Bernard Haye

Control by TSH of a phospholipase A2 activity, a limiting factor in the biosynthesis of prostaglandins in the thyroid

Tbyreostimuhn QTSH) stimulates the thyroid sdenyi-cyclase and increases the level of cyclic 3*-S’adcnosine monophosphate (c-AMP)_ The model of Sutherland, in which c-AMP is the second messenger, explains many effects of T§H [ t 1. On the creher hand, the prostagtandins (EYE2 and PGFZa) which are abondane in the ehyroid [2], seimulate the thyroid adenyl-cyciase f3--51. Recently Burke 161 has shown an increase in the ineracellu%ar concentration of thy&d proseaglandins under the infh.rence of TSH, The hypothesis of Kuehl [7-S], according to which the action of the luteinizin~ hormone @JI) on the ovary adenyl-cyclase svould necessarily involve a stimulation of prostaglandins biosynthesis, has been applied to the ehyroid by Burke [4-g] _ Qr; the other side, several authors have reached the conclusion that the release of prostaglandins from a tissue was due to a neo-synthesis and not to a depletion of storage material [lo, I 9 1: The efficiency of the prostagiandin synthetase seems to depend only on the amount of suhstrate available, as indeed the addition of arachidonic acid causes a rapid formation of ‘prostaglandins [ 12,13] . The polyunsaturated fatty acids, precursors of the prostaghmdins, exist at a low concentration as free acids, but their concentration is not negligible in the membrane phosphohpids. It has been suggested that the a&hey ofendogenous phospholipases A could be the factor regulating the iriosynthesis of prostagEandins [ 14, IS] _ It has been demonstrated that phospholipids can play the role of precursors in the biosynthesis of prostaglandins [ 16, 171 and that prior treatment with a venom phosphoIipase A increases the conversion yield [ 15,17f . fn this work we have studied 3;~ one hand whether the prostaglandins are obligatory intermediates in ihe action of TSH on the adenyl-cycLse, as suggested by Burke, and on the other hand if there is a TSH-depenclent control mechanism of the production of E, and F2ar prostaglandins from thyrrjid phosphohpids. ‘We have obtained the following results: i) TSH increases the biosynthesis of prostagiandins in the thyroid in vifro; we thus confirm the results of Burke et al. [6] using a different methodology. This effect of TCSH is abolished by indomethacin and aspirin, which are inhibitors of proseaglandin synthetaso

Tissue inhibitor of metalloproteinases-1 signalling pathway leading to erythroid cell survival

Tissue inhibitors of metalloproteinases (TIMP) are specific inhibitors of matrix metalloproteinases (MMPs) and thus participate in maintaining the balance between extracellular matrix deposition and degradation in several physio-pathological processes. Nevertheless, TIMP must be regarded as multifunctional proteins involved in cell growth, angiogenesis and apoptosis. The molecular mechanisms induced by TIMP remain largely unknown. In the present study, we provide evidence that TIMP-1 induces a significant anti-apoptotic effect in the human erythroleukaemic cell line UT-7 and in the murine myeloid cell line 32D. Using specific kinases inhibitors, we show that TIMP-1-mediated cell survival is dependent upon Janus kinase (JAK) 2 and phosphoinositide 3-kinase (PI 3-kinase) activities. By transient transfection of dominant-negative Akt in UT-7 cells, we demonstrate that this kinase is crucial for the TIMP-1 anti-apoptotic effect. Moreover, TIMP-1 enhances specific phosphorylation of both Akt and Bad (Bcl-2/Bcl-X(L)-antagonist, causing cell death) in a PI 3-kinase-dependent manner and, besides, controls the level of the anti-apoptotic protein Bcl-X(L). We conclude that TIMP-1 induces haematopoietic cell survival via the JAK2/PI 3-kinase/Akt/Bad pathway.

Differential Induction of Grapevine Defenses by Two Strains of Botrytis cinerea

ABSTRACT Even though Botrytis cinerea, the causal agent of gray mold, is a highly variable fungus with strains displaying very different degrees of virulence toward one given host plant species, no study has yet shown any correlation between the lack of aggressiveness of one given strain and its ability to stimulate a defense response from its host. Strains of B. cinerea collected from different host plant species were screened for their pathogenicity on grapevine to select two strains with similar morphological characteristics but different levels of virulence. In grapevine leaves, the less aggressive strain, T4, enhanced the accumulation of many defense products including secondary metabolites and the pathogenesis-related proteins, chitinase and beta-1,3-glucanase. Interestingly, secondary metabolites were formed in cells around a small group of dead cells. When compared with T4, the more aggressive strain, T8, had larger necrotic spots, no secondary metabolite biosynthesis, and accumulations of chitinases and beta-1,3-glucanases that were more delayed, yet only slightly weaker. The culture fluids of both strains mimicked the differential effect of each isolate in stimulating chitinase activity when infiltrated into grapevine leaves.

Chitinases of the grapevine (Vitis vinifera L.): five isoforms induced in leaves by salicylic acid are constitutively expressed in other tissues

Using a colored substrate assay, chitinase activity was detected in various grapevine (Vitis vinifera L.) tissues such as winter-resting stem inter-nodes, roots, berries and leaves. The highest specific activity was found in the berries and was about 10 times higher than the one extracted from the leaves. Leaf wounding and salicylic acid treatment were able to rise the basal leaf activity by factors of 4.9 and 5.5, respectively. Up to 13 chitinase isoforms (4 basic and 9 acidic) could be identified in various untreated grapevine tissues by a combination of native IEF and PAGE. Untreated leaves were found to express six (four basic and two acidic) isoforms. Wounding of leaf tissue resulted in the appearance of four new acidic isoforms which have also been identified in berry extracts, one of them being present in the roots. Treatment of wounded leaves with 1 mM salicylic acid provoked a slight further increase of these four chitinases and greatly stimulated three of the basic constitutive isoforms. One of the latter was leaf specific while the two other ones were present in the stems, roots and berries.

Annexin A1 processing is associated with caspase-dependent apoptosis in BZR cells

Annexins are widely distributed and have been described in lung as well as in other cells and tissues. Annexin I (ANX AI) is a member of the calcium‐dependent phospholipid binding protein family. Besides its anti‐inflammatory function, ANX AI has been involved in several mechanisms such as the Erk repression pathway or apoptosis. To investigate the role of ANX AI on apoptosis in broncho‐alveolar cells, we have constructed a plasmid containing the ANX AI full length cDNA. Transfected BZR cells displayed a higher level of both forms of ANX AI (37 and 33 kDa) as well as a decrease in cell viability (two‐fold versus cells transfected with an empty vector). In order to analyse the endogenous ANX AI processing during stimulus‐induced apoptosis, BZR cells were treated with a commonly used inducer, i.e. C2 ceramides. In these conditions, microscopic analysis revealed chromatin condensation in dying cells and the Bcl‐2, Bcl‐xL/Bax mRNA balance was altered. Caspase‐3 is one of the key executioners of apoptosis, being responsible for the cleavage of many proteins such as the nuclear enzyme poly(ADP‐ribose) polymerase (PARP). We demonstrate that caspase‐3 was activated after 4 h treatment in the presence of ceramide leading to the cleavage of PARP. Dose–response experiments revealed that cell morphology and viability modifications following ceramide treatment were accompanied by an increase in endogenous ANX AI processing. Interestingly, in both ceramide and transfection experiments, the ANX AI cleaved form was enhanced whereas pre‐treatment with the caspase inhibitor Z‐VAD‐fmk abolished ANX AI cleavage. In conclusion, this study demonstrates a complex regulatory role of caspase‐dependent apoptosis where ANX AI is processed at the N‐terminal region which could give susceptibility to apoptosis upon ceramide treatment.

Activation of Raf-1 and mitogen-activated protein kinases by erythropoietin and inositolphosphate-glycan in normal erythroid progenitor cells: involvement of protein kinase C.

In this work, we show that erythropoietin and inositolphosphate-glycan activate Raf-1 and the mitogen-activated protein kinases (MAP kinases) in normal erythropoietin-responsive cells. Using a protein kinase C (PKC) activator such as the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate and the PKC inhibitor GF109203X, we investigated a possible involvement of PKC during activation of Raf-1 and MAP kinase by erythropoietin or inositolphosphate-glycan. We found that erythropoietin increased MAP kinase level with a maximum stimulation reached at 5-10 min. Inositolphosphate-glycan and 12-O-tetradecanoyl-phorbol-13-acetate increased MAP kinase activity in the same manner. This activity was inhibited by cell preincubation with GF109203X. Two MAP kinase isoforms were present in erythroid progenitor cells, the 44 and 42 kDa proteins. We report here that erythropoietin, inositolphosphate-glycan, and 12-O-tetradecanoyl-phorbol-13-acetate activated only the p44 form (erk-1) of MAP kinase and the Raf-1 protein. GF109203X was used at a concentration which inhibited by 50% erythroid colonie (CFU-E) proliferation and differentiation induced by erythropoietin or inositolphosphate-glycan. These results support the hypothesis that erythropoietin and inositolphosphate-glycan activate Raf-1 and MAP kinases in normal erythroid progenitor cells and suggest that this activation involves PKC.

Existence of two pools of prostaglandins during stimulation of the thyroid by TSH

In a previous work [l] we have shown that Kuehl’s hypothesis [2,3], taken over by Burke [4,5], according to which the prostaglandins would represent an obligatory intermediate in the hormonal stimulation of adenyl cyclase, could not be applied to the stimulation of the thyroid by TSH. Our results have been simultaneously confirmed by those of Wolff [6]. Finally, Burke himself was puzzled by the observation [7] that dibutyryl adenosine monophosphate cyclic (DBC) was able to increase the level of prostaglandins in rat or mouse thyroid. We decided to study the relations which could exist between the process of prostaglandin biosynthesis that we have described, where TSH stimulates a phospholipase A2 which preferentially liberates arachidonate from phosphatidyl-inositol, and the effect of DBC. The results that we have obtained show the independence of the two pathways. DBC or CAMP have no effect on phospholipase AZ, but they activate a lipase which liberates arachidonate from neutral lipids. It is therefore possible to distinguish two pools of prostaglandins, one which is pre-CAMP and not compulsory, the other which is post-CAMP and is a consequence of the activation of adenyl-cyclase.

Control by thyrotropin of the production by thyroid cells of an inositol phosphate-glycan

The increased turnover of phosphatidylinositol promoted by thyrotropin (TSH) in pig thyroid tissue does not seem to be caused by an increased production of inositol tris-phosphate. We have explored another possibility, the synthesis of an inositol phosphate-glycan (IP-gly). Our results show that thyroid cells in culture produced this substance from a precursor phosphatidylinositol-glycan (Gly-PI). The obtained IP-gly seemed, by its analytical and biological properties, to be identical, or similar, to the previously described insulin mediator.

The elastin peptides-mediated induction of pro-collagenase-1 production by human fibroblasts involves activation of MEK/ERK pathway via PKA- and PI3K-dependent signaling

Elastin peptides, such as κ‐elastin (kE), bind to the elastin receptor at the cell surface of human dermal fibroblasts and stimulate collagenase‐1 expression at the gene and protein levels. Using specific inhibitors and phosphospecific antibodies, we show here that the binding of elastin peptides to their receptor activates the extracellular signal‐regulated kinase (ERK) pathway; this activation is essential for the induction of pro‐collagenase‐1 production. Moreover, protein kinase A (PKA) and phosphatidylinositol 3‐kinase (PI3K) signaling were found to participate in ERK activation. Concomitantly, we demonstrate that stimulation by elastin peptides leads to enhanced DNA binding of activator protein‐1 (AP‐1). Our data indicate that the up‐regulation of collagenase‐1 following treatment of fibroblasts with elastin peptides results from a cross‐talk between PKA, PI3K and the ERK signaling pathways and that this regulation is accompanied by activation of AP‐1 transcription factors.

Tetradecanoyl phorbol-13-acetate counteracts the responsiveness of cultured thyroid cells to thyrotropin

We have studied the effects of TPA on the metabolism of porcine thyroid cells cultured for 1-4 days in the absence (control cells) and in the presence of 0.1 mU/ml TSH (TSH cells). The phospholipid turnover, evaluated after a 2 hr incorporation of 32P-phosphate into phospholipids, is markedly modified by the presence of TPA (1.5 microM, 2 hr) in the incubation medium of control and TSH treated cells. The total incorporation is 3-4 times higher than untreated cells, the labelling of phosphatidylinositol (PI) is slightly decreased or unchanged whereas that of phosphatidylcholine (PC) is strongly increased. The increased labelling of PI, promoted by an acute TSH treatment is counteracted by TPA. This TPA effect is not observed when prelabelled cells are challenged for 5 min with the drug. A similar effect is observed when 10 nM TPA is added in the culture medium for 20 hr. The addition of TPA does not affect significantly the protein iodine content in 3 or 4 days control cells incubated for 45 min or 2 hr with 125I-iodine, but dramatically decreases the very high iodination rate of TSH cells. We have tested the TPA effect on the cyclic AMP accumulation for the last 5 min of a 2 hr incubation. TPA inhibits by about 50-80% the stimulation evoked by TSH and only by 10% that evoked by forskolin (0.1 mM). These results suggest a possible link between the PC turnover and the adenylate cyclase responsiveness to TSH and the iodination rate.