c-Fos induction by gut hormones and extracellular ATP in osteoblastic-like cell lines

It is widely accepted that the c-Fos gene has a role in proliferation and differentiation of bone cells. ATP-induced c-Fos activation is relevant to bone homeostasis, because nucleotides that are present in the environment of bone cells can contribute to autocrine/paracrine signalling. Gut hormones have previously been shown to have an effect on bone metabolism. In this study, we used the osteoblastic Saos-2 cell line transfected with a c-Fos-driven reporter stimulated with five gut hormones: glucose inhibitory peptide (GIP), glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), ghrelin and obestatin, in the presence or absence of ATP. In addition, TE-85 cells were used to determine the time course of c-Fos transcript induction following stimulation with GLP-1, and GLP-2 with or without ATP, using reverse transcription qPCR. The significant results from the experiments are as follows: higher level of c-Fos induction in presence of GIP, obestatin (p = 0.019 and p = 0.011 respectively), and GIP combined with ATP (p < 0.001) using the luciferase assay; GLP-1 and GLP-2 combined with ATP (p = 0.034 and p = 0.002, respectively) and GLP-2 alone (p < 0.001) using qPCR. In conclusion, three of the gut peptides induced c-Fos, providing a potential mechanism underlying the actions of these hormones in bone which can be directed or enhanced by the presence of ATP

Fine-Tuning Roles of Endogenous Brain-Derived Neurotrophic Factor, TrkB and Sortilin in Colorectal Cancer Cell Survival

International audienceBACKGROUND: Neurotrophin receptors were initially identified in neural cells. They were recently detected in some cancers in association with invasiveness, but the function of these tyrosine kinase receptors was not previously investigated in colorectal cancer (CRC) cells. METHODS AND FINDINGS: We report herein that human CRC cell lines synthesize the neural growth factor Brain-derived neurotrophic factor (BDNF) under stress conditions (serum starvation). In parallel, CRC cells expressed high- (TrkB) and low-affinity (p75(NTR)) receptors at the plasma membrane, whereas TrkA and TrkC, two other high affinity receptors for NGF and NT-3, respectively, were undetectable. We demonstrate that BDNF induced cell proliferation and had an anti-apoptotic effect mediated through TrkB, as assessed by K252a, a Trk pharmacologic inhibitor. It suppressed both cell proliferation and survival of CRC cells that do not express TrkA nor TrkC. In parallel to the increase of BDNF secretion, sortilin, a protein acting as a neurotrophin transporter as well as a co-receptor for p75(NTR), was increased in the cytoplasm of primary and metastatic CRC cells, which suggests that sortilin could regulate neurotrophin transport in these cells. However, pro-BDNF, also detected in CRC cells, was co-expressed with p75(NTR) at the cell membrane and co-localized with sortilin. In contrast to BDNF, exogenous pro-BDNF induced CRC apoptosis, which suggests that a counterbalance mechanism is involved in the control of CRC cell survival, through sortilin as the co-receptor for p75(NTR), the high affinity receptor for pro-neurotrophins. Likewise, we show that BDNF and TrkB transcripts (and not p75(NTR)) are overexpressed in the patients' tumors by comparison with their adjacent normal tissues, notably in advanced stages of CRC. CONCLUSION: Taken together, these results highlight that BDNF and TrkB are essential for CRC cell growth and survival in vitro and in tumors. This autocrine loop could be of major importance to define new targeted therapies

P2Y2 and P2Y6 receptor activation elicits intracellular calcium responses in human adipose-derived mesenchymal stromal cells

Adipose tissue contains self-renewing multipotent cells termed mesenchymal stromal cells. In situ, these cells serve to expand adipose tissue by adipogenesis, but their multipotency has gained interest for use in tissue regeneration. Little is known regarding the repertoire of receptors expressed by adipose-derived mesenchymal stromal cells (AD-MSCs). The purpose of this study was to undertake a comprehensive analysis of purinergic receptor expression. Mesenchymal stromal cells were isolated from human subcutaneous adipose tissue and confirmed by flow cytometry. The expression profile of purinergic receptors was determined by quantitative real-time PCR and immunocytochemistry. The molecular basis for adenine and uracil nucleotide-evoked intracellular calcium responses was determined using Fura-2 measurements. All the known subtypes of P2X and P2Y receptors, excluding P2X2, P2X3 and P2Y12 receptors, were detected at the mRNA and protein level. ATP, ADP and UTP elicited concentration-dependent calcium responses in mesenchymal cells (N = 7–9 donors), with a potency ranking ADP (EC50 1.3 ± 1.0 μM) > ATP (EC50 2.2 ± 1.1 μM) = UTP (3.2 ± 2.8 μM). Cells were unresponsive to UDP (< 30 μM) and UDP-glucose (< 30 μM). ATP responses were attenuated by selective P2Y2 receptor antagonism (AR-C118925XX; IC50 1.1 ± 0.8 μM, 73.0 ± 8.5% max inhibition; N = 7 donors), and UTP responses were abolished. ADP responses were attenuated by the selective P2Y6 receptor antagonist, MRS2587 (IC50 437 ± 133nM, 81.0 ± 8.4% max inhibition; N = 6 donors). These data demonstrate that adenine and uracil nucleotides elicit intracellular calcium responses in human AD-MSCs with a predominant role for P2Y2 and P2Y6 receptor activation. This study furthers understanding about how human adipose-derived mesenchymal stromal cells can respond to external signalling cues

Diverse effect of BMP-2 homodimer on mesenchymal progenitors of different origin

Bone morphogenetic protein-2 (BMP-2), is a potential factor to enhance osseointegration of dental implants. However, the appropriate cellular system to investigate the osteogenic effect of BMP-2 in vitro in a standardized manner still needs to be defined. The aim of this study was to examine the effect of BMP-2 on the cell proliferation and osteogenic differentiation of human osteogenic progenitors of various origins: dental pulp stem cells (DPSC), human osteosarcoma cell line (Saos-2) and human embryonic palatal mesenchymal cell line (HEPM). For induction of osteogenic differentiation, cell culture medium was supplemented with BMP-2 homodimer alone or in combination with conventionally used differentiation inducing agents. Differentiation was monitored for 6-18 days. To assess differentiation, proliferation rate, alkaline phosphatase activity, calcium deposition and the expression level of osteogenic differentiation marker genes (Runx2, BMP-2) were measured. BMP-2 inhibited cell proliferation in a concentration and time-dependent manner. In a concentration which caused maximal cell proliferation, BMP-2 did not induce osteogenic differentiation in any of the tested systems. However, it had a synergistic effect with the osteoinductive medium in both DPSC and Saos-2, but not in HEPM cells. We also found that the differentiation process was faster in Saos-2 than in DPSCs. Osteogenic differentiation could not be induced in the osteoblast progenitor HEPM cells. Our data suggest that in a concentration that inhibits proliferation the differentiation inducing effect of BMP-2 is evident only in the presence of permissive osteoinductive components. beta-glycerophosphate, was identified interacting with BMP-2 in a synergistic manner

Cytotoxic effects of rabbit anti-thymocyte globulin preparations on primary human thymic epithelial cells

BACKGROUND: Graft-versus-host disease (GvHD) presents a major cause for morbidity and mortality after allogeneic hematopoietic stem cell transplantation (alloHSCT). Rabbit anti-thymocyte globulin (rATG) treatment reduces the incidence of GvHD after alloHSCT. However, delayed immune reconstitution following rATG treatment, partly due to hampered thymic function, is being discussed. The present study aimed at elucidating possible cytotoxic effects of two commonly used rATG preparations on cultured human thymic stroma, especially thymic epithelial cells (TECs). METHODS: A primary thymic epithelial cell culture was established and the binding and cytotoxicity of two rATG preparations to the aforementioned cells were assessed by flow cytometry and immunofluorescence analyses. The release of several cytokines by cultured TSCs in response to rATG was analyzed via multiplex ELISA. RESULTS: Both preparations showed a comparable dose-dependent binding to TECs and exerted a similar complement-independent, dose-dependent cytotoxicity. rATG exposure further resulted in hampered secretion of IL-7, IL-15 and IL-6, cytokines being involved in thymic T cell development and proliferation. Pretreatment with keratinocyte growth factor (KGF) diminished rATG-induced cytotoxicity of TECs and restored their IL-7 and IL-15 secretion. CONCLUSIONS: Cytotoxic effects on TECs link a rATG-induced thymic damage to the delayed T cell reconstitution witnessed after rATG treatment. Our data support a combination treatment of rATG and thymus-protective strategies such as KGF in order to simultaneously offer sufficient GvHD prophylaxis and overcome delayed T cell reconstitution due to thymic damage