The farnesoid X receptor negatively regulates osteoclastogenesis in bone remodeling and pathological bone loss

Farnesoid X receptor (FXR, NR1H4) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. Since the role of FXR in osteoclast differentiation remains ill-defined, we investigated the biological function of FXR on osteoclastogenesis, using FXR-deficient mice. We demonstrated that FXR deficiency increases osteoclast formation in vitro and in vivo. First, FXR deficiency was found to accelerate osteoclast formation via down-regulation of c-Jun N-terminal kinase (JNK) 1/2 expression. Increased expression of peroxisome proliferator-activated receptor (PPAR)γ and peroxisome proliferator-activated receptor gamma coactivator 1 (PGC- 1)β seems to mediate the pro-osteoclastogenic effect of FXR deficiency via the JNK pathway. In addition, we found that FXR deficiency downregulated the expression of interferon-β (IFN-β), a strong inhibitor of osteoclastogenesis, via receptor activator of nuclear factor-kappaB ligand (RANKL). We further suggested that interference of IFN-β expression by FXR deficiency impaired the downstream JAK3-STAT1 signaling pathways, which in turn increased osteoclast formation. Finally, FXR deficiency accelerated unloading- or ovariectomy-induced bone loss in vivo. Thus, our findings demonstrate that FXR is a negative modulator in osteoclast differentiation and identify FXR as a potential therapeutic target for postmenopausal osteoporosis and unloadinginduced bone loss

Plasma membrane calcium ATPase regulates bone mass by fine-tuning osteoclast differentiation and survival

The precise regulation of Ca(2+) dynamics is crucial for proper differentiation and function of osteoclasts. Here we show the involvement of plasma membrane Ca(2+) ATPase (PMCA) isoforms 1 and 4 in osteoclastogenesis. In immature/undifferentiated cells, PMCAs inhibited receptor activator of NF-κB ligand–induced Ca(2+) oscillations and osteoclast differentiation in vitro. Interestingly, nuclear factor of activated T cell c1 (NFATc1) directly stimulated PMCA transcription, whereas the PMCA-mediated Ca(2+) efflux prevented NFATc1 activation, forming a negative regulatory loop. PMCA4 also had an anti-osteoclastogenic effect by reducing NO, which facilitates preosteoclast fusion. In addition to their role in immature cells, increased expression of PMCAs in mature osteoclasts prevented osteoclast apoptosis both in vitro and in vivo. Mice heterozygous for PMCA1 or null for PMCA4 showed an osteopenic phenotype with more osteoclasts on bone surface. Furthermore, PMCA4 expression levels correlated with peak bone mass in premenopausal women. Thus, our results suggest that PMCAs play important roles for the regulation of bone homeostasis in both mice and humans by modulating Ca(2+) signaling in osteoclasts

Plasma membrane calcium ATPase regulates bone mass by fine-tuning osteoclast differentiation and survival.

The precise regulation of Ca2+ dynamics is crucial for proper differentiation and function of osteoclasts. Here we show the involvement of plasma membrane Ca2+ ATPase (PMCA) isoforms 1 and 4 in osteoclastogenesis. In immature/undifferentiated cells, PMCAs inhibited receptor activator of NF-B ligand–induced Ca2+ oscillations and osteoclast differentiation in vitro. Interestingly, nuclear factor of activated T cell c1 (NFATc1) directly stimulated PMCA transcription, whereas the PMCA-mediated Ca2+ efflux prevented NFATc1 activation, forming a negative regulatory loop. PMCA4 also had an anti-osteoclastogenic effect by reducing NO, which facilitates preosteoclast fusion. In addition to their role in immature cells, increased expression of PMCAs in mature osteoclasts prevented osteoclast apoptosis both in vitro and in vivo. Mice heterozygous for PMCA1 or null for PMCA4 showed an osteopenic phenotype with more osteoclasts on bone surface. Furthermore, PMCA4 expression levels correlated with peak bone mass in premenopausal women. Thus, our results suggest that PMCAs play important roles for the regulation of bone homeostasis in both mice and humans by modulating Ca2+ signaling in osteoclasts.OAIID:oai:osos.snu.ac.kr:snu2012-01/102/0000026258/8SEQ:8PERF_CD:SNU2012-01EVAL_ITEM_CD:102USER_ID:0000026258ADJUST_YN:NEMP_ID:A076310DEPT_CD:861CITE_RATE:10.264FILENAME:J Cell Biol-2012-Kim-ATPase.pdfDEPT_NM:치의학과EMAIL:[email protected]_YN:YCONFIRM:

Tissue Engineering an In Vitro Model of Osteoporosis

Postmenopausal osteoporosis is a skeletal disorder characterised by bone loss. Declining oestrogen levels postmenopause disrupt bone remodelling by over-stimulating resorption. Although the disorder is currently studied in animals, we should aim to minimise their use. Therefore, this thesis explored the feasibility of developing an in vitro model of postmenopausal osteoporosis using tissue engineering principles. The response of three osteoblast cell lines, MC3T3-E1, MLOA5, and IDG-SW3, to oestrogen was explored, finding only MC3T3-E1 was stimulated by the hormone. The ability of RAW264.7 to undergo osteoclastogenesis was strongly influenced by seeding density and proliferation. Additionally, tartrate-resistant acid phosphatase (TRAP) activity could be suppressed by oestrogen exposure. Due to its ability to support osteoclastogenesis in co-culture, IDG-SW3 was the most suitable osteoblast cell line for the model. Bone-matrix deposition over 28 days on three scaffolds (PolyHIPE, polyurethane, Biotek) was compared to select the most appropriate for the model. PolyHIPE and polyurethane scaffolds supported significantly more matrix deposition than the Biotek. Mineralisation on the scaffold could be detected by micro-computed tomography; however, the presence of PBS interfered with this. Due to its cellular performance and ease of manufacture, the polyurethane scaffold was identified as the most suitable for the model. Changes in mineral content, TRAP and alkaline phosphatase activity were confirmed as markers for osteoclast and osteoblast activity in co-culture. RAW264.7 pre-treatment with oestrogen to mimic pre-menopause had lasting effects on their ability to undergo osteoclastogenesis. 2D co-cultures using oestrogen withdrawal to mimic menopause resulted in increased resorption, analogous to the effect seen in vivo. From the conditions assessed in 3D co-cultures, no equivalent response was observed. This thesis demonstrates it is possible to imitate the onset of postmenopausal osteoporosis in vitro. However, a 3D system that uses human cells and longer time periods is necessary to provide a valid alternative to animal models

Dichotomic role of NAADP/two-pore channel 2/Ca2+ signaling in regulating neural differentiation of mouse embryonic stem cells

Poster Presentation - Stem Cells and Pluripotency: abstract no. 1866The mobilization of intracellular Ca2+stores is involved in diverse cellular functions, including cell proliferation and differentiation. At least three endogenous Ca2+mobilizing messengers have been identified, including inositol trisphosphate (IP3), cyclic adenosine diphosphoribose (cADPR), and nicotinic adenine acid dinucleotide phosphate (NAADP). Similar to IP3, NAADP can mobilize calcium release in a wide variety of cell types and species, from plants to animals. Moreover, it has been previously shown that NAADP but not IP3-mediated Ca2+increases can potently induce neuronal differentiation in PC12 cells. Recently, two pore channels (TPCs) have been identified as a novel family of NAADP-gated calcium release channels in endolysosome. Therefore, it is of great interest to examine the role of TPC2 in the neural differentiation of mouse ES cells. We found that the expression of TPC2 is markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebound during the late stages of neurogenesis. Correspondingly, perturbing the NAADP signaling by TPC2 knockdown accelerates mouse ES cell differentiation into neural progenitors but inhibits these neural progenitors from committing to the final neural lineage. Interestingly, TPC2 knockdown has no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Overexpression of TPC2, on the other hand, inhibits mouse ES cell from entering the neural lineage. Taken together, our data indicate that the NAADP/TPC2-mediated Ca2+signaling pathway plays a temporal and dichotomic role in modulating the neural lineage entry of ES cells; in that NAADP signaling antagonizes ES cell entry to early neural progenitors, but promotes late neural differentiation.postprin

Deciphering the Mechanism and Roles of Muscarinic and Purinergic Calcium Signals in the Human Colonic Epithelium

The human large intestine is lined by a monolayer of polarized epithelial cells, which form invaginations known as ‘Crypts of Lieberkühn’ and serves as a barrier between the gut luminal contents and the systemic circulation. In order to maintain this barrier, the epithelium constantly renews and replenishes itself, a process known as tissue homeostasis. This process is driven by multipotent intestinal stem cells residing at the base of crypts which give rise to specialised epithelial cells. Loss of tissue homeostasis is associated with an increased risk of major intestinal diseases, such as colorectal cancer. Stem cell-driven tissue renewal is regulated by luminal and systemic factors. Luminal-sensing of gut contents – nutrients, microbes, and their metabolites – triggers the release of hormones, cytokines and chemokines from the epithelium and gut immune cells. Higher levels of control that modulate the epithelium and integrate luminal inputs are exerted at the basal pole by neurotransmitters, such as acetylcholine and ATP. These neurotransmitters affect gut immunity and motility, mucus and fluid and electrolyte secretion, proliferation, and cancer development. This thesis is interested in expanding our understanding of how the colonic epithelium integrates signalling inputs. Calcium is a signal integrator that regulates gut homeostasis. In drosophila, external signalling molecules causes calcium oscillations which influences intestinal stem cells. In mouse, carbachol – a non-hydrolysable analogue of acetylcholine – increases cytosolic calcium concentrations and modulates bicarbonate secretion. Calcium signalling also has a complex role on the development and progression of cancer. However, the spatial-temporal status of calcium signals in primary human intestinal epithelium remains unclear. This thesis elucidates components of the calcium signalling toolkit and investigates the spatial-temporal status of calcium signals in cultured human intestinal crypts and organoids. It also explores the physiological roles of calcium signalling in maintaining gut tissue homeostasis and investigates the status of calcium signalling in colorectal cancer

The role of the tumour microenvironment in the phenotype of pituitary adenomas

PhD ThesisNon-neoplastic cells in the tumour microenvironment (TME) influence tumoural aggressiveness and oncogenic mechanisms. Little is known about the TME in pituitary adenomas (PAs). This work aimed to characterise the TME of PAs and its effects in tumour aggressiveness and oncogenic mechanisms, focusing on the cytokine network, infiltrating immune cells and PA-associated fibroblasts (TAFs). To study the cytokine secretion of tumour/non-tumoural cells, cytokine bead arrays were performed on culture supernatants. PA-infiltrating immune cells, angiogenesis, epithelial-to-mesenchymal transition (EMT) and matrix metalloproteinases were assessed by immunohistochemistry. In vitro pituitary tumour–macrophage/TAF interactions were assessed by conditioned medium (CM) of GH3 (pituitary tumour) and RAW264.7 (macrophage) cell lines, as well as primary TAFs, in terms of morphology, migration, invasion and EMT activation. IL-8, CCL2, CCL3, CCL4, CXCL10, CCL22 and CXCL1 were the main PA-derived cytokines, which facilitate macrophage, neutrophil and T lymphocyte recruitment. More FOXP3+ T cells, lower CD8:CD4 or CD8:FOXP3 ratios and deleterious immune phenotype (CD68hiCD4hiFOXP3hiCD20hi) correlated with tumour proliferation, whereas M2:M1 ratio correlated with microvessel density and area. Invasive PAs had higher TAF-derived IL-6 levels, whereas TAFs from PA with more vessels and increased proliferation secreted more CCL2, both inhibited by pasireotide. GH3 cell-CM increased macrophage chemotaxis, while macrophage-CM/TAF-CM changed morphology, migration, invasion and EMT in GH3 cells. These data support that different TME elements affect PA tumourigenesis and aggressiveness. Data from different in vitro cell models suggest that AIP deficiency may not lead to differential cytokine secretion, and thus unlike to play a crucial role in the cytokine secretory function. The clinical study revealed that AIPmut and MEN1mut PA phenotypes are variable, including highly aggressive but also indolent cases such as prospectively-diagnosed AIPmut PAs, which are less aggressive and associated with more favourable clinical outcomes comparing to clinically-presenting AIPmut PAs, highlighting the benefits of AIP genetic and clinical screenings