Eric Raspé

THYROTROPIN ACTIVATES BOTH THE CYCLIC AMP AND THE PIP2 CASCADES IN CHO CELLS EXPRESSING THE HUMAN CDNA OF TSH RECEPTOR

The effect of thyrotropin (TSH) on cyclic AMP accumulation, phosphatidylinositol bisphosphate (PIP2) hydrolysis and [Ca2+]i rise has been studied in CHO cells stably transfected with human TSH receptor (hTSHR) cDNA. In human thyroid slices, TSH activates these two intracellular cascades with a higher affinity for the adenylate cyclase activation (from 0.1 to 1 mU/ml TSH) than for phospholipase C activation (from 1 to 10 mU/ml TSH). The CHO cells transfected with the recently cloned cDNA of human TSH receptor respond in the same way to TSH. They respond between 0.1 and 1 mU/ml TSH for cyclic AMP accumulation and between 1 and 10 mU/ml TSH for inositol monophosphate (IP1) increase. In these same cells, TSH 10 mU/ml, but not forskolin (10 microM), or dibutyryl cyclic AMP (100 microM), clearly enhances intracellular calcium concentration [( Ca2+]i). Our results demonstrate unequivocally that a single transcription unit has the potential to encode receptor molecules coupled to both cascades.

Molecular cloning, functional expression and pharmacological characterization of a human bradykinin B2 receptor gene.

The gene encoding a putative G protein-coupled receptor (HG10) was cloned from human genomic DNA by low stringency PCR and found to be homologous to the recently described rat bradykinin B2 receptor. The receptor was expressed in xenopus oocytes and stably transfected CHO cell lines. Binding studies demonstrated that HG10 encodes a high affinity BK receptor with an apparent Kd of 150 pM. Displacement by BK agonists and antagonists allowed the characterization of the receptor as a B2 subtype. Functional coupling to the Ca(2+)-phosphatidylinositol cascade was demonstrated in transfected CHO cells where inositol phosphates accumulation and intracellular calcium concentration were elevated in response to BK stimulation. The agonistic and antagonistic properties of BK analogs do not match strictly the pharmacological profile described for the rat or guinea pig B2 receptor subtypes or the putative B3 subtype. This discrepancy is attributed either to species variability or to differences in the coupling efficiency of receptors to the transduction cascade in different cell types. From our results, the existence of B3 receptors and of B2 subtypes appears questionable.

Coexpression of P2Y and P2U Receptors on Aortic Endothelial Cells : Comparison of Cell Localization and Signaling Pathways

Depending on the vascular bed considered, the actions of ATP on the endothelium are mediated by either P2Y or P2U receptors. The two types of receptors seem to coexist on bovine aortic endothelial cells, where they are both coupled to phospholipase C. In this study, we have investigated whether they are truly coexpressed on the same cells and whether their signaling pathways diverge beyond phospholipase C activation. Measurements of [Ca2+]i in single cells showed that almost all bovine aortic endothelial cells are responsive to both 2-methylthio-ATP (2MeSATP), an agonist of P2Y receptors, and UTP, an agonist of P2U receptors. UTP stimulated the release of prostacyclin from freshly isolated bovine aortic endothelial cells, even when they were exposed to cycloheximide at the time of their collection: this indicates that P2U receptors must already be expressed on endothelial cells in situ and do not appear during cell culture. The time course of inositol phosphate (InsP) accumulation and the relative proportion of Ins(1,4,5)P3, Ins(1,3,4,5)P4, and Ins(1,3,4)P3 were similar in cells stimulated by 2MeSATP or UTP. UTP and 2MeSATP both stimulated the hydrolysis of phosphatidylcholine by phospholipase D, as reflected by the release of [3H]choline from prelabeled cells. The responses to both agents were blocked after downregulation of protein kinase C, resulting from a prolonged exposure to phorbol 12-myristate 13-acetate: this blockade occurred at a step distal to phospholipase C activation. A single difference between the two pathways has been identified: the effect of 2MeSATP on InsP3 was significantly more inhibited after a short exposure to phorbol 12-myristate 13-acetate than that of UTP.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP, bradykinin, TRH and TSH activate the Ca2+-phosphatidylinositol cascade of human thyrocytes in primary culture

We have recently shown that adenosine triphosphate (ATP), bradykinin and thyrotropin-releasing hormone (THR) increase the ([Ca2+]i) of human thyrocytes in primary culture. We show here that these agents also stimulate the generation of [3H]-inositol monophosphate (IP1), inositol bisphosphate (IP2) and inositol trisphosphate (IP3). The stimulation of IP3 generation followed two distinct kinetics: it was sustained when the cells were triggered with ATP and transient when the response was elicited by TRH or bradykinin. In addition, we have shown that under the appropriate experimental conditions, high thyroid-stimulating hormone (TSH) concentrations were also able to stimulate human thyrocyte IP1, IP2 and IP3 accumulation and to increase their [Ca2+]i. These data suggest that ATP, bradykinin, TRH and high TSH concentrations activate the Ca(2+)-phosphatidylinositol cascade of human thyrocytes. Since this cascade plays a crucial role in the control of protein iodination, ATP, TRH and bradykinin could be important regulators of thyroid hormone synthesis in human thyrocytes.

Lack of correlation between the activation of the Ca2+ phosphatidylinositol cascade and the regulation of DNA synthesis in the dog thyrocyte

Changes in the [Ca2+]i and/or activation of phospholipase C are thought to participate in the control by several growth factors of the mammalian cell proliferation. It has even been claimed that activation of the Ca(2+)-phosphatidylinositol cascade is sufficient to elicit cell proliferation [Jackson et al. (1988) Nature 335, 437-440; Julius et al. (1989) Science 244, 1057-1062]. In this work, we have evaluated the control of DNA synthesis by this cascade in a differentiated epithelial cell model: the dog thyrocyte in primary culture. We first observed that potent activators of the dog thyrocyte (2+)-phosphatidylinositol cascade such as carbachol or bradykinin failed to promote the onset of DNA synthesis in these cells. Moreover, carbachol inhibited the mitogenic effect of thyroid stimulating hormone (TSH) and of epidermal growth factor (EGF). The mitogenic effect of EGF was also reduced by bradykinin. Nevertheless, carbachol enhanced the expression of the protooncogenes c-fos and c-myc mRNAs. The time course of this enhancement was identical to the time course for the induction of c-fos and c-myc mRNAs by phorbol esters or EGF. On the other hand, in most experiments, TSH and EGF were able to trigger the onset of dog thyrocyte DNA synthesis without affecting their intracellular free Ca2+ concentration [Ca2+]i, 45Ca2+ efflux, or inositol phosphate generation. In several experiments, TSH increased the dog thyrocyte 45Ca2+ release and promoted a rise in the [Ca2+]i or the inositol phosphate accumulation but these effects were weak. In contrast to the effect of carbachol, the TSH effects on the [Ca2+]i and the 45Ca2+ efflux appeared slowly, were sustained, and were extremely sensitive to extracellular Ca2+ depletion. They were observed at hormone concentrations higher than the concentration achieving maximal stimulation of DNA synthesis. Similarly, in a few experiments, a slight increase in the [Ca2+]i or in the inositol trisphosphate generation were provoked by EGF. However, these modifications were not associated with an increased mitogenic potency of EGF. Finally, in all experiments, fetal calf serum slightly accelerated the dog thyrocyte 45Ca2+ efflux and increased their inositol phosphate generation.(ABSTRACT TRUNCATED AT 400 WORDS)

Pendrin: the Thyrocyte Apical Membrane Iodide Transporter?

In the thyroid, the transport of iodide from the extracellular space to the follicular lumen requires two steps: the transport in the cell at the basal side and in the lumen at the apical side. The first step is mediated by the Na+/I- symporter (NIS). In most reviews and textbooks, the second step is presented as mediated by pendrin. In this review, we analyze this assumption. There are several arguments supporting the concept that indeed pendrin plays an important role in thyroid physiology. However, biochemical, clinical and histological data on the thyroid of a patient with Pendred syndrome do not suggest an essential role in iodide transport, which is corroborated by the lack of a thyroid phenotype in pendrin knockout mice. Experiments in vivo and in vitro on polarized and unpolarized cells show that iodide is transported transport of iodide at the apex of the thyroid cell. Moreover, ectopic expression of pendrin in transfected non-thyroid cells is capable of mediating iodide efflux. It is concluded that pendrin may participate in the iodide efflux into thyroid lumen but not as the unique transporter. Moreover, another role of pendrin in mediating Cl-/HCO3- exchange and controlling luminal pH is suggested.

P2-purinergic receptors in vascular endothelial cells: from concept to reality.

ATP exerts at least 2 actions on arterial endothelial cells: it stimulates the release of endothelium-derived relaxing factor, a still unidentified vasodilator, and of prostacyclin, a potent inhibitor of platelet aggregation. A study of agonist specificity indicates that these responses are mediated by P2-purinergic receptors. We have now demonstrated that in these cells, the P2-receptors are coupled to a phospholipase C hydrolysing phosphatidylinositol-bisphosphate and that this coupling involves a pertussis toxin-sensitive GTP-binding regulatory protein.

Carbamylcholine, TRH, PGF2 alpha and fluoride enhance free intracellular Ca++ and Ca++ translocation in dog thyroid cells.

Effects on Ca++ translocation and [Ca++]i were studied in dog thyroïd cell monolayers using both 45Ca++ efflux and the indicator quin-2. Carbamylcholine, a non hydrolysable analog of acetylcholine, through muscarinic receptors, and to a lesser extent TRH and PGF2 alpha increased both these parameters. [Ca++]i increased by 171, 100 and 75% respectively over a basal level of 66 +/- 17 nM (mean +/- SD). The response to carbamylcholine was biphasic. A transient increase in [Ca++]i was followed by a more sustained phase where the [Ca++]i was slightly higher than the basal level. Only the first phase was insensitive to extracellular Ca++ depletion. This phase is probably due to a release of Ca++ from an intracellular store. NaF also induced a sustained rise in [Ca++]i dependent on extracellular Ca++ and affected 45Ca++ efflux. Our data provide direct evidence of an implication of intracellular Ca++ in the response of dog thyroïd cells to all these agents.

Transducing Systems in the Control of Human Thyroid Cell Function, Proliferation and Differentiation

Our laboratory has been involved in the study of thyroid regulation at the cellular level for many years. The complex picture emerging from these studies leads to conclusions of general relevance. The regulation of the thyroid cell was once a classical example of the concept one hormone — one cell type — one intracellular secondary messenger with its pleiotypic effects. It should now rather be considered as a network of crosslinked regulatory steps where the extracellular and intracellular signal-molecules act on their receptors as bits of information in an electronic circuit, i.e., express on/off regulations with no definite general physiological meaning per se. Such networks differ from one cell type to another and for a given cell type from one species to another. In the case of the thyroid many apparent discrepancies in the literature are explained if this is taken into account. In this presentation, we wish to draw mainly on the results of our group to illustrate this point with regard to the regulation of function, proliferation and differentiation of the thyroid cell.

CDK4 phosphorylation status and a linked gene expression profile predict sensitivity to palbociclib

Cyclin D‐CDK4/6 are the first CDK complexes to be activated in the G1 phase in response to oncogenic pathways. The specific CDK4/6 inhibitor PD0332991 (palbociclib) was recently approved by the FDA and EMA for treatment of advanced ER‐positive breast tumors. Unfortunately, no reliable predictive tools are available for identifying potentially responsive or insensitive tumors. We had shown that the activating T172 phosphorylation of CDK4 is the central rate‐limiting event that initiates the cell cycle decision and signals the presence of active CDK4. Here, we report that the profile of post‐translational modification including T172 phosphorylation of CDK4 differs among breast tumors and associates with their subtypes and risk. A gene expression signature faithfully predicted CDK4 modification profiles in tumors and cell lines. Moreover, in breast cancer cell lines, the CDK4 T172 phosphorylation best correlated with sensitivity to PD0332991. This gene expression signature identifies tumors that are unlikely to respond to CDK4/6 inhibitors and could help to select a subset of patients with HER2‐positive and basal‐like tumors for clinical studies on this class of drugs.