Expression of the foraging gene in adult Drosophila melanogaster.

The foraging gene in Drosophila melanogaster, which encodes a cGMP-dependent protein kinase, is a highly conserved, complex gene with multiple pleiotropic behavioral and physiological functions in both the larval and adult fly. Adult foraging expression is less well characterized than in the larva. We characterized foraging expression in the brain, gastric system, and reproductive systems using a T2A-Gal4 gene-trap allele. In the brain, foraging expression appears to be restricted to multiple sub-types of glia. This glial-specific cellular localization of foraging was supported by single-cell transcriptomic atlases of the adult brain. foraging is extensively expressed in most cell types in the gastric and reproductive systems. We then mapped multiple cis-regulatory elements responsible for parts of the observed expression patterns by a nested cloned promoter-Gal4 analysis. The mapped cis-regulatory elements were consistently modular when comparing the larval and adult expression patterns. These new data using the T2A-Gal4 gene-trap and cloned foraging promoter fusion GAL4's are discussed with respect to previous work using an anti-FOR antibody, which we show here to be non-specific. Future studies of foraging's function will consider roles for glial subtypes and peripheral tissues (gastric and reproductive systems) in foraging's pleiotropic behavioral and physiological effects.

Copper-induced activation of TRP channels promotes extracellular calcium entry and activation of CaMK, PKA, PKC, PKG and CBLPK leading to increased expression of antioxidant enzymes in Ectocarpus siliculosus.

The existence of functional Transient Receptor Potential (TRP) channels was analyzed in Ectocarpus siliculosus using agonists of human TRPs and specific antagonists of TRPA1, TRPC5, TRPM8 and TRPV; intracellular calcium was detected for 60 min. Increases in intracellular calcium were observed at 13, 29, 39 and 50-52 min, which appeared to be mediated by the activation of TRPM8/V1 at 13 min, TRPV1 at 29 min, TRPA1/V1 at 39 min and TRPA1/C5 at 50-52 min. In addition, intracellular calcium increases appear to be due to extracellular calcium entry, not requiring protein kinase activation. On the other hand, 2.5 µM copper exposure induced increased intracellular calcium at 13, 29, 39 and 51 min, likely due to the activation of a TRPA1/V1 at 13 min, TRPA1/C5/M8 at 29 min, TRPC5/M8 at 39 min, and a TRPC5/V1 at 51 min. The increases in intracellular calcium induced by copper were due to extracellular calcium entry and required protein kinase activation. Furthermore, from 3 to 24 h, copper exposure induced an increase in the level of transcripts encoding antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase, glutathione reductase and peroxiredoxin. The described upregulation decreased with inhibitors of CaMK, PKA, PKC, PKG and CBLPK, as well as with a mixture of TRP inhibitors. Thus, copper induces the activation of TRP channels allowing extracellular calcium entry as well as the activation of CaMK, PKA, PKC, PKG and CBLPK leading to increased expression of genes encoding antioxidant enzymes in E. siliculosus.

Expression and purification of the natively disordered and redox sensitive metal binding regions of Mycobacterium tuberculosis protein kinase G.

Mycobacterium tuberculosis protein kinase G (PknG) is secreted into host macrophages to block lysosomal degradation. The catalytic domain (∼147-405) is C-terminally flanked by a tetratricopeptide repeat domain (TPRD). The preceding rubredoxin-like metal-binding motif (RD, ∼74-147) mediates PknG redox regulation. The N-terminal ∼75 residues were predicted to show no regulatory secondary structure (NORS) and harbor the only site (T63) phosphorylated in vivo. Deletions or mutations in the NORS or the redox-sensitive RD significantly decrease the survival function. Here, we show that the RD appears only to be present in the folded, metal-bound state if ZnCl2 is added upon induction of protein expression in minimal medium. Since factor Xa cleaves at the end of its recognition site (IEGR), a modified expression plasmid for PknG1-147 was obtained by mutating the N-terminal thrombin to a factor Xa recognition site. This allows preparing PknG1-147 with its native N-terminus. We further present a fast approach to generate expression plasmids for only the NORS or the RD by site-directed mutagenesis of the expression plasmid for His-tagged PknG1-147. An expression plasmid for PknG1-75 was obtained by introducing a stop codon at position 76 and one for PknG74-174 by introducing a factor Xa recognition site before position 74. SDS-PAGE analysis shows that all fragments are highly expressed in E. coli and can be purified to high purity. Thereby, the established preparation protocols pave the route for the NMR structural characterization of PknG regulation by its N-terminal regions, which is demonstrated by the recorded initial (1)H-(15)N-HSQC spectra.

Development of a transgenic Plasmodium berghei line (Pb pfpkg) expressing the P. falciparum cGMP-dependent protein kinase, a novel antimalarial drug target.

With the inevitable selection of resistance to antimalarial drugs in treated populations, there is a need for new medicines to enter the clinic and new targets to progress through the drug discovery pipeline. In this study we set out to develop a transgenic rodent model for testing inhibitors of the Plasmodium falciparum cyclic GMP-dependent kinase in vivo. A model was needed that would allow us to investigate whether differences in amino acid sequence of this enzyme between species influences in vivo efficacy. Here we report the successful development of a transgenic P. berghei line in which the cyclic GMP-dependent protein kinase (PKG) was replaced by the P. falciparum orthologue. We demonstrate that the P. falciparum orthologue was able to functionally complement the endogenous P. berghei pkg gene throughout blood stage development and early sexual development. However, subsequent development in the mosquito was severely compromised. We show that this is due to a defect in the female lineage of the transgenic by using genetic crosses with both male and female deficient P. berghei lines. This defect could be due to expression of a female-specific target in the mosquito stages of P. berghei that cannot be phosphorylated by the P. falciparum kinase. Using a previously reported anti-coccidial inhibitor of the cyclic GMP-dependent protein kinase, we show no difference in in vivo efficacy between the transgenic and control P. berghei lines. This in vivo model will be useful for screening future generations of cyclic GMP-dependent protein kinase inhibitors and allowing us to overcome any species-specific differences in the enzyme primary sequence that would influence in vivo efficacy in the rodent model. The approach will also be applicable to in vivo testing of other antimalarial compounds where the target is known.

PKG in honey bees: spatial expression, Amfor gene expression, sucrose responsiveness, and division of labor.

Division of labor is a hallmark of social insects. In honey bees, division of labor involves transition of female workers from one task to the next. The most distinct tasks are nursing (providing food for the brood) and foraging (collecting pollen and nectar). The brain mechanisms regulating this form of behavioral plasticity have largely remained elusive. Recently, it was suggested that division of labor is based on nutrition-associated signaling pathways. One highly conserved gene associated with food-related behavior across species is the foraging gene, which encodes a cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG). Our analysis of this gene reveals the presence of alternative splicing in the honey bee. One isoform is expressed in the brain. Expression of this isoform is most pronounced in the mushroom bodies, the subesophageal ganglion, and the corpora allata. Division of labor and sucrose responsiveness in honey bees correlate significantly with foraging gene expression in distinct brain regions. Activating PKG selectively increases sucrose responsiveness in nurse bees to the level of foragers, whereas the same treatment does not affect responsiveness to light. These findings demonstrate a direct link between PKG signaling in distinct brain areas and division of labor. Furthermore, they demonstrate that the difference in sensory responsiveness between nurse bees and foragers can be compensated for by activating PKG. Our findings on the function of PKG in regulating specific sensory responsiveness and social organization offer valuable indications for the function of the cGMP/PKG pathway in many other insects and vertebrates.

cGMP-dependent protein kinase Iα transfection inhibits hypoxia-induced migration, phenotype modulation and annexins A1 expression in human pulmonary artery smooth muscle cells.

Our previous work has demonstrated that the cellular phenotype changes of human pulmonary artery smooth muscle cells (PASMCs) play an important role during pulmonary vascular remodelling. However, little is known about the role of PASMCs phenotype modulation in the course of hypoxia-induced migration and its behind molecular mechanisms. In this study, we have shown that cGMP-dependent protein kinase (PKG) Iα transfection significantly attenuated the hypoxia-induced down-regulation of the expressions of SM-α-actin, MHC and calponin. Hypoxia-induced PASMC migration was also suppressed by PKGIα overexpression. Furthermore, this overexpression attenuated ANX A1 upregulation under hypoxic conditions. All those effects were reversed by a PKG inhibitor KT5823. Our data indicate that manipulating upstream entity e.g., PKGIa, may have a potential therapeutic value to prevent hypoxia-associated pulmonary arterial remodeling for pulmonary hypertension development.

A molecular readout of long-term olfactory adaptation in C. elegans.

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann(1,2). Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor(3) for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,(4) uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC(4). By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC(5). Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC(6,7). Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A guanosine 3',5'-cyclic monophosphate (cGMP) reporter system based on the G-kinase/CREB/CRE signal transduction pathway.

Guanylate cyclases constitute a gene family of enzymes that synthesize the second messenger guanosine 3',5'-cyclic monophosphate (cGMP) and play important roles in diverse physiological functions. Here we report a novel, simple and highly sensitive method for measurement intracellular cGMP concentrations using a cAMP-responsive element (CRE) and cGMP-dependent protein kinase (cGK). Transient transfection of the CRE reporter plasmid, encoding a luciferase reporter gene under the control of a modified promoter containing a CRE, and a cGK expression vector into HEK293 cells followed by treatment with 8-bromo-cGMP showed a dose dependent increase in luciferase activity. Moreover, HEK293 cells expressing GC-A or GC-B natriuretic peptide receptors and harboring this reporter system responded to specific ligands in a dose dependent manner. Our results indicate that this reporter gene method enables high throughput screening of receptor-type GC selective agonists in the treatment of cardiovascular diseases and homeostatic dysfunctions.

Fibronectin upregulates cGMP-dependent protein kinase type Iß through C/EBP transcription factor activation in contractile cells.

The nitric oxide (NO)-soluble guanylate cyclase (sGC) pathway exerts most of its cellular actions through the activation of the cGMP-dependent protein kinase (PKG). Accumulation of extracellular matrix is one of the main structural changes in pathological conditions characterized by a decreased activity of this pathway, such as hypertension, diabetes, or aging, and it is a well-known fact that extracellular matrix proteins modulate cell phenotype through the interaction with membrane receptors such as integrins. The objectives of this study were 1) to evaluate whether extracellular matrix proteins, particularly fibronectin (FN), modulate PKG expression in contractile cells, 2) to analyze the mechanisms involved, and 3) to evaluate the functional consequences. FN increased type I PKG (PKG-I) protein content in human mesangial cells, an effect dependent on the interaction with ß(1)-integrin. The FN upregulation of PKG-I protein content was due to increased mRNA expression, determined by augmented transcriptional activity of the PKG-I promoter region. Akt and the transcription factor CCAAT enhancer-binding protein (C/EBP) mediated the genesis of these changes. FN also increased PKG-I in another type of contractile cell, rat vascular smooth muscle cells (RVSMC). Tirofiban, a pharmacological analog of FN, increased PKG-I protein content in RVSMC and rat aortic walls and magnified the hypotensive effect of dibutyryl cGMP in conscious Wistar rats. The present results provide evidence of a mechanism able to increase PKG-I protein content in contractile cells. Elucidation of this novel mechanism provides a rationale for future pharmacotherapy in certain vascular diseases.

Sildenafil treatment in vivo stimulates Leydig cell steroidogenesis via the cAMP/cGMP signaling pathway.

Sildenafil citrate (Viagra), a cGMP-selective phosphodiesterase (PDE) inhibitor, is widely used to treat erectile dysfunction and pulmonary arterial hypertension. In contrast to its well established action on erectile dysfunction, little is known on the action of sildenafil on cGMP/cAMP signaling and testicular steroidogenesis. This study was designed to assess the effects of prolonged sildenafil treatment on NO synthase-dependent signaling and steroidogenic function of rat Leydig cells. Male adult rats were treated with Viagra (1.25 mg/kg body wt) daily for 30 days. In our studies, serum testosterone and ex vivo testosterone production significantly increased in sildenafil-treated animals. Human chorionic gonadotropin-stimulated testosterone production and cAMP accumulation were also significantly higher in Leydig cells obtained from sildenafil-treated rats. The expression of soluble guanylyl cyclase (GUCY1) subunits (Gucy1a1, Gucy1b1) significantly increased; cAMP-specific Pde4a, cGMP-specific Pde6c, and dual Pde1c and Nos2 were inhibited and expression of Nos3, protein kinase G1 (Pkg1), and Pde5 remained unchanged. Treatment of purified Leydig cells with NO donor caused a dose-dependent increase in both testosterone and cGMP production. Testosterone and cGMP production was significantly higher in Leydig cells obtained from sildenafil-treated animals. The stimulatory effect of NO donor was significantly enhanced by saturating concentrations of hCG in both Leydig cells obtained from control and sildenafil-treated animals. Occurrence of mature steroidogenic acute regulatory protein also increased in sildenafil treated animals in accord with increased cAMP and cGMP production. In summary, inhibition of PDE activity during prolonged sildenafil treatment increased serum testosterone level and Leydig cells' steroidogenic capacity by coordinated stimulatory action on cAMP and cGMP signaling pathway.

Expression and distribution of cyclic AMP- and cyclic GMP-binding protein kinases in the human vagina- an immunohistochemical study.

INTRODUCTION: In contrast to research findings describing the localization of nitric oxide synthases (NOS), guanylyl cyclases, and cyclic adenosine monophosphate (cAMP)- and cyclic guanosine monophosphate (cGMP)-degrading phosphodiesterase isoenzymes in the human vagina, the distribution of proteins known as major targets for cyclic nucleotides has not yet been evaluated. cAMP- and cGMP-dependent protein kinases (cAK, cGKI) have been identified as important receptors for cyclic nucleotides downstream the signaling cascades. AIM: To investigate, by means of immunohistochemistry, the expression of cAK and cGKI in relation to endothelial NOS (eNOS), vasoactive intestinal polypeptide (VIP), and protein gene product 9.5 (PGP 9.5) in the human vagina. MAIN OUTCOME MEASURES: Expression and distribution of cAK and cGKI(alpha,beta) in relation to eNOS, VIP, and PGP 9.5 in human vaginal tissue. METHODS: Immunohistochemical techniques were applied to sections of human vaginal full wall specimens in order to evaluate the presence of cAK and cGKI(alpha,beta) in relation to VIP, PGP 9.5, and eNOS, respectively. Western blot analyses were conducted using cytosolic supernatants of homogenized specimens of the vaginal wall and epithelium. RESULTS: Immunostaining specific for cGKIbeta was observed in vascular and nonvascular smooth muscle of the vagina. In the endothelial layer, cGKIbeta was found colocalized with eNOS. In contrast, no signals indicating cGKIalpha were registered. cAK-positive subepithelial vessels were found to be innervated by a dense meshwork of PGP-containing varicose nerve fibers, some of which presented expression of VIP. The expression of cAK and cGKIbeta was confirmed by Western blotting. CONCLUSIONS: Our results demonstrate the expression of cAK and cGKIbeta in the human vagina. The colocalization with VIP and eNOS underlines the significance of both the cAMP and GMP pathway in the control of human vaginal vascular and nonvascular smooth muscle.

Protein kinase G as a therapeutic target for the treatment of metastatic colorectal cancer.

Colorectal cancer is a leading cause of cancer-related death in the world and there is an urgent need for new strategies to combat this disease. Findings from several independent laboratories have converged on cGMP signaling as an exciting new therapeutic target, but the mechanisms remain controversial. A key intracellular effector of cGMP is protein kinase G (PKG). This article reviews the scientific literature concerning PKG effects on tumor development and progression, and discusses possible strategies for its exploitation in future cancer therapies. Studies from several independent laboratories have described novel anti-tumor effects of PKG in colon cancer cells that include inhibition of tumor growth and angiogenesis. While more preclinical research is warranted to better understand signaling mechanisms, these properties support the notion that PKG is a novel cancer target.

Regulation of cGMP-dependent protein kinase expression by Rho and Kruppel-like transcription factor-4.

Type I cGMP-dependent protein kinase (PKG I) plays a major role in vascular homeostasis by mediating smooth muscle relaxation in response to nitric oxide, but little is known about the regulation of PKG I expression in smooth muscle cells. We found opposing effects of RhoA and Rac1 on cellular PKG I expression: (i) cell density-dependent changes in PKG I expression varied directly with Rac1 activity and inversely with RhoA activity; (ii) RhoA activation by calpeptin suppressed PKG I, whereas RhoA down-regulation by small interfering RNA increased PKG I expression; and (iii) PKG I promoter activity was suppressed in cells expressing active RhoA or Rho-kinase but was enhanced in cells expressing active Rac1 or a dominant negative RhoA. Sp1 consensus sequences in the PKG I promoter were required for Rho regulation and bound nuclear proteins in a cell density-dependent manner, including the Krüppel-like factor 4 (KLF4). KLF4 was identified as a major trans-acting factor at two proximal Sp1 sites; active RhoA suppressed KLF4 DNA binding and trans-activation potential on the PKG I promoter. Experiments with actin-binding agents suggested that RhoA could regulate KLF4 via its ability to induce actin polymerization. Regulation of PKG I expression by RhoA may explain decreased PKG I levels in vascular smooth muscle cells found in some models of hypertension and vascular injury.

A xanthine-based epithelium-dependent airway relaxant KMUP-3 (7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) increases respiratory performance and protects against tumor necrosis factor-alpha-induced tracheal contraction, involving nitric oxide release and expression of cGMP and protein kinase G.

KMUP-3 (7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) was investigated in guinea pig tracheal smooth muscle. Intratracheal instillation of tumor necrosis factor (TNF)-alpha (0.01 mg/kg/300 microl) induced bronchoconstriction, increases of lung resistance, and decreases of dynamic lung compliance. Instillation of KMUP-3 (0.5-2.0 mg/kg) reversed this situation. In isolated trachea precontracted with carbachol, KMUP-3 (10-100 microM)-caused relaxations were attenuated by epithelium removal and by pretreatments with an inhibitor of K(+) channel, tetraethylammonium (10 mm); K(ATP) channel, glibenclamide (1 microM); voltage-dependent K(+) channel, 4-aminopyridine (100 microM); Ca(2+)-dependent K(+) channel, charybdotoxin (0.1 microM) or apamin (1 microM); soluble guanylate cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1one (ODQ, 1 microM); nitric-oxide (NO) synthase, N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM); and adenylate cyclase, SQ 22536 [9-(terahydro-2-furanyl)-9H-purin-6-amine] (100 microM). KMUP-3 (0.01-100 microM) induced increases of cGMP and cAMP in primary culture of tracheal smooth muscle cells (TSMCs). The increase in cGMP by KMUP-3 was reduced by ODQ and L-NAME; the increase in cAMP was reduced by SQ 22536. Western blot analysis indicated that KMUP-3 (1 microM) induced expression of protein kinase A (PKA)(ri) and protein kinase G (PKG)(1alpha 1beta) in TSMCs.SQ 22536 inhibited KMUP-3-induced expression of (PKA)(ri). On the contrary, ODQ inhibited KMUP-3-induced expression of PKG(1alpha 1beta) In epithelium-intact trachea, KMUP-3 increased the NO release. Activation of sGC, NO release, and inhibition of phosphodiesterases in TSMCs by KMUP-3 may result in increases of intracellular cGMP and cAMP, which subsequently activate PKG and PKA, efflux of K(+) ion, and associated reduction in Ca(2+) influx in vitro, indicating the action mechanism to protect against TNF-alpha-induced airway dysfunction in vivo.

An anti-tumor role for cGMP-dependent protein kinase.

This study compared Type-1 cGMP-dependent protein kinase (PKG) expression in normal and tumor tissues and examined PKG function in tumor growth. Studies with a cDNA array revealed that PKG expression was reduced in many tumors compared to respective normal tissue. This decrease in PKG expression was confirmed using quantitative RT-PCR and western blotting of matched colon specimens from normal epithelium and tumor tissue, and also in colon derived cell lines where luciferase reporter analysis revealed that the decreased expression occurred at the transcriptional level. Using SW620 colon carcinoma cells engineered for inducible expression of PKG1beta, it was found that exogenous PKG1beta lead to decreased tumor growth and invasiveness in nude mouse xenografts.

Activation of protein kinase G up-regulates expression of 15-lipoxygenase-1 in human colon cancer cells.

Recent studies indicate that the induction of apoptosis in human colon cancer cells by certain nonsteroidal antiinflammatory drugs involves increased expression of 15-LOX-1 and synthesis of its major product 13-S-hydroxyoctadecadienoic acid (13-S-HODE). Evidence was obtained that this occurs via a cyclooxygenase-2 (COX-2)-independent mechanism, but the actual mechanism of induction of 15-LOX-1 by these compounds is not known. There is extensive evidence that treatment of SW480 human colon cancer cells with sulindac sulfone (Exisulind, Aptosyn) or the related derivative OSI-461, both of which inhibit cyclic GMP (cGMP)-phosphodiesterases but lack COX-2 inhibitory activity, causes an increase in intracellular levels of cGMP, thus activating protein kinase G (PKG), which then activates pathways that lead to apoptosis. Therefore, in the present study, we examined the effects of various agents that cause increased cellular levels of cGMP on the expression of 15-LOX-1 in SW480 human colon cancer cells. Treatment of the cells with Exisulind, sulindac sulfide, OSI-461, the guanylyl cyclase activator YC-1, or the cell-permeable cGMP compound 8-para-chlorophenylthio-cGMP (8-pCPT-cGMP) caused an increase in cellular levels of 15-LOX-1. Exisulind, OSI-461, and 8-pCPT-cGMP also increased mRNA levels of 15-LOX-1, suggesting that the effects were at the level of transcription. The cGMP-phosphodiesterase inhibitors and YC-1 increased the production of 13-S-HODE, which is the linoleic acid metabolite of 15-LOX-1. Treatment of SW480 cells with the PKG inhibitor Rp-8-pCPT-cGMP blocked Exisulind-induced 15-LOX-1 expression. Furthermore, derivatives of SW480 cells that were engineered to stably overexpress wild-type PKG Ibeta displayed increased cellular levels of 15-LOX-1 when compared with vector control cells. Taken together, these results provide evidence that the cGMP/PKG pathway can play an important role in the induction of 15-LOX-1 expression by nonsteroidal antiinflammatory drugs and related agents.

Neutrophil dysfunction in guanosine 3',5'-cyclic monophosphate-dependent protein kinase I-deficient mice.

The regulation of neutrophil functions by Type I cGMP-dependent protein kinase (cGKI) was investigated in wild-type (WT) and cGKI-deficient (cGKI-/-) mice. We demonstrate that murine neutrophils expressed cGKIalpha. Similar to the regulation of Ca2+ by cGKI in other cells, there was a cGMP-dependent decrease in Ca2+ transients in response to C5a in WT, but not cGKI-/- bone marrow neutrophils. In vitro chemotaxis of bone marrow neutrophils to C5a or IL-8 was significantly greater in cGKI-/- than in WT. Enhanced chemotaxis was also observed with cGKI-/- peritoneal exudate neutrophils (PE-N). In vivo chemotaxis with an arachidonic acid-induced inflammatory ear model revealed an increase in both ear weight and myeloperoxidase (MPO) activity in ear punches of cGKI-/- vs WT mice. These changes were attributable to enhanced vascular permeability and increased neutrophil infiltration. The total extractable content of MPO, but not lysozyme, was significantly greater in cGKI-/- than in WT PE-N. Furthermore, the percentage of MPO released in response to fMLP from cGKI-/- (69%) was greater than that from WT PE-N (36%). PMA failed to induce MPO release from PE-N of either genotype. In contrast, fMLP and PMA released equivalent amounts of lysozyme from PE-N. However, the percentage released was less in cGKI-/- (approximately 60%) than in WT (approximately 90%) PE-N. Superoxide release (maximum velocity) revealed no genotype differences in responses to PMA or fMLP stimulation. In summary, these results show that cGKIalpha down-regulates Ca2+ transients and chemotaxis in murine neutrophils. The regulatory influences of cGKIalpha on the secretagogue responses are complex, depending on the granule subtype.

In the rostral ventrolateral medulla, the 70-kDa heat shock protein (HSP70), but not HSP90, confers neuroprotection against fatal endotoxemia via augmentation of nitric-oxide synthase I (NOS I)/protein kinase G signaling pathway and inhibition of NOS II/peroxynitrite cascade.

Heat shock proteins (HSPs) represent a group of highly conserved intracellular proteins that participate in protective adaptation against cellular stress. We evaluated the neuroprotective role of the 70-kDa HSP (HSP70) and the 90-kDa HSP (HSP90) at the rostral ventrolateral medulla (RVLM), the medullary origin of sympathetic vasomotor tone, during fatal endotoxemia. In Sprague-Dawley rats maintained under propofol anesthesia, Escherichia coli lipopolysaccharide (30 mg/kg, i.v.) induced a decrease (phase I), followed by an increase (phase II; "pro-life" phase) and a secondary decrease (phase III; "pro-death" phase) in the power density of the vasomotor component of systemic arterial pressure spectrum, along with progressive hypotension or bradycardia. Proteomic and Western blot analyses revealed that whereas HSP70 expression in the RVLM was significantly augmented during phases I and II and returned to baseline during phase III endotoxemia, HSP90 protein expression remained constant. The increase in HSP70 level was significantly blunted on pretreatment with microinjection of the transcription inhibitor actinomycin D or protein synthesis inhibitor cycloheximide into the bilateral RVLM. Functional blockade of HSP70 in the RVLM by an anti-HSP70 antiserum or prevention of synthesis by an antisense hsp70 oligonucleotide exacerbated mortality or potentiated the cardiovascular depression during experimental endotoxemia, alongside significantly reduced nitric-oxide synthase (NOS) I or protein kinase G (PKG) level or augmented NOS II or peroxynitrite level in the RVLM. We conclude that whereas HSP90 is ineffective, de novo synthesis of HSP70 in the RVLM may confer neuroprotection during fatal endotoxemia by preventing cardiovascular depression via enhancing the sympathoexcitatory NOS I/PKG signaling pathway and inhibiting the sympathoinhibitory NOS II/peroxynitrite cascade in the RVLM.

Upstream stimulatory factors (USF-1/USF-2) regulate human cGMP-dependent protein kinase I gene expression in vascular smooth muscle cells.

Cyclic GMP-dependent protein kinase I plays a pivotal role in regulating smooth muscle cell relaxation, growth, and differentiation. Expression of the enzyme varies greatly in smooth muscle and in other tissues and cell types, yet little is known regarding the mechanisms regulating cGMP-dependent protein kinase gene expression. The present work was undertaken to characterize the mechanisms controlling kinase gene expression in vascular smooth muscle cells. A 2-kb human cGMP-dependent protein kinase I 5'-noncoding promoter sequence was characterized by serial deletion, and functional studies demonstrated that a 591-bp 5'-promoter construct possessed the highest activity compared with all other constructs generated from the larger promoter. Analysis of the sequence between -472 and -591 bp from the transcriptional start site revealed the existence of two E-like boxes known to bind upstream stimulatory factors. Electrophoretic mobility shift assays and functional studies using luciferase reporter gene assays identified upstream stimulatory factors as the transcription factors bound to the E-boxes in the 591-bp promoter. Site-directed mutagenesis of the E-boxes abolished the binding of upstream stimulatory factor proteins and decreased the activity of the cGMP-dependent protein kinase I 591-bp promoter, thus confirming the involvement of these transcription factors in mediating gene expression. Cotransfection experiments demonstrated that overexpression of upstream stimulatory factors 1 and 2 increased cGMP-dependent protein kinase I promoter activity. Collectively, these data suggest that the human proximal cGMP-dependent protein kinase I promoter is regulated by tandem E-boxes that bind upstream stimulatory factors.

Expression and function of cGMP-dependent protein kinase type I during medaka fish embryogenesis.

We isolated and characterized cDNA clones (PKG Ialpha and PKG Ibeta) for medaka fish cGMP-dependent protein kinase (PKG) Ialpha and Ibeta, and demonstrated that both are expressed in the embryos after late gastrula stage. Whole-mount in situ hybridization using each isoform-specific probe revealed that the transcripts of the PKG Ialpha gene were present in the spinal cord and gill arch, whereas those of the PKG Ibeta gene were only weakly expressed in these organs, but highly expressed in the otic vesicles. Injection of PKG Ialpha-specific morpholino antisense oligonucleotides (Ialpha-MO) into two-cell stage medaka fish embryos caused severe abnormalities in the developing embryos, such as the development of a hammer-like head, fusion of the developing eyes, and degeneration of cells around the eyes, whereas injection of PKG Ibeta-specific morpholino antisense oligonucleotides (Ibeta-MO) caused fewer abnormalities in the embryos, even when injected at higher concentrations than Ialpha-MO. The PKG I-overexpressing embryos exhibited smaller eyes and enlargement of the forebrain, a phenotype similar to that observed in the cAMP-dependent protein kinase (PKA)-depressed embryos. In the PKG-deficient embryos, a sonic hedgehog (shh)-target gene, HNF-3beta, was expressed weakly, and this phenotype was similar to that observed in the PKA-overexpressing embryos suggesting that the cGMP/PKG signaling pathway is involved in some steps of shh signaling. We also demonstrated that Gli proteins, shh-downstream molecules, are phosphorylated by the NO/cGMP signaling pathway, probably by PKG in NG108-15 neuroblastoma cells. These results imply that PKG and PKA share common substrates and work in an opposite manner during the early embryogenesis of medaka fish.