Etelcalcetide decreases the PTH-calcium setpoint without changing maximum and minimum PTH secretion in mice with primary hyperparathyroidism.

INTRODUCTION: Etelcalcetide binds to the extracellular domain of the calcium-sensing receptor (CaSR), while cinacalcet binds to the 7-transmembrane domain of the CaSR; however, it is unknown, whether etelcalcetide has similar effects to cinacalcet on parathyroid hormone (PTH) secretion. MATERIALS AND METHODS: The PTH-calcium setpoint and maximum and minimum PTH secretion were determined using an 'in vivo setpoint analyses.' The PTH-calcium setpoint was obtained in a mouse model of primary hyperparathyroidism (PC) and wild-type (WT) mice, with PC mice divided into two groups. The setpoint was obtained after 7 days of etelcalcetide (3.0 mg/kg BW/day) or vehicle administration via anosmotic pump. After 7 days of crossover administration, the setpoint was obtained again. Parathyroid glands were obtained after crossover administration, and CaSR expression was analyzed by immunohistochemistry. RESULTS: Etelcalcetide administration significantly decreased the setpoint from 9.03 ± 0.56 mg/dL to 6.80 ± 0.28 mg/dL, which was restored to 8.81 ± 0.38 mg/dL after vehicle administration. In the second group of mice, vehicle administration did not alter the setpoint (8.84 ± 0.69 mg/dL to 8.98 ± 0.63 mg/dL), but subsequent etelcalcetide administration significantly decreased it to 7.10 ± 0.72 mg/dL. There was no significant change in maximum and minimum PTH secretion. Expression levels of parathyroid CaSR were lower in PC mice than in WT mice; however, no significant differences were observed between the two mouse groups. CONCLUSION: Etelcalcetide decreased the PTH-calcium setpoint without changing maximum and minimum PTH secretion in PC mice, suggesting that like cinacalcet, etelcalcetide has calcimimetic potency.

Effect of etelcalcetide on parathyroid hormone secretion by primary hyperparathyroidism patient-derived primary parathyroid cells.

INTRODUCTION: Etelcalcetide (Parsabiv®, AMG 416/ONO-5163) is a novel allosteric modulator for the calcium-sensing receptor approved for hemodialysis patients with secondary hyperparathyroidism of uremia. Etelcalcetide reduced parathyroid hormone levels in hemodialysis patients with secondary hyperparathyroidism of uremia in clinical studies. However, its direct effect on parathyroid hormone secretion in human parathyroid cells remains unknown. This study aimed to determine if etelcalcetide suppresses parathyroid hormone secretion by human parathyroid cells in vitro. MATERIALS AND METHODS: We prepared primary cell cultures from human parathyroid tissue and determined calcium-sensing receptor expression levels by immunohistochemistry. Pathyroid tumors were removed from fourteen patients with primary hyperparathyrodism. Parathyroid tissue was dispersed with collagenase, resuspended in culture medium, incubated for 2 h with etelcalcetide and Ca2+, and the medium was then collected. Final etelcalcetide concentrations in the medium were 0.005-50 µmol/L. Levels of human parathyroid hormone in the medium were determined by enzyme-linked immunosorbent assay. RESULTS: In eight of the fourteen parathyroid cell cultures, extracellular Ca2+ reduced parathyroid hormone levels. In four of the eight parathyroid cell cultures which responded extracellular Ca2+, etelcalcetide reduced hormone secretion with the 50% effective concentrations of 0.57, 20.8, 0.42, and 0.57 µmol/L. Expression levels of the calcium-sensing receptor were significantly lower in primary hyperparathyroidism patient-derived parathyroid tissues compared with controls. CONCLUSION: This is the first report that etelcalcetide directly reduced parathyroid hormone secretion from the primary cultured human parathyroid cells from patients with primary hyperparathyroidism. To verify this conclusion, further studies are needed using secondary hyperparathyroidism patient-derived parathyroid cells.

Pharmacology of Parsabiv® (etelcalcetide, ONO-5163/AMG 416), a novel allosteric modulator of the calcium-sensing receptor, for secondary hyperparathyroidism in hemodialysis patients.

Etelcalcetide hydrochloride (Parsabiv®, ONO-5163/AMG 416) is an allosteric modulator of the calcium (Ca)-sensing receptor that was originally produced by KAI Pharmaceuticals Inc. (now Amgen Inc.). It has recently been approved as the first intravenous calcimimetic agent for secondary hyperparathyroidism (SHPT) in many countries. Etelcalcetide is an intravenous injectable drug that can be administered and eliminated through the dialysis circuit in chronic kidney disease patients. In the present study, we evaluated the in vitro pharmacological profile and in vivo parathyroid hormone (PTH)- and Ca-lowering activities of etelcalcetide in a rat 5/6 nephrectomy model of chronic renal insufficiency with SHPT. Etelcalcetide increased the intracellular Ca concentration in HEK-293T cells expressing human Ca-sensing receptor with an EC50 value (95% confidence interval) of 0.53 µM (0.28-1.0 µM) and suppressed PTH secretion from rat parathyroid gland cells with 0.36 µM (0.24-0.54 µM) by activating Ca-sensing receptor. The specificity of etelcalcetide was evaluated by examining its ability to stimulate or inhibit radioligand binding to a panel of 34 off-target proteins. There were no significant changes in the presence of 10 µM etelcalcetide. Furthermore, in a rat 5/6 nephrectomy model of chronic renal insufficiency with SHPT, single intravenous administration of etelcalcetide at 0.3, 1.0, and 3.0 mg/kg decreased plasma PTH and serum Ca levels. Taken together, the present findings identify etelcalcetide as a calcimimetic with potent PTH- and Ca-lowering effects via Ca-sensing receptor agonist activity.

[The Discovery, Research and Development of Etelcalcetide Hydrochloride, the World 1st Intravenous Calcimimetics.]

Etelcalcetide hydrochloride is the first intravenous calcimimetics agent for secondary hyperparathyroidism (SHPT). Etelcalcetide hydrochloride is to be administered through dialysis circuit by physician or medical staff upon completion of dialysis and such administration is expected to reduce the burden of medication in patients. From the nonclinical study results, etelcalcetide functions as an allosteric activator of calcium-sensing receptor(CaSR). Etelcalcetide suppressed PTH secretion both in vitro and in vivo. In a rat model of chronic renal insufficiency, etelcalcetide suppressed SHPT disorders, such as parathyroid gland hypertrophy, bone disorder, and ectopic calcification. In conclusion, etelcalcetide hydrochloride is expected to exhibit therapeutic effect against each SHPT condition by decreasing blood PTH concentrations via CaSR-agonist activity in the clinical situation.

Role of calnexin in the ER quality control and productive folding of CFTR; differential effect of calnexin knockout on wild-type and DeltaF508 CFTR.

Cystic fibrosis (CF) is caused by the mutation in CF transmembrane conductance regulator (CFTR), a cAMP-dependent Cl(-) channel at the plasma membrane of epithelium. The most common mutant, DeltaF508 CFTR, has competent Cl(-) channel function, but fails to express at the plasma membrane since it is retained in the endoplasmic reticulum (ER) by the ER quality control system. Here, we show that calnexin (CNX) is not necessary for the ER retention of DeltaF508 CFTR. Our data show that CNX knockout (KO) does not affect the biosynthetic processing, cellular localization or the Cl(-) channel function of DeltaF508 CFTR. Importantly, cAMP-induced Cl(-) current in colonic epithelium from CNX KO/DeltaF508 CFTR mice was comparable with that of DeltaF508 CFTR mice, indicating that CNX KO failed to rescue the ER retention of DeltaF508 CFTR in vivo. Moreover, we show that CNX assures the efficient expression of WT CFTR, but not DeltaF508 CFTR, by inhibiting the proteasomal degradation, indicating that CNX might stimulate the productive folding of WT CFTR, but not DeltaF508 CFTR, which has folding defects.

Phosphatidic acid metabolism regulates the intracellular trafficking and retrotranslocation of CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR) is transported to the plasma membrane from endoplasmic reticulum (ER) through the Golgi. Crucial to these trafficking events is the role of not only the proteinous factors but also the membrane lipids. However, the involvement of lipids, such as phospholipids, on the regulation of CFTR trafficking has been largely unexplored. Here, we show that the inhibition of phospholipase D (PLD)-mediated phosphatidic acid (PA) formation by 1-butanol inhibited the maturation and export of CFTR from the ER. Exogenously added PA reversed these effects. Moreover, knock down of PLD1 by small interfering RNA decreased the expression of mature CFTR. Interestingly, sustaining the level of PA, by the addition of excess PA in the presence of PA phosphatase inhibitor, attenuated the transport of CFTR from the Golgi to plasma membrane and the retrograde transport of DeltaF508 CFTR to the cytoplasm, a necessary step for the ER-associated degradation of DeltaF508 CFTR. These results indicated that the metabolism of PA modulated the intracellular dynamics and trafficking of CFTR.

The bacterium, nontypeable Haemophilus influenzae, enhances host antiviral response by inducing Toll-like receptor 7 expression: evidence for negative regulation of host anti-viral response by CYLD.

The incidence of mixed viral/bacterial infections has increased recently because of the dramatic increase in antibiotic-resistant strains, the emergence of new pathogens, and the resurgence of old ones. Despite the relatively well-known role of viruses in enhancing bacterial infections, the impact of bacterial infections on viral infections remains unknown. In this study, we provide direct evidence that nontypeable Haemophilus influenzae (NTHi), a major respiratory bacterial pathogen, augments the host antiviral response by up-regulating epithelial Toll-like receptor 7 (TLR7) expression in vitro and in vivo. Moreover, NTHi induces TLR7 expression via a TLR2-MyD88-IRAK-TRAF6-IKK-NF-kappaB-dependent signaling pathway. Interestingly, CYLD, a novel deubiquitinase, acts as a negative regulator of TLR7 induction by NTHi. Our study thus provides new insights into a novel role for bacterial infection in enhancing host antiviral response and further identifies CYLD for the first time as a critical negative regulator of host antiviral response.

Curcumin enhances cystic fibrosis transmembrane regulator expression by down-regulating calreticulin.

Curcumin has been reported to correct cystic fibrosis caused by the DeltaF508 mutation of the cystic fibrosis transmembrane regulator (CFTR) but its mechanistic action remains unclear. We have recently demonstrated that the ER chaperone calreticulin (CRT) negatively regulates the CFTR cell surface expression and activity. Thus, we aimed at determining whether CRT mediates the effect of curcumin on CFTR. We show here that the treatment with curcumin of Chinese hamster ovary cells suppressed CRT expression and increased wild-type CFTR but did not affect DeltaF508 CFTR expression. However, we determined that although curcumin did not augment DeltaF508 CFTR expression, it enhanced the functional competence of DeltaF508 CFTR induced by 26 degrees C incubation. Knock down of CRT by siRNA at low-temperature had a similar effect. Our findings suggest that the positive effect of curcumin on CFTR expression is mediated through the down-regulation of CRT, a negative regulator of CFTR.

Bafilomycin A1-sensitive pathway is required for the maturation of cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis (CF) is the most common lethal genetic disease in Caucasians caused by the trafficking defects of CF transmembrane conductance regulator (CFTR), which is a cAMP-dependent Cl- channel at the plasma membrane. The trafficking pathway of CFTR is thought to be non-conventional because CFTR maturation is inhibited by the dysfunction of syntaxin 13, which is involved in protein recycling via endosomal pathway. In this study, to clarify whether the endosomal trafficking is required for CFTR maturation, we utilized a specific vacuolar H+-ATPase inhibitor, bafilomycin A1 (BafA1), which inhibits the protein trafficking from early endosome. Our data showed that low concentration of BafA1 (50 nM) decreased the expression of mature CFTR but induced the accumulation of immature CFTR in the juxta-nuclear region containing an early endosome marker. Pulse-chase analysis showed that BafA1 inhibited the maturation of CFTR, but it slightly stabilized immature CFTR. These results indicate that BafA1-sensitive pathway is required for CFTR maturation and emphasize that endosomal trafficking pathway might be involved in the maturation of CFTR.

Calreticulin facilitates the cell surface expression of ABCG5/G8.

ATP-binding cassette (ABC) G5 (G5) and ABCG8 (G8) heterodimerize and function as sterol transporter that promote biliary excretion of neutral sterols. Both G5 and G8 interact with a lectin-like chaperone, calnexin (CNX), in the endoplasmic reticulum (ER) but the significance of this interaction remains unclear. Here, we show that not only CNX, but also its homologue calreticulin (CRT), is involved in the biosynthesis of G5/G8 sterol transporter. Both CNX and CRT interacted with immature forms of G5 and G8, and stimulated their productive folding by inhibiting their degradation. Interestingly, CRT predominantly enhanced the cell surface expression of mature G5/G8 whereas CNX did not have a similar effect. Inhibitors of N-glycan processing indicated that quality control of G5 and G8 might be differentially regulated in the ER. These findings clarify the role of CNX and CRT in the biosynthesis and quality control of G5/G8 sterol transporter.

Calreticulin negatively regulates the cell surface expression of cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent Cl- channel at the plasma membrane, and its malfunction results in cystic fibrosis, the most common lethal genetic disease in Caucasians. Quality control of CFTR is strictly regulated by several molecular chaperones. Here we show that calreticulin (CRT), which is a lectin-like chaperone in the endoplasmic reticulum (ER), negatively regulates the cell surface CFTR. RNA interference-based CRT knockdown induced the increase of CFTR expression. Consistently, this effect was observed in vivo. CRT heterozygous (CRT+/-) mice had a higher endogenous expression of CFTR than the wild-type mice. Moreover, CRT overexpression induced cell surface expression of CRT, and it significantly decreased the cell surface expression and function of CFTR. CRT overexpression destabilized the cell surface CFTR by enhancing endocytosis, leading to proteasomal degradation. Deletion of the carboxyl domain of CRT, which results in its ER export, increased the negative effect and enhanced the interaction with CFTR. Thus, CRT in the post-ER compartments may act as a negative regulator of the cell surface CFTR.

Characterization of the trafficking pathway of cystic fibrosis transmembrane conductance regulator in baby hamster kidney cells.

To examine the unknown trafficking pathway of the cystic fibrosis transmembrane conductance regulator (CFTR) from the endoplasmic reticulum (ER), we utilized baby hamster kidney cells stably expressing CFTR fused with green fluorescent protein. CFTR trafficking from the ER was visualized and analyzed by immunocytochemical analyses. Here we show that CFTR was exported from the ER to the cis-Golgi and early endosome, suggesting that CFTR transport in the early secretory pathway may utilize a non-conventional pathway. This CFTR trafficking pathway may be a target for pharmacological modulation that selectively stimulates CFTR transport.

Delta F508 CFTR pool in the endoplasmic reticulum is increased by calnexin overexpression.

The most common cystic fibrosis transmembrane conductance regulator (CFTR) mutant in cystic fibrosis patients, Delta F508 CFTR, is retained in the endoplasmic reticulum (ER) and is consequently degraded by the ubiquitin-proteasome pathway known as ER-associated degradation (ERAD). Because the prolonged interaction of Delta F508 CFTR with calnexin, an ER chaperone, results in the ERAD of Delta F508 CFTR, calnexin seems to lead it to the ERAD pathway. However, the role of calnexin in the ERAD is controversial. In this study, we found that calnexin overexpression partially attenuated the ERAD of Delta F508 CFTR. We observed the formation of concentric membranous bodies in the ER upon calnexin overexpression and that the Delta F508 CFTR but not the wild-type CFTR was retained in the concentric membranous bodies. Furthermore, we observed that calnexin overexpression moderately inhibited the formation of aggresomes accumulating the ubiquitinated Delta F508 CFTR. These findings suggest that the overexpression of calnexin may be able to create a pool of Delta F508 CFTR in the ER.

Calnexin Delta 185-520 partially reverses the misprocessing of the Delta F508 cystic fibrosis transmembrane conductance regulator.

Abnormal retention of Delta F508 CFTR (cystic fibrosis transmembrane conductance regulator) in the endoplasmic reticulum is a major cause of cystic fibrosis (CF). We show that calnexin Delta 185-520 but not calnexin can partially reverse the mislocalization of Delta F508 CFTR. This 256-amino acid protein has neither the transmembrane domain nor the P domain of calnexin. Calnexin Delta 185-520 interacted with CFTR directly, and was secreted into the extracellular compartment over time. Forty-eight hours after transfection into CHO cells, calnexin Delta 185-520 increased the conversion of immature Delta F508 CFTR into mature Delta F508 CFTR. In immortalized human CF cell lines expressing Delta F508 CFTR, a halide efflux assay showed that calnexin Delta 185-520 partially restored CFTR function. These data indicate that calnexin Delta 185-520 may give a clue to develop the therapeutic way of cystic fibrosis with Delta F508 CFTR.