Calcium-sensing Receptor Expression Research Articles

Effects of evocalcet on parathyroid calcium-sensing receptor and vitamin D receptor expression in uremic rats.

Little is known about the effect of the recently developed calcimimetic evocalcet (Evo) on parathyroid calcium-sensing receptor (CaSR) and vitamin D receptor (VDR) expression. We examined the effects of Evo and cinacalcet (Cina) on CaSR and VDR expression in 5/6 nephrectomized Sprague-Dawley rats fed a high-phosphorus diet for 4 weeks to develop secondary hyperparathyroidism (SHPT). These uremic rats were divided into 4 groups-baseline control (Nx4W) and groups with additional treatment with either the Vehicle, Evo, or Cina for 2 weeks; normal rats were used as normal controls (NC). Blood parameters and parathyroid tissue were analyzed. CaSR and VDR expression levels were determined using immunohistochemistry. The degree of kidney injury and hyperphosphatemia was similar in the uremic groups (Nx4W, Vehicle, Cina, and Evo). Serum parathyroid hormone levels were significantly higher in the Nx4W and Vehicle groups than in the NC group. This increase was significantly suppressed in the Cina and Evo groups compared with that in the Vehicle group. Serum calcium levels were significantly and equally lower in the Cina and Evo groups relative to those in the Vehicle group. CaSR expression was significantly lower in the Nx4W and Vehicle groups than in the NC group. This downregulation was of an equally lesser magnitude in the Cina and Evo groups. A similar trend was observed for VDR expression. These results indicate that Evo and Cina treatment can increase parathyroid CaSR and VDR expression in uremic rats with SHPT, which could provide better control of mineral and bone disorder markers.

CaSR modulates proliferation of the superficial zone cells in temporomandibular joint cartilage via the PTHrP nuclear localization sequence.

The superficial zone cells in mandibular condylar cartilage are proliferative. The present purpose was to delineate the relation of calcium-sensing receptor (CaSR) and parathyroid hormone-related peptide nuclear localization sequence (PTHrP87-139 ), and their role in the proliferation behaviors of the superficial zone cells. A gain- and loss-of-function strategy were used in an in vitro fluid flow shear stress (FFSS) model and an in vivo bilateral elevation bite model which showed mandibular condylar cartilage thickening. CaSR and PTHrP87-139 were modulated through treating the isolated superficial zone cells with activator/SiRNA and via deleting CaSR or parathyroid hormone-related peptide (PTHrP) gene in mice with the promoter gene of proteoglycan 4 (Prg4-CreERT2 ) in the tamoxifen-inducible pattern with or without additional injection of Cinacalcet, the CaSR agonist, or PTHrP87-139 peptide. FFSS stimulated CaSR and PTHrP expression, and accelerated proliferation of the Prg4-expressing superficial zone cells, in which process CaSR acted as an up-streamer of PTHrP. Proteoglycan 4 specific knockout of CaSR or PTHrP reduced the cartilage thickness, suppressed the proliferation and early differentiation of the superficial zone cells, and inhibited cartilage thickening and matrix production promoted by bilateral elevation bite. Injections of CaSR agonist Cinacalcet could not improve the phenotype caused by PTHrP mutation. Injections of PTHrP87-139 peptide rescued the cartilage from knockout of CaSR gene. CaSR modulates proliferation of the superficial zone cells in mandibular condylar cartilage through activation of PTHrP nuclear localization sequence. Our data support the therapeutic target of CaSR in promoting PTHrP production in superficial zone cartilage.

Leucine and arginine enhance milk fat and milk protein synthesis via the CaSR/Gi/mTORC1 and CaSR/Gq/mTORC1 pathways.

Amino acids (AAs) not only constitute milk protein but also stimulate milk synthesis through the activation of mTORC1 signaling, but which amino acids that have the greatest impact on milk fat and protein synthesis is still very limited. In this study, we aimed to identify the most critical AAs involved in the regulation of milk synthesis and clarify how these AAs regulate milk synthesis through the G-protein-coupled receptors (GPCRs) signaling pathway. In this study, a mouse mammary epithelial cell line (HC11) and porcine mammary epithelial cells (PMECs) were selected as study subjects. After treatment with different AAs, the amount of milk protein and milk fat synthesis were detected. Activation of mTORC1 and GPCRs signaling induced by AAs was also investigated. In this study, we demonstrate that essential amino acids (EAAs) are crucial to promote lactation by increasing the expression of genes and proteins related to milk synthesis, such as ACACA, FABP4, DGAT1, SREBP1, α-casein, β-casein, and WAP in HC11 cells and PMECs. In addition to activating mTORC1, EAAs uniquely regulate the expression of calcium-sensing receptor (CaSR) among all amino-acid-responsive GPCRs, which indicates a potential link between CaSR and the mTORC1 pathway in mammary gland epithelial cells. Compared with other EAAs, leucine and arginine had the greatest capacity to trigger GPCRs (p-ERK) and mTORC1 (p-S6K1) signaling in HC11 cells. In addition, CaSR and its downstream G proteins Gi, Gq, and Gβγ are involved in the regulation of leucine- and arginine-induced milk synthesis and mTORC1 activation. Taken together, our data suggest that leucine and arginine can efficiently trigger milk synthesis through the CaSR/Gi/mTORC1 and CaSR/Gq/mTORC1 pathways. We found that the G-protein-coupled receptor CaSR is an important amino acid sensor in mammary epithelial cells. Leucine and arginine promote milk synthesis partially through the CaSR/Gi/mTORC1 and CaSR/Gq/mTORC1 signaling systems in mammary gland epithelial cells. Although this mechanism needs further verification, it is foreseeable that this mechanism may provide new insights into the regulation of milk synthesis.

1617-P: Small Intestinal Protein Sensing Mechanism in Feeding and Glucose Regulation

Protein activates small intestinal calcium sensing receptor (CaSR) and/or peptide transporter 1 (PepT1) to increase GLP-1, but the role in glucose and feeding regulation remains elusive. We performed intravenous glucose tolerance test and refeeding studies and infused 8% (w/v) casein hydrolysate into the upper small intestine (USI) or ileum of male rats. We found that USI casein vs. saline infusion increased glucose tolerance in chow (p<0.01) and HF-fed rats (p<0.05) and suppressed feeding in chow (p<0.01) but not HF rats. We inhibited CaSR via injecting lentivirus expressing shRNA of CaSR into the USI (reduced expression by ~40%) and CaSR knockdown negated casein to increase glucose tolerance but not to lower feeding (p<0.01). Instead, viral-mediated PepT1 knockdown by ~50% negated casein to lower feeding as similar to previously reported for glucose control. Thus, CaSR and PepT1 are required for casein in the USI to increase glucose tolerance, while PepT1 but not CaSR mediates casein to lower feeding, in chow rats. The differential role of PepT1 vs CaSR may explain why HF disrupts casein to regulate feeding but not glucose tolerance as HF inhibited PepT1 expression (p<0.05) but increased CaSR expression (p<0.05) in the USI. Ileal casein vs saline infusion increased glucose tolerance (p<0.01) and lowered feeding (p<0.01). Ileal CaSR inhibition negated casein vs saline to increase glucose tolerance and lower feeding, whereas ileal PepT1 inhibition failed to do so, indicating that ileal casein regulates glucose tolerance and food intake via PepT1-independent but CaSR-dependent pathways in chow rats. HF feeding inhibited ileal CaSR expression by ~60% (p<0.05) in association with a disruption in ileal casein action on feeding but surprisingly not on glucose control (p<0.01), suggesting that ileal casein could even regulate glucose tolerance independent of CaSR. In sum, we discover differential role of CaSR vs. PepT1 in the USI and ileum that alter feeding and glucose regulatory abilities of protein sensing. Disclosure D.Barros: None. R.J.W.Li: None. Y.Lim: None. S.Zhang: None. R.Kuah: None. A.Gao: None. T.K.Lam: None. Funding Canadian Institutes of Health Research (PJT183901)

Effects of calcium-free ageing on ethanol-induced activation and developmental potential of mouse oocytes.

Although ethanol treatment is widely used to activate oocytes, the underlying mechanisms are largely unclear. Roles of intracellular calcium stores and extracellular calcium in ethanol-induced activation (EIA) of oocytes remain to be verified, and whether calcium-sensing receptor (CaSR) is involved in EIA is unknown. This study showed that calcium-free ageing (CFA) in vitro significantly decreased intracellular stored calcium (sCa) and CaSR expression, and impaired EIA, spindle/chromosome morphology and developmental potential of mouse oocytes. Although EIA in oocytes with full sCa after ageing with calcium does not require calcium influx, calcium influx is essential for EIA of oocytes with reduced sCa after CFA. Furthermore, the extremely low EIA rate in oocytes with CFA-downregulated CaSR expression and the fact that inhibiting CaSR significantly decreased the EIA of oocytes with a full complement of CaSR suggest that CaSR played a significant role in the EIA of ageing oocytes. In conclusion, CFA impaired EIA and the developmental potential of mouse oocytes by decreasing sCa and downregulating CaSR expression. Because mouse oocytes routinely treated for activation (18 h post hCG) are equipped with a full sCa complement and CaSR, the present results suggest that, while calcium influx is not essential, CaSR is required for the EIA of oocytes.

The role of calcium-sensing receptor in ginsenoside Rg1 promoting reendothelialization to inhibit intimal hyperplasia after balloon injury

Calcium-sensing receptor (CaSR) is a G protein-coupled receptor, widely distributed in various tissues, including vascular endothelial cells and smooth muscle cells, which plays an important role in the migration and homing of stem/progenitor cells and the proliferation of tissue cells. Restenosis after Percutaneous coronary intervention (PCI) seriously affects its prognosis and application. Our previous research has found that ginsenoside Rg1 (GS-Rg1) can inhibit the occurrence of restenosis after balloon injury of the common carotid artery in rats, but the mechanism is still unclear. In this study, it was found that GS-Rg1 (4, 8, 16 mg/kg) inhibited vascular restenosis caused by balloon injury, and mobilize endothelial progenitor cells (EPCs) to promote reendothelialization and inhibit intimal hyperplasia, which significantly reduced after administration of CaSR antagonist NPS 2143. Interestingly, CaSR and its downstream JNK, P38 were highly expressed in the proliferative intima and participated in the abnormal proliferation of vascular smooth muscle cells mediated by smooth muscle progenitor cells (SMPCs). GS-Rg1 inhibited intimal hyperplasia, while it decreased the expression of CaSR, JNK, and P38. This might relate to the distribution of CaSR and the facilitation of GS-Rg1 on the vascular endothelial repair. It is concluded that CaSR plays a key role in GS-Rg1 promoting reendothelialization to inhibit intimal hyperplasia after balloon Injury.

Establishment and characterization of long-term human primary parathyroid tumor subclones derived from Indian PHPT.

The continuous cell line of epithelial human parathyroid cells has been proven difficult. Previously, PTH-C1 cell line was only established rat parathyroid tissue cell line known to express the parathyroid hormone-related peptide (Pthrp) gene. The paucity of continuous cell line of human parathyroid cells secreting parathyroid hormone (PTH) has imposed hurdle in in vitro assessment of the mechanisms involved in the control of parathyroid cell function and proliferation. The primary cell cultures of human parathyroid cells were derived from parathyroid adenoma tissue biopsy (n = 5). The cells were subsequently subcultured to maintained primary subclones. Karyotyping analysis was performed to analyze the genotypic identity of derived subclones. The expression of calcium-sensing receptor (CaSR) and intact parathyroid hormone (iPTH) were analyzed using immunocytochemistry and immunofluorescence. In the present study, we have used a defined condition medium to generate the continuous culture of human parathyroid cells derived from patients with parathyroid adenoma due to primary hyperparathyroidism. The subcultured primary subclones were maintained epithelial and polygonal morphology, doubling time of approximately 25h, displaying a diploid chromosome number, and secretion of PTH. This cell line produces PTH and expresses the calcium-sensing receptor (CaSR) known to be involved in parathyroid function. Altogether these findings indicate the uniqueness of the human parathyroid cell line as an in vitro model for cellular and molecular studies on parathyroid physiopathology.

Osteocalcin modulates parathyroid cell function in human parathyroid tumors.

The bone matrix protein osteocalcin (OC), secreted by osteoblasts, displays endocrine effects. We tested the hypothesis that OC modulates parathyroid tumor cell function. Primary cell cultures derived from parathyroid adenomas (PAds) and HEK293 cells transiently transfected with the putative OC receptor GPRC6A or the calcium sensing receptor (CASR) were used as experimental models to investigate γ-carboxylated OC (GlaOC) or uncarboxylated OC (GluOC) modulation of intracellular signaling. In primary cell cultures derived from PAds, incubation with GlaOC or GluOC modulated intracellular signaling, inhibiting pERK/ERK and increasing active β-catenin levels. GlaOC increased the expression of PTH, CCND1 and CASR, and reduced CDKN1B/p27 and TP73. GluOC stimulated transcription of PTH, and inhibited MEN1 expression. Moreover, GlaOC and GluOC reduced staurosporin-induced caspase 3/7 activity. The putative OC receptor GPRC6A was detected in normal and tumor parathyroids at membrane or cytoplasmic level in cells scattered throughout the parenchyma. In PAds, the membrane expression levels of GPRC6A and its closest homolog CASR positively correlated; GPRC6A protein levels positively correlated with circulating ionized and total calcium, and PTH levels of the patients harboring the analyzed PAds. Using HEK293A transiently transfected with either GPRC6A or CASR, and PAds-derived cells silenced for CASR, we showed that GlaOC and GluOC modulated pERK/ERK and active β-catenin mainly through CASR activation. Parathyroid gland emerges as a novel target of the bone secreted hormone osteocalcin, which may modulate tumor parathyroid CASR sensitivity and parathyroid cell apoptosis.

Primary and secondary hyperparathyroidism present different expressions of calcium-sensing receptor

BackgroundDecreased calcium-sensing receptor (CaSR) has been observed in hyperparathyroidism (HPT) without a known mechanism. The purpose of this study was to evaluate the expression of CaSR in primary (PHPT) and secondary (SHPT) subtypes.MethodsImmunohistochemical (IHC) staining and quantitative real-time PCR (qRT-PCR) assay were used to measure the differences in expression of CaSR protein and gene in PHPT and SHPT human samples, compared to matched controls.ResultsCaSR protein was differentially downregulated in SHPT and PHPT compared to normal parathyroid tissues (2.42 ± 0.5 vs. 3.2 ± 0.62, P < 0.05; 1.8 ± 0.83 vs. 3.2 ± 0.62, P < 0.05, respectively). Furthermore, SHPT tissues exhibited significantly higher levels of CaSR mRNA (0.29 ± 0.23 vs. 0.01 ± 0.12, P < 0.05) and protein (2.42 ± 0.5 vs. 1.8 ± 0.83, P < 0.05) than those in PHPT tissue samples.ConclusionDepressed CaSR expression was a critical pathological hallmark of HPT. We found a differential decline of CaSR, in terms of both mRNA and protein levels, in PHPT and SHPT human samples. We think that CaSR dysregulation occurred at the very beginning of disease onset in PHPT, while a similar pathological scenario appeared at the later stage of SHPT. Future studies should be directed to dissect the mechanistic involvement of CaSR in PHPT and SHPT in order to bring treatment precisions in HPT management.

Characterizing the Role of Calcium Sensing Receptor in the Progression of Obesity-Mediated Aggressive Prostate Cancer Phenotype

Obesity increases the risk of advanced prostate cancer (PCa). The calcium sensing receptor (CaSR) has been shown to be responsive to obesity-mediated cytokines and is upregulated in metastatic PCa. This study used a novel in vitro approach, involving the exposure of PCa cells to sera, from obese or normal weight males, and to CaSR inhibitor NPS-2143. Cell viability was determined using MTT assay. MMP-9 activity and invasion were assessed using zymography and invasion chambers, respectively. Microscopy was used to visualize EMT proteins. qRT-PCR and immunoblot analysis were used to quantify changes in genes and proteins important for tumorigenesis. Exposure to obese sera increased the proliferation, and the invasive capacity of PCa cells and de-localized epithelial-mesenchymal transition markers, which were attenuated with CaSR inhibition. Exposure to obese sera upregulated mRNA expression of PTHrP and protein expression of COX-2, IL-6, and CaSR. Inhibition of CaSR downregulated the mRNA expression of PTHrP and RANK, and protein expression of pERK and TNF-α. Obesity was shown to increase invasion and upregulate the expression of genes and proteins involved in PCa tumorigenesis. CaSR inhibition downregulated the expression of several of these factors. Thus, CaSR is a potentially important protein to target in obesity-mediated PCa progression.

The CaSR/TRPV4 coupling mediates pro-inflammatory macrophage function.

Although calcium-sensing receptor (CaSR) and transient receptor potential vanilloid 4 (TRPV4) channels are functionally expressed on macrophages, it is unclear if they work coordinately to mediate macrophage function. The present study investigates whether CaSR couples to TRPV4 channels and mediates macrophage polarization via Ca2+ signaling. The role of CaSR/TRPV4/Ca2+ signaling was assessed in lipopolysaccharide (LPS)-treated peritoneal macrophages (PMs) from wild-type (WT) and TRPV4 knockout (TRPV4 KO) mice. The expression and function of CaSR and TRPV4 in PMs were analyzed by immunofluorescence and digital Ca2+ imaging. The correlation factors of M1 polarization, CCR7, IL-1β, and TNFα were detected using q-PCR, western blot, and ELISA. We found that PMs expressed CaSR and TRPV4, and CaSR activation-induced marked Ca2+ signaling predominately through extracellular Ca2+ entry, which was inhibited by selective pharmacological blockers of CaSR and TRPV4 channels. The CaSR activation-induced Ca2+ signaling was significantly attenuated in PMs from TRPV4 KO mice compared to those from WT mice. Moreover, the CaSR activation-induced Ca2+ entry via TRPV4 channels was inhibited by blocking phospholipases A2 (PLA2)/cytochromeP450 (CYP450) and phospholipase C (PLC)/Protein kinase C (PKC) pathways. Finally, CaSR activation promoted the expression and release of M1-associated cytokines IL-1β and TNFɑ, which were attenuated in PMs from TRPV4 KO mice. We reveal a novel coupling of the CaSR and TRPV4 channels via PLA2/CYP450 and PLC/PKC pathways, promoting a Ca2+ -dependent M1 macrophage polarization. Modulation of this coupling and downstream pathways may become a potential strategy for the prevention/treatment of immune-related disease.

Amlodipine downregulates gene expression that involved in the signaling pathways of coagulation process in COVID-19 patients: An observational clinical study.

The SARS-CoV-2 virus has the property of activating the coagulation process, which is responsible for producing thrombotic events which is considered as one of the most serious COVID-19 complications. Hypertension is a hazard factor for COVID-19 complications, and people who are treated with calcium entry blockers may halt the occurrence of thrombotic events. to evaluate the effect of amlodipine on some genes involved in the activation of the coagulation procedure in COVID-19 patients with hypertensive. observational, cross-sectional study. This study was carried out in the Department of Pharmacy at Al-Kut University College in Wasit, Iraq, in conjunction with Al Zahraa Hospital from June 2021 to March 2022. A total of 45 COVID-19 patients participated in this study who were grouped into as follows: Group I (n = 23) who had no previous history of hypertension and Group II (n = 22) who had previous hypertension and were treated with amlodipine. Expression of the calcium-sensing receptor (CaSR), coagulation factor V (F5), and methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1 Like (MTHFD1L) genes was determined. P values were calculated by Chi-square test for categorized facts and the Mann-Whitney test for incessant data. P ≤ 0.05 was considered statistically significant. Group II patients had significantly lower levels of CaSR, F5, and MTHFD1L gene expression compared with the corresponding levels in Group I patients. The expression level of MTHFD1L was elevated significantly in patients who had currently high blood pressure compared with normotensive patients in both the groups. Amlodipine is preferred in hypertensive patients who have COVID-19 because it attenuates the levels of gene expression that have an impact on the coagulation process.

Role of calcium-sensing receptor in regulating activation susceptibility of postovulatory aging mouse oocytes.

The mechanisms underlying postovulatory oocyte aging (POA) remain largely unknown. The expression of the calcium-sensing receptor (CaSR) in mouse oocytes and its role in POA need to be explored. Our objective was to observe CaSR expression and its role in the susceptibility to activating stimuli (STAS) in POA mouse oocytes. The results showed that, although none of the newly ovulated oocytes were activated, 40% and 94% of the oocytes recovered 19 and 25 h after human chorionic gonadotropin (hCG) injection were activated, respectively, after ethanol treatment. The level of the CaSR functional dimer protein in oocytes increased significantly from 13 to 25 h post hCG. Thus, the CaSR functional dimer level was positively correlated with the STAS of POA oocytes. Aging in vitro with a CaSR antagonist suppressed the elevation of STAS, and cytoplasmic calcium in oocytes recovered 19 h post hCG, whereas aging with a CaSR agonist increased STAS, and cytoplasmic calcium of oocytes recovered 13 h post hCG. Furthermore, the CaSR was more important than the Na-Ca2+ exchanger in regulating oocyte STAS, and T- and L-type calcium channels were inactive in aging oocytes. We conclude that the CaSR is involved in regulating STAS in POA mouse oocytes, and that it is more important than the other calcium channels tested in this connection.

High-dose remifentanil exacerbates myocardial ischemia-reperfusion injury through activation of calcium-sensing receptor-mediated pyroptosis.

Background: The aim of this study was to investigate whether calcium-sensing receptor (CaSR) was involved in HRF-mediated exacerbation of MI/R injury through NLRP3 inflammasome activation and pyroptosis. Methods: In vivo, a rat MI/R model was established by ligating the left coronary artery, and short-term HRF exposure was induced during reoxygenation. Then, TUNEL, H&E, Masson staining, immunohistochemical (IHC) and serum levels of lactate dehydrogenase (LDH) and creatine kinase isoenzyme (CK), as well as the expression levels of CaSR and pyroptosis-related proteins in heart tissues, were measured. H9c2 cells were cultured to create a hypoxia/reoxygenation (H/R) model and exposed to different concentrations of RF. After pretreatment with the CaSR activator gadolinium chloride (GdCl3) and inhibitor NPS2143 in the H/R model and treatment with HRF, we compared cellular viability, TUNEL, cytosolic [Ca2+]i, the levels of LDH and CK, pyroptosis-related proteins and CaSR in H9c2 cells. We further researched the mechanisms of CaSR-mediated pyroptosis in the H/R+HRF model by CaSR-shRNA, Ac-YVAD-CMK, MCC950 and NAC. Results: We found that HRF significantly increased CaSR expression, rate of cell death, levels of CK and LDH, and exacerbated pyroptosis in MI/R model. In vitro, HRF increased CaSR expression, decreased viability, enhanced cytosolic [Ca2+]i and exacerbated pyroptosis in H/R cells. Pretreated with GdCl3 worsen these changes, and NPS2143, MCC950, Ac-YVAD-CMK, NAC and sh-CaSR can reversed these effects. Conclusion: Exposure to HRF for a short time exacerbates MI/R-induced injury by targeting CaSR to increase cytosolic [Ca2+]i and ROS levels, which mediate the NLRP3 inflammasome and pyroptosis.

A rescue diet raises the plasma calcium concentration and ameliorates rheumatoid arthritis in mice: Role of CaSR-mediated inhibition of osteoclastogenesis.

Calcium modulates bone cell recruitment, differentiation, and function by binding to the calcium-sensing receptor (CaSR). However, the function of CaSR induced by high extracellular calcium (Ca2+ e ) in the regulation of osteoclast formation in rheumatoid arthritis (RA) remains unknown. Here, we used TNFα-transgenic (TNFTG ) RA mice and their wildtype (WT) littermates fed a normal or a rescue diet (high calcium, high phosphorus, and high lactose diet, termed rescue diet) to compare their joint bone phenotypes. In comparison to TNFTG mice fed the normal diet, articular bone volume and cartilage area are increased, whereas inflamed area, eroded surface, TRAP+ surface, and osteoclast-related genes expression are decreased in TNFTG mice fed the rescue diet. Besides, TNFTG mice fed the rescue diet were found to exhibit more CaSR+ area and less NFATc1+ /TRAP+ area. Furthermore, at normal Ca2+ e concentrations, osteoclast precursors (OCPs) from TNFTG mice formed more osteoclasts than OCPs from WT mice, but the number of osteoclasts gradually decreased when the Ca2+ e concentration increased. Meanwhile, the expression of CaSR increased responding to a high level of Ca2+ e , whereas the expression of NF-κB/NFATc1 signaling molecules decreased. At last, the knockdown of CaSR blocked the inhibition of osteoclast differentiation attributed to high Ca2+ e . Taken together, our findings indicate that high Ca2+ e inhibits osteoclast differentiation in RA mice partially through the CaSR/NF-κB/NFATc1 pathway.

The calcium-sensing receptor (CaSR) regulates zebrafish sensorimotor decision making via a genetically defined cluster of hindbrain neurons.

Decision making is a fundamental nervous system function that ranges widely in complexity and speed of execution. We previously established larval zebrafish as a model for sensorimotor decision making and identified the G-protein-coupled calcium-sensing receptor (CaSR) to be critical for this process. Here, we report that CaSR functions in neurons to dynamically regulate the bias between two behavioral outcomes: escapes and reorientations. By employing a computational guided transgenic strategy, we identify a genetically defined neuronal cluster in the hindbrain as a key candidate site for CaSR function. Finally, we demonstrate that transgenic CaSR expression targeting this cluster consisting of a few hundred neurons shifts behavioral bias in wild-type animals and restores decision making deficits in CaSR mutants. Combined, our data provide a rare example of a G-protein-coupled receptor that biases vertebrate sensorimotor decision making via a defined neuronal cluster.

The calcium-sensing receptor in inflammation: Recent updates.

The Calcium-Sensing Receptor (CaSR) is a member of the class C of G-proteins coupled receptors (GPCRs), it plays a pivotal role in calcium homeostasis by directly controlling calcium excretion in the kidneys and indirectly by regulating parathyroid hormone (PTH) release from the parathyroid glands. The CaSR is found to be ubiquitously expressed in the body, playing a plethora of additional functions spanning from fluid secretion, insulin release, neuronal development, vessel tone to cell proliferation and apoptosis, to name but a few. The present review aims to elucidate and clarify the emerging regulatory effects that the CaSR plays in inflammation in several tissues, where it mostly promotes pro-inflammatory responses, with the exception of the large intestine, where contradictory roles have been recently reported. The CaSR has been found to be expressed even in immune cells, where it stimulates immune response and chemokinesis. On the other hand, CaSR expression seems to be boosted under inflammatory stimulus, in particular, by pro-inflammatory cytokines. Because of this, the CaSR has been addressed as a key factor responsible for hypocalcemia and low levels of PTH that are commonly found in critically ill patients under sepsis or after burn injury. Moreover, the CaSR has been found to be implicated in autoimmune-hypoparathyroidism, recently found also in patients treated with immune-checkpoint inhibitors. Given the tight bound between the CaSR, calcium and vitamin D metabolism, we also speculate about their roles in the pathogenesis of severe acute respiratory syndrome coronavirus-19 (SARS-COVID-19) infection and their impact on patients' prognosis. We will further explore the therapeutic potential of pharmacological targeting of the CaSR for the treatment and management of aberrant inflammatory responses.

Elevation of spermine remodels immunosuppressive microenvironment through driving the modification of PD-L1 in hepatocellular carcinoma

BackgroundSpermine is frequently elevated in tumor tissues and body fluids of cancer patients and is critical for cancer cell proliferation, migration and invasion. However, the immune functions of spermine in hepatocellular carcinoma progression remains unknown. In the present study, we aimed to elucidate immunosuppressive role of spermine in hepatocellular carcinoma and to explore the underlying mechanism.MethodsWhole-blood spermine concentration was measured using HPLC. Human primary HCC tissues were collected to examine the expression of CaSR, p-Akt, β-catenin, STT3A, PD-L1, and CD8. Mouse model of tumorigenesis and lung metastasis were established to evaluate the effects of spermine on hepatocellular carcinoma. Western blotting, immunofluorescence, real time PCR, digital Ca2+ imaging, and chromatin immunoprecipitation assay were used to investigate the underlying mechanisms by which spermine regulates PD-L1 expression and glycosylation in hepatocellular carcinoma cells.ResultsBlood spermine concentration in the HCC patient group was significantly higher than that in the normal population group. Spermine could facilitate tumor progression through inducing PD-L1 expression and decreasing the CD8+ T cell infiltration in HCC. Mechanistically, spermine activates calcium-sensing receptor (CaSR) to trigger Ca2+ entry and thereby promote Akt-dependent β-catenin stabilization and nuclear translocation. Nuclear β-catenin induced by spermine then activates transcriptional expression of PD-L1 and N-glycosyltransferase STT3A, while STT3A in turn increases the stability of PD-L1 through inducing PD-L1 protein N-glycosylation in HCC cells.ConclusionsThis study reveals the crucial function of spermine in establishing immune privilege by increasing the expression and N-glycosylation of PD-L1, providing a potential strategy for the treatment of hepatocellular carcinoma.7ng36xqHia8rCQn62itWxRVideo

MiR-1249 on Endothelial Extracellular Vesicles Mediates Cigarette Smoke-Induced Pulmonary Hypertension by Inhibiting HDAC10 (Histone Deacetylase 10)-NFκB (Nuclear Factor κB)-CaSR (Calcium-Sensing Receptor) Cascade.

Overproduction of endothelial extracellular vesicles (eEVs) is correlated with pulmonary hypertension progression, but the precise mechanism remains largely unclear. MicroRNA-chip and real-time polymerase chain reaction were conducted to screen and validate microRNA profiles in blood plasma eEVs of rats and human with or without cigarette smoking. Pulmonary artery smooth muscle cells were cultured to study signaling pathways. Pulmonary hypertension phenotypes were evaluated in wild-type and calcium-sensing receptor knockout rats to identify the pathophysiological significance of the microRNA pathway. MicroR-1249 was predominant highly expressed in eEVs from plasma of rats exposed to cigarette smoking, and confirmed in eEVs from plasma of human smokers as well as in eEVs from cigarette smoke extract-treated pulmonary artery endothelial cells, but not in cigarette smoke extract-treated pulmonary artery smooth muscle cells. In cultured pulmonary artery smooth muscle cells, microR-1249 downregulated the expression of histone deacetylase 10, which in turn enhanced the acetylated form of NFκB (nuclear factor κB) level and its nuclear translocation leading to increased expression of calcium-sensing receptor. In rats, the repression of microR-1249 in eEVs by microR-1249 inhibitor, histone deacetylase 10 overexpression, or calcium-sensing receptor knockout profoundly inhibited the proliferative capacities and diminished apoptosis-resistance of pulmonary artery smooth muscle cells and pulmonary hypertension development in rats intravenously administrated with eEVs preparation from cigarette smoke extract-treated pulmonary artery endothelial cells. Cigarette smoke-enriched microR-1249 in endothelial extracellular vesicles facilitates the hyperproliferative and antiapoptotic status of pulmonary artery smooth muscle cells promoting pulmonary hypertension evolution through the inhibition of histone deacetylase 10-NFκB-calcium-sensing receptor cascade.

Investigation into Antiepileptic Effect of Ganoderic Acid A and Its Mechanism in Seizure Rats Induced by Pentylenetetrazole.

Ganoderic acid A (GAA) exhibited neuron protection in in vitro epilepsy study, but no study has been done in vivo. Rats were administered (i.p.) pentylenetetrazole daily for 28 days to induce seizure. Rats with grade II or above of epileptic score were divided into three groups and given placebo, sodium valproate, or GAA treatment, respectively, for 7 days. The electrical signals of brain were monitored with electroencephalography (EGG); epileptic behavior was assessed using the Racine scale; morphological changes and apoptosis rate of cortical neurons were assessed with H&E staining and TUNEL staining, respectively. Protein expression of calcium-sensing receptor, p-ERK, p-JNK, and p-p38 in hippocampal tissue and Bcl-2, cleaved caspase-3, and Bax in cortical tissues was observed by Western blot and immunohistochemistry assay, respectively. After GAA treatment, apparent seizure-like EEG with significant arrhythmic disorder and spike waves was reduced or disappeared, and wave amplitude of EEG was reduced significantly. GAA showed similar effect with sodium valproate treatments on epilepsy. There were an apparent improvement of the epileptic behavior and a significant increase in the epileptic latency and shortening of the epileptic duration in the treatment group compared to control. GAA treatment ameliorated the nuclear pyknosis of neurons which appeared seriously in the epilepsy group. GAA treatment significantly reduced the cortical neuron apoptosis of epilepsy and the expression of calcium-sensing receptor, p-P38, p-JNK, cleaved caspase-3, and Bax but increased the expression of both p-ERK and Bcl-2. In conclusion, GAA treatment showed strong antiepileptic effect by decreasing apoptosis in cortical neuron and the expression of calcium-sensing receptor and stimulating the MAPK pathway.