyroid
DOI:10.1138/2001031
There have been two recent contributions that will help us eventually construct a wire diagram of the parathyroid (PT), one from Kifor et al. in Boston () and the other from Quitterer et al. in Wurzburg, Germany (). They both utilized dispersed PT cells, one of bovine and the other of human source, and both admirably exploited the conserved physiology of HEK293 cells stably transfected with the calcium-sensing receptor (CaR) (HEKCaR cells). In PT cells, activation of the CaR by extracellular calcium leads to the Gq/11-dependent activation of the phosphatidylinositol-specific phospholipase C (PI-PLC), causing accumulation of inositol 1,4,5-triphosphate (IP3) and 1,2-sn-diacylglycerol and promoting rapid release of Ca2+ from its intracellular stores. Activation of phospholipases A2 (PLA2) and D by high Ca2+ is indirect, utilizing CaR-mediated, PLC-dependent activation of protein kinase C (PKC) (). The CaR also signals in the PT cell via a pertussis toxin sensitive G protein, probably an isoform of Gi, leading to decreased activation of adenylate cyclase and decreased cAMP levels. The net result of these two G protein-dependent pathways is a high Cao2+ induced decrease in PTH secretion.
Quitterer et al. studied events closer to the cell surface, so let us first consider their contribution (). Their model was the effect of a persistently low serum magnesium to decrease PTH secretion. This model has been known to the clinician for many years: the patient with a low serum calcium, Mg2+ and PTH who does not respond to large doses of intravenous calcium but corrects his calcium and serum PTH after an infusion of Mg2+. There has never been a good explanation. Quitterer et al. studied the relationship between magnesium deficiency, PTH secretion and CaR-mediated signaling. They first showed that PTH release from dispersed human PT cells was decreased after exposure to a low Mg2+, analogous to the human situation. Interestingly, the effect of a low Mg2+ to inhibit PTH secretion is not preserved in the rat. They then showed that in both dispersed human PT cells as well as in the HEKCaR cells, a low Mg2+ enhanced both CaR mediated signaling pathways, namely there were increased concentrations of inositol phosphates and decreased concentration of cAMP. The role of the CaR in the effect of Mg2+ depletion was confirmed by experiments where the CaR was desensitized by chronic exposure to a high Ca2+ concentration or its signal transduction partially disrupted by the use of pertussis toxin. These results indicated that magnesium acts in the CaR-G protein pathway, rather than affecting the secretory mechanism for PTH itself. Mutations of the CaR such as inactivating mutations responsible for Familial Hypocalciuric Hypercalcemia (FHH) and activating mutations responsible for autosomal dominant hypoparathyroidism have been well characterized (). Inactivating and activating mutations of the CaR were transfected into HEK293 cells and the effect of a low Mg2+ on signal transduction was preserved, indicating that Mg2+ binding site responsible for the inhibition of PTH secretion is not the same as the extracellular binding site for Ca2+ and Mg2+. They then showed that the effect of a low Mg2+ was due to its action on the intracellular side of the CaR, at the CaR-G-protein interface. A decrease in Mg2+ concentration increased the rate of GTPγS binding to recombinant Gαi-protein. In addition, they showed that Mg2+ inhibited the basal guanine nucleotide exchange of wild type Gαi GTP-binding protein, but not of a Gαi mutant with impaired Mg2+ binding. To complete the story they showed that Mg2+ deficiency leads to a decrease in intracellular Mg2+. They were able to conclude that the paradoxical block of PTH release under Mg2+ deficiency is mediated through a novel mechanism involving an increase in the activity of Gα subunits of heterotrimeric G-proteins.
Kifor et al. have taken us deeper into the PT cell by studying the effects of activation of the CaR on the MAP kinase pathway (). As quoted earlier, the effect of the CaR is on the Gq/11-PI-PLC pathway to activate PKC as well as the Gi pathway. Secondary to these effects is the activation of PLA2 and the subsequent release of free arachidonic acid and its metabolism to biologically active mediators such as hydroxyperoxyeicosatetranoic acid (HPETE) or hydroxyeicosatetranoic acid (HETE), which may then decrease PTH secretion. Kifor et al. showed the centrality of MAP kinase (MAPK) to the effects of the PKC and Gi pathways to phosphorylate and activate cPLA2. They studied the regulation by the CaR on the phosphorylation of the MAPK, ERK1 and ERK2 (extracellular signal-regulated kinase), because they are known to able to phosphorylate cPLA2. They utilized dispersed bovine PT cells and the HEKCaR cells and showed that increased Cao2+ or a calcimimetic drug led to phosphorylation of ERK1/2. The use of specific inhibitors showed that this effect was mediated by both the Gi tyrosine kinase pathway as well as by the Gq/11-PI-PLC pathway to activate PKC. High Ca2+ increased serine phosphorylation of cPLA2 in both PT and HEKCaR cells and this was inhibited by a selective MAPK inhibitor. They have clearly shown the importance of MAPK in cPLA2 activation.
The mechanism of how an elevated Cao2+ acts on the CaR and its subsequent signal transduction via cPLA2 to decrease PTH secretion is now becoming clearer. It therefore suggests that in the situation of a decreased Cao2+ there is a relaxed CaR and unrestrained secretion of PTH. Is the PT cell designed to synthesize and secrete PTH and only stops doing so when constrained by the CaR? We still await the results of further experiments to fine-wire the PT and it is comforting to know that the HEKCaR cells so faithfully reproduce the physiology of the PT. The use of these cells to study the effect of the CaR on PTH mRNA stability () and components of the cell cycle machinery will also be of great interest (). In the meantime these two studies provide food for thought.
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