The calcium-sensing receptor (CaSR) is suggested to mediate the antiproliferative effects of calcium in colon. However, in colorectal cancer (CRC) the expression of the CaSR is silenced and the underlying mechanisms leading to its loss are poorly understood. We investigated whether loss of the CaSR expression in colorectal tumors is caused by DNA hypermethylation and imbalance of transcriptionally permissive/repressive histone alterations. We observed significantly lower CaSR mRNA expression (n = 65, p < 0.001) in colorectal tumors compared with the adjacent mucosa from the same patient. Immunofluorescence staining confirmed downregulation of the CaSR protein also. The CaSR promoter was methylated to a greater extent in tumors compared with adjacent mucosa as determined by bisulfite sequencing (n = 20, p < 0.01) and by pyrosequencing (n = 45, p < 0.001), and methylation correlated inversely with mRNA expression (n = 20, ρ = -0.310, p < 0.05 and n = 45, ρ = -0.588, p < 0.001). Treatments with 5-aza-2'-deoxycytidine (DAC), a DNA methyltransferase inhibitor and/or with two different histone deacetylase inhibitors, trichostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA) restored the expression of CaSR in colon cancer cells. Restored CaSR expression in Coga1A and HT29 cells was functional. Inhibition of lysine-specific demethylase 1 (LSD1) to prevent demethylation of mono- and dimethylated H3K4, increased CaSR expression only marginally. Our data show that hypermethylation of the CaSR promoter and H3K9 deacetylation, but not H3K4me2 demethylation are important factors that cause silencing of the CaSR in colorectal cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype that often metastasizes to the brain. Leptomeningeal disease (LMD), a devastating brain metastasis common in TNBC, has limited treatment options. We sought to test whether the common anti-helminthic drug mebendazole (MBZ) may be effective against murine TNBC LMD.

Epidemiological studies suggest an inverse correlation between dietary calcium (Ca(2+)) and vitamin D intake and the risk of colorectal cancer (CRC). It has been shown in vitro that the active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25-D3) can upregulate expression of the calcium-sensing receptor (CaSR). In the colon, CaSR has been suggested to regulate proliferation of colonocytes. However, during tumorigenesis colonic CaSR expression is downregulated and we hypothesized that the loss of CaSR could influence the anti-tumorigenic effects of Ca(2+) and vitamin D. Our aim was to assess the impact of CaSR expression and function on the anti-neoplastic effects of 1,25-D3 in colon cancer cell lines. We demonstrated that in the healthy colon of mice, high vitamin D diet (2500 IU/kg diet) increased expression of differentiation and apoptosis markers, decreased expression of proliferation markers and significantly upregulated CaSR mRNA expression, compared with low vitamin D diet (100 IU/kg diet). To determine the role of CaSR in this process, we transfected Caco2-15 and HT29 CRC cells with wild type CaSR (CaSR-WT) or a dominant negative CaSR mutant (CaSR-DN) and treated them with 1,25-D3 alone, or in combination with CaSR activators (Ca(2+) and NPS R-568). 1,25-D3 enhanced the anti-proliferative effects of Ca(2+) and induced differentiation and apoptosis only in cells with a functional CaSR, which were further enhanced in the presence of NPS R-568, a positive allosteric modulator of CaSR. The mutant CaSR inhibited the anti-tumorigenic effects of 1,25-D3 suggesting that the anti-neoplastic effects of 1,25-D3 are, at least in part, mediated by the CaSR. Taken together, our data provides molecular evidence to support the epidemiological observation that both, vitamin D and calcium are needed for protection against malignant transformation of the colon and that their effect is modulated by the presence of a functional CaSR. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

Epidemiological studies suggest a correlation between vitamin D deficiency and colorectal cancer (CRC) incidence. The majority of sporadic tumors develop from premalignant lesions with aberrant activation of the Wnt/β-catenin signaling pathway. The adenoma cell line LT97 harbors an adenomatous polyposis coli (APC) mutation leading to constitutively active Wnt signaling. In these cells, expression of Wnt target genes leads to increased survival capacity. We hypothesized that 1,25-dihydroyvitamin D3 (1,25-D3), the active form of vitamin D3, promotes differentiation by modulating β-catenin/T-cell factor (TCF) 4-mediated gene transcription. The effect of dietary vitamin D on colonic Wnt signaling was investigated in mice fed either with 100 IU or 2500 IU vitamin D/kg diet. We examined the effect of 1,25-D3 on differentiation by measuring alkaline phosphatase activity. We analyzed mRNA expression of Wnt target genes by real time qRT-PCR. The impact of 1,25-D3 on β-catenin and TCF4 protein expression was assessed by western blot and immunohistochemistry. In LT97 cells, 1,25-D3 increased cellular differentiation and reduced nuclear β-catenin levels. Further, 1,25-D3 decreased mRNA expression of the Wnt target genes BCL-2, Cyclin D1, Snail1, CD44 and LGR5. In healthy colon of mice fed with high vitamin D diet, the mRNA levels of Wnt5a and ROR2, that promote degradation of β-catenin, were upregulated whereas β-catenin and TCF4 protein expression were decreased. In conclusion, 1,25-D3 inhibits Wnt signaling even in nonmalignant cells underlining its importance in protection against colorectal tumorigenesis and early tumor progression. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

Our previous studies showed that the 1,25-dihydroxyvitamin D (1,25-D3) catabolizing enzyme, 1,25-dihydoxyvitamin D 24 hydroxylase (CYP24A1) was overexpressed in colorectal tumours and its level correlated with increased proliferation. We hypothesised that cells overexpressing CYP24A1 have growth advantage and a diet rich in vitamin D and soy would restore sensitivity to the anti-tumourigenic effects of vitamin D. Soy contains genistein, a natural CYP24A1 inhibitor. To determine causality between CYP24A1 and tumour growth, we established xenografts in male SCID mice with HT29 cells stably overexpressing either GFP-tagged CYP24A1 or GFP. Mice were fed with either high (2500 IU D3/kg) or low vitamin D (100 IU D3/kg) diet in the presence or absence of soy (20% diet). In vitro, cells overexpressing CYP24A1 grew faster than controls. 1,25-D3, the active vitamin D metabolite, reduced cell number only in the presence of the CYP24A1 inhibitor VID400. Regardless of the amount of vitamin D in the diet, xenografts overexpressing CYP24A1 grew faster, were heavier and more aggressive. Soy reduced tumour volume only in the control xenografts, while the tumours overexpressing CYP24A1 were larger in the presence of dietary soy. In conclusion, we demonstrate that CYP24A1 overexpression results in increased aggressiveness and proliferative potential of colorectal tumours. Irrespective of the dietary vitamin D3, dietary soy is able to increase tumour volume when tumours overexpress CYP24A1, suggesting that combination of vitamin D3 and soy could have an anti-tumourigenic effect only if CYP24A1 levels are normal.

With aging, there is progressive loss of tissue homeostasis and functional reserve, leading to an impaired response to stress and an increased risk of morbidity and mortality. A key mediator of the cellular response to damage and stress is the transcription factor NF-κB. We demonstrated previously that NF-κB transcriptional activity is upregulated in tissues from both natural aged mice and in a mouse model of a human progeroid syndrome caused by defective repair of DNA damage (ERCC1-deficient mice). We also demonstrated that genetic reduction in the level of the NF-κB subunit p65(RelA) in the Ercc1-/∆ progeroid mouse model of accelerated aging delayed the onset of age-related pathology including muscle wasting, osteoporosis, and intervertebral disk degeneration. Here, we report that the largest fraction of NF-κB -expressing cells in the bone marrow (BM) of aged (>2 year old) mice (C57BL/6-NF-κBEGFP reporter mice) are Gr-1+ CD11b+ myeloid-derived suppressor cells (MDSCs). There was a significant increase in the overall percentage of MDSC present in the BM of aged animals compared with young, a trend also observed in the spleen. However, the function of these cells appears not to be compromised in aged mice. A similar increase of MDSC was observed in BM of progeroid Ercc1-/∆ and BubR1H/H mice. The increase in MDSC in Ercc1-/∆ mice was abrogated by heterozygosity in the p65/RelA subunit of NF-κB. These results suggest that NF-κB activation with aging, at least in part, drives an increase in the percentage of MDSCs, a cell type able to suppress immune cell responses.

Colorectal cancer is one of the most common cancers in industrialised societies. Epidemiological studies, animal experiments, and randomized clinical trials have shown that dietary factors can influence all stages of colorectal carcinogenesis, from initiation through promotion to progression. Calcium is one of the factors with a chemoprophylactic effect in colorectal cancer. The aim of this study was to understand the molecular mechanisms of the anti-tumorigenic effects of extracellular calcium ([Ca2+]o) in colon cancer cells. Gene expression microarray analysis of colon cancer cells treated for 1, 4, and 24h with 2mM [Ca2+]o identified significant changes in expression of 1571 probe sets (ANOVA, p<10-5). The main biological processes affected by [Ca2+]o were DNA replication, cell division, and regulation of transcription. All factors involved in DNA replication-licensing were significantly downregulated by [Ca2+]o. Furthermore, we show that the calcium-sensing receptor (CaSR), a G protein-coupled receptor is a mediator involved in this process. To test whether these results were physiologically relevant, we fed mice with a standard diet containing low (0.04%), intermediate (0.1%), or high (0.9%) levels of dietary calcium. The main molecules regulating replication licensing were inhibited also in vivo, in the colon of mice fed high calcium diet. We show that among the mechanisms behind the chemopreventive effect of [Ca2+]o is inhibition of replication licensing, a process often deregulated in neoplastic transformation. Our data suggest that dietary calcium is effective in preventing replicative stress, one of the main drivers of cancer and this process is mediated by the calcium-sensing receptor.

Non-alcoholic fatty liver disease (NAFLD) is a common metabolic disorder in obese individuals. Adenine nucleotide translocase (ANT) exchanges ADP/ATP through the mitochondrial inner membrane, and Ant2 is the predominant isoform expressed in the liver. Here we demonstrate that targeted disruption of Ant2 in mouse liver enhances uncoupled respiration without damaging mitochondrial integrity and liver functions. Interestingly, liver specific Ant2 knockout mice are leaner and resistant to hepatic steatosis, obesity and insulin resistance under a lipogenic diet. Protection against fatty liver is partially recapitulated by the systemic administration of low-dose carboxyatractyloside, a specific inhibitor of ANT. Targeted manipulation of hepatic mitochondrial metabolism, particularly through inhibition of ANT, may represent an alternative approach in NAFLD and obesity treatment.

Substance use has become a critical health concern during the COVID-19 pandemic, and emerging attention has been paid to people with the persistent symptoms of COVID-19 (COVID-19 long haulers) due to their high vulnerability. However, scant research has investigated their substance use and relevant psychosocial factors. The current study was to (1) examine substance use behaviors (i.e., legal drug use, illicit drug use, and non-medical use of prescription drugs); and (2) assessed their associations with psychiatric symptoms (i.e., depression, anxiety, and post-traumatic stress disorder) and psychosocial factors (i.e., personal mastery and social support) among COVID-19 long haulers.

The calcium sensing receptor (CaSR), a calcium-binding G protein-coupled receptor is expressed also in tissues not directly involved in calcium homeostasis like the colon. We have previously reported that CaSR expression is down-regulated in colorectal cancer (CRC) and that loss of CaSR provides growth advantage to transformed cells. However, detailed mechanisms underlying these processes are largely unknown.

In colorectal cancer (CRC) the vitamin D catabolizing enzyme 1,25-dihydroxyvitamin D 24-hydroxylase (CYP24A1) is overexpressed with a potentially significant, positive impact on the catabolism of 1,25-dihydroxyvitamin D3 (1,25-D3 ). However, the underlying mechanism of CYP24A1 overexpression is poorly understood. In the present study, we investigated possible causes including hypomethylation of the CYP24A1 promoter, amplification of the CYP24A1 gene locus (20q13.2), and altered expression of CYP24A1-specific transcription factors. We quantified CYP24A1 gene copy-number, performed bisulfite sequencing of the CYP24A1 promoter to assess DNA methylation, and measured mRNA expression of CYP24A1, 25-hydroxyvitamin D 1α-hydroxylase (CYP27B1), vitamin D receptor (VDR) and retinoid X receptor (RXR). We found that 77 (60%) out of 127 colorectal tumors showed increased CYP24A1 gene copy-number and that more than 6 copies of CYP24A1 correlated positively with CYP24A1 mRNA expression suggestive of a causal relationship. No differences in CYP24A1 promoter methylation were found between tumor tissue and adjacent mucosa from the same patient or between tissues with high or low mRNA expression, thus excluding DNA hypomethylation as a possible cause of CYP24A1 overexpression in CRC. Furthermore, mRNA expression of several factors involved in replication licensing positively correlated with CYP24A1 mRNA expression, raising the possibility that CYP24A1 overexpression might favor increased proliferation in tumors by suppressing local 1,25-D3 levels. We conclude that high copy-number gain is a key determinant of CYP24A1 overexpression in CRC. Other postulated causes of CYP24A1 overexpression including promoter hypomethylation and enhanced VDR and/or RXR expression do not appear to be involved.

Inflammatory bowel disease increases the odds of developing colitis-associated cancer. We hypothesized that Western-style diet (WD) aggravates azoxymethane (AOM)/dextran sulfate sodium salt (DSS)-induced colitis-associated tumorigenesis and that switching to the standard AIN93G diet will ameliorate disease symptoms even after cancer initiation. Female BALB/c mice received either WD (WD group) or standard AIN93G diet (AIN group) for the whole experimental period. After five weeks, the mice received 12.5 mg/kg AOM intraperitoneally, followed by three DSS cycles. In one group of mice, the WD was switched to AIN93G the day before starting the first DSS cycle (WD/AIN group). Feeding the WD during the whole experimental period aggravated colitis symptoms, shortened the colon (p < 0.05), changed microbiota composition and increased tumor promotion. On molecular level, the WD reduced proliferation (p < 0.05) and increased expression of the vitamin D catabolizing enzyme Cyp24a1 (p < 0.001). The switch to the AIN93G diet ameliorated this effect, reflected by longer colons, fewer (p < 0.05) and smaller (p < 0.01) aberrant colonic crypt foci, comparable with the AIN group. Our results show that switching to a healthy diet, even after cancer initiation is able to revert the deleterious effect of the WD and could be an effective preventive strategy to reduce colitis symptoms and prevent tumorigenesis.

The generation of new adipocytes from precursor cells (adipogenesis) has implications for systemic metabolism and is a commonly used model for studying the process of cell differentiation in vitro. Previous studies from us and others suggested that the peripheral circadian clock can influence adipogenesis in vitro, but the mechanisms driving this activity and the relevance for adipogenesis in vivo are unknown. Here we reveal that mouse adipocyte precursor cells (APCs) contain a circadian clock that oscillates in vivo. We expose context-specific features of the clock in APCs: expression of the canonical core clock component Per1 does not significantly oscillate, whereas the lesser-understood paralog Per3 has a prominent rhythm. We discovered that deletion of Per3 promotes adipogenesis in vivo by a clock output pathway in which PER3 and BMAL1 directly regulate Klf15 expression. These findings demonstrate that Per3 has a major role in the APC clock and regulates adipogenesis in vivo.

The mitochondrial ADP/ATP carrier (AAC) is a major transport protein of the inner mitochondrial membrane. It exchanges mitochondrial ATP for cytosolic ADP and controls cellular production of ATP. In addition, it has been proposed that AAC mediates mitochondrial uncoupling, but it has proven difficult to demonstrate this function or to elucidate its mechanisms. Here we record AAC currents directly from inner mitochondrial membranes from various mouse tissues and identify two distinct transport modes: ADP/ATP exchange and H+ transport. The AAC-mediated H+ current requires free fatty acids and resembles the H+ leak via the thermogenic uncoupling protein 1 found in brown fat. The ADP/ATP exchange via AAC negatively regulates the H+ leak, but does not completely inhibit it. This suggests that the H+ leak and mitochondrial uncoupling could be dynamically controlled by cellular ATP demand and the rate of ADP/ATP exchange. By mediating two distinct transport modes, ADP/ATP exchange and H+ leak, AAC connects coupled (ATP production) and uncoupled (thermogenesis) energy conversion in mitochondria.

The calcium-sensing receptor (CaSR) plays a pivotal role in systemic calcium metabolism by regulating parathyroid hormone secretion and urinary calcium excretion. The CaSR is ubiquitously expressed, implying a wide range of functions regulated by this receptor. Abnormal CaSR function affects the development of both calciotropic disorders such as hyperparathyroidism, and non-calciotropic disorders such as cardiovascular disease and cancer, which are the leading causes of mortality worldwide. The CaSR is able to bind a plethora of ligands; it interacts with multiple G protein subtypes, and regulates highly divergent downstream signalling pathways, depending on the cellular context. The CaSR is a key regulator for such diverse processes as hormone secretion, gene expression, inflammation, proliferation, differentiation, and apoptosis. Due to this pleiotropy, the CaSR is able to regulate cell fate and is implicated in the development of many types of benign or malignant tumours of the breast, prostate, parathyroid, and colon. In cancer, the CaSR appears to have paradoxical roles, and depending on the tissue involved, it is able to prevent or promote tumour growth. In tissues like the parathyroid or colon, the CaSR inhibits proliferation and induces terminal differentiation of the cells. Therefore, loss of the receptor, as seen in colorectal or parathyroid tumours, confers malignant potential, suggestive of a tumour suppressor role. In contrast, in prostate and breast tumours the expression of the CaSR is increased and it seems that it favours metastasis to the bone, acting as an oncogene. Deciphering the molecular mechanism driving the CaSR in the different tissues could lead to development of new allosteric drug compounds that selectively target the CaSR and have therapeutic potential for cancer. This article is part of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen.

Trithorax proteins and long-intergenic noncoding RNAs are critical regulators of embryonic stem cell pluripotency; however, how they cooperatively regulate germ layer mesoderm specification remains elusive. We report here that HoxBlinc RNA first specifies Flk1(+) mesoderm and then promotes hematopoietic differentiation through regulation of hoxb pathways. HoxBlinc binds to the hoxb genes, recruits Setd1a/MLL1 complexes, and mediates long-range chromatin interactions to activate transcription of the hoxb genes. Depletion of HoxBlinc by shRNA-mediated knockdown or CRISPR-Cas9-mediated genetic deletion inhibits expression of hoxb genes and other factors regulating cardiac/hematopoietic differentiation. Reduced hoxb expression is accompanied by decreased recruitment of Set1/MLL1 and H3K4me3 modification, as well as by reduced chromatin loop formation. Re-expression of hoxb2-b4 genes in HoxBlinc-depleted embryoid bodies rescues Flk1(+) precursors that undergo hematopoietic differentiation. Thus, HoxBlinc plays an important role in controlling hoxb transcription networks that mediate specification of mesoderm-derived Flk1(+) precursors and differentiation of Flk1(+) cells into hematopoietic lineages.

The calcium-sensing receptor (CaSR) is a family C G-protein-coupled receptor that plays a pivotal role in extracellular calcium homeostasis. The CaSR is also highly expressed in pancreatic islet α- and β-cells that secrete glucagon and insulin, respectively. To determine whether the CaSR may influence systemic glucose homeostasis, we characterized a mouse model with a germline gain-of-function CaSR mutation, Leu723Gln, referred to as Nuclear flecks (Nuf). Heterozygous- (CasrNuf/+) and homozygous-affected (CasrNuf/Nuf) mice were shown to have hypocalcemia in association with impaired glucose tolerance and insulin secretion. Oral administration of a CaSR antagonist compound, known as a calcilytic, rectified the glucose intolerance and hypoinsulinemia of CasrNuf/+ mice and ameliorated glucose intolerance in CasrNuf/Nuf mice. Ex vivo studies showed CasrNuf/+ and CasrNuf/Nuf mice to have reduced pancreatic islet mass and β-cell proliferation. Electrophysiological analysis of isolated CasrNuf/Nuf islets showed CaSR activation to increase the basal electrical activity of β-cells independently of effects on the activity of the adenosine triphosphate (ATP)-sensitive K+ (KATP) channel. CasrNuf/Nuf mice also had impaired glucose-mediated suppression of glucagon secretion, which was associated with increased numbers of α-cells and a higher α-cell proliferation rate. Moreover, CasrNuf/Nuf islet electrophysiology demonstrated an impairment of α-cell membrane depolarization in association with attenuated α-cell basal KATP channel activity. These studies indicate that the CaSR activation impairs glucose tolerance by a combination of α- and β-cell defects and also influences pancreatic islet mass. Moreover, our findings highlight a potential application of targeted CaSR compounds for modulating glucose metabolism.

Coordinated activation of muscle stem cells (known as satellite cells) is critical for postnatal muscle growth and regeneration. The muscle stem cell niche is central for regulating the activation state of satellite cells, but the specific extracellular signals that coordinate this regulation are poorly understood. Here we show that macrophages at sites of muscle injury induce activation of satellite cells via expression of Adamts1. Overexpression of Adamts1 in macrophages in vivo is sufficient to increase satellite cell activation and improve muscle regeneration in young mice. We demonstrate that NOTCH1 is a target of ADAMTS1 metalloproteinase activity, which reduces Notch signaling, leading to increased satellite cell activation. These results identify Adamts1 as a potent extracellular regulator of satellite cell activation and have significant implications for understanding the regulation of satellite cell activity and regeneration after muscle injury.Satellite cells are crucial for growth and regeneration of skeletal muscle. Here the authors show that in response to muscle injury, macrophages secrete Adamts1, which induces satellite cell activation by modulating Notch1 signaling.

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The inverse correlation between dietary calcium intake and the risk of colorectal cancer (CRC) is well known, but poorly understood. Expression of the calcium-sensing receptor (CaSR), a calcium-binding G protein-coupled receptor is downregulated in CRC leading us to hypothesize that the CaSR has tumor suppressive roles in the colon. The aim of this study was to understand whether restoration of CaSR expression could reduce the malignant phenotype in CRC. In human colorectal tumors, expression of the CaSR negatively correlated with proliferation markers whereas loss of CaSR correlated with poor tumor differentiation and reduced apoptotic potential. In vivo, dearth of CaSR significantly increased expression of proliferation markers and decreased levels of differentiation and apoptotic markers in the colons of CaSR/PTH double knock-out mice confirming the tumor suppressive functions of CaSR. In vitro CRC cells stably overexpressing wild-type CaSR showed significant reduction in proliferation, as well as increased differentiation and apoptotic potential. The positive allosteric modulator of CaSR, NPS R-568 further enhanced these effects, whereas treatment with the negative allosteric modulator, NPS 2143 inhibited these functions. Interestingly, the dominant-negative mutant (R185Q) was able to abrogate these effects. Our results demonstrate a critical tumor suppressive role of CaSR in the colon. Restoration of CaSR expression and function is linked to regulation of the balance between proliferation, differentiation, and apoptosis and provides a rationale for novel strategies in CRC therapy.