The resolution of acute inflammation induced by cyclic AMP is dependent on annexin A1.

Annexin A1 (AnxA1) is a glucocorticoid-regulated protein known for its anti-inflammatory and pro-resolving effects. We have shown previously that the cAMP-enhancing compounds rolipram (ROL; a PDE4 inhibitor) and Bt2cAMP (a cAMP mimetic) drive caspase-dependent resolution of neutrophilic inflammation. In this follow-up study, we investigated whether AnxA1 could be involved in the pro-resolving properties of these compounds using a model of LPS-induced inflammation in BALB/c mice. The treatment with ROL or Bt2cAMP at the peak of inflammation shortened resolution intervals, improved resolution indices, and increased AnxA1 expression. In vitro studies showed that ROL and Bt2cAMP induced AnxA1 expression and phosphorylation, and this effect was prevented by PKA inhibitors, suggesting the involvement of PKA in ROL-induced AnxA1 expression. Akin to these in vitro findings, H89 prevented ROL- and Bt2cAMP-induced resolution of inflammation, and it was associated with decreased levels of intact AnxA1. Moreover, two different strategies to block the AnxA1 pathway (by using N-t-Boc-Met-Leu-Phe, a nonselective AnxA1 receptor antagonist, or by using an anti-AnxA1 neutralizing antiserum) prevented ROL- and Bt2cAMP-induced resolution and neutrophil apoptosis. Likewise, the ability of ROL or Bt2cAMP to induce neutrophil apoptosis was impaired in AnxA-knock-out mice. Finally, in in vitro settings, ROL and Bt2cAMP overrode the survival-inducing effect of LPS in human neutrophils in an AnxA1-dependent manner. Our results show that AnxA1 is at least one of the endogenous determinants mediating the pro-resolving properties of cAMP-elevating agents and cAMP-mimetic drugs.

A novel mechanism for the inhibition of type 2 iodothyronine deiodinase by tumor necrosis factor α: involvement of proteasomal degradation.

Thyroxine (T4) needs to be converted to 3,5,3'-triiodothyronine (T3) by iodothyronine deiodinase to exert its biological activity. Recent studies revealed the presence of type 2 iodothyronine deiodinase (D2) in human thyroid tissue, human skeletal muscle and other tissues, suggesting that D2 is involved in maintaining plasma T3 level in human. Tumor necrosis factor α (TNFα) is an inflammatory cytokine of which production is elevated in patients with nonthyroidal illness. Although several lines of evidence suggest the causal role of TNFα in nonthyroidal illness, detailed nature of the effect of TNFα on D2 remains unclear. In the present study, we identified D2 activity and D2 mRNA in TCO-1 cells, which were derived from human anaplastic thyroid carcinoma, and studied the mechanisms involved in the regulation of D2 expression by TNFα. The characteristics of the deiodinating activity in TCO-1 cells were compatible with those of D2 and Northern analysis demonstrated that D2 mRNA was expressed in TCO-1cells. D2 activity and D2 mRNA expression were rapidly increased by dibutyryl cAMP ((Bu)2cAMP). TNFα showed an inhibitory effect on (Bu)2cAMP-stimulated D2 activity in spite of little effect on (Bu)2cAMP-stimulated D2 mRNA expression. MG132, a proteasome inhibitor abolished TNFα suppression of D2 activity whereas BAY11-7082 or 6-amino-4-(4-phenoxyphenylethylamino) quinazoline, inhibitors of nuclear factor-κB (NF-κB) failed to attenuate the effect of TNFα on D2 activity. These data suggest that a posttranslational mechanism through proteasomal degradation but not NF-κB activation is involved in the suppression of D2 by TNFα.

Na+/Ca2+ exchanger inhibitors inhibit neurite outgrowth in PC12 cells.

To elucidate the role of Na(+)/Ca(2+) exchanger (NCX) in neurite outgrowth, we investigated the effects of NCX inhibitors on neurite outgrowth in PC12 cells. KB-R7943 and 3',4'-dichlorobenzamil, NCX inhibitors, inhibited the neurite outgrowth caused by nerve growth factor (NGF). NCX inhibitors inhibited the neurite outgrowth caused by dibutylyl cAMP, which rapidly reorganizes the cytoskeleton. KB-R7943 inhibited the neurite outgrowth caused by Y-27632, an inhibitor of Rho kinase (ROCK) that regulates actin. However, NCX inhibitors did not inhibit NGF-induced phosphorylation of extracellular signal-regulated kinase. These results suggest that NCX inhibitor affects downstream of the Rho-ROCK signal transduction pathways in neurite outgrowth.

[Interaction of signal cascades induced by cAMP and prolactin in bovine oocyte-cumulus complexes].

Prolactin (PRL) is one of the pituitary hormones participating in the control of mammalian folliculo- and oogenesis. In the present study, the joint effect of PRL (50 ng/ml) and dibutyryl cAMP (dbcAMP, 1 mM) on oocyte maturation and the morphologic-functional state of surrounding cumulus cells was investigated in vitro. It has been shown that PRL totally suppresses the braking impact of dbcAMP on meiosis reinitiation and the completion of oocyte nuclear maturation. Furthermore, PRL partly inhibited cumulus expansion induced by dbcAMP, although it exerted the opposite effect in the control medium. In the presence of PRL, the inhibitory impact of dbcAMP on the proliferative activity of cumulus cells and on the PRL-elicited braking of destructive processes in the cells has been found. In cumulus cells, mRNA expression of PRL receptor long isoform was revealed by the RT-PCR method. The data obtained suggest an interaction of signal cascades induced by PRL and cAMP in bovine oocyte-cumulus complexes, with the coupling site of these cascades in oocytes being apparently different from that in cumulus cells.

Sphingosine kinase 1 is involved in dibutyryl cyclic AMP-induced granulocytic differentiation through the upregulation of extracellular signal-regulated kinase, but not p38 MAP kinase, in HL60 cells.

The role of sphingosine kinase (SPHK) in the dibutyryl cyclic AMP (dbcAMP)-induced granulocytic differentiation of HL60 cells was investigated. During differentiation, SPHK activity was increased, as were mRNA and protein levels of SPHK1, but not of SPHK2. Pretreatment of HL60 cells with N,N-dimethylsphingosine (DMS), a potent SPHK inhibitor, completely blocked dbcAMP-induced differentiation. The phosphorylation of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 MAPK was also increased during dbcAMP-induced differentiation. Pretreatment of HL60 cells with the MEK inhibitor, U0126, but not the p38 MAPK inhibitor, SB203580, completely suppressed dbcAMP-induced ERK1/2 activation and granulocytic differentiation, but did not affect the increase in SPHK activity. DMS inhibited dbcAMP-induced ERK1/2 activation, but had little effect on p38 MAPK activation. DMS had no effect on the dbcAMP-induced membrane translocation of protein kinase C (PKC) isozymes, and PKC inhibitors had no significant effect on ERK activation. The overexpression of wild-type SPHK1, but not dominant negative SPHK1, resulted in high basal levels of ERK1/2 phosphorylation and stimulated granulocytic differentiation in HL60 cells. These data show that SPHK1 participates in the dbcAMP-induced differentiation of HL60 cells by activating the MEK/ERK pathway.

Neurotrophins elevate cAMP to reach a threshold required to overcome inhibition by MAG through extracellular signal-regulated kinase-dependent inhibition of phosphodiesterase.

Inhibitors of regeneration in myelin, such as myelin-associated glycoprotein (MAG), play an important role in preventing regeneration after CNS injury. Elevation of cAMP, either with dibutyryl-cAMP (db-cAMP) or by priming with a variety of neurotrophins, overcomes inhibition by MAG and myelin. However, activation of cAMP is not generally regarded as a signaling pathway for neurotrophins. Here we show that the NGF-like neurotrophins overcome inhibition by MAG by activating tyrosine kinase receptors. We also show that activation of extracellular signal-regulated kinase (Erk) by BDNF is required to overcome inhibition by MAG, and that activated Erk transiently inhibits phosphodiesterase 4 (PDE4), the enzyme that hydrolyzes cAMP. Inhibition of PDE4 then allows cAMP to increase and so initiates the pathway to overcome inhibition. Furthermore, we also show that basal levels of Erk activation and basal cAMP levels contribute to the effects of db-cAMP by pushing the combined levels of cAMP above a threshold required to overcome inhibition. Together, these results not only show how NGF-like neurotrophins can elevate cAMP and overcome inhibition but also point to a novel mechanism of cross talk in neurons from the Erk to the cAMP signaling pathways.

Effect of volatile anesthetics on steroidogenesis in isolated bovine adrenocortical fasciculata cells.

To examine effects of volatile anesthetics (VAs) on steroidogenesis, cell suspensions of isolated bovine adrenocortical cells were incubated with several steroidogenic agents in the presence or absence of halothane and sevoflurane. The adrenocortical cells were dispersed by trypsin digestion of bovine adrenal cortex. The cortisol level was measured fluorometrically. VAs inhibited adrenocorticotropic hormone-, acetylcholine-, angiotensin-II-, and KCl-stimulated steroidogenesis in a concentration-dependent manner with extracellular Ca(2+). However, dibutyryl cyclic adenosine monophosphate-stimulated steroidogenesis was not inhibited by VAs. These results suggest that VAs inhibit steroidogenesis by blocking Ca(2+)-influx from the extracellular space without influencing the action of intracellular cyclic nucleotides.

Opposite nociceptive effects of the arginine/NO/cGMP pathway stimulation in dermal and subcutaneous tissues.

1. Nitric oxide has been described either as pronociceptive or antinociceptive. In this investigation, using an electronic pressure-metre, the intradermal and the subcutaneous effects of prostaglandin E(2) (PGE(2)) and agents that mimic or inhibit the arginine/NO/cGMP pathway were compared. 2. The hypernociceptive effect of the intradermal injection of PGE(2) (100 ng) was immediate, peaking within 15-30 min and returning to basal values in 45-60 min. The subcutaneous injection of PGE(2) induced a hypernociception with a delayed peak (3 h) plateauing for 4-6 h. 3. Intradermal administration of 3-morpholino-sydnonimine-hydrochloride (SIN-1) enhanced, while its subcutaneous administration inhibited, subcutaneous hypernociception induced by PGE(2). This inhibition was prevented by ODQ (8 micro g) but not by NG-monomethyl-L-arginine (L-NMMA) (50 micro g). 4. Intradermal but not subcutaneous administration of L-arginine (1-100 micro g), SIN-1 (1-100 micro g) and dibutyrylguanosine 3':5'-cyclic monophosphate (db cGMP) (0.1-100 micro g) induced an early (15-30 min) dose-dependent hypernociceptive effect. Intradermal pretreatment with NG-monomethyl-L-arginine (L-NMMA; 50 micro g) inhibited the hypernociception induced by L-Arg (10 micro g), but not that induced by SIN-1 (10 micro g) or db cGMP (10 micro g). 5. Intradermal injection of ODQ (8 micro g) antagonized the hypernociception induced by L-arginine and SIN-1, but not that induced by db cGMP. 6. Considering (a) the different time course of intradermal and subcutaneous PGE(2)-induced hypernociception, (b) the opposite nociceptive effect of intradermal and subcutaneous administration of SIN-1 (db cGMP) as well as the arginine/NO/cGMP pathway, the existence of different subsets of nociceptive primary sensory neurons in which the arginine/NO/cGMP pathway plays opposing roles is suggested. This hypothesis would explain the apparent contradictory observations described in the literature.

cAMP-dependent activation of the renal-specific Na+-K+-2Cl- cotransporter is mediated by regulation of cotransporter trafficking.

The murine apical bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter gene (mBSC1) exhibits two spliced isoform products that differ at the COOH-terminal domain. A long COOH-terminal isoform (L-mBSC1) encodes the Na(+)-K(+)-2Cl(-) cotransporter, and a short isoform (S-mBSC1) exerts a dominant-negative effect on L-mBSC1 cotransporter activity that is abrogated by cAMP. However, the mechanism of this dominant-negative effect was not clear. In this study, we used confocal microscopic analysis of an enhanced green fluorescent protein (EGFP) fusion construct (L-mBSC1-EGFP) expressed to characterize the surface expression of the L-BSC1 isoform in Xenopus laevis oocytes. Functional expression was also assessed in L-mBSC1-injected oocytes by measuring the bumetanide-sensitive (86)Rb(+) uptake. Oocytes injected with L-mBSC1-EGFP cRNA developed a distinct plasma membrane-associated fluorescence that colocalized with the fluorescent membrane dye FM 4-64. The fluorescence intensity in L-mBSC1-EGFP oocytes did not change after cAMP was added to the extracellular medium. In contrast, L-mBSC1-EGFP fluorescence intensity was reduced in a dose-dependent manner, with coexpression of S-mBSC1. The inhibitory effect of S-mBSC1 was abrogated by cAMP. Finally, the exocytosis inhibitor colchicine blocked the effect of cAMP on the L-mBSC1-EGFP/S-mBSC1-coinjected oocytes. All changes in L-mBSC1 surface expression correlated with modification of bumetanide-sensitive (86)Rb(+) uptake. Our data suggest that the dominant-negative effect of S-mBSC1 on L-mBSC1 transport function is due to the effects of the cotransporter on trafficking.

Secretion of keratinocyte growth factor by cultured human endometrial stromal cells is induced through a cyclic adenosine monophosphate-dependent pathway.

OBJECTIVE: To evaluate the effects of known modulators of endometrial function on the production of keratinocyte growth factor by endometrial stromal cells. DESIGN: The effects of dibutyryl-cyclic adenosine monophosphate (db-cAMP), 12-O-tetradecanoylphorbol 13-acetate (TPA), ethinyl estradiol-17alpha (EE), and medroxyprogesterone acetate (MPA) on the secretion of keratinocyte growth factor by endometrial stromal cells were investigated. SETTING: Research laboratory at a university medical school. PATIENT(S): Eleven endometrial specimens in the late proliferative phase. INTERVENTION(S): Endometrial stromal cells were incubated for 24 hours with db-cAMP, TPA, EE, or MPA. MAIN OUTCOME MEASURE(S): The concentration of keratinocyte growth factor in the culture media was measured using an ELISA. RESULT(S): Small amounts of keratinocyte growth factor were detected in the culture media of unstimulated endometrial stromal cells. The production of keratinocyte growth factor by endometrial stromal cells was stimulated with db-cAMP in a dose-dependent manner. The stimulatory effect of db-cAMP was inhibited by Rp-adenosine 3',5'-cyclic monophosphothioate. None of TPA, EE, nor MPA affected the keratinocyte growth factor production by these cells. CONCLUSION(S): These results suggest that a cAMP-dependent pathway may play an important role in the regulation of keratinocyte growth factor production by endometrial stromal cells. Keratinocyte growth factor secreted by endometrial stromal cells may be involved in the regeneration of the endometrium during the normal menstrual cycle and early pregnancy.

The inhibition of phosphatidylinositol-3-kinase induces neurite retraction and activates GSK3.

It has been extensively described that neuronal differentiation involves the signalling through neurotrophin receptors to a Ras-dependent mitogen-activated protein kinase (MAPK) cascade. However, signalling pathways from other neuritogenic factors have not been well established. It has been reported that cAMP may activate protein kinase (PKA), and it has been shown that PKA-mediated stimulation of MAPK pathway regulates not only neuritogenesis but also survival. However, extracellular regulated kinases (ERKs) mediated pathways are not sufficient to explain all the processes which occur in neuronal differentiation. Our present data show that: in cAMP-mediated neuritogenesis, using the SH-SY5Y human neuroblastoma cell line, there exists a link between the activation of PKA and stimulation of phosphatidylinositol 3-kinase (PI3K). Both kinase activities are essential to the initial elongation steps. Surprisingly, this neuritogenic process appears to be independent of ERKs. While the activity of PI3K is essential for elongation and maintenance of neurites, its inhibition causes retraction. In this neurite retraction process, GSK3 is activated. Using both a pharmacological approach and gene transfer of a dominant negative form of GSK3, we conclude that this induced retraction is a GSK3-dependent process which in turn appears to be a common target for transduction pathways involved in lysophosphatidic acid-mediated and PI3K-mediated neurite retraction.

The inhibitory effects of lead on steroidogenesis in MA-10 mouse Leydig tumor cells.

Lead is an environmental and occupational pollutant. It has been reported that lead affects the male reproductive system in humans and animals. However, the cellular mechanism of the adverse effect of lead on Leydig cell steroidogenesis remains unknown. To clarify whether lead has a direct effect on Leydig cells and how lead affects Leydig cells, MA-10 cells, a mouse Leydig tumor cell line, were exploited in this study. Lead acetate significantly inhibited hCG- and dbcAMP-stimulated progesterone production in MA-10 cells at 2 h. Steroid production stimulated by hCG or dbcAMP were reduced by lead. The mechanism of lead in reducing MA-10 cell steroidogenesis was further investigated. The expression of Steroidogenic Acute Regulatory (StAR) protein and the activities of P450 side-chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) enzymes were detected. Cells were treated with dbcAMP, 22R-hydroxycholesterol or pregnenolone alone or in combination with lead acetate ranging from 10(-8) to 10(-5) M for 2 h. The expression of StAR protein stimulated by dbcAMP was suppressed by lead at about 50%. Progesterone productions treated with 22R-hydroxycholesterol or pregnenolone were reduced 30-40% in lead-treated MA-10 cells. These data suggest that lead directly inhibited steroidogenesis by decreasing StAR protein expression and the activities of P450scc and 3beta-HSD enzymes with a dose-response trend in MA-10 cells. Moreover, cadmium, a calcium channel blocker, abolished inhibitory effect of lead on MA-10 cell steroid production. This indicates that lead might act on calcium channel to regulate MA-10 cell steroidogenesis.

W-5 and quin 2-AM reverse the inhibitory effect of insulin on lipolysis due to dibutyryl cAMP.

The effects of W-5, a weak calmodulin antagonist, and quin 2-AM, a cell permeant calcium chelator, on lipolysis and antilipolytic activity of insulin were studied in isolated rat adipocytes. We have previously shown that W-7, a strong calmodulin antagonist, suppresses the inhibitory effect of insulin on lipolysis due to dibutyryl cAMP (Bt2cAMP) in a dose-dependent manner [H. Goko, A. Matsuoka, Diabetes Res. Clin. Prac. 19 (1993) 177-181] and verapamil, a calcium antagonist, potentiates lipolysis due to Bt2cAMP. Like W-7, W-5 suppressed the antilipolytic action of insulin on lipolysis due to Bt2cAMP in a dose-dependent manner. However, when lipolysis was potentiated with 3-isobutyryl-1-methylxanthine (IBMX), W-5 did not suppress the antilipolytic action of insulin. At the same time, like verapamil, W-5 also potentiated lipolysis due to Bt2cAMP in a dose-dependent manner. Thus W-5 has the pharmaceutical effects of both W-7 and verapamil. The chelation of intracellular Ca2+ in adipocytes with quin 2-AM also produced a dose-dependent potentiation of lipolysis due to Bt2cAMP and suppression of the antilipolytic action of insulin on lipolysis due to Bt2cAMP. These effects of quin 2-AM are the same as those of W-5. Therefore, our results suggest that the cytoplasmic Ca2+ plays a pivotal role in mediating the potentiation of lipolysis and antilipolytic action of insulin when lipolysis is induced by Bt2cAMP in rat adipocytes and that W-5 appears to exert its pharmaceutical effects through the inhibition of intracellular calcium-dependent steps other than calmodulin.

Inhibition of Ca2+-independent phospholipase A2 by bromoenol lactone attenuates prostaglandin generation induced by interleukin-1 beta and dibutyryl cAMP in rat mesangial cells.

Cytokine-induced prostaglandin generation in rat mesangial cells has been suggested to be dependent on the expression of secretory phospholipase A2 (sPLA2). In the present study, we investigated the possible involvement of Ca2+-independent phospholipase A2 (iPLA2) in the generation. The results showed that bromoenol lactone, a relatively selective iPLA2 inhibitor, significantly attenuated prostaglandin E2 generation induced by interleukin-1beta and dibutyryl cAMP in parallel with the inhibition of iPLA2 activity. However, the inhibitor did not affect sPLA2 release upon stimulation, activities of sPLA2 or cytosolic phospholipase A2, or Ca2+ ionophore-induced arachidonic acid liberation. These results suggest that prostaglandin E2 generation upon stimulation may be partially mediated by iPLA2 in addition to sPLA2.

Prostaglandin E2 stimulates IL-8 gene expression in human colonic epithelial cells by a posttranscriptional mechanism.

Intestinal mucosal epithelial cells produce IL-8, a neutrophil chemoattractant that contributes to mucosal inflammation in various infectious and inflammatory diseases. However, the mediators involved and the molecular regulation of IL-8 production are poorly understood. As PGE2 is central in gut inflammation and modulates a variety of mucosal epithelial cell functions, we determined whether PGE2 can affect the expression of IL-8. Exogenous PGE2 induced the accumulation of IL-8 mRNA and protein production in a dose- and time-dependent manner in T84 human colonic epithelial cells. Forskolin and dibutyryl cAMP, which increase intracellular cAMP, stimulated IL-8 in a fashion similar to that of PGE2. PGE2 and PGE2 receptor agonists coupling through EP4 receptors elevated intracellular cAMP and up-regulated IL-8 mRNA expression by activating protein kinase A. Unlike PMA, PGE2 and forskolin did not increase IL-8 gene transcription. However, PGE2, forskolin, and PMA enhanced the stability of IL-8 mRNA transcripts, suggesting the involvement of posttranscriptional regulation. Chloramphenicol acetyltransferase reporter gene transfection studies confirmed the presence of a PGE2 responsive cis-element(s) in the IL-8 3' untranslated region. Furthermore, dexamethasone inhibited PGE2-, forskolin-, and dibutyryl cAMP-induced, but not PMA-induced, IL-8 protein production. These results highlight a novel role for PGE2 in up-regulating IL-8 gene expression by colonic epithelial cells, which may contribute to exacerbation of inflammation in the gastrointestinal tract.

Differential effects of lead and cAMP on development and activities of Th1- and Th2-lymphocytes.

Lead (Pb) is known to have detrimental effects on the central nervous, hematopoietic, renal, and immune systems. Herein, it is demonstrated that Pb can skew T cell reactivities by preferentially enhancing the development of Th2 cells and inhibiting the development of Th1 cells. When naive splenic CD4+ T cells from DO11.10 ovalbumin-specific transgenic (OVA-tg) mice or OVA-tg/RAG2-/- mice were developed in vitro in the presence of Pb, preferential skewing toward Th2 cells was evident. The Pb-driven skewing toward Th2 was blocked significantly in the presence of exogenous IL-12 or anti-IL-4 mAbs. Although Pb and dibutyryl cAMP (dbcAMP) appear to have similar effects on the development and reactivity of Th1 cells, unlike Pb, dbcAMP did not enhance Th2 development/activity. Further evidence of Pb's differential T cell effects was observed, in that regardless of the activation stimuli (Ag/APC; anti-CD3; PMA + ionomycin), the addition of PbCl2 consistently resulted in significant inhibition of IFN gamma production by a Th1 clone and in increased IL-4 production by a Th2 clone. In vitro addition of IL-12 overcame Pb's inhibition of Th1 cells. Th1 cells treated with a phosphodiesterase inhibitor had significantly elevated [cAMP]i levels following anti-CD3 activation in the presence of Pb, suggesting that Pb may inhibit Th1 development by enhancing adenylate cyclase activity and elevating the [cAMP]i level. Similar to Pb, a low concentration (10 microM) of dbcAMP inhibited IFN gamma production by Th1, which was prevented by IL-12; however, inhibition of protein kinase A activity by KT5720 did not reverse these effects. These results indicate that the environmental toxicant Pb can modify immune reactivities by significantly altering the differentiation of precursor or naive Th cells as well as by directly inhibiting Th1 cells and stimulating Th2 cells.

Regulation of fibronectin gene expression by cyclic AMP and phorbol myristate acetate in HT-1080 human fibrosarcoma cells.

We studied the regulation of fibronectin (FN) gene expression by cAMP and phorbol-12-myristate-13-acetate (PMA) in HT-1080 human fibrosarcoma cells. Dibutyryl cAMP increased FN synthesis and mRNA levels, while PMA inhibited the cAMP-induced FN synthesis. In transient transfection assays, cAMP increased FN promoter activity, while PMA paradoxically enhanced the cAMP-induced promoter activity. Stable transfection experiments, however, showed that neither cAMP or PMA alone nor together affected FN promoter activity. These results suggest that PMA antagonizes the cAMP-induced FN gene expression and that both the action of cAMP and the inhibition of its action by PMA may occur at the posttranscriptional level in HT-1080 cells.

Regulation of fibronectin gene expression by cyclic AMP and phorbol myristate acetate in HT-1080 human fibrosarcoma cells

We studied the regulation of fibronectin (FN) gene expression by cAMP and phorbol-12-myristate-13-acetate (PMA) in HT-1080 human fibrosarcoma cells. Dibutyryl cAMP increased FN synthesis and mRNA levels, while PMA inhibited the cAMP-induced FN synthesis. In transient transfection assays, cAMP increased FN promoter activity, while PMA paradoxically enhanced the cAMP-induced promoter activity. Stable transfection experiments, however, showed that neither cAMP or PMA alone nor together affected FN promoter activity. These results suggest that PMA antagonizes the cAMP-induced FN gene expression and that both the action of cAMP and the inhibition of its action by PMA may occur at the posttranscriptional level in HT-1080 cells.

Antilipolytic actions of vanadate and insulin in rat adipocytes mediated by distinctly different mechanisms.

Vanadate, which mimics the biological effects of insulin, also inhibits lipolysis in rat adipocytes. Here we demonstrate that the antilipolytic effect of vanadate differs from that of insulin at least by the five following criteria: 1) vanadate inhibits lipolysis mediated by high (supraphysiological) concentrations of catecholamines; 2) vanadate antagonizes (Bu)2cAMP-mediated lipolysis; 3) vanadate antagonizes isobutylmethylxanthine-dependent lipolysis, 4) vanadate inhibits lipolysis mediated by okadaic acid; and 5) wortmannin, which blocks the antilipolytic effect of insulin, fails to block vanadate-mediated antilipolysis. Vanadate does activate phosphoinositol 3-kinase, and wortmannin blocks this activation. Our working hypothesis assumes that all of the insulin-like effects of vanadate, including antilipolysis, are initiated by the inhibition of protein phosphotyrosine phosphatases (PTPases). Among documented PTPase inhibitors we found that VOSO4 (oxidation state +4), several organic vanadyl compounds (+4), zinc (Zn2+), tungstate (W), and molybdate (Mo) also had antilipolytic activity. The order of potency was vanadyl acetylacetonate > or = VOSO4 > or = NaVO3 > or = vanadyl-dipicolinate > Zn2+ >> W > Mo, and it correlated better with the inhibition of adipose membranal-PTPases in cell-free experiments. We have concluded that the antilipolytic effect of vanadate is 1) mechanistically distinct from that of insulin, 2) independent of phosphoinositol 3-kinase activation, and 3) independent of the lipolytic cascade. We also strongly suggest that the antilipolytic effect of vanadate emanates from inhibiting adipose membranal, rather than cytosolic PTPases, and present preliminary data showing distinct differences in catalysis between these two PTPase categories. Overall, the study indicates that antilipolysis can be manifested via alternative, insulin-independent, signal-transducing pathways.

Follitropin action on the transferrin gene in Sertoli cells is mediated by cAMP-responsive-element-binding-protein and antagonized by chicken ovalbumin-upstream-promoter-transcription factor.

The transcription of the transferrin (Tf) gene is induced by follitropin via cAMP in rat Sertoli cells. We previously demonstrated that the cAMP-responsive-element-binding protein (CREB) interacts on the proximal region II (PRII) of the human Tf promoter (Suire et al., 1995). The PRII region is identified as essential for cAMP inducibility of the Tf promoter and contains a CCAAT box. This unexpected result led us to study the relation that exists between CREB and the PRII site. In the liver, CCAAT/enhancer-binding (C/EBP) proteins act at the PRII site. Although these factors are absent in Sertoli cells, their overexpression in Sertoli cells disturbs basal and induced transcription. C/EBP alpha and delta were able to stimulate the basal transcription driven by the -100 to +39 region, placed upstream of the chloramphenicol acetyltransferase (CAT) gene. However, only C/EBP alpha allowed the cAMP-inducible expression. The Ka of CREB bZIP (254-327), a deleted form of CREB, for the CRE site (3.92 x 10(8)M-1) and for the PRII site (1.38 x 10(8)M-1) were determined using the surface plasmon resonance (SPR) method. The Ka values were similar, although the derived kinetics were different: higher ka and kd of CREB for the PRII site were found compared with the CRE site. Since we observed important dissociation kinetics, we hypothesized that the binding of CREB to the PRII site is stabilized by CREB-binding protein (CBP) or by chicken-ovalbumin-upstream-promoter transcription factor (COUP-TF) binding to PRI site near to PRII. However, we observed that the overexpression of CBP in Sertoli cells did not potentiate the basal and cAMP-stimulated activity of CREB of the -100 to +39Tf-CAT construct. In basal and cAMP-stimulated conditions, COUP-TF appeared to repress the transcription driven by the -100 to +39 region in a specific manner. These results demonstrate a direct action of CREB on hTf promoter, which is antagonized by COUP-TF and may explain the transcriptional regulation of Tf by follitropin, via cAMP.