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Thrombospondin-1, a multi-modular matrix protein is able to interact with a variety of matrix proteins and cell-surface receptors. Thus it is multifunctional. In this work, we examined the role of thrombospondin-1 in ceramide-induced thyroid apoptosis. We focused on the VVM containing sequence localized in the C-terminal domain of the molecule. Primary cultured thyroid cells synthesize thrombospondin-1 depending on their morphological organization. As it leads thyrocytes to organize into monolayers before inducing apoptosis ceramide can modulate this organization. Here, we established that C(2)-ceramide treatment decreased thrombospondin-1 expression by interfering with the adenylyl cyclase pathway, thus leading to apoptosis. Furthermore, we demonstrated that the thrombospondin-1-derived peptide 4N1 (RFYVVMWK) abolished ceramide-induced thyroid cell death by preventing intracellular cAMP levels from dropping. Finally, we reported that 4N1-mediated inhibition of ceramide-induced apoptosis was consistently associated with a down-regulation of the caspase-3 processing. Integrin-associated protein receptor (IAP or CD47) was identified as a molecular relay mediating the observed 4N1 effects. Taken together, our results shed light for the first time on anti-apoptotic activities of the thrombospondin-1-derived peptide 4N1 and provide new information on how thrombospondin-1 may control apoptosis of non-tumoral cells.

Camptothecin and doxorubicin belong to a family of anticancer drugs that exert cytotoxic effects by triggering apoptosis in various cell types. However there have only been few investigations showing that matricellular proteins like thrombospondin-1 (TSP-1) could be involved in the underlying mechanism of this cytotoxicity. In this report, using Hoechst reagent staining, reactive oxygen species production and caspase-3 activity measurement, we determined that both camptothecin and doxorubicin induced apoptosis in human thyroid carcinoma cells (FTC-133). On the one hand, we demonstrated that camptothecin and doxorubicin inhibited TSP-1 expression mainly occurring at the transcriptional level. On the other hand, drug-induced apoptosis determined by western blot analysis for PARP cleavage and caspase-3 activity measurement, was significantly decreased in presence of exogenous TSP-1. In order to identify the sequence responsible for this effect, we used the CD47/IAP-binding peptide 4N1 (RFYVVMWK), derived from the C-terminal domain of TSP-1, and known to play a role in apoptosis. Thus, in presence of 4N1, camptothecin and doxorubicin-induced pro-apoptotic activity was considerably inhibited. These findings suggest that induction of apoptosis by camptothecin or doxorubicin in FTC-133 cells is greatly dependent by a down-regulation of TSP-1 expression and shed new light on a possible role for TSP-1 in drug resistance.

Interleukin-1 beta (IL-1beta) is an important regulator of the thyroid cell function. This cytokine has been largely described to trigger an important biological signaling cascade: the sphingomyelin/ceramide pathway. In this report, we show that IL-1beta induces the transient activation of a neutral sphingomyelinase in porcine thyroid cells. Moreover, IL-1beta and ceramides are demonstrated to inhibit the TSH-induced cAMP production via the implication of alphaGi subunit of the adenylyl cyclase system. This crosstalk between cAMP and ceramide pathways constitutes a preponderant process in the TSH-controlled differentiation state of thyrocytes. All these results argue for the involvement of ceramides and IL-1beta in the thyroid function regulation, leading to a cell dedifferentiated state.

Thrombospondins belong to a family of extracellular matrix (ECM) proteins widely found from embryonic to adult tissues. The modular structure of thrombospondins contains a series of peptide sequences implicated in a multiplicity of biological functions. Extracellular matrix undergoes important alterations under proteolysis that occurs in pathological processes like tumorigenesis. An elevated secretion of thrombospondin 1 (TSP1) is often observed in tumors and is sometimes considered as a predictive factor. However, the role of TSP1 in cancer progression remains controversial and must be carefully apprehended. The regulation of cell adhesion, proliferation, apoptosis by TSP1 is examined in the present review and it is clear from the literature and from our investigations that TSP1 presents both stimulatory and inhibitory effects. The exposition of cryptic sites upon conformational changes can partially explain this contradiction. More interestingly, the analysis of TSP1-directed intracellular signaling pathways activated through specific receptors or supramolecular receptors docking systems may be useful to discriminate the precise function of TSP1 in tumor progression. The central role played by TSP1 in the control of matrix-degrading enzyme activation and catabolism reveals attractive tracks of research and highlights the involvement of the lipoprotein receptor-related protein (LRP) receptor in these events. Therefore, TSP1-derived peptides constitute a source of potentially active matrikins which could provide essential tools in cancer therapy.

In emission tomography, quantification of brain tracer uptake, metabolism or binding requires knowledge of the cerebral input function. Traditionally, this is achieved with arterial blood sampling. We propose a noninvasive alternative via the use of a blood vessel time-activity curve (TAC) extracted directly from dynamic positron emission tomography (PET) scans by cluster analysis. Five healthy subjects were injected with the 5HT2A- receptor ligand [18F]-altanserin and blood samples were subsequently taken from the radial artery and cubital vein. Eight regions-of-interest (ROI) TACs were extracted from the PET data set. Hierarchical K-means cluster analysis was performed on the PET time series to extract a cerebral vasculature ROI. The number of clusters was varied from K = 1 to 10 for the second of the two-stage method. Determination of the correct number of clusters was performed by the 'within-variance' measure and by 3D visual inspection of the homogeneity of the determined clusters. The cluster-determined input curve was then used in Logan plot analysis and compared with the arterial and venous blood samples, and additionally with one of the currently used alternatives to arterial blood sampling, the Simplified Reference Tissue Model (SRTM) and Logan analysis with cerebellar TAC as an input. There was a good agreement (P < 0.05) between the values of Distribution Volume (DV) obtained from the K-means-clustered input function and those from the arterial blood samples. This work acts as a proof-of-principle that the use of cluster analysis on a PET data set could obviate the requirement for arterial cannulation when determining the input function for kinetic modelling of ligand binding, and that this may be a superior approach as compared to the other noninvasive alternatives. © 2003 Elsevier Inc. All rights reserved.