Guanylyl cyclases and signaling by cyclic GMP.

Guanylyl cyclases are a family of enzymes that catalyze the conversion of GTP to cGMP. The family comprises both membrane-bound and soluble isoforms that are expressed in nearly all cell types. They are regulated by diverse extracellular agonists that include peptide hormones, bacterial toxins, and free radicals, as well as intracellular molecules, such as calcium and adenine nucleotides. Stimulation of guanylyl cyclases and the resultant accumulation of cGMP regulates complex signaling cascades through immediate downstream effectors, including cGMP-dependent protein kinases, cGMP-regulated phosphodiesterases, and cyclic nucleotide-gated ion channels. Guanylyl cyclases and cGMP-mediated signaling cascades play a central role in the regulation of diverse (patho)physiological processes, including vascular smooth muscle motility, intestinal fluid and electrolyte homeostasis, and retinal phototransduction. Topics addressed in this review include the structure and chromosomal localization of the genes for guanylyl cyclases, structure and function of the members of the guanylyl cyclase family, molecular mechanisms regulating enzymatic activity, and molecular sequences coupling ligand binding to catalytic activity. A brief overview is presented of the downstream events controlled by guanylyl cyclases, including the effectors that are regulated by cGMP and the role that guanylyl cyclases play in cell physiology and pathophysiology.

Differential regulation of cyclooxygenases 1 and 2 by interleukin-1 beta, tumor necrosis factor-alpha, and transforming growth factor-beta 1 in human lung fibroblasts.

In the present studies we found that incubation of human lung fibroblasts with transforming growth factor-beta 1 (TGF-beta 1) potentiated the interleukin-1 beta (IL-1 beta) and/or tumor necrosis factor-alpha (TNF-alpha)-stimulated production of prostaglandin E2 (PGE2). Analysis of fibroblast proteins showed the induction of cyclooxygenase-1 (Cox-1) by TGF-beta 1 and the induction of Cox-2 by IL-1 beta and TNF-alpha. The levels of transcripts for Cox-1 were minimally modified by IL-1 beta or TNF-alpha, however, they were increased by 12-fold by TGF-beta 1. Transcripts for Cox-2 were induced by IL-1 beta or TNF-alpha and their induction was potentiated by TGF-beta 1. TGF-beta 1 alone did not induce Cox-2 transcripts. In vitro transcription assays showed that IL-1 beta and TNF-alpha increased the transcription of the Cox-2 gene, whereas TGF-beta 1 had no effect. Addition of TGF-beta did not increase further the transcription of Cox-2 in IL-1 beta-treated cells, but increased the stability of the corresponding transcripts. The transcription rate of the Cox-1 gene was not increased by any of the cytokines studied. In summary, we demonstrate that the potentiation of PGE2 production by TGF-beta 1 in IL-1 beta and TNF-alpha-treated fibroblasts is the result of transcriptional stimulation of the Cox-2 gene by IL-1 beta and TNF-alpha and the stabilization of the resulting transcripts by TGF-beta 1.

Developmental regulation of various annexins in the embryonic palate of the mouse: dexamethasone affects expression of annexin-1.

The annexins are a group of structurally related proteins implicated in a number of cellular processes, including growth, membrane fusion, and the effects of glucocorticoids on cellular physiology, signal transduction, and regulation of activities of phospholipase A2. Though their exact role in cellular physiology is not clear, their properties make them candidate proteins for signal transduction pathways by which growth factors and glucocorticoids modulate development of the palate. We have determined the exact cellular location and development expression of various annexins in the embryonic murine palate as a first step in assessing their developmental function. Western blot analysis revealed an increased accumulation of selected annexins in elevated palates compared to vertical (unelevated) ones. This was particularly striking for lipocortin I1 (annexin I), whose mRNA accumulated as well. Lipocortin I was expressed primarily in the apical portion of the palatal epithelium at early stages of development, but throughout the epithelium at later stages. Also, there was increased immunoreactivity for lipocortin I in the mesenchyme as development proceeded. Immunoreactivity for the endonexins (annexins IV and V) was found in the palatal epithelium and mesenchyme, whereas immunoreactivity for the 67-kDa calelectrin (annexin VI) was found only in the mesenchyme. Treatment of pregnant A/J strain mice with a cleft-palate inducing regimen of dexamethasone stimulated accumulation of lipocortin I protein and mRNA, but not lipocortin II (annexin II) protein. In contrast, the same regimen of dexamethasone did not affect levels of lipocortin I protein in palates of the glucocorticoid-less sensitive C57BL/6J strain mouse embryo. These data permit the suggestion that lipocortin I plays some critical, but as yet undefined, role in modulating ontogeny of the murine palate.

The secretory phospholipase A2 gene is a candidate for the Mom1 locus, a major modifier of ApcMin-induced intestinal neoplasia.

Mutations in the APC gene are responsible for various familial and sporadic colorectal cancers. Min mice carry a dominant mutation in the homolog of the Apc gene and develop multiple adenomas throughout their small and large intestine. Quantitative trait loci studies have identified a locus, Mom1, which maps to the distal region of chromosome 4, that dramatically modifies Min-induced tumor number. We report here the identification of a candidate gene for Mom1. The gene for secretory type II phospholipase A2 (Pla2s) maps to the same region that contains Mom1 and displays 100% concordance between allele type and tumor susceptibility. Expression and sequence analysis revealed that Mom1 susceptible strains are most likely null for Pla2s activity. Our results indicate that Pla2s acts as a novel gene that modifies polyp number by altering the cellular microenvironment within the intestinal crypt.

The annexins: specific markers of midline structures and sensory neurons in the developing murine central nervous system.

The annexins are a family of cytoplasmic proteins that have been shown to have numerous actions within a cell. Recent evidence suggests that at least one of these proteins plays a role in the development of the central nervous system (CNS). The present study examines the temporal expression and spatial distribution of annexins I, II, IV, V, and VI during development and at maturity in the murine CNS by immunocytochemical analysis. The results demonstrate that annexins I, II and IV exhibit clear immunolabeling in the murine CNS with distinct patterns of temporal and spatial expression. Annexin IV is the first annexin to be expressed on embryonic day (E) 9.5 while annexin I is the last to be expressed (E11.5). Annexins I, II and IV are found in the floor plate region, but to differing rostrocaudal extents. Annexin I has a very restricted distribution, only present in the midline raphe of the brainstem. Annexin II is present in the spinal cord, brainstem and mesencephalon. Annexin IV has the widest midline distribution, being observed in the floor and roof plates of the developing CNS. Additionally, antibodies against annexin II and IV immunolabel most dorsal root and sensory ganglion cells and their axons. During early postnatal development, immunolabeling with each antibody gradually disappears in many structures, and only first order sensory neurons and their fibers are immunopositive for annexins II and IV at weaning. Three functions of the annexins are suggested by the present findings: (1) to help establish the midline structures of the floor and roof plates, (2) to help direct the decussation of sensory fibers, and (3) to regulate some aspect of sensory neuron processing, such as signal transduction.

Epidermal growth factor coordinately regulates the expression of prostaglandin G/H synthase and cytosolic phospholipase A2 genes in embryonic mouse cells.

Confluent, primary cultures of mouse embryo palate mesenchyme (MEPM) cells are refractory to activation of phospholipase A2 (PLA2) by the calcium ionophore A23187. However, treatment of these cultures with epidermal growth factor (EGF) permits the cells to activate PLA2 in response to A23187. We have developed this finding by exploring molecular mechanisms by which growth factors modulate mobilization and metabolism of arachidonic acid. We found chronic treatment (> 6 h) of confluent MEPM cells with EGF (a) increases their ability to metabolize exogenous arachidonic acid to prostaglandin E2 (PGE2) and (b) stimulated constitutive expression of activities of PLA2 and cyclooxygenase (CyOx). Immunoprecipitation of [35S]proteins and Western blot analysis revealed EGF treatment stimulated synthesis and accumulation of PLA2c, CyOx-1, and CyOx-2. Northern hybridization analysis revealed EGF increased the steady-state levels of a transcript for the high molecular weight, cytosolic PLA2 (PLA2c), and both the 2.8- and 4.2-kb transcripts for CyOx-1 and CyOx-2, respectively. In vitro nuclear transcription assays showed a parallel increase in the transcription rate of the genes corresponding to CyOx-1 and PLA2c, but not CyOx-2, in response to EGF. Treatment with EGF had no effect on either synthesis of the low molecular weight, group II PLA2, accumulation of its transcript, or the transcription rate of its gene. Coordinate regulation of activities of PLA2 and CyOx in response to EGF did not parallel the mitogenic effects of EGF on confluent MEPM cells.

Oxidant-sensitive protein phosphorylation in endothelial cells.

Reactive oxygen is an important regulator of vascular cell biology; however, the mechanisms involved in transducing signals from oxidants in endothelial cells are poorly defined. Because protein phosphorylation is a major mechanism for signal transduction, cultured aortic endothelial cells were exposed to nonlethal concentrations of H2O2 to examine oxidant-sensitive changes in phosphorylation state. Addition of H2O2 increases the phosphorylation of the heat shock protein 27 (HSP27) within 2 min. This response is maximal by 20 min and remains constant for more than 45 min. Levels of intracellular free Ca2+ in endothelial cells did not change following addition of 100 microM H2O2, nor did the ability of the cells to respond to bradykinin. H2O2-induced phosphorylations were either not affected or were slightly increased in cells pretreated with PKC inhibitors (H-8, staurosporin, or calphostin c). Two-dimensional analysis of phosphoproteins from homogenates of 32P-labeled cells revealed that phorbol myristate acetate (PMA) did not cause the same degree of HSP27 phosphorylation as H2O2. Simultaneous addition of 10 eta M PMA and 50 microM H2O2 decreased the oxidant-stimulated phosphorylation of the most acidic HSP27 isoform. These data suggest that signal transduction for H2O2-sensitive endothelial cell responses are not only independent of PKC, but may also be suppressed by the action of the kinase.

Differential eicosanoid synthesis by murine fetal thymic non-lymphoid cells.

The temporal patterns of synthesis of prostaglandin (PG)E2 and PGI2 by organ-cultured fetal thymic lobes and the cell population(s) responsible for synthesis of such products within the murine fetal thymus have been investigated. Embryonic day 14 thymic lobes were organ-cultured in defined media for 14 days and the media were collected every 24 h and replaced with fresh media. Collected media were processed for quantitation of either PGE2 or PGI2. Lobes were also cultured in 2'-deoxyguanosine (1.35 mmol/L) to produce an enriched non-lymphoid population. The per cent cyclooxygenase-positive cells within non-lymphoid cell-enriched lobes as well as the capacity of such lobes to synthesize either PGE2 or PGI2 were determined and compared with that of intact thymic lobes. Results demonstrate that fetal thymic lobes, in vitro, differentially synthesize PGI2 and synthesize PGE2 at a constant rate. Moreover, lobes enriched for non-lymphoid cells contain a greater percentage of cyclooxygenase-positive cells and synthesize increased amounts of eicosanoids per 10(4) cells compared with controls.

Synthesis of platelet activating factor and metabolism of related lipids in embryonic cells.

Primary cultures of mouse embryo palate mesenchyme (MEPM) cells incubated with 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphocholine ([3H])lyso-PAF) incorporated radiolabel into 1-radyl-2-acyl-sn-glycero-3-phosphocholine (PC) and -phosphoethanolamine (PE). The radiolabeled PC was insensitive to hydrolysis with HCl fumes, whereas at least 82% of the 3H found in the PE was hydrolyzed to 3H-aldehydes by such treatment. Treatment of the PC with Vitride produced [3H]alkylglycerol; similar treatment of the PE produced [3H]alk-1-enylglycerol. None of the radiolabeled products yielded fatty alcohol upon reduction with Vitride. These findings indicate the radiolabeled PC was 1-O-alkyl-linked whereas the PE contained predominantly 1-O-alk-1'-enyl species with smaller amounts of 1-O-alkyl species. Homogenates of MEPM cells which had been prelabeled with [3H]lyso-PAF and [14C]arachidonic acid produced 14C-fatty acid, [3H]lyso-PC, and [3H]alkylglycerol when incubated at selected values of pH and concentrations of calcium. There was no accumulation of [3H]lyso-PE in the various incubation mixtures. Stimulation of MEPM cells with the ionophore A23187 in the presence of calcium and [3H]acetate resulted in the production of 3H-platelet-activating factor (PAF), identified by its migration with authentic PAF and its conversion to 1-O-[3H]alkyl-2,3-diacetylglycerol upon treatment with phospholipase C and acetic anhydride. These studies demonstrate that: (i) MEPM cells are able to incorporate [3H]lyso-PAF into 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine, the storage form of PAF, and into 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (PE plasmalogen); (ii) endogenous 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine can serve as a substrate for phospholipase A2 in homogenates; and (iii) MEPM cells have the ability to synthesize PAF, thus raising the possibility that this compound may play a role in modulating the physiology of these embryonic cells.

Expression of prostaglandin G/H synthase (cyclooxygenase) during murine fetal thymic development.

Fetal thymic lobes in organ culture have been shown to have the capacity to metabolize [14C]arachidonic acid (AA) to prostaglandins (PGs), including 6-ketoPGF1 alpha, PGF2 alpha, PGE2, and PGA2. Inhibition of AA metabolism results in inhibition of growth and Thy 1 expression during thymic organ culture. We report herein that freshly-isolated fetal thymic lobes also have the capacity to metabolize [14C]AA to PGs and HETEs at Days 14 and 16 of prenatal murine development. RNA encoding phospholipase A2, which liberates arachidonic acid from membrane phospholipids, and cyclooxygenase (prostaglandin G/H synthase), the first enzyme involved in the conversion of AA to PGs, are expressed during thymic development. We have localized the cyclooxygenase protein to stromal cells in the fetal and adult thymus. Exogenous AA or an analogue of PGI2 (iloprost) stimulated growth of fetal thymocytes in organ culture. These findings, together with our studies of the morphology of thymic lobes cultured with inhibitors of arachidonate metabolism, support the hypothesis that PGs are required for thymocyte proliferation during thymic development.

Thymic eicosanoid profiles and cyclooxygenase localization in prenatal and postnatal CBA/J mice.

Previous work in our laboratory has implicated arachidonic acid metabolites as modulators of fetal murine thymocyte development in vitro. Therefore, we determined whether distinct temporal patterns existed with regard to the capacity for eicosanoid synthesis by fetal and postnatal thymic lobes which might relate to early proliferative and differentiation events within the thymus. Thin layer chromatography was used to demonstrate differential synthesis of 15-HETE, 5-HETE, PGD2, 6-keto PGF1 alpha, and PGF2 alpha by both fetal and postnatal thymic lobes. In contrast, PGE2 and TXB2 were synthesized at a constant level. These findings were in part substantiated by radioimmunoassay of culture media for PGE2 and 6-keto PGF1 alpha following fetal thymic organ culture for 7 or 8 days. In addition, we were able to demonstrate immunoreactive cells for cyclooxygenase in early embryonic and postnatal thymic lobes. Taken together, the above findings permit the suggestion that metabolites of arachidonic acid modulate development of thymocytes and, in turn, their capacity for synthesis is modulated as the thymus develops.

Epidermal growth factor modulates release of arachidonic acid from embryonic cells.

The calcium ionophore A23187 stimulates release of free [3H]arachidonic acids from radiolabeled cultures of MEPM cells which are growing, but not from those which are confluent. However, when confluent MEPM cells are pretreated with EGF or PMA, release of [3H]arachidonic acids does occur in response to A23187. Since neither EGF nor PMA themselves stimulate release of [3H]arachidonic acids from these cells, but do activate protein kinase C, these data support the hypothesis that protein kinase C modulates the activities of phospholipid hydrolases in MEPM cells.

Dexamethasone does not interfere with hormone-sensitive PI hydrolysis.

Serum, but not epidermal growth factor (EGF), stimulated the release of radiolabeled inositol phosphates from human embryo palate mesenchyme (HEPM) cells prelabeled with [3H]-myoinositol. Pretreatment of cells with 10(-6) M dexamethasone (DEX) for 48 h had no effect on the release of inositol phosphates in response to serum. Furthermore, although treatment of the glucocorticoid-sensitive A/J strain of mouse embryo palate mesenchyme (MEPM) cells with 10(-6) M DEX inhibited their proliferation by 40%, it had no effect on the activity of phospholipase(s) C. However, DEX did enhance the incorporation of [3H]-myoinositol into membrane lipids. We interpret these data to mean that 1) serum factors enhance metabolism of inositol lipids in HEPM cells, 2) DEX does not interfere with the primary events by which agonists utilize metabolism of inositol lipids as a mechanism for transmembrane signaling, and 3) DEX may affect synthesis of phosphoinositides, as reported by Grove et al. (Biochem. Biophys. Res. Commun. 110:200-207, 1983; J. Craniofac. Genet. Dev. Biol. Suppl. 2:285-292, 1986).

Epidermal growth factor alters metabolism of inositol lipids and activity of protein kinase C in mouse embryo palate mesenchyme cells.

Epidermal growth factor (EGF) stimulated mouse embryo palate mesenchyme (MEPM) cells (1) to incorporate [32P]O4(3-) into phosphatidylinositol (PI), phosphatidylcholine, and phosphatidic acid over a period of 60 min; 2) to incorporate [32P]O4(3-) into polyphosphoinositides as a function of time; and 3) to incorporate [32P]O4(-3) into PI, only, as a function of concentration when the period of stimulation was kept short. EGF stimulated the release of radiolabeled inositol phosphates from MEPM cells that had been radiolabeled with [3H]myoinositol. The release of inositol 1-phosphate was sustained over a period of at least 60 min, whereas the release of inositol 1,4-bisphosphate and inositol trisphosphate peaked during the first 10 min of stimulation. EGF also stimulated phosphorylation of an Mr 80,000 protein whose pI, phosphopeptide map, and phosphoamino acid pattern were identical to those of an Mr 80,000 protein phosphorylated in response to phorbol 12-myristate 13-acetate. Mobilization or metabolism of arachidonic acid was not stimulated under the same conditions that permitted EGF to alter inositol lipid metabolism. We interpret these data to mean that 1) in contrast to the findings with some cell lines, alterations in inositol lipid metabolism may be part of the signalling mechanism for EGF in embryonic cells; 2) EGF is capable of activating inositol-dependent signalling pathways leading to activation of protein kinase C in MEPM cells; and 3) mobilization and metabolism of arachidonic acid are not an inherent part of this signalling mechanism.

Relation between arachidonic acid metabolism and development of thymocytes in fetal thymic organ cultures.

Because products of arachidonic acid metabolism, particularly the PG, have been implicated as modulators of growth and differentiation of adult thymocytes, we investigated relations between metabolism of arachidonic acid and growth, as well as differentiation, of thymocytes during fetal thymic organ culture. Fetal thymic cells synthesized immunoreactive PGE2 during organ culture and were found to be capable of metabolizing exogenous arachidonic acid to products that cochromatographed with authentic 6-keto-PGF1 alpha, PGE2, PGF2 alpha. Synthesis of these products and growth and expression of Thy-1 and Lyt-1 Ag were inhibited by culture of fetal thymic lobes with indomethacin, a cyclooxygenase inhibitor, as well as meclofenamate and eicosatetraynoic acid, inhibitors of cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism. Only indomethacin inhibited expression of Lyt-2. Culture with eicosatetraynoic acid also inhibited the capacity of thymic lobes to synthesize 15-hydroxyeicosatetraenoic acid-like products. The inhibitory effects of indomethacin on growth and expression of Thy-1 were partially reversed by simultaneous addition of arachidonic acid. Thus, fetal thymic cells appear to require an intact cyclooxygenase, and possibly lipoxygenase, pathway of arachidonic acid metabolism for growth and differentiation. These data also provide evidence that Lyt-1 and Lyt-2 may be regulated by different requirements with respect to arachidonic acid metabolism.

Effects of epidermal growth factor and phorbol 12-myristate 13-acetate on protein phosphorylation in mouse embryo palate mesenchyme cells in vitro.

Epidermal growth factor (EGF) or phorbol 12-myristate 13-acetate (PMA) stimulated mouse embryo palate mesenchyme (MEPM) cells to incorporate [32P]O(3-)4 into a protein with an apparent molecular weight of 80 kDa, in vitro. Agents known to elevate intracellular levels of cyclic AMP did not stimulate phosphorylation of this phosphoprotein. Since there is a significant amount of evidence obtained with other cells indicating that phosphorylation of such an 80-kDa phosphoprotein reflects specifically the activation of protein kinase C in response to PMA and other agents, including mitogens, these findings raise the possibility that EGF may activate protein kinase C in MEPM cells.

Cyclic adenosine-3',5'-monophosphate alters hydrolysis of phospholipids by mouse embryo palate mesenchyme cells.

The purpose of this study was to determine whether inhibition of release of arachidonic acid from mouse embryo palate mesenchyme (MEPM) cells in response to cAMP is due to a selected or generalized inhibition of hydrolysis of esterified pools of arachidonic acid. The calcium ionophore A23187 proved to be a useful probe of phospholipid hydrolases in MEPM cells, since it stimulated release of radiolabeled fatty acids from phospholipids of prelabeled MEPM cells as a function of the length of exposure, concentration, and concentration of Ca2+ in the medium. Elevation of intracellular levels of cAMP by treatment with (-) isoproterenol resulted in the inhibition of release of radiolabeled arachidonic acid in response to A23187. Analysis by quantitative gas-liquid chromatography revealed the source of the arachidonic acid released in response to the ionophore to be 1,2-diradyl-sn-glycero-3-phosphoethanolamine; elevation of intracellular levels of cAMP inhibited hydrolysis of this substrate, but may have stimulated hydrolysis of 1,2-diradyl-sn-glycero-3-phosphocholine. These findings permit the conclusions that 1) the ionophore stimulates activities of selected phospholipases A in MEPM cells and 2) cAMP modulates certain phospholipases A in MEPM cells in a specific manner.

Effects of retinoic acid treatment on release of arachidonic acid by chondrogenic cells in response to ionophore A23187.

Chondrogenic differentiation in mouse limb bud mesenchymal cells cultured at high density was suppressed by supplementation of the medium with retinoic acid in a dose-dependent fashion. Cells prelabeled with (3H) arachidonic acid were treated with 0.3 microgram/ml retinoic acid. Treatment with retinoic acid increased the (3H) fatty acid in the triglyceride fraction. Furthermore, treatment with retinoic acid enhanced the release of (3H) fatty acid upon stimulation of these cells with the divalent ionophore A23187. These data permit the suggestion that there may be a correlation between altered lipid metabolism and retinoic acid's ability to disrupt chondrogenic differentiation.

Metabolism of prostaglandin E2 by mouse embryo palate mesenchyme cells in vitro.

Mouse embryo palate mesenchyme cells synthesize a number of prostaglandins, particularly prostaglandin E2 (PGE2). However, the ability of such cells to metabolize prostaglandins was unknown. By use of radiolabeled PGE2 we determined that palate mesenchyme cells have little ability to degrade that prostaglandin in vitro but are able to metabolize products formed from its spontaneous degradation.

Cyclic AMP inhibits the release of prostaglandins and arachidonic acid from cultures of mouse embryo palate mesenchyme cells.

Mouse embryo palate mesenchyme (MEPM) cells are able to synthesize and respond to prostaglandins. However, mechanisms that regulate their synthesis in these cells are not known. Cyclic adenosine 3',5' monophosphate (cAMP) has been implicated as being involved in differentiation of the palate, accumulates in MEPM cells in response to stimulation with selected prostaglandins, and has been found to modulate synthesis of prostaglandins by other cells and tissues. Therefore, we have investigated whether cAMP modulates synthesis of prostaglandins by MEPM mesenchyme cells and partially characterized the metabolic site at which such modulation occurs. We found that treatment of MEPM cells with various agents to stimulate a seven- to 100-fold increase in intracellular levels of cAMP inhibited release of various prostaglandins by at least 50%. Similarly, elevation of intracellular levels of cAMP inhibited release of radiolabeled arachidonic acid from membrane phospholipids by as much as 27%. The inhibitory effects of cAMP on release of prostaglandins from MEPM cells could be almost completely overcome by the addition of arachidonic acid to the culture medium. We interpret these data to mean that there is a regulatory cycle in MEPM cells in which intracellular levels of cAMP regulates synthesis of prostaglandins and prostaglandins regulate accumulation of cAMP and regulation of synthesis of prostaglandins by cAMP is predominantly through inhibition of a phospholipase.