Insulin-like growth factor binding proteins increase intracellular calcium levels in two different cell lines.

BACKGROUND: Insulin-like growth factor binding proteins (IGFBPs) are six related secreted proteins that share IGF-dependent and -independent functions. If the former functions begin to be well described, the latter are somewhat more difficult to investigate and to characterize. At the cellular level, IGFBPs were shown to modulate numerous processes including cell growth, differentiation and apoptosis. However, the molecular mechanisms implicated remain largely unknown. We previously demonstrated that IGFBP-3, but not IGFBP-1 or IGFBP-5, increase intracellular calcium concentration in MCF-7 cells (Ricort J-M et al. (2002) FEBS lett 527: 293-297). METHODOLOGY/PRINCIPAL FINDINGS: We perform a global analysis in which we studied, by two different approaches, the binding of each IGFBP isoform (i.e., IGFBP-1 to -6) to the surface of two different cellular models, MCF-7 breast adenocarcinoma cells and C2 myoblast proliferative cells, as well as the IGFBP-induced increase of intracellular calcium concentration. Using both confocal fluorescence microscopy and flow cytometry analysis, we showed that all IGFBPs bind to MCF-7 cell surface. By contrast, only four IGFBPs can bind to C2 cell surface since neither IGFBP-2 nor IGFBP-4 were detected. Among the six IGFBPs tested, only IGFBP-1 did not increased intracellular calcium concentration whatever the cellular model studied. By contrast, IGFBP-2, -3, -4 and -6, in MCF-7 cells, and IGFBP-3, -5 and -6, in C2 proliferative cells, induce a rapid and transient increase in intracellular free calcium concentration. Moreover, IGFBP-2 and -3 (in MCF-7 cells) and IGFBP-5 (in C2 cells) increase intracellular free calcium concentration by a pertussis toxin sensitive signaling pathway. CONCLUSIONS: Our results demonstrate that IGFBPs are able to bind to cell surface and increase intracellular calcium concentration. By characterizing the IGFBPs-induced cell responses and intracellular couplings, we highlight the cellular specificity and complexity of the IGF-independent actions of these IGF binding proteins.

G protein-coupled receptor kinase 2 and group I metabotropic glutamate receptors mediate inflammation-induced sensitization to excitotoxic neurodegeneration.

OBJECTIVE: The concept of inflammation-induced sensitization is emerging in the field of perinatal brain injury, stroke, Alzheimer disease, and multiple sclerosis. However, mechanisms underpinning this process remain unidentified. METHODS: We combined in vivo systemic lipopolysaccharide-induced or interleukin (IL)-1ß-induced sensitization of neonatal and adult rodent cortical neurons to excitotoxic neurodegeneration with in vitro IL-1ß sensitization of human and rodent neurons to excitotoxic neurodegeneration. Within these inflammation-induced sensitization models, we assessed metabotropic glutamate receptors (mGluR) signaling and regulation. RESULTS: We demonstrate for the first time that group I mGluRs mediate inflammation-induced sensitization to neuronal excitotoxicity in neonatal and adult neurons across species. Inflammation-induced G protein-coupled receptor kinase 2 (GRK2) downregulation and genetic deletion of GRK2 mimicked the sensitizing effect of inflammation on excitotoxic neurodegeneration. Thus, we identify GRK2 as a potential molecular link between inflammation and mGluR-mediated sensitization. INTERPRETATION: Collectively, our findings indicate that inflammation-induced sensitization is universal across species and ages and that group I mGluRs and GRK2 represent new avenues for neuroprotection in perinatal and adult neurological disorders.

Activation of microglial N-methyl-D-aspartate receptors triggers inflammation and neuronal cell death in the developing and mature brain.

OBJECTIVE: Activated microglia play a central role in the inflammatory and excitotoxic component of various acute and chronic neurological disorders. However, the mechanisms leading to their activation in the latter context are poorly understood, particularly the involvement of N-methyl-D-aspartate receptors (NMDARs), which are critical for excitotoxicity in neurons. We hypothesized that microglia express functional NMDARs and that their activation would trigger neuronal cell death in the brain by modulating inflammation. METHODS AND RESULTS: We demonstrate that microglia express NMDARs in the murine and human central nervous system and that these receptors are functional in vitro. We show that NMDAR stimulation triggers microglia activation in vitro and secretion of factors that induce cell death of cortical neurons. These damaged neurons are further shown to activate microglial NMDARs and trigger a release of neurotoxic factors from microglia in vitro, indicating that microglia can signal back to neurons and possibly induce, aggravate, and/or maintain neurologic disease. Neuronal cell death was significantly reduced through pharmacological inhibition or genetically induced loss of function of the microglial NMDARs. We generated Nr1 LoxP(+/+) LysM Cre(+/-) mice lacking the NMDAR subunit NR1 in cells of the myeloid lineage. In this model, we further demonstrate that a loss of function of the essential NMDAR subunit NR1 protects from excitotoxic neuronal cell death in vivo and from traumatic brain injury. INTERPRETATION: Our findings link inflammation and excitotoxicity in a potential vicious circle and indicate that an activation of the microglial NMDARs plays a pivotal role in neuronal cell death in the perinatal and adult brain.

Structural localization and expression of CXCL12 and CXCR4 in rat heart and isolated cardiac myocytes.

CXCL12 (SDF-1), which binds CXCR4, is involved in several physiological and pathophysiological processes. In heart, this axis seems to play a key role in cardiogenesis and is involved in the neovascularization of ischemic tissues. Rats have three known CXCL12 mRNA isoforms, of which only alpha and gamma are present in the normal heart. However, little is known about CXCL12 protein expression and localization. We investigated the pattern of protein expression and the localization of both CXCR4 and CXCL12 in the heart, using isolated cardiomyocytes and a rat myocardial infarction model. Western blots showed that cardiomyocytes contained a specific 67-kDa CXCR4 isoform and a 12-kDa CXCL12 isoform. Confocal and electron microscopy clearly showed that CXCR4 was present at the plasmalemma and CXCL12 in continuity of the Z-line, in the proximal part of T-tubules. In conclusion, we provide the first description of the expression and fine localization of CXCR4 and CXCL12 proteins in normal rat heart and cardiomyocytes. These results suggest that the CXCL12/CXCR4 axis may be involved in cardiomyocyte calcium homeostasis regulation. Our work and the well-known chemoattraction properties of the CXCL12/CXCR4 axis highlight the importance of deciphering the function of this axis in both normal and pathological hearts.

Commutators of PAR-1 signaling in cancer cell invasion reveal an essential role of the Rho-Rho kinase axis and tumor microenvironment.

We recently reported that proteinase-activated receptors type I (PAR-1) are coupled to both negative and positive invasion pathways in colonic and kidney cancer cells cultured on collagen type I gels. Here, we found that treatments with the cell-permeant analog 8-Br-cGMP and the soluble guanylate cyclase activator BAY41-2272, and Rho kinase (ROK) inhibition by Y27632 or a dominant negative form of ROK lead to PAR-1-mediated invasion through differential Rac1 and Cdc42 signaling. Hypoxia or the counteradhesive matricellular protein SPARC/BM-40 (SPARC: secreted protein acidic rich in cysteine) overexpressed during cancer progression also commutated PAR-1 to cellular invasion through the cGMP/protein kinase G (PKG) cascade, RhoA inactivation, and Rac1-dependent or -independent signaling. Cultured primary cancer cells isolated from peritoneal and pleural effusions from patients with colon cancer or other malignant tumors harbored PAR-1, as shown by RT-PCR and FACS analyses. These malignant effusions also contained high levels of activated thrombin and fibrin, and induced a proinvasive response in HCT8/S11 human colorectal cancer cells. Our data underline the essential role of the tumor microenvironment and of several commutators targeting cGMP/PKG signaling and the RhoA-ROK axis in the control of PAR-1 proinvasive activity and metastatic potential of cancer cells in distant organs and peritoneal or pleural cavities. We also add new insights into the mechanisms linking the coagulation mediators thrombin and PAR-1 in the context of blood coagulation disorders and venous thrombosis often observed in cancer patients, as described in 1865 by Armand Trousseau.

VIP and PACAP induce selective neuronal differentiation of mouse embryonic stem cells.

The capacity of embryonic stem cells (ES cells) to differentiate into neuronal cells represents a potential source for neuronal replacement and a model for studying factors controlling early stages of neuronal differentiation. Various molecules have been used to induce such differentiation but so far neuropeptides acting via functional G-protein-coupled receptors (GPCRs) have not been investigated. Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are neuropeptides expressed in early development which affect neuronal precursor proliferation and neuronal differentiation. VIP and PACAP share two common receptors (VPAC1 and VPAC2 receptors) while only PACAP binds with high affinity to PAC1 receptors. The aim of the study was to determine whether VIP and PACAP could produce functional neuronal differentiation of ES cells. Mouse ES cells were allowed to aggregate in embryoid bodies (EBs) in the presence or not of VIP and PACAP for 1 week. VIP and PACAP potently increased the proportion of EB-derived cells expressing specifically a neuronal phenotype shown by immunocytochemistry and neurite outgrowth without altering glial cell number. Binding and RT-PCR analyses demonstrated the presence of VPAC2 and PAC1 receptors on ES cells. Accordingly, both peptides increased cyclic AMP and intracellular calcium. In contrast, EB-derived cells only expressed a functional PAC1 receptor, suggesting a switch in GPCR phenotype during ES cell differentiation. These original data demonstrate that functional GPCRs for VIP and PACAP are present on ES cells and that these neuropeptides may induce their differentiation into a neuronal phenotype. It opens an exciting new field for neuropeptide regulation of tissue ontogenesis.

The toxicity of H2O2 on the ionic homeostasis of airway epithelial cells in vitro.

Oxygen species may be formed in the air spaces of the respiratory tract in response to environmental pollution such as particulate matter. The mechanisms and target molecules of these oxidants are still mainly unknown but may involve modifications of the ionic homeostasis in epithelial cells. Cytosolic concentrations of Ca2+ (Fura2) and Na+ (SBFI) and short-circuit current (Isc) were followed in primary cultures of human nasal epithelial cells and in the cell line 16HBE14o- after exposure to H2O2 or *OH (H2O2 + Fe2+). Cells were grown on glass coverslips for ionic imaging or on permeable snapwell inserts for Isc studies. Exposure of the apical as well as the basal side of the cultures to H2O2 or *OH induced a concentration-dependent transient increase in Isc which is due to a transient secretion of Cl-. Cai also increased transiently with approximately the same kinetics. The response was dependent on the release of calcium from intracellular stores. Nai on the contrary increased steadily over more than an hour. When the apical membrane was permeabilized with gramicidin, *OH inhibited the Na+ current (a measure of Na(+)-K(+)-ATPase activity in the baso-lateral membrane). The arrest of the pump was significant after 30 min exposure to oxidant. On the other hand no increase in the apical or baso-lateral sodium conductances could be detected. The progressive arrest of the Na+/K(+)-pump may contribute to the sustained elevation of Nai. This strong modification in the cellular ionic homeostasis may participate in the stress response of the respiratory epithelium through alterations in signal transduction pathways.

CCN3 and calcium signaling.

The CCN family of genes consists presently of six members in human (CCN1-6) also known as Cyr61 (Cystein rich 61), CTGF (Connective Tissue Growth Factor), NOV (Nephroblastoma Overexpressed gene), WISP-1, 2 and 3 (Wnt-1 Induced Secreted Proteins). Results obtained over the past decade have indicated that CCN proteins are matricellular proteins, which are involved in the regulation of various cellular functions, such as proliferation, differentiation, survival, adhesion and migration. The CCN proteins have recently emerged as regulatory factors involved in both internal and external cell signaling. CCN3 was reported to physically interact with fibulin-1C, integrins, Notch and S100A4. Considering that, the conformation and biological activity of these proteins are dependent upon calcium binding, we hypothesized that CCN3 might be involved in signaling pathways mediated by calcium ions.In this article, we review the data showing that CCN3 regulates the levels of intracellular calcium and discuss potential models that may account for the biological effects of CCN3.

Characterization and functionality of CXCR4 chemokine receptor and SDF-1 in human corneal fibroblasts.

PURPOSE: The aim of this study was to investigate whether cultured human corneal fibroblasts express functional chemokine CXCR4 receptors on their cell surface and to determine the presence of its specific ligand, SDF-1 (CXCL12), in human corneal fibroblasts. METHODS: Human corneal fibroblast cultures were obtained using human donor corneas. CXCR4 receptors were characterized using binding studies and autoradiography with [125I]SDF-1. The functionality of CXCR4 receptors was assessed by intracellular calcium measurement using a dynamic imaging microscopy system. CXCR4 and SDF-1 mRNA were detected in human corneal fibroblasts using reverse transcriptase polymerase chain reaction (RT-PCR). The CXCR4 protein was detected by western blot analysis. RESULTS: [125I]SDF-1 specifically bound to cultured corneal fibroblasts with a KD value of 8.3+/-1.2 nM. The presence of CXCR4 was confirmed by autoradiography of the radioligand on slices of corneal stroma. SDF-1 induced a rapid and transient intracellular calcium increase in cultured corneal fibroblasts that was blocked by the specific antagonist bicyclam. Moreover, a 48 kDa protein was detected by western blot analysis of corneal fibroblast extracts, using a specific CXCR4 polyclonal antibody. RT-PCR showed the expression of both CXCR4 and SDF-1 mRNAs in human corneal fibroblasts. CONCLUSIONS: These results indicate for the first time that cultured human corneal fibroblasts express the chemokine receptors CXCR4 and its ligand SDF-1. This latter might exert physiological effects on the cornea and could be involved in pathological conditions such as corneal angiogenesis.

Effects of endothelin-1 on epithelial ion transport in human airways.

Endothelin-1 (ET-1) exerts many biological effects in airways, including bronchoconstriction, airway mucus secretion, cell proliferation, and inflammation. We investigated the effect of ET-1 on Na absorption and Cl secretion in human bronchial epithelial cells. Addition of 10(-7) M ET-1 had no effect on the inhibition of the short circuit current (Isc) induced by amiloride, a Na channel blocker. Addition of 10(-7) M ET-1 to the apical bath in the presence of amiloride increased Isc in cultured human bronchial epithelial cells studied in Ussing chambers. No effect was observed when ET-1 was added to basolateral bath, indicating that the involved ET-1 receptors are likely present only in the apical membrane of the cells. Use of Cl-free solutions and bumetanide reduced the ET-1-induced increases in Isc, indicating that ET-1 stimulates Cl secretion. The ET-1-induced increase in Isc was prevented by exposure to the ETB receptor antagonist BQ-788 but not to the ETA receptor antagonist BQ-123. ET-1 did not raise intracellular Ca levels, but increased the intracellular concentration of cAMP. These findings indicate that ET-1 is a Cl secretagogue in human airways and acts presumably through apically located ETB receptors and activation of the cAMP pathway.

Insulin-like growth factor binding protein-3 increases intracellular calcium concentrations in MCF-7 breast carcinoma cells.

Insulin-like growth factor binding protein-3, IGFBP-3, specifically binds to IGFs with high affinity, but it is also capable of modulating the IGF-I signalling pathway or inducing apoptosis independently of its binding to IGFs. The molecular mechanisms underlying the action of IGFBP-3 have not been elucidated. In this study, we have demonstrated that binding of IGFBP-3 to a cell surface receptor in MCF-7 breast carcinoma cells induces a rapid and transient increase in intracellular free calcium. This increase was mediated via a pertussis toxin-sensitive pathway, indicating that the IGFBP-3 receptor may be specifically coupled to a Gi protein. The effect of IGFBP-3 on calcium concentrations was dose-dependent and also occurred when IGFBP-3 was complexed with either IGF-I or heparin, suggesting that the receptor binding site is probably located in the least conserved central domain of IGFBP-3. Neither IGFBP-1, nor IGFBP-5 (structurally the closest to IGFBP-3) altered intracellular calcium concentrations. These results provide evidence that a specific intracellular signal is triggered by IGFBP-3 binding to a cell surface receptor.

Expression of neurotensin receptors in human corneal keratocytes.

PURPOSE: The purpose of the study was to investigate whether cultured human keratocytes express the neurotensin receptors (NTR1, NTR2, and NTR3), to determine the presence of neurotensin (NT) in keratocytes, and to assess the influence of NT on these cells. METHODS: Human keratocytes were cultured in medium treated with various concentrations (10(-7)-10(-9) M) of JMV449 (a weakly degradable NT agonist). Cell proliferation and viability were analyzed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay. Apoptosis was studied by nucleus labeling with a fluorescent dye and cold light fluorometry. NT, NTR1, NTR2, and NTR3 mRNA were detected in human keratocytes by means of reverse transcriptase-polymerase chain reaction (RT-PCR). NTR1 protein was detected by Western blot analysis. Functionality of NTR1 was assessed by intracellular calcium ([Ca2+]i) measurement with a dynamic imaging microscopy system. RESULTS: RT-PCR and Western blot analysis showed the expression of the NTR1 (mRNA and protein) and NTR3 mRNA in human corneal keratocytes. NT and NTR2 mRNA were undetectable. JMV449 induced a rapid and transient [Ca2+]i increase in human corneal keratocytes that was blocked by the specific antagonist SR48692. JMV449 significantly increased cell proliferation and viability after 72, 96, and 120 hours of culture, with a maximum effect at 10(-7) M (P < 0.005). Finally, JMV449 decreased keratocyte apoptosis, whatever the concentration used (P < 0.005). CONCLUSIONS: These results indicate that cultured human keratocytes express NTR1 and NTR3 and that NT may exert physiological effects on cornea such as regulation of keratocyte proliferation and apoptosis.

Europium-labeled epidermal growth factor and neurotensin: novel probes for receptor-binding studies.

We investigated the possibility of labeling two biologically active peptides, epidermal growth factor (EGF) and neurotensin (NT), with europium (Eu)-diethylenetriaminepentaacetic acid. More specifically, we tested them as probes in studying receptor binding using time-resolved fluorescence of Eu3+. The relatively simple synthesis yields ligands with acceptable binding characteristics similar to isotopically labeled derivatives. The binding affinity (Kd) of labeled Eu-EGF to human A431 epidermal carcinoid cells was 3.6 +/- 1.2 nM, similar to the reported Kd values of EGF, whereas the Kd of Eu-NT to human HT29 colon cancer cells (7.4 +/- 0.5 nM) or to Chinese hamster ovary (CHO) cells transfected with the high-affinity NT receptor (CHO-NT1) were about 10-fold higher than the Kd values of NT. The bioactivity of the Eu-labeled EGF as determined by stimulation of cultured murine D1 hematopoietic cell proliferation was nearly the same as that obtained with native EGF. The maximal stimulation of Ca2+ influx with NT and Eu-NT in CHO-NT1 cells was similar, but the respective K0.5 values were 20 pM and 1 nM, corresponding to differences in the binding affinities previously described. The results of these studies indicate that Eu labeling of peptide hormones and growth factor molecules ranging from 10(3) to 10(5) Da can be conveniently accomplished. Importantly, the Eu-labeled products are stable for approximately 2 years and are completely safe for laboratory use compared to the biohazardous radioligands. Thus, Eu-labeled peptides present an attractive alternative for commonly used radiolabeled ligands in biological studies in general and in receptor assays in particular.