Cardiac hypertrophy is inhibited by a local pool of cAMP regulated by phosphodiesterase 2

Rationale: Chronic elevation of 3'-5'-cyclic adenosine monophosphate (cAMP) levels has been associated with cardiac remodelling and cardiac hypertrophy. However, enhancement of particular aspects of cAMP/protein kinase A (PKA) signalling appears to be beneficial for the failing heart. cAMP is a pleiotropic second messenger with the ability to generate multiple functional outcomes in response to different extracellular stimuli with strict fidelity, a feature that relies on the spatial segregation of the cAMP pathway components in signalling microdomains. Objective: How individual cAMP microdomains impact on cardiac pathophysiology remains largely to be established. The cAMP-degrading enzymes phosphodiesterases (PDEs) play a key role in shaping local changes in cAMP. Here we investigated the effect of specific inhibition of selected PDEs on cardiac myocyte hypertrophic growth. Methods and Results: Using pharmacological and genetic manipulation of PDE activity we found that the rise in cAMP resulting from inhibition of PDE3 and PDE4 induces hypertrophy whereas increasing cAMP levels via PDE2 inhibition is anti-hypertrophic. By real-time imaging of cAMP levels in intact myocytes and selective displacement of PKA isoforms we demonstrate that the anti-hypertrophic effect of PDE2 inhibition involves the generation of a local pool of cAMP and activation of a PKA type II subset leading to phosphorylation of the nuclear factor of activated T cells (NFAT). Conclusions: Different cAMP pools have opposing effects on cardiac myocyte cell size. PDE2 emerges as a novel key regulator of cardiac hypertrophy in vitro and in vivo and its inhibition may have therapeutic applications

Implication de cascades de signalisation et de gênes qu'elles induisent dans la prolifération des cellules thyroïdiennes

Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Identification of differentially expressed genes in thyrotropin stimulated dog thyroid cells by the cDNA-AFLP technique.

In dog thyrocytes in primary culture, thyrotropin (TSH), through cAMP, positively controls proliferation and differentiation. As until now, the key events and the genes involved in the action of TSH remain largely uncharacterized, our goal was to identify new differentially expressed genes in TSH-induced thyroid proliferation. Using cDNA-AFLP, we visualized 105 different transcripts showing significant differential expression during the stimulation of dog thyrocytes with TSH for different times, in the presence of insulin. Northern blot and RT-PCR analyses confirmed the pattern expression of 5 clones encoding known proteins: thrombospondine 1, TNFr1, RhoE, RalB, and annexin A2. These regulations provide molecular counterparts of in vivo physiological effects of TSH: angiogenesis (decreased thrombospondin 1), decreased apoptosis (decreased TNFR1) and actin filament disruption, macropinocytosis and thyroid hormone secretion (decreased RhoE).Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

The influence of anionic lipids on SHIP2 phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase activity.

The SH2 domain containing inositol 5-phosphatase 2 (SHIP2) catalyzes the dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) to phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P(2)) and participates in the insulin signalling pathway in vivo. In a comparative study of SHIP2 and the phosphatase and tensin homologue deleted on chromosome 10 (PTEN), we found that their lipid phosphatase activity was influenced by the presence of vesicles of phosphatidylserine (PtdSer). SHIP2 PtdIns(3,4,5)P(3) 5-phosphatase activity was greatly stimulated in the presence of vesicles of PtdSer. This effect appears to be specific for di-C8 and di-C16 fatty acids of PtdIns(3,4,5)P(3) as substrate. It was not observed with inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)) another in vitro substrate of SHIP2, nor with Type I Ins(1,4,5)P(3)/Ins(1,3,4,5)P(4) 5-phosphatase activity, an enzyme which acts on soluble inositol phosphates. Vesicles of phosphatidylcholine (PtdCho) stimulated only twofold PtdIns(3,4,5)P(3) 5-phosphatase activity of SHIP2. Both a minimal catalytic construct and the full length SHIP2 were sensitive to the stimulation by PtdSer. In contrast, PtdIns(3,4,5)P(3) 5-phosphatase activity of the Skeletal muscle and Kidney enriched Inositol Phosphatase (SKIP), another member of the mammaliam Type II phosphoinositide 5-phosphatases, was not sensitive to PtdSer. Our enzymatic data establish a specificity in the control of SHIP2 lipid phosphatase activity with PtdIns(3,4,5)P(3) as substrate which is depending on the fatty acid composition of the substrate.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Role of the different mitogen-activated protein kinase subfamilies in the stimulation of dog and human thyroid epithelial cell proliferation by cyclic adenosine 5'-monophosphate and growth factors.

We have investigated the role of the different classes of MAPKs, i.e. ERKs, c-Jun N-terminal kinases (JNKs), and p38 MAPK in the proliferation of dog and human thyroid epithelial cells (thyrocytes) in primary cultures. In these cells, TSH, acting through cAMP, epidermal growth factor, hepatocyte growth factor (HGF), and phorbol 12-myristate 13-acetate induce DNA synthesis. With the exception of HGF, all of these factors require the presence of insulin for mitogenic effects to be expressed. We found that TSH and forskolin are without effect on the phosphorylation and activity of the different classes of MAPKs. In contrast, all the cAMP-independent growth factors, whereas without effect on the phosphorylation and activity of JNKs and p38 MAPK, stimulated the ERKs. This effect was strong and sustained in response to HGF, epidermal growth factor and 12-myristate 13-acetate but weak and transient in response to insulin. Moreover, whereas in stimulated cells DNA synthesis was inhibited by PD 098059, an inhibitor of MAPK kinase 1 and consequently of ERKs, it was not modified by SB 203580, an inhibitor of p38 MAPK. Taken together, these data 1) exclude a role of JNKs and p38 MAPK in the proliferation of dog and human thyrocytes; 2) suggest that the mitogenic action of the cAMP-independent agents requires a strong and sustained activation of both ERKs and phosphatidylinositol 3-kinase/protein kinase B as realized by HGF alone or by the other agents together with insulin; and 3) show that TSH and cAMP do not activate ERKs but that the weak activation of ERKs by insulin is nevertheless necessary for DNA synthesis to occur.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Phosphatidylinositol 3-kinase, protein kinase B and ribosomal S6 kinases in the stimulation of thyroid epithelial cell proliferation by cAMP and growth factors in the presence of insulin.

The proliferation of most normal cells depends on the co-operation of several growth factors and hormones, each with a specific role, but the key events involved in the action of each necessary stimulant remain largely uncharacterized. In the present study, the pathways involved in the mechanism(s) of co-operation have been investigated in primary cultures of dog thyroid epithelial cells. In this physiologically relevant system, thyroid stimulating hormone (TSH) acting through cAMP, epidermal growth factor (EGF) and phorbol esters (such as PMA) induce DNA synthesis. Their effect requires stimulation of the insulin-like growth factor-1 (IGF-1) receptor by either IGF-1 or insulin, which are not themselves mitogenic agents. In contrast, hepatocyte growth factor (HGF) is itself fully mitogenic. The results of the study demonstrate that cAMP, EGF, HGF and PMA stimulate p70 ribosomal S6 kinase (p70 S6 kinase). However, insulin/IGF-1 also stimulate p70 S6 kinase. Thus stimulation of p70 S6 kinase might be necessary, but is certainly not sufficient, for the induction of DNA synthesis and is not specific for any stimulated pathway. In contrast, phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase B (PKB) activation by insulin and HGF is strong and sustained, whereas it is weak and transient with EGF and absent in the presence of TSH or PMA. These findings suggest that: (i) stimulation of PI 3-kinases and/or PKB is not involved in the cAMP-dependent pathways leading to thyrocyte proliferation, or in the action of PMA, (ii) the stimulation of the PI 3-kinase/PKB pathway may account for the permissive action of insulin/IGF-1 in the proliferation of these cells, and (iii) the stimulation of this pathway by HGF may explain why this agent does not require insulin or IGF-1 for its mitogenic action.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Association between th SH2-containig inositol polyphosphate 5-phosphatase 2 (SHIP2) and the adaptor protein APS has an impact on biochemical properties of both partners

info:eu-repo/semantics/nonPublishe

Activation of the small G protein Rap1 in dog thyroid cells by both cAMP-dependent and -independent pathways

Thyrotropin, through a cAMP-dependent pathway, stimulates function, differentiation, and proliferation of dog and human thyroid cells. Our previous findings suggested that, in addition to PKA activation, another cAMP-dependent mechanism is involved in TSH action. In this work, we assess whether the newly identified cAMP-Epac-Rap1 cascade is involved in TSH-cAMP-mediated effects in dog thyroid cells. We first demonstrate that TSH and forskolin strongly activate Rap1 in a PKA-independent manner. However, activation of Rap1 is not specific for TSH or cAMP. Indeed, carbachol, TPA, insulin, or EGF, which activate different cAMP-independent cascades, all independently activate Rap1. Rap1 is therefore a common step in all these cascades which exert various effects on proliferation, differentiation, and function of thyroid cells. Moreover, the microinjection of the Rap1 protein alone or in combination with the catalytic C subunit of PKA fails to induce proliferation or expression of thyroglobulin.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe