Elevated cyclic AMP suppresses ConA-induced MT1-MMP expression in MDA- MB-231 human breast cancer cells

We have previously reported that induction of MMP-2 activation by Concanavalin A (ConA) in MDA-MB-231 human breast cancer cells involves both transcriptional and post-transcriptional mechanisms, and that the continuous presence of ConA is required for MMP-2 activation (Yu et al. Cancer Res, 55, 3272-7, 1995). In an effort to identify signal transduction pathways which may either contribute to or modulate this mechanism, we found that three different cAMP-inducing agents, cholera toxin (CT), forskolin (FSK), and 3- isobutyl-1-methylxanthine (IBMX) partially inhibited ConA-induced MT1-MMP expression and MMP-2 activation in MDA-MB-231 cells. Combinations of CT or FSK with IBMX exhibited additive effects on reduction of MT1-MMP mRNA expression and MMP-2 activation. Agents which increase cAMP levels appeared to target transcriptional aspects of ConA induction, reducing MT1-MMP mRNA and protein in parallel with the reduced MMP-2 activation. In the absence of ConA, down-regulation of constitutive production of MT1-MMP mRNA and protein was observed, indicating that cAMP acts independently of ConA. These observations may help to elucidate factors regulating MT1-MMP expression, which may be pivotal to the elaboration of invasive machinery on the cell surface.

Localization of fibronectin in the rat testis : effects of serum and dibutyryl cyclic AMP on fibronectin deposition by peritubular myoid cells in culture

The peritubular zone of the rat testis has an extensive extracellular matrix (ECM). Fibronectin (FN) is distributed primarily in the basal lamina of the seminiferous tubule boundary tissue and is synthesized by peritubular myoid cells. Several extracellular changes are mediated by growth factors and these changes occur at the time of hormone mediated testicular development, particularly in the peritubular zone. The effects of serum or dibutyryl cyclic AMP (cAMP) on FN production by the mesenchymal peritubular myoid cells were evaluated. Rats of various ages (10, 15, 20, 40 and 80 days) were employed for immunofluorescent localization of rat testicular FN in frozen sections. In all age groups tested, FN was primarily present in a broad layer around each seminiferous tubule, and blood vessel, and in variable distribution throughout the interstitial stroma. By day 20 there was no clear distinction in FN staining between the peritubular zone and the interstitial tissue. This indicates an involvement of FN in the ECM developments which occur in the peritubular zone of the testis at this time. The peritubular myoid cells were isolated from 20-22 day old rat testis and cultured on glass coverslips. These cells were grown to confluence with 10% fetal calf serum (FCS) in medium until day 4 and then subcultured to have secondary monocultures maintained with or without serum. By means of immunofluorescence and cytochemistry using avidin-biotin peroxidase complex it was observed that peritubular myoid cells were positive for FN and most of the FN was localized in the perinuclear region. Subcultured peritubular myoid cells maintained for 4 days in medium containing FCS developed an extensive interconnecting FN matrix. In the presence of 0.5 mM cAMP in culture, FN became localized along the filamentous process of peritubular myoid cells and more prominently in the areas of triangulated multi-cell aggregates as well as on the surface of the contracted small spherical cells. The addition of cAMP in the presence of FCS, also caused a noticeable change in the staining pattern; FN was detected along the filamentous process developing into a complex network of cells encased in an extensive matrix. It would appear that the translocation of FN in the cytoplasmic extensions of peritubular myoid cells may be a direct consequence of morphological changes associated with metabolic regulation of cAMP. This may also be related to the puberty associated development of in vivo changes in the ECM produced by peritubular myoid cells.

A Mycobacterial Cyclic AMP Phosphodiesterase That Moonlights as a Modifier of Cell Wall Permeability

Mycobacterium tuberculosis utilizes many mechanisms to establish itself within the macrophage, and bacterially derived cAMP is important in modulating the host cellular response. Although the genome of M. tuberculosis is endowed with a number of mammalian-like adenylyl cyclases, only a single cAMP phosphodiesterase has been identified that can decrease levels of cAMP produced by the bacterium. We present the crystal structure of the full-length and sole cAMP phosphodiesterase, Rv0805, found in M. tuberculosis, whose orthologs are present only in /the genomes of slow growing and pathogenic mycobacteria. The dimeric core catalytic domain of Rv0805 adopts a metallophosphoesterase fold, and the C-terminal region builds the active site and contributes to multiple substrate utilization.Localization of Rv0805 to the cell wall is dependent on its C terminus, and expression of either wild type or mutationally inactivated Rv0805 in M. smegmatis alters cell permeability to hydrophobic cytotoxic compounds. Rv0805 may therefore play a key role in the pathogenicity of mycobacteria, not only by hydrolyzing bacterial cAMP, but also by moonlighting as a protein that can alter cell wall functioning.

Cyclic AMP in Mycobacteria: Characterization and Functional Role of the Rv1647 Ortholog in Mycobacterium smegmatis{triangledown}

Mycobacterial genomes are endowed with many eukaryote-like nucleotide cyclase genes encoding proteins that can synthesize 3',5'-cyclic AMP (cAMP). However, the roles of cAMP and the need for such redundancy in terms of adenylyl cyclase genes remain unknown. We measured cAMP levels in Mycobacterium smegmatis during growth and under various stress conditions and report the first biochemical and functional characterization of the MSMEG_3780 adenylyl cyclase, whose orthologs in Mycobacterium tuberculosis (Rv1647) and Mycobacterium leprae (ML1399) have been recently characterized in vitro. MSMEG_3780 was important for producing cAMP levels in the logarithmic phase of growth, since the {Delta}MSMEG_3780 strain showed lower intracellular cAMP levels at this stage of growth. cAMP levels decreased in wild-type M. smegmatis under conditions of acid stress but not in the {Delta}MSMEG_3780 strain. This was correlated with a reduction in MSMEG_3780 promoter activity, indicating that the effect of the reduction in cAMP levels on acid stress was caused by a decrease in the transcription of MSMEG_3780. Complementation of the {Delta}MSMEG_3780 strain with the genomic integration of MSMEG_3780 or the Rv1647 gene could restore cAMP levels during logarithmic growth. The Rv1647 promoter was also acid sensitive, emphasizing the biochemical and functional similarities in these two adenylyl cyclases. This study therefore represents the first detailed biochemical and functional analysis of an adenylyl cyclase that is important for maintaining cAMP levels in mycobacteria and underscores the subtle roles that these genes may play in the physiology of the organism.

Cyclic AMP-induced Conformational Changes in Mycobacterial Protein Acetyltransferases

The activities of a number of proteins are regulated by the binding of cAMP and cGMP to cyclic nucleotide binding (CNB) domains that are found associated with one or more effector domains with diverse functions. Although the conserved architecture of CNB domains has been extensively studied by x-ray crystallography, the key to unraveling the mechanisms of cAMP action has been protein dynamics analyses. Recently, we have identified a novel cAMP-binding protein from mycobacteria, where cAMP regulates the activity of an associated protein acetyltransferase domain. In the current study, we have monitored the conformational changes that occur upon cAMP binding to the CNB domain in these proteins, using a combination of bioluminescence resonance energy transfer and amide hydrogen/deuterium exchange mass spectrometry. Coupled with mutational analyses, our studies reveal the critical role of the linker region (positioned between the CNB domain and the acetyltransferase domain) in allosteric coupling of cAMP binding to activation of acetyltransferase catalysis. Importantly, major differences in conformational change upon cAMP binding were accompanied by stabilization of the CNB and linker domain alone. This is in contrast to other cAMP-binding proteins, where cyclic nucleotide binding has been shown to involve intricate and parallel allosteric relays. Finally, this powerful convergence of results from bioluminescence resonance energy transfer and hydrogen/deuterium exchange mass spectrometry reaffirms the power of solution biophysical tools in unraveling mechanistic bases of regulation of proteins in the absence of high resolution structural information.

The precision with which single cells of Dictyostelium discoideum can locate a source of cyclic AMP

When stimulated by a point source of cyclic AMP, a starved amoeba of Dictyostelium discoideum responds by putting out a hollow balloon-like membrane extension followed by a pseudopod. The effect of the stimulus is to influence the position where either of these protrusions is made on the cell rather than to cause them to be made. Because the pseudopod forms perpendicular to the cell surface, its location is a measure of the precision with which the cell can locate the cAMP source. Cells beyond 1 h of starvation respond non-randomly with a precision that improves steadily thereafter. A cell that is starved for 1-2 h can locate the source accurately 43% of the time; and if starved for 6-7 h, 87% of the time. The response always has a high scatter; population-level heterogeneity reflects stochasticity in single cell behaviour. From the angular distribution of the response its maximum information content is estimated to be 2-3 bits. In summary, we quantitatively demonstrate the stochastic nature of the directional response and the increase in its accuracy over time.

Cyclic AMP-dependent protein lysine acylation in mycobacteria regulates fatty acid and propionate metabolism

Acetylation of lysine residues is a posttranslational modification that is used by both eukaryotes and prokaryotes to regulate a variety of biological processes. Here we identify multiple substrates for the cAMP-dependent protein lysine acetyltransferase from Mycobacterium tuberculosis (KATmt). We demonstrate that a catalytically important lysine residue in a number of FadD (fatty acyl CoA synthetase) enzymes is acetylated by KATmt in a cAMP-dependent manner and that acetylation inhibits the activity of FadD enzymes. A sirtuin-like enzyme can deacetylate multiple FadDs, thus completing the regulatory cycle. Using a strain deleted for the KATmt ortholog in Mycobacterium bovis Bacillus Calmette-Guerin (BCG), we show for the first time that acetylation is dependent on intracellular cAMP levels. KATmt can utilize propionyl CoA as a substrate and, therefore, plays a critical role in alleviating propionyl CoA toxicity in mycobacteria by inactivating acyl CoA synthetase (ACS). The precision by which mycobacteria can regulate the metabolism of fatty acids in a cAMP-dependent manner appears to be unparalleled in other biological organisms and is ideally suited to adapt to the complex environment that pathogenic mycobacteria experience in the host.

Cyclic AMP in Mycobacteria: the second messenger comes first

Cyclic AMP (cAMP) has emerged as a pivotal molecule for signalling in all life forms. Mycobacterial genomes have been found to encode for numerous proteins that are involved in cAMP generation, degradation and utilization. Many of these proteins have domain organizations unique to mycobacteria. This review summarizes recent advances in mechanisms of cAMP synthesis and degradation, focusing on the processes by which cAMP modulates mycobacterial signalling. We explore its impact on the physiology of the organism and on the discourse between M. tuberculosis and its host.

Nutrient- and hypotonicity-induced insulin secretion: effects of cyclic AMP analogs, IBMX, tenidap and 5-nitro-2-(3-phenylpropylamino)benzoic acid

info:eu-repo/semantics/published

IGF-1 or insulin, and the TSH cyclic AMP cascade separately control dog and human thyroid cell growth and DNA synthesis, and complement each other in inducing mitogenesis.

The regular doubling of cell mass, and therefore of cell protein content, is required for repetitive cell divisions. Preliminary observations have shown that in dog thyrocytes insulin induces protein accumulation but not DNA synthesis, while TSH does not increase protein accumulation but triggers DNA synthesis in the presence of insulin. We show here that EGF and phorbol myristate ester complement insulin action in the same way. HGF is the only factor activating both protein accumulation and DNA synthesis. The effects of insulin on protein accumulation and in permitting the TSH effect are reproduced by IGF-1 and are mediated, at least in part by the IGF-1 receptor. The concentration effect curves are similar for both effects. Similar results are obtained in human thyrocytes. They reflect true cell growth, as shown by increases in RNA content and cell size. Carbachol and fetal calf serum also stimulate protein synthesis and accumulation without triggering DNA synthesis, but they are not permissive for the mitogenic effects of TSH or of the general adenylate cyclase activator, forskolin. Moreover the mitogenic effect of TSH greatly decreased in cells deprived of insulin for 2 days although these cells remain hypertrophic. Hypertrophy may therefore be necessary for cell division, but it is not sufficient to permit it. Three different mechanisms can therefore be distinguished in the mitogenic action of TSH: (1) the increase of cell mass (hypertrophy) induced by insulin or IGF-1; (2) the permissive effect of insulin or IGF-1 on the mitogenic effect of TSH which may involve both the increase of cell mass and the induction of specific proteins such as cyclin D3 and (3) the mitogenic effect of the TSH cyclic AMP cascade proper.

Effects of the novel (Pro(3))GIP antagonist and exendin(9-39)amide on GIP- and GLP-1-induced cyclic AMP generation, insulin secretion and postprandial insulin release in obese diabetic (ob/ob) mice: evidence that GIP is the major physiological incretin

AIMS/HYPOTHESIS: This study examined the biological effects of the GIP receptor antagonist, (Pro3)GIP and the GLP-1 receptor antagonist, exendin(9-39)amide.

METHODS: Cyclic AMP production was assessed in Chinese hamster lung fibroblasts transfected with human GIP or GLP-1 receptors, respectively. In vitro insulin release studies were assessed in BRIN-BD11 cells while in vivo insulinotropic and glycaemic responses were measured in obese diabetic ( ob/ ob) mice.

RESULTS: In GIP receptor-transfected fibroblasts, (Pro(3))GIP or exendin(9-39)amide inhibited GIP-stimulated cyclic AMP production with maximal inhibition of 70.0+/-3.5% and 73.5+/-3.2% at 10(-6) mol/l, respectively. In GLP-1 receptor-transfected fibroblasts, exendin(9-39)amide inhibited GLP-1-stimulated cyclic AMP production with maximal inhibition of 60+/-0.7% at 10(-6) mol/l, whereas (Pro(3))GIP had no effect. (Pro(3))GIP specifically inhibited GIP-stimulated insulin release (86%; p<0.001) from clonal BRIN-BD11 cells, but had no effect on GLP-1-stimulated insulin release. In contrast, exendin(9-39)amide inhibited both GIP and GLP-1-stimulated insulin release (57% and 44%, respectively; p<0.001). Administration of (Pro(3))GIP, exendin(9-39)amide or a combination of both peptides (25 nmol/kg body weight, i.p.) to fasted (ob/ob) mice decreased the plasma insulin responses by 42%, 54% and 49%, respectively (p<0.01 to p<0.001). The hyperinsulinaemia of non-fasted (ob/ob) mice was decreased by 19%, 27% and 18% (p<0.05 to p<0.01) by injection of (Pro3)GIP, exendin(9-39)amide or combined peptides but accompanying changes of plasma glucose were small.

CONCLUSIONS/INTERPRETATION: These data show that (Pro(3))GIP is a specific GIP receptor antagonist. Furthermore, feeding studies in one commonly used animal model of obesity and diabetes, (ob/ob) mice, suggest that GIP is the major physiological component of the enteroinsular axis, contributing approximately 80% to incretin-induced insulin release.

Cyclic AMP production and insulin releasing activity of synthetic fragment peptides of glucose-dependent insulinotropic polypeptide

Synthetic fragment peptides of glucose-dependent insulinotropic polypeptide (GIP) were evaluated for their ability to elevate cellular cAMP production and stimulate insulin secretion. In GIP receptor transfected CHL cells, GIP(4-42) and GIP(17-30) dose-dependently inhibited GIP-stimulated cAMP production (40 +/- 8%; p <0.01 and 15 +/- 6%; p <0.05, respectively), while GIP(1-16) exerted very weak agonist effects on cAMP production. In the clonal pancreatic beta-cell line, BRIN-BD11, GIP(1-16) demonstrated weak insulin releasing activity compared with native GIP. In contrast, GIP(4-42) and GIP (17-30) weakly antagonized the insulin releasing activity of the native peptide (23 +/- 6%; p <0.05 and 11 +/- 3%, respectively). These data demonstrate the critical role of the N-terminus and the involvement of regions of the C-terminal domain in generating full biological potency of GIP.