The calcium-sensing receptor modulates the prostaglandin E2 pathway in intestinal inflammation.

Introduction: The prostaglandin E2 (PGE2) pathway is one of the main mediators of intestinal inflammation. As activation of the calcium-sensing receptor (CaSR) induces expression of inflammatory markers in the colon, we assessed the impact of the CaSR on the PGE2 pathway regulation in colon cancer cells and the colon in vitro and in vivo. Methods and Results: We treated CaSR-transfected HT29 and Caco-2 colon cancer cell lines with different orthosteric ligands or modulators of the CaSR and measured gene expression and PGE2 levels. In CaSR-transfected HT29CaSR-GFP and Caco-2CaSR-GFP cells, the orthosteric CaSR ligand spermine and the positive allosteric CaSR modulator NPS R-568 both induced an inflammatory state as measured by IL-8 gene expression and significantly increased the expression of the PGE2 pathway key enzymes cyclooxygenase (COX)-2 and/or prostaglandin E2 synthase 1 (PGES-1). Inhibition of the CaSR with the calcilytic NPS 2143 abolished the spermine- and NPS R-568-induced pro-inflammatory response. Interestingly, we observed cell-line specific responses as e.g. PGES-1 expression was affected only in HT29CaSR-GFP but not in Caco-2CaSR-GFP cells. Other genes involved in the PGE2 pathway (COX-1, or the PGE2 receptors) were not responsive to the treatment. None of the studied genes were affected by any CaSR agonist in GFP-only transfected HT29GFP and Caco-2GFP cells, indicating that the observed gene-inducing effects of spermine and R-568 were indeed mediated by the CaSR. In vivo, we had previously determined that treatment with the clinically approved calcimimetic cinacalcet worsened symptoms in a dextran sulfate sodium (DSS)-induced colitis mouse model. In the colons of these mice, cinacalcet significantly induced gene expression of PGES-2 and the EP3 receptor, but not COX-2; while NPS 2143 increased the expression of the PGE2-degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Importantly, neither treatment had any effect on the colons of non-DSS treated mice. Discussion: Overall, we show that activation of the CaSR induces the PGE2 pathway, albeit with differing effects in vitro and in vivo. This may be due to the different microenvironment in vivo compared to in vitro, specifically the presence of a CaSR-responsive immune system. Since calcilytics inhibit ligand-mediated CaSR signaling, they may be considered for novel therapies against inflammatory bowel disease.

Hormonal regulation of mammary gland development and lactation.

Lactation is critical to infant short-term and long-term health and protects mothers from breast cancer, ovarian cancer and type 2 diabetes mellitus. The mammary gland is a dynamic organ, regulated by the coordinated actions of reproductive and metabolic hormones. These hormones promote gland development from puberty onwards and induce the formation of a branched, epithelial, milk-secreting organ by the end of pregnancy. Progesterone withdrawal following placental delivery initiates lactation, which is maintained by increased pituitary secretion of prolactin and oxytocin, and stimulated by infant suckling. After weaning, local cytokine production and decreased prolactin secretion trigger large-scale mammary cell loss, leading to gland involution. Here, we review advances in the molecular endocrinology of mammary gland development and milk synthesis. We discuss the hormonal functions of the mammary gland, including parathyroid hormone-related peptide secretion that stimulates maternal calcium mobilization for milk synthesis. We also consider the hormonal composition of human milk and its associated effects on infant health and development. Finally, we highlight endocrine and metabolic diseases that cause lactation insufficiency, for example, monogenic disorders of prolactin and prolactin receptor mutations, maternal obesity and diabetes mellitus, interventions during labour and delivery, and exposure to endocrine-disrupting chemicals such as polyfluoroalkyl substances in consumer products and other oestrogenic compounds.

Protocol for an observational study investigating hormones triggering the onset of sustained lactation: the INSIGHT study.

INTRODUCTION: Lactation is a hormonally controlled process that promotes infant growth and neurodevelopment and reduces the long-term maternal risk of diabetes, cardiovascular disease and breast cancer. Hormones, such as prolactin and progesterone, mediate mammary development during pregnancy and are critical for initiating copious milk secretion within 24-72 hours post partum. However, the hormone concentrations mediating lactation onset are ill defined. METHODS AND ANALYSIS: The primary objective of the investigating hormones triggering the onset of sustained lactation study is to establish reference intervals for the circulating hormone concentrations initiating postpartum milk secretion. The study will also assess how maternal factors such as parity, pregnancy comorbidities and complications during labour and delivery, which are known to delay lactation, may affect hormone concentrations. This single-centre observational study will recruit up to 1068 pregnant women over a 3-year period. A baseline blood sample will be obtained at 36 weeks' gestation. Participants will be monitored during postpartum days 1-4. Lactation onset will be reported using a validated breast fullness scale. Blood samples will be collected before and after a breastfeed on up to two occasions per day during postpartum days 1-4. Colostrum, milk and spot urine samples will be obtained on a single occasion. Serum hormone reference intervals will be calculated as mean±1.96 SD, with 90% CIs determined for the upper and lower reference limits. Differences in hormone values between healthy breastfeeding women and those at risk of delayed onset of lactation will be assessed by repeated measures two-way analysis of variance or a mixed linear model. Correlations between serum hormone concentrations and milk composition and volume will provide insights into the endocrine regulation of milk synthesis. ETHICS AND DISSEMINATION: Approval for this study had been granted by the East of England-Cambridgeshire and Hertfordshire Research Ethics Committee (REC No. 20/EE/0172), by the Health Research Authority (HRA), and by the Oxford University Hospitals National Health Service Foundation Trust. The findings will be published in high-ranking journals and presented at national and international conferences. TRIAL REGISTRATION NUMBER: ISRCTN12667795.

Effects of pharmacological calcimimetics on colorectal cancer cells over-expressing the human calcium-sensing receptor.

The calcium-sensing receptor (CaSR) is a ubiquitously expressed multifunctional G protein-coupled receptor. Several studies reported that the CaSR plays an anti-inflammatory and anti-tumorigenic role in the intestine, and that it is down-regulated during colorectal carcinogenesis. We hypothesized that positive allosteric CaSR modulators (type II calcimimetics) selectively targeting the intestinal cells could be used for the treatment of intestinal pathologies. Therefore, the aim of this study was to determine the effect of pharmacological stimulation of CaSR on gene expression in vitro and on tumor growth in vivo. We stably transduced two colon cancer cell lines (HT29 and Caco2) with lentiviral vectors containing either the CaSR fused to GFP or GFP only. Using RNA sequencing, RT-qPCR experiments and ELISA, we determined that CaSR over-expression itself had generally little effect on gene expression in these cells. However, treatment with 1 µM of the calcimimetic NPS R-568 increased the expression of pro-inflammatory factors such as IL-23α and IL-8 and reduced the transcription of various differentiation markers in the cells over-expressing the CaSR. In vivo, neither the presence of the CaSR nor p.o. treatment of the animals with the calcimimetic cinacalcet affected tumor growth, tumor cell proliferation or tumor vascularization of murine HT29 xenografts. In summary, CaSR stimulation in CaSR over-expressing cells enhanced the expression of inflammatory markers in vitro, but was not able to repress colorectal cancer tumorigenicity in vivo. These findings suggest potential pro-inflammatory effects of the CaSR and type II calcimimetics in the intestine.

Nutritional and Pharmacological Targeting of the Calcium-Sensing Receptor Influences Chemically Induced Colitis in Mice.

The calcium-sensing receptor (CaSR) is the main regulator of extracellular Ca2+ homeostasis. It has diverse functions in different tissues, including the intestines. Intestine-specific knockout of the CaSR renders mice more susceptible to dextran sulphate sodium (DSS)-induced colitis. To test our hypothesis that the CaSR reduces intestinal inflammation, we assessed the effects of nutritional and pharmacological agonists of the CaSR in a colitis model. We treated female Balb/C mice with dietary calcium and protein (nutritional agonists of the CaSR) or pharmacological CaSR modulators (the agonists cinacalcet and GSK3004774, and the antagonist NPS-2143; 10 mg/kg), then induced colitis with DSS. The high-protein diet had a strong pro-inflammatory effect-it shortened the colons (5.3 ± 0.1 cm vs. 6.1 ± 0.2 cm normal diet, p < 0.05), lowered mucin expression and upregulated pro-inflammatory cytokines, such as interferon-γ, (4.2-fold, p < 0.05) compared with the normal diet. Cinacalcet reduced mucin expression, which coincided with an increase in tumor necrosis factor-α (4.4-fold, p < 0.05) and IL-6 (4.9-fold, p < 0.05) in the plasma, compared with vehicle. The CaSR antagonist, NPS-2143, significantly reduced the cumulative inflammation score compared with the vehicle control (35.3 ± 19.1 vs. 21.9 ± 14.3 area under the curve, p < 0.05) and reduced infiltration of inflammatory cells. While dietary modulation of the CaSR had no beneficial effects, pharmacological inhibition of the CaSR may have the potential of a novel add-on therapy in the treatment of inflammatory bowel diseases.

Calcium-sensing receptor in colorectal inflammation and cancer: Current insights and future perspectives.

The extracellular calcium-sensing receptor (CaSR) is best known for its action in the parathyroid gland and kidneys where it controls body calcium homeostasis. However, the CaSR has different roles in the gastrointestinal tract, where it is ubiquitously expressed. In the colon, the CaSR is involved in controlling multiple mechanisms, including fluid transport, inflammation, cell proliferation and differentiation. Although the expression pattern and functions of the CaSR in the colonic microenvironment are far from being completely understood, evidence has been accumulating that the CaSR might play a protective role against both colonic inflammation and colorectal cancer. For example, CaSR agonists such as dipeptides have been suggested to reduce colonic inflammation, while dietary calcium was shown to reduce the risk of colorectal cancer. CaSR expression is lost in colonic malignancies, indicating that the CaSR is a biomarker for colonic cancer progression. This dual anti-inflammatory and anti-tumourigenic role of the CaSR makes it especially interesting in colitis-associated colorectal cancer. In this review, we describe the clinical and experimental evidence for the role of the CaSR in colonic inflammation and colorectal cancer, the intracellular signalling pathways which are putatively involved in these actions, and the possibilities to exploit these actions of the CaSR for future therapies of colonic inflammation and cancer.

Potent Inactivation-Dependent Inhibition of Adult and Neonatal NaV1.5 Channels by Lidocaine and Levobupivacaine.

BACKGROUND: Cardiotoxic effects of local anesthetics (LAs) involve inhibition of NaV1.5 voltage-gated Na channels. Metastatic breast and colon cancer cells also express NaV1.5, predominantly the neonatal splice variant (nNaV1.5) and their inhibition by LAs reduces invasion and migration. It may be advantageous to target cancer cells while sparing cardiac function through selective blockade of nNaV1.5 and/or by preferentially affecting inactivated NaV1.5, which predominate in cancer cells. We tested the hypotheses that lidocaine and levobupivacaine differentially affect (1) adult (aNaV1.5) and nNaV1.5 and (2) the resting and inactivated states of NaV1.5. METHODS: The whole-cell voltage-clamp technique was used to evaluate the actions of lidocaine and levobupivacaine on recombinant NaV1.5 channels expressed in HEK-293 cells. Cells were transiently transfected with cDNAs encoding either aNaV1.5 or nNaV1.5. Voltage protocols were applied to determine depolarizing potentials that either activated or inactivated 50% of maximum conductance (V½ activation and V½ inactivation, respectively). RESULTS: Lidocaine and levobupivacaine potently inhibited aNaV1.5 (IC50 mean [SD]: 20 [22] and 1 [0.6] µM, respectively) and nNaV1.5 (IC50 mean [SD]: 17 [10] and 3 [1.6] µM, respectively) at a holding potential of -80 mV. IC50s differed significantly between lidocaine and levobupivacaine with no influence of splice variant. Levobupivacaine induced a statistically significant depolarizing shift in the V½ activation for aNaV1.5 (mean [SD] from -32 [4.6] mV to -26 [8.1] mV) but had no effect on the voltage dependence of activation of nNaV1.5. Lidocaine had no effect on V½ activation of either variant but caused a significantly greater depression of maximum current mediated by nNaV1.5 compared to aNaV1.5. Similar statistically significant shifts in the V½ inactivation (approximately -10 mV) occurred for both LAs and NaV1.5 variants. Levobupivacaine (1 µM) caused a significantly greater slowing of recovery from inactivation of both variants than did lidocaine (10 µM). Both LAs caused approximately 50% tonic inhibition of aNaV1.5 or nNaV1.5 when holding at -80 mV. Neither LA caused tonic block at a holding potential of either -90 or -120 mV, voltages at which there was little steady-state inactivation. Higher concentrations of either lidocaine (300 µM) or levobupivacaine (100 µM) caused significantly more tonic block at -120 mV. CONCLUSIONS: These data demonstrate that low concentrations of the LAs exhibit inactivation-dependent block of NaV1.5, which may provide a rationale for their use to safely inhibit migration and invasion by metastatic cancer cells without cardiotoxicity.