Molecular mechanisms involved in gastrin-mediated regulation of cAMP-responsive promoter elements.

In the present study, we explore the role of cAMP-responsive (CRE) promoter elements in gastrin-mediated gene activation. By using the minimal CRE promoter reporter plasmid, pCRELuc, we show that gastrin can activate CRE. This activation is blocked by H-89 and GF 109203x, which inhibit protein kinases A and C, respectively. Moreover, Ca(2+)-activated pathways seem to be involved, because the calmodulin inhibitor W-7 reduced gastrin-mediated activation of pCRELuc. Deletion of CRE from the c-fos promoter rendered this promoter completely unresponsive to gastrin, indicating that CRE plays a central role in c-fos transactivation. Interestingly, gastrin-induced expression of the inducible cAMP early repressor (ICER), a gene that is known to be regulated by CRE promoter elements, was not reduced by H-89, W-7, or GF 109203x. Furthermore, bandshift analyses indicated that the region of the ICER promoter containing the CRE-like elements CARE 3-4 binds transcription factors that are not members of the CRE-binding protein-CRE modulator protein-activating transcription factor, or CREB/CREM/ATF-1, family. Our results underline the significance of the CRE promoter element in gastrin-mediated gene regulation and indicate that a variety of signaling mechanisms are involved, depending on the CRE promoter context.

Atypical lambda/iota PKC conveys 5-lipoxygenase/leukotriene B4-mediated cross-talk between phospholipase A2s regulating NF-kappa B activation in response to tumor necrosis factor-alpha and interleukin-1beta.

The transcription factor nuclear factor kappaB (NF-kappaB) plays crucial roles in a wide variety of biological functions such as inflammation, stress, and immune responses. We have shown previously that secretory nonpancreatic (snp) and cytosolic (c) phospholipase A(2) (PLA(2)) regulate NF-kappaB activation in response to tumor necrosis factor (TNF)-alpha or interleukin (IL)-1beta activation and that a functional coupling mediated by the 5-lipoxygenase (5-LO) metabolite leukotriene B(4) (LTB(4)) exists between snpPLA(2) and cPLA(2) in human keratinocytes. In this study, we have further investigated the mechanisms of PLA(2)-modulated NF-kappaB activation with respect to specific kinases involved in TNF-alpha/IL-1beta-stimulated cPLA(2) phosphorylation and NF-kappaB activation. The protein kinase C (PKC) inhibitors RO 31-8220, Gö 6976, and a pseudosubstrate peptide inhibitor of atypical PKCs attenuated arachidonic acid release, cPLA(2) phosphorylation, and NF-kappaB activation induced by TNF-alpha or IL-1beta, thus indicating atypical PKCs in cPLA(2) regulation and transcription factor activation. Transfection of a kinase-inactive mutant of lambda/iotaPKC in NIH-3T3 fibroblasts completely abolished TNF-alpha/IL-1beta-stimulated cellular arachidonic acid release and cPLA(2) activation assayed in vitro, confirming the role of lambda/iotaPKC in cPLA(2) regulation. Furthermore, lambda/iotaPKC and cPLA(2) phosphorylation was attenuated by phosphatidyinositol 3-kinase (PI3-kinase) inhibitors, which also reduced NF-kappaB activation in response to TNF-alpha and IL-1beta, indicating a role for PI3-kinase in these processes in human keratinocytes. TNF-alpha- and IL-1beta-induced phosphorylation of lambda/iotaPKC was attenuated by inhibitors toward snpPLA(2) and 5-LO and by an LTB(4) receptor antagonist, suggesting lambda/iotaPKC as a downstream effector of snpPLA(2) and 5-LO/LTB(4) the LTB(4) receptor. Hence, lambda/iotaPKC regulates snpPLA(2)/LTB(4)-mediated cPLA(2) activation, cellular arachidonic acid release, and NF-kappaB activation induced by TNF-alpha and IL-1beta. In addition, our results demonstrate that PI3-kinase and lambda/iotaPKC are involved in cytokine-induced cPLA(2) and NF-kappaB activation, thus identifying lambda/iotaPKC as a novel regulator of cPLA(2).

Functional coupling between secretory and cytosolic phospholipase A2 modulates tumor necrosis factor-alpha- and interleukin-1beta-induced NF-kappa B activation.

Tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta are potent activators of the transcription factor NF-kappaB, induced during inflammatory conditions. We have previously shown that both secretory and cytosolic phospholipase A(2) (PLA(2)) are involved in TNF-alpha- and IL-1beta-induced NF-kappaB activation. In this study, we have addressed the mechanism of PLA(2) involvement with respect to downstream arachidonic acid (AA) metabolites and the functional coupling between PLA(2)s mediating NF-kappaB activation. We show that in addition to inhibitors of secretory and cytosolic PLA(2)s, 5-lipoxygenase inhibitors attenuate TNF-alpha- and IL-1beta-stimulated NF-kappaB activation. Exogenous addition of leukotriene B(4) (LTB(4)) restored NF-kappaB activation reduced by 5-lipoxygenase inhibitors or an LTB(4) receptor antagonist, thus identifying LTB(4) as a mediator in signaling to NF-kappaB. TNF-alpha- and IL-1beta-induced AA release from cellular membranes was accompanied by phosphorylation of cytosolic PLA(2). Inhibitors of secretory PLA(2) and of 5-lipoxygenase/LTB(4) functionality markedly reduced AA release and nearly completely abolished cytosolic PLA(2) phosphorylation. This demonstrates that secretory PLA(2), through 5-lipoxygenase metabolites, is an essential upstream regulator of cytosolic PLA(2) and AA release. Our results therefore suggest the existence of a functional link between secretory and cytosolic PLA(2) in cytokine-activated keratinocytes, providing a molecular explanation for the participation of both secretory and cytosolic PLA(2) in arachidonic acid signaling and NF-kappaB activation in response to proinflammatory cytokines.

Mildly oxidized LDL induces expression of group IIa secretory phospholipase A(2) in human monocyte-derived macrophages.

Phospholipase A(2)s (PLA(2)s) constitute a family of enzymes that hydrolyze fatty acids of membrane phospholipids, thus initiating the synthesis of proinflammatory mediators. Various PLA(2)s have been detected in human atherosclerotic arteries (advanced lesions); however, only the secretory group of PLA(2) has been shown to specifically hydrolyze low density lipoprotein (LDL)-associated phospholipids and, as such, may play a potential role in atherogenesis. In the present study, we investigated the expression pattern of group IIa, IV, and V PLA(2)s in human macrophages, which are the key cells involved in the onset and perpetuation of atherosclerosis. Immunohistochemical staining by double labeling showed that the secretory nonpancreatic PLA(2) (snpPLA(2)) is detectable in macrophages in the intima of early atherosclerotic lesions. Reverse transcription-polymerase chain reaction analysis of RNA extracted from human monocytes clearly showed that expression of group IV PLA(2) was enhanced during differentiation into macrophages, with an onset of induction at days 2 to 3 of differentiation. Group V snpPLA(2) was constitutively expressed on differentiation, whereas the detection of group IIa snpPLA(2) was dependent on both differentiation and subsequent stimulation of macrophages. Indeed, the transcription of group IIa snpPLA(2) in macrophages was induced by treatment with minimally modified or mildly oxidized LDL, whereas native, extensively oxidized, or acetylated LDL had no effect. To our knowledge, this is the first report describing induction of group IIa snpPLA(2) expression in human monocyte-derived macrophages. The mRNA levels of cytosolic PLA(2) group IV and snpPLA(2) group V remained unchanged on LDL treatment. Thus, our results show that the expression of distinct PLA(2) enzymes is regulated not only during differentiation of monocytes into macrophages but also on exposure of macrophages to distinct LDL species. Consequently, our results indicate a potential role for both cytosolic and secretory PLA(2) enzymes in inflammation and in macrophage functions related to atherosclerosis, with a specific role for group IIa snpPLA2 in LDL scavenging.

Identification of novel phosphorylation sites in hormone-sensitive lipase that are phosphorylated in response to isoproterenol and govern activation properties in vitro.

Hormone-sensitive lipase (HSL) is the rate-limiting enzyme in lipolysis. Stimulation of rat adipocytes with isoproterenol results in phosphorylation of HSL and a 50-fold increase in the rate of lipolysis. In this study, we used site-directed mutagenesis and two-dimensional phosphopeptide mapping to show that phosphorylation sites other than the previously identified Ser-563 are phosphorylated in HSL in response to isoproterenol stimulation of 32P-labeled rat adipocytes. Phosphorylation of HSL in adipocytes in response to isoproterenol and in vitro phosphorylation of HSL containing Ser --> Ala mutations in residues 563 and 565 (S563A, S565A) with protein kinase A (PKA), followed by tryptic phosphopeptide mapping resulted in two tryptic phosphopeptides. These tryptic phosphopeptides co-migrated with the phosphopeptides released by the same treatment of F654HPRRSSQGVLHMPLYSSPIVK675 phosphorylated with PKA. Analysis of the phosphorylation site mutants, S659A, S660A, and S659A,S660A disclosed that mutagenesis of both Ser-659 and Ser-660 was necessary to abolish the activation of HSL toward a triolein substrate after phosphorylation with PKA. Mutation of Ser-563 to alanine did not cause significant change of activation compared with wild-type HSL. Hence, our results demonstrate that in addition to the previously identified Ser-563, two other PKA phosphorylation sites, Ser-659 and Ser-660, are present in HSL and, furthermore, that Ser-659 and Ser-660 are the major activity controlling sites in vitro.

Partial purification and identification of hormone-sensitive lipase from chicken adipose tissue.

HSL from chicken adipose tissue exhibits remarkable activation upon phosphorylation with cAMP-dependent protein kinase (cAMP-PK) compared to HSL from rat and human adipose tissue. In order to characterize the chicken HSL enzyme, it was purified 3500 fold from a chicken adipose tissue homogenate using pH 5.2 precipitation and anion-exchange chromatography. The purified chicken HSL was identified as an 86 kDa protein using Western blot analysis. The HSL diacylglycerol lipase activity was inhibited by 98% upon incubation with anti-rat HSL antiserum, and the specific activity of chicken HSL was estimated to be approximately the same as for the rat enzyme. Furthermore, the 86 kDa polypeptide was phosphorylated by cAMP-PK to about the same stoichiometry as for the recombinant rat enzyme. Hence, our results demonstrate that HSL from chicken adipose tissue is comparable in size and specific activity to HSL from mammalian species, and not a smaller 42 kDa polypeptide with 1000-fold lower specific activity as previously reported (Berglund, L., Khoo, J. C., Jensen, D., and Steinberg, D., 1980 J. Biol. Chem. 255, 5420-5428).

13C-n.m.r. studies of the acetylation sequences in partially N-deacetylated chitins (chitosans).

Chitosans obtained under homogeneous conditions of N-deacetylation, with degrees of N-deacetylation between 46% and 94%, were depolymerised and their 125-MHz 13C-n.m.r. spectra have been interpreted. The sequence of 2-acetamido-2-deoxy-beta-D-glucopyranose (GlcNAc) and 2-amino-2-deoxy-beta-D-glucopyranose (GlcN) residues influenced the chemical shifts, and the diad and triad frequencies have been calculated. Chitosans that were N-deacetylated under homogeneous and heterogeneous conditions gave values for the diad and triad frequencies that were consistent with a random arrangement of GlcN and GlcNAc residues.

Determination of the degree of N-acetylation and the distribution of N-acetyl groups in partially N-deacetylated chitins (chitosans) by high-field n.m.r. spectroscopy.

The composition and sequence of 2-acetamido-2-deoxy-beta-D-glucose (GlcNAc) and 2-amino-2-deoxy-beta-D-glucose (GlcN) residues in partially N-deacetylated chitosans, prepared under homogeneous and heterogeneous conditions, have been determined by 1H-n.m.r. spectroscopy. It was necessary to depolymerise the chitosan slightly by treatment with nitrous acid before spectroscopy. A sequence-dependent deshielding of H-1 of the GlcNAc residues made it possible to determine the proportions of the four possible diads. Chitosan prepared by N-deacetylation under homogeneous conditions gave values for the diad frequencies that were roughly consistent with a random distribution of the N-acetyl groups. Samples prepared under heterogeneous conditions have a frequency of the GlcNAc-GlcNAc diad slightly higher than for a random (Bernoullian) distribution. The chitosans, prepared under both homogeneous and heterogeneous conditions, with a degree of acetylation of 50% were soluble at neutral pH.