Platelet-derived growth factor negatively regulates the insulin-like growth factor signaling pathway through the coordinated action of phosphatidylinositol 3-kinase and protein kinase C beta I

AbstractWe recently described that epidermal and fibroblast growth factors (EGF and FGF) regulate the IGF-I signaling pathway at the level of IRS-1 through the cooperative action of two independent signaling pathways; one dependent on phosphatidylinositol 3-kinase (PI 3-kinase) and the other on protein kinase D1 (PKD1) (Karam et al. [22]). To determine whether this mechanism could be generalized to another tyrosine kinase receptor-dependent growth factor, the effect of platelet-derived growth factor (PDGF) on the IGF-I signaling pathway was studied. PDGF inhibited IGF-I-stimulated IRS-1 tyrosine phosphorylation and subsequent IGF-I-induced PI 3-kinase activity, and stimulated IRS-1 serine 307 phosphorylation. These effects were mediated through a PI 3-kinase-dependent but extracellular signal-regulated kinase (ERK)-independent signaling pathway. However, PDGF-induced IRS-1 serine 307 phosphorylation was not sufficient per se to inhibit the IGF-I signaling but required another independent pathway. Noteworthy, although acutely stimulated by PDGF, and contrary to what we previously described (Karam et al. [22]), PKD1 did not associate with IRS-1and did not inhibit the IGF-I signaling in response to PDGF. However, we identified PKCβI as a new regulatory partner of PI 3-kinase for PDGF-induced inhibition of the IGF-I signaling pathway. Therefore, our results reinforce the idea that a coordinated action of two independent pathways seems absolutely necessary to negatively regulate IRS-1. Moreover, they also demonstrated that, depending of the cross-talk considered, subtle and specific regulatory mechanisms occur at the level of IRS-1 and that a unique regulatory model is not conceivable

Etude du rôle de la protéine kinase D1 dans les intercommunications entre les voies de signalisation des récepteurs à activité tyrosine kinase et dans la prolifération des cellules tumorales mammaires MCF-7

La protéine kinase D1, PKD1, est une nouvelle sérine/thréonine kinase activée par de nombreux mitogènes et dérégulée dans de nombreux types de cancers dont le cancer du sein, ce qui suggère un rôle de cette kinase dans la prolifération cellulaire et la tumorigenèse. Cependant, le rôle précis et les cibles de PKD1 ne sont pas encore bien connus. Au cours de ce travail, nous avons tout d abord démontré que PKD1 est activée par les facteurs de croissance épidermique (EGF) et fibroblastique (FGF) et qu elle régule la voie de signalisation de l insulin-like Growth Factor-I (IGF-I). D autre part, nos résultats démontrent que PKD1 favorise les propriétés pro-prolifératives et pro-tumorales des cellules MCF-7 dérivées d un adénocarcinome mammaire humain estrogéno-dépendant. Ces mécanismes mettent en jeu des voies de signalisation dépendantes de protéines kinases (la voie MEK/ERK) et hormonales (la voie estrogène/REa). Ainsi, l ensemble de ce travail fait apparaître PKD1 comme une nouvelle cible thérapeutique anti-tumorale potentielle.Protein kinase D1, PKD1, is a novel serine/threonine kinase which can be activated by mitogens and whose expression is altered in many tumors such as breast cancer, suggesting a role for this kinase in cancer development. However, its precise role and targets are still unclear. Our study identified PKD1 as a new regulatory kinase implicated in the control of IGF-I signal transduction pathway. Furthermore, we showed that PKD1 enhances estrogen-dependent MCF-7 breast cancer cell proliferation and tumorigenesis through the regulation of MEK/ERK and estrogen/ERa pathways. Thus, this work may define PKD1 as a novel potential anti-tumor therapeutic target.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

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

The Role of GH/IGF Axis in Dento-Alveolar Complex from Development to Aging and Therapeutics: A Narrative Review

International audienceThe GH/IGF axis is a major regulator of bone formation and resorption and is essential to the achievement of normal skeleton growth and homeostasis. Beyond its key role in bone physiology, the GH/IGF axis has also major pleiotropic endocrine and autocrine/paracrine effects on mineralized tissues throughout life. This article aims to review the literature on GH, IGFs, IGF binding proteins, and their respective receptors in dental tissues, both epithelium (enamel) and mesenchyme (dentin, pulp, and tooth-supporting periodontium). The present review re-examines and refines the expression of the elements of the GH/IGF axis in oral tissues and their in vivo and in vitro mechanisms of action in different mineralizing cell types of the dento-alveolar complex including ameloblasts, odontoblasts, pulp cells, cementoblasts, periodontal ligament cells, and jaw osteoblasts focusing on cell-specific activities. Together, these data emphasize the determinant role of the GH/IGF axis in physiological and pathological development, morphometry, and aging of the teeth, the periodontium, and oral bones in humans, rodents, and other vertebrates. These advancements in oral biology have elicited an enormous interest among investigators to translate the fundamental discoveries on the GH/IGF axis into innovative strategies for targeted oral tissue therapies with local treatments, associated or not with materials, for orthodontics and the repair and regeneration of the dento-alveolar complex and oral bones

IGFBP-1, -3, -5 and -6, but not IGFBP-2 and -4, associate with C2 cell surface.

<p>A. C2 cells were incubated for 1 hour at 4°C with or without IGFBP-1 to -6 (5 µg.mL^-1), fixed and incubated with specific primary antibodies and fluorescent secondary antibodies as indicated in Materials and Methods. Staining profiles were analyzed by FACS and mean fluorescence intensities (MFI) quantified. Data presented are representative of three independent experiments. B. C2 cells growing on coverslips were incubated with IGFBP-1 to -6 (5 µg.mL^-1) and then fixed without detergent. Cells were incubated with specific antibodies directed against IGFBP-1 to -6 processed with secondary antibodies stained either with Alexa 488 (IGFBP-1, -3, -4, -5) or Alexa 633 (IGFBP-2, -6). For each incubation condition, upper panel shows confocal fluorescent images and lower panel shows differential interference contrast images. Results are representative of four independent experiments.</p

IGFBP-2, -3, -4 and -6 increase intracellular calcium concentrations in MCF-7 cells.

<p>MCF-7 cells cultured on glass coverslips were incubated with Fura-2/AM. A. After background recording for 40 seconds to determine basal intracellular calcium concentrations as described in Methods, cells were incubated with IGFBP-6 (20 nM), then with IGFBP-3 (20 nM) and then with thapsigargin (Tp) (1 µM). The results of a typical experiment are shown in which the whole field (red line) was analysed and intracellular calcium quantified. The slides from left to right show representative views of the cells (a) before addition of IGFBP-6, (b) after addition of IGFBP-6, (c) after addition of IGFBP-3, and (d) after addition of thapsigargin. The results presented are representative of 5 independent experiments. B and C. Same experiments as in panel A excepted that cells were incubated with either 20 nM IGFBP-2 (panel B) or 20 nM IGFBP-4 (panel C). Slides show representative views of the cells after addition of IGFBP-2 or -4, respectively. Results presented are representative of 4 independent experiments. D. Quantitative analysis of the calcium response (maximal calcium response - basal calcium level) obtained for IGFBP-2, -4 and -6 in calcium free (empty bars) or calcium containing (hatched bars) medium. Results are the means ± SEM for three to five independent experiments. E. Dose-response curves for intracellular calcium concentrations were established as described in Materials and Methods. Values are expressed as percentages of the maximal response measured with 50 nM IGFBP-2 and are the mean for two independent experiments. F. Graphs present the average values of the intracellular calcium concentration modulated by addition of 20 nM of IGFBP-2, -4 or -6 following thapsigargin (1 µM) treatment in calcium free or containing medium as indicated.</p

IGFBPs increase intracellular calcium concentration via pertussis toxin-sensitive and -insensitive signaling pathways in MCF-7 and C2 proliferative cells.

<p>S: sensitive, NS: non sensitive.</p

IGFBP-1 to -6 associate with MCF-7 cell surface.

<p>A. MCF-7 cells were incubated for 1 hour at 4°C with or without IGFBP-1 to -6 (5 µg.mL^-1), fixed and incubated with specific primary antibodies and fluorescent secondary antibodies as indicated in Materials and Methods. Staining profiles were analyzed by FACS and mean fluorescence intensities (MFI) quantified. Data presented are representative of four independent experiments. B. MCF-7 cells growing on coverslips were incubated with IGFBP-1 to -6 (5 µg.mL^-1) and then fixed without detergent. Cells were incubated with specific antibodies directed against IGFBP-1 to -6 processed with secondary antibodies stained either with Alexa 488 (IGFBP-1, -3, -4, -5) or Alexa 633 (IGFBP-2, -6). For each incubation condition, upper panel shows confocal fluorescent images and lower panel shows differential interference contrast images. Results are representative of three independent experiments.</p

Protein Kinase D1 (PKD1) Is a New Functional Non-Genomic Target of Bisphenol A in Breast Cancer Cells

International audienceExposure to bisphenol A (BPA), one of the most widespread endocrine disruptors present in our environment, has been associated with the recent increased prevalence and severity of several diseases such as diabetes, obesity, autism, reproductive and neurological defects, oral diseases, and cancers such as breast tumors. BPA is suspected to act through genomic and non-genomic pathways. However, its precise molecular mechanisms are still largely unknown. Our goal was to identify and characterize a new molecular target of BPA in breast cancer cells in order to better understand how this compound may affect breast tumor growth and development. By using in vitro (MCF-7, T47D, Hs578t, and MDA-MB231 cell lines) and in vivo models, we demonstrated that PKD1 is a functional non-genomic target of BPA. PKD1 specifically mediates BPAinduced cell proliferation, clonogenicity, and anchorage-independent growth of breast tumor cells. Additionally, low-doses of BPA (≤10 −8 M) induced the phosphorylation of PKD1, a key signature of its activation state. Moreover, PKD1 overexpression increased the growth of BPA-exposed breast tumor xenografts in vivo in athymic female Swiss nude (Foxn1 nu/nu) mice. These findings further our understanding of the molecular mechanisms of BPA. By defining PKD1 as a functional target of BPA in breast cancer cell proliferation and tumor development, they provide new insights into the pathogenesis related to the exposure to BPA and other endocrine disruptors acting similarly