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NO-sensitive guanylyl cyclase β1 subunit is peripherally associated to chromosomes during mitosis. Novel role in chromatin condensation and cell cycle progression
Abstract
NO-sensitive guanylyl cyclase (GC(NO)), the major NO target, is involved in important regulatory functions in the cardiovascular, gastrointestinal and central nervous systems. GC(NO) exists as heterodimers of alpha(1/2) and beta1 subunits. Deletion of the obligate beta1 dimerizing partner abrogates NO/cGMP signaling and shortens the life span of KO mice. Localization studies in the CNS have shown that beta1 is more widespread than alpha subunits and in some areas is the only GC(NO) subunit expressed, suggesting that beta1 may have GC(NO)-independent functions. GC(NO) is predominantly cytosolic, but association to membranes and other intracellular structures has been described. Here, we show localization of beta1 in cytoplasm and nucleus of cells expressing alpha subunits and GC(NO) activity (astrocytes, C6 cells), as well as in cells devoid of alpha subunits and GC(NO) activity (microglia). In both cell types beta1 associates peripherally to chromosomes in all phases of mitosis. Immunodepletion of beta1 in C6 cells enhances chromatin condensation in an in vitro assay. Moreover, silencing beta1 by siRNA induces cell cycle re-entry as determined by flow cytometry, and increases proliferation rate in a MTT-assay, whereas infection with beta1-containing adenovirus has the opposite effect. These actions are independent of cGMP formation. We postulate that beta1 is a multifunctional protein that regulates chromatin condensation and cell cycle progression, in addition to being an obligate monomer in functional GC(NO) heterodimers.
... In contrast, the sGCβ1 role in cGMP-independent functions has been much less investigated. sGCβ1 was shown to associate with chromosomes during mitosis in glial cells thereby impeding cell division and promoting cell cycle arrest [15]. sGCβ1 gene promoter region was also found to be hyperacetylated and hypermethylated in breast cancer cells, and restoration of sGCβ1 subunit expression decreases cell proliferation and migration [16,17]. ...
... Here we demonstrated that the reduction in cell viability was independent of cGMP production, since sGC enzymatic activity inhibition by ODQ did not avoid the effects J o u r n a l P r e -p r o o f observed after sGCβ1 overexpression in the current model. Previous evidence obtained in a glioblastoma model supports our findings [15,40]. Another study reported that restoration of sGC activity decreased cell growth and viability in breast cancer tumor cell lines. ...
... Confocal microscopy revealed that sGCα1 is located in cytoplasm, whereas sGCβ1 exhibited cytosolic as well as nuclear localization for both cell types. This finding concords with results reported on glia [15], differentiating embryonic cells [41] and U87 glioblastoma cells [40]. All this evidence strengthens the concept that sGC1 [10][11][12] and sGC1 subunits have independent roles beyond forming a heterodimeric enzyme. ...
Endometrial and cervical cancer are among the most frequently diagnosed malignancies globally. Nitric oxide receptor-soluble guanylyl cyclase (sGC) is a heterodimeric enzyme composed of two subunits, α1 and β1. Previously we showed that sGCα1 subunit promotes cell survival, proliferation, and migration, but the role of sGCβ1 subunit has not been addressed. The aim of the present work was to study the impact of sGCβ1 restoration in proliferation, survival, migration, and cell signaling in endometrial and cervical cancer cells. We found that sGCβ1 transcript levels are reduced in endometrial and cervical tumors vs normal tissues. We confirmed nuclear enrichment of sGCβ1, unlike sGCα1. Overexpression of sGCβ1 reduced cell viability and augmented apoptotic index. Cell migration and invasion were also negatively affected. All these sGCβ1-driven effects were independent of sGC enzymatic activity. sGCβ1 reduced the expression of epithelial-to-mesenchymal transition factors such as N-cadherin and β-catenin and increased the expression of E-cadherin. sGCβ1 impacted signaling in endometrial and cervical cancer cells through significant downregulation of Akt pathway affecting some of its main targets such as GSK-3β and c-Raf. Our results show for the first time that sGCβ1 exerts several antiproliferative actions in ECC-1 and HeLa cell lines by targeting key regulatory pathways.
... Genes of a1 and b1 subunits lack of estrogen response elements (ERE) in their sequences, but they include consensus sites for binding of Sp-1, c-Myb and NFk-B, among others, in their promoter regions [34]. These factors are regulated by E2353637 so it could be possible that E2 regulates sGC subunit expression through modulation of these proteins. Although sGC a1 and b1 promoter regions have many binding sites for different factors in common, they yield independent activities, which together with their separated location in the chromosome, support the hypothesis of independent regulation of both genes [38]. ...
... These facts could explain the differential response of both genes to E2 action [39]. Evidence provided by us, together with reports of temporal unbalance in a1 and b1 expression [8] and participation of a1 and b1 individually in cell cycle regulation [34,35] support the hypotheses that the imbalance of sGC subunits expression exceed the solely regulation of enzimatic sGC activity. It was previously reported that a1 acts in prostate cancer via a novel pathway that does not depend on b1 and that its expression correlates with advanced prostate cancer. ...
... Thus, a1 has been proposed as an important mediator of the procarcinogen effects of androgens [34]. On the other hand, b1 was found associated with chromosomes during mitosis in neural cells negatively regulating cell cycle progression [35]. Thus, besides comprising sGC enzyme, a1 and b1 independently appear as multifunctional proteins with key roles in cell processes. ...
17β-estradiol (E2) regulates hormonal release as well as proliferation and cell death in the pituitary. The main nitric oxide receptor, nitric oxide sensitive- or soluble guanylyl cyclase (sGC), is a heterodimer composed of two subunits, α and β, that catalyses cGMP formation. α1β1 is the most abundant and widely expressed heterodimer, showing the greater activity. Previously we have shown that E2 decreased sGC activity but exerts opposite effects on sGC subunits increasing α1 and decreasing β1 mRNA and protein levels. In the present work we investigate the mechanisms by which E2 differentially regulates sGC subunits' expression on rat anterior pituitary gland. Experiments were performed on primary cultures of anterior pituitary cells from adult female Wistar rats at random stages of estrous cycle. After 6 h of E2 treatment, α1 mRNA and protein expression is increased while β1 levels are down-regulated. E2 effects on sGC expression are partially dependent on de novo transcription while de novo translation is fully required. E2 treatment decreased HuR mRNA stabilization factor and increased AUF1 p37 mRNA destabilization factor. E2-elicited β1 mRNA decrease correlates with a mRNA destabilization environment in the anterior pituitary gland. On the other hand, after 6 h of treatment, E2-BSA (1 nM) and E2-dendrimer conjugate (EDC, 1 nM) were unable to modify α1 or β1 mRNA levels, showing that nuclear receptor is involved in E2 actions. However, at earlier times (3 h), 1 nM EDC causes a transient decrease of α1 in a PI3k-dependent fashion. Our results show for the first time that E2 is able to exert opposite actions in the anterior pituitary gland, depending on the activation of classical or non-classical pathways. Thus, E2 can also modify sGC expression through membrane-initiated signals bringing to light a new point of regulation in NO/sGC pathway.
... A smaller fraction of stained cells (*12%) demonstrated intense filamentous staining, indicating that a1 sGC protein is likely to be associated with some intracellular structures in these cells (Fig. 4, left pane). The b1 sGC subunit was shown to be associated with chromosomes during mitosis and was localized to the perinuclear region in a number of cell lines [27]. Thus, we have assessed subcellular localization of sGC a1 and b1 subunits in differentiated H9 cells by confocal microscopy. ...
... 5 indicate that a1 sGC is predominantly localized in the cytosol. On the contrary, the b1 sGC subunit demonstrated both perinuclear and cytosolic localization supporting the hypothesis that sGC subunits might participate in differentiation process as hetero-and mono-dimers [27]. ...
... We used confocal microscopy to evaluate nuclear localization of a1 and b1 sGC subunits in differentiating H9 cells. Nuclear and perinuclear localization of sGC subunits has been demonstrated in ES cell-derived cardiomyocytes [12], hepatocytes [33], and astrocytes [27], where it was proposed to play an important role in regulation of transcription and proliferation via both cGMP-dependent and cGMP-independent signaling. Our data support the report of nuclear localization of the monomeric b1 subunit [27], as we found that a1 sGC signal is preferentially in the cytosolic regions (Fig. 5, green), whereas the b1 sGC staining (Fig. 5, red) was located both in nuclei and cytosol. ...
Nitric oxide (NO), an important mediator molecule in mammalian physiology, initiates a number of signaling mechanisms by activating the enzyme soluble guanylyl cyclase (sGC). Recently, a new role for NO/cyclic guanosine monophosphate signaling in embryonic development and cell differentiation has emerged. The changes in expression of NO synthase isoforms and various sGC subunits has been demonstrated during human and mouse embryonic stem (ES) cells differentiation. Previously, our laboratory demonstrated that nascent α1 sGC transcript undergoes alternative splicing and that expression of α1 sGC splice forms directly affects sGC activity. Expression of sGC splice variants in the process of human ES (hES) cells differentiation has not been investigated. In this report, we demonstrate that α1 sGC undergoes alternative splicing during random hES differentiation for the first time. Our results indicate that C-α1 sGC splice form is expressed at high levels in differentiating cells and its intracellular distribution varies from canonical α1 sGC subunit. Together, our data suggest that alternative splicing of sGC subunits is associated with differentiation of hES cells.
... Intriguingly there is recent evidence to suggest that both the isolated a 1 subunit and the isolated b 1 subunit have a separate function from cGMP signalling: the a 1 subunit seems to act as an important mediator of the procarcinogenic effect of androgens [7,8]. The b 1 subunit has been suggested to regulate chromatin condensation and cell cycle progression [9]. ...
... In addition our data show that the a 2 subunit also forms homodimers. Thus coexpression of a and b subunits will always lead to a mixture of active NOsGC-heterodimers and catalytically inactive NOsGC homodimers with other potential functions [7][8][9]. To isolate pure heterodimeric enzymes, we cloned constructs that guarantee a 1:1 stochiometry between a and b subunits and rule out the presence of homodimers. ...
... The knockout animals clearly demonstrate interdependence of the stability of the a and b subunits [19]. Third in a transgenic overexpression model homodimers of isolated a or isolated b subunits will form in addition to heterodimers and there is recent evidence to suggest that they have a separate function from cGMP signaling: it has been suggested that the b 1 subunit is a multifunctional protein that regulates chromatin condensation and cell cycle progression in addition to being an obligate component of functional NOsGC-heterodimers [9]. Published data also indicates that the a 1 subunit acts in prostate cancer via a novel pathway that does not depend on the b 1 subunit [7] and is especially important in androgen-independent growth of prostate cancer cells [8]. ...
Nitric oxide sensitive guanylyl cyclase (NOsGC) is a heterodimeric enzyme consisting of an α and a β subunit. Two heterodimeric enzymes are known to be important for NO-signalling in humans: α(1)/β(1) and α(2)/β(1). No difference had so far been detected with respect to their pharmacological properties, but as we show in the present paper the new drugs cinaciguat and ataciguat activate the α(1)/β(1) form more effectively. Recent evidence suggests that homodimeric complexes of α and β subunits exist in vivo and that these non-heterodimerizing subunits have a separate function from cGMP signaling. To isolate the effect of the α(1)/β(1) or α(2)/β(1) heterodimeric enzyme in overexpression experiments from potential effects of non-heterodimerizing α(1), β(1) or α(2) subunits, we cloned constructs that guarantee a 1:1 stochiometry between α and β subunits and rule out the presence of homodimers. The carboxy-terminus of the β(1) subunit was directly fused to the amino-terminus of either the α(1) or α(2) subunit. The two different "conjoined" NOsGCs faithfully reproduced the biochemical and pharmacological properties of the α(1)/β(1) and α(2)/β(1) heterodimeric enzymes including the differential activation by ciguat-activators. Conjoined NOsGCs can be used for isoform specific overexpression in transgenic animals and therapeutic overexpression may be an application in the future. In both cases possible side effects of homodimeric α or β subunits are avoided. Crystallization with the goal of structure determination may also be easier for conjoined NOsGCs because enzyme preparations are more homogenous and are free of "contaminating" homodimers.
... In situ proximity ligation assay shows close proximity of sGC and PDI Thus far, the interaction between sGC and PDI was shown to be the result of thiol-disulfide exchange by Western blot, thiol titer and PDI-Flag IP. In addition, PDI inhibited sGC NOstimulated activity Although PDI is typically found in the ER, other subcellular locations are known [14][15][16][17][18]; and, sGC while "soluble," is also found in non-cytosolic subcellular locations [10,31,32]. In order for the redox interaction to be physiologically relevant, we sought to confirm this interaction in smooth muscle cells (SMC). ...
... Yet, PDI is also found at other cellular locations and under certain conditions, including a recently described thiol-dependent interaction with the subunit p47 of NADPH oxidase in the cytosol, ( [14,17]; reviewed in [16,18]). Of note, sGC, which is described as a "soluble" protein, has subcellular locations other than the cytosol including the plasma membrane and nucleus [10,31,32]. Interestingly, mammalian form sGC α1β1 has a high number of conserved Cys (2.8% total amino acid content). ...
Nitric oxide (NO) binds to the receptor, soluble guanylyl cyclase (sGC), stimulating cGMP production. The NO-sGC-cGMP pathway is a key component in the cardiovascular system. Discrepancies in sGC activation and deactivation in vitro vs. in vivo have led to a search for endogenous factors that regulate sGC or assist in cellular localization. In our previous work, which identified Hsp70 as a modulator of sGC, we determined that protein disulfide isomerase (PDI) bound to an sGC-affinity matrix. We here establish and characterize this interaction. Incubation of purified PDI with semi-purified sGC, both reduced and oxidized, resulted in different migration patterns on non-reducing Western blots indicating a redox component to the interaction. In sGC-infected COS-7 cells, transfected Flag-tagged PDI and PDI-CXXS (redox active site "trap-mutant") pulled down sGC. This PDI-sGC complex was resolved by reductant, confirming a redox interaction. PDI inhibited NO-stimulated sGC activity in COS-7 lysates; however, a PDI redox inactive mutant, PDI-SXXS, did not. Together, these data unveil a novel mechanism of sGC redox modulation via thiol-disulfide exchange. Finally, in smooth muscle cells, endogenous PDI and sGC co-localize by in situ proximity ligation assay, suggesting biological relevance. PDI-dependent redox regulation of sGC NO-sensitivity may provide a secondary control over vascular homeostasis.
... The three NOS isoforms have all been observed in cell nuclei under pathophysiological conditions [99][100][101][102][103]. Guanylyl cyclase [104], subunits of the sGC [105], cGMP production [105], calmodulin [106] and tetrahydrobiopterin biosynthetic enzymes [107] have also been detected in nuclei, suggesting that nuclear production of nitric oxide and the subsequent activation of its downstream targets is possible. ...
... The three NOS isoforms have all been observed in cell nuclei under pathophysiological conditions [99][100][101][102][103]. Guanylyl cyclase [104], subunits of the sGC [105], cGMP production [105], calmodulin [106] and tetrahydrobiopterin biosynthetic enzymes [107] have also been detected in nuclei, suggesting that nuclear production of nitric oxide and the subsequent activation of its downstream targets is possible. ...
The effects of nitric oxide in biological systems depend on its steady-state concentration and where it is being produced. The organ where nitric oxide is produced is relevant, and within the organ, which types of cells are actually contributing to this production seem to play a major determinant of its effect. Subcellular compartmentalization of specific nitric oxide synthase enzymes has been shown to play a major role in health and disease. Pathophysiological conditions affect the cellular expression and localization of nitric oxide synthases, which in turn alter organ cross talk. In this study, we describe the compartmentalization of nitric oxide in organs, cells, and subcellular organelles and how its localization relates to several relevant clinical conditions. Understanding the complexity of the compartmentalization of nitric oxide production and the implications of this compartmentalization in terms of cellular targets and downstream effects will eventually contribute toward the development of better strategies for treating or preventing pathological events associated with the increase, inhibition, or mislocalization of nitric oxide production.
... Thus, loss of the subunit in LNCaP cells recapitulates functional sGC loss. Under androgen-replete conditions, the sGC KO cells displayed enhanced proliferation compared to their WT counterparts (Fig 3F, left), consistent with cell cycle-inhibitory effects previously reported for the subunit 29 . More significantly, we found that the LNCaP SB0 KO cell population behaved similarly to the LNCaP SB5 variants 4 : they were able to proliferate under androgen-deprived conditions (Fig. 3F, right), displayed overall lower levels of senescence-associated beta-galactosidase (SA-beta-gal) activity (Fig. 3G) and lower p16 INK4a under prolonged AD culture (Fig. 3H). ...
Castration-resistant prostate cancer (CRPC) is fatal and therapeutically under-served. We describe a novel CRPC-restraining role for the vasodilatory soluble guanylyl cyclase (sGC) pathway. We discovered that sGC subunits are dysregulated during CRPC progression and its catalytic product, cyclic GMP (cGMP), is lowered in CRPC patients. Abrogating sGC heterodimer formation in castration-sensitive prostate cancer (CSPC) cells inhibited androgen deprivation (AD)-induced senescence, and promoted castration-resistant tumor growth. We found sGC is oxidatively inactivated in CRPC. Paradoxically, AD restored sGC activity in CRPC cells through redox-protective responses evoked to protect against AD-induced oxidative stress. sGC stimulation via its FDA-approved agonist, riociguat, inhibited castration-resistant growth, and the anti-tumor response correlated with elevated cGMP, indicating on-target sGC activity. Consistent with known sGC function, riociguat improved tumor oxygenation, decreasing the PC stem cell marker, CD44, and enhancing radiation-induced tumor suppression. Our studies thus provide the first evidence for therapeutically targeting sGC via riociguat to treat CRPC.
Statement of significance
Prostate cancer is the second highest cancer-related cause of death for American men. Once patients progress to castration-resistant prostate cancer, the incurable and fatal stage, there are few viable treatment options available. Here we identify and characterize a new and clinically actionable target, the soluble guanylyl cyclase complex, in castration-resistant prostate cancer. Notably we find that repurposing the FDA-approved and safely tolerated sGC agonist, riociguat, decreases castration-resistant tumor growth and re-sensitizes these tumors to radiation therapy. Thus our study provides both new biology regarding the origins of castration resistance as well as a new and viable treatment option.
... The present study revealed that sGCβ1 alone can migrate into the nucleus, thus impacting malignant cellular signaling to change the course of tumor growth. Consistent with this finding, the studies of sGCβ1 in rat brain astrocytes and glioma cells demonstrated that sGCβ1 is localized in the nucleus and is associated with the chromosomes during mitosis, regulating chromatin condensation and cell cycle progression in a cGMP-independent manner [45]. ...
Simple Summary
A marked reduction in soluble guanylyl cyclase β1 (sGCβ1) transcript is characteristic for human glioma specimens. Restoring the expression of sGCβ1 inhibited the aggressive course of glioblastoma in an orthotopic xenograft mouse model. The present study is the first to reveal that sGCβ1 migrated into the nucleus and interacted with the promoter of the TP53 gene. sGCβ1 overexpression impacted signaling in glioblastoma multiforme, including the promotion of nuclear accumulation of p53, a marked reduction in cyclin-dependent kinase 6 (CDK6), and a significant decrease in integrin α6. Antitumor effect of sGCβ1 was not associated with enzymatic activity of sGC.
Abstract
Malignant glioma is the most common and deadly brain tumor. A marked reduction in the levels of sGC (soluble guanylyl cyclase) transcript in the human glioma specimens has been revealed in our previous studies. In the present study, restoring the expression of sGCβ1 alone repressed the aggressive course of glioma. The antitumor effect of sGCβ1 was not associated with enzymatic activity of sGC since overexpression of sGCβ1 alone did not influence the level of cyclic GMP. Additionally, sGCβ1-induced inhibition of the growth of glioma cells was not influenced by treatment with sGC stimulators or inhibitors. The present study is the first to reveal that sGCβ1 migrated into the nucleus and interacted with the promoter of the TP53 gene. Transcriptional responses induced by sGCβ1 caused the G0 cell cycle arrest of glioblastoma cells and inhibition of tumor aggressiveness. sGCβ1 overexpression impacted signaling in glioblastoma multiforme, including the promotion of nuclear accumulation of p53, a marked reduction in CDK6, and a significant decrease in integrin α6. These anticancer targets of sGCβ1 may represent clinically important regulatory pathways that contribute to the development of a therapeutic strategy for cancer treatment.
... When sGC α and β subunits were expressed separately in eukaryotic cells, they formed homodimers, although they did so to a much lesser extent when the two different subunits were co-expressed (38). Predominant sGCβ or sGCα expression appears to take place in certain cells or tissues (39)(40)(41)(42)(43), and both sGC subunits can display reduced buildup under stress conditions (44)(45)(46)(47)(48)(49). Finally, although many H-NOX protein family members have attached catalytic domains (50,51) they typically function as monomers or as homodimers rather than heterodimers (15,21,51), distinguishing sGC in this respect. ...
Soluble guanylate cyclase (sGC) is a heme-containing heterodimeric enzyme that generates many molecules of cGMP in response to its ligand NO; sGC thereby acts as an amplifier in NO-driven biological signaling cascades. Because sGC helps regulate the cardiovascular, neuronal, and gastrointestinal systems through its cGMP production, boosting sGC activity and preventing or reversing sGC inactivation are important therapeutic and pharmacologic goals. Work over the last two decades is uncovering the processes by which sGC matures to become functional, how sGC is inactivated, and how sGC is rescued from damage. A diverse group of small molecules and proteins have been implicated in these processes, including NO itself, reactive oxygen species, cellular heme, cell chaperone Hsp90, and various redox enzymes as well as pharmacologic sGC agonists. This review highlights their participation and provides an update on the processes that enable sGC maturation, drive its inactivation, or assist in its recovery in various settings within the cell, in hopes of reaching a better understanding of how sGC function is regulated in health and disease.
... It has even been suggested that non-heterodimerizing subunits have a separate function from cGMP signaling: The a 1 subunit is thought to act as a mediator of the procarcinogenic effect of androgens [13][14][15]. The b 1 subunit has been implicated in chromatin condensation and cell cycle progression [16]. To isolate the effects of a 1 /b 1 and a 2 /b 1 heterodimeric enzyme complexes from effects of non-heterodimerizing a and b 1 subunits, we cloned and characterized conjoined NOsGCs [3,17]. ...
Nitric oxide-sensitive guanylyl cyclase is a heterodimeric enzyme consisting of an α and a β subunit. Two different α subunits (α1 and α2) give rise to two heterodimeric enzymes α1/β1 and α2/β1. Both coexist in a wide range of tissues including blood vessels and the lung, but expression of the α2/β1 form is generally much lower and approaches levels similar to the α1/β1 form in the brain only. In the present paper, we show that the α2/β1 form interacts with Lin7a in mouse brain synaptosomes based on co-precipitation analysis. In HEK293 cells, we found that the overexpressed α2/β1 form, but not the α1/β1 form is directed to calcium-insensitive cell-cell contacts. The isolated PDZ binding motif of an amino-terminally truncated α2 subunit was sufficient for cell-cell contact localization. For the full length α2 subunit with the PDZ binding motif this was only the case in the heterodimer configuration with the β1 subunit, but not as isolated α2 subunit. We conclude that the PDZ binding motif of the α2 subunit is only accessible in the heterodimer conformation of the mature nitric oxide-sensitive enzyme. Interaction with Lin7a, a small scaffold protein important for synaptic function and cell polarity, can direct this complex to nectin based cell-cell contacts via MPP3 in HEK293 cells. We conclude that heterodimerization is a prerequisite for further protein-protein interactions that direct the α2/β1 form to strategic sites of the cell membrane with adjacent neighbouring cells. Drugs increasing the nitric oxide-sensitivity of this specific form may be particularly effective.
... One of the subunits − 1 interacts with chromosomes during mitosis (Fig. 2c). Therefore, a nuclear pathway of NO-activated sGC, involving interactions with folding of chromatin and chromosomes during mitosis, has also been proposed (Pifarré et al., 2009). Despite extensive research, the precise transport mechanism of the 1 subunit of sGC to the nucleus remains unclear. ...
Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) activates signaling pathways responsible for smooth muscle cell relaxation, leading to vasodilation and thus plays an important role in controlling vascular homeostasis, thrombosis and inflammation. Recent studies indicate that S-nitrosothiols produced in vivo as well as synthetic ones might be important reservoirs of NO. Based on a broad range of NO functions within the living organisms, this review highlights the impact of S-nitrosothiols on cardiovascular cell cycle. The cell membrane transport and the decomposition patterns responsible of S-nitrosothiols actions are presented. The effects of NO delivery through S-nitrosothiols have a significant potential in cardiovascular diseases with various underlying causes. The challenges related to their application in the pharmacotherapy of patients with various cardiovascular diseases are also discussed.
... However, the exact function of nuclear nNOS has remained elusive even though compelling evidence has shown that many of the factors that are involved in its activity (e.g. sGC subunits, calmodulin and enzymes involved in tetrahydrobiopterin synthesis) are present in the nucleus (Bachs et al., 1992;Elzaouk et al., 2004;Pifarré et al., 2009). Besides regulating transcription by interacting with transcription factors (i.e. by the formation of nNOS-Sp1 complex), we have found that, during myocyte differentiation, nuclear nNOS-derived NO functions by driving mitochondrial biogenesis . ...
Neuronal nitric oxide synthase (nNOS) and peroxisome proliferator activated receptor γ co-activator 1α (PGC-1α) are two fundamental factors involved in the regulation of skeletal muscle cell metabolism. nNOS exists as several alternatively spliced variants, each having a specific pattern of subcellular localisation. Nitric oxide (NO) functions as a second messenger in signal transduction pathways that lead to the expression of metabolic genes involved in oxidative metabolism, vasodilatation and skeletal muscle contraction. PGC-1α is a transcriptional coactivator and represents a master regulator of mitochondrial biogenesis by promoting the transcription of mitochondrial genes. PGC-1α can be induced during physical exercise, and it plays a key role in coordinating the oxidation of intracellular fatty acids with mitochondrial remodelling. Several lines of evidence demonstrate that NO could act as a key regulator of PGC-1α expression; however, the link between nNOS and PGC-1α in skeletal muscle remains only poorly understood. In this Commentary, we review important metabolic pathways that are governed by nNOS and PGC-1α, and aim to highlight how they might intersect and cooperatively regulate skeletal muscle mitochondrial and lipid energetic metabolism and contraction.
... [44] Recent results point to separate effects of the single subunits related to androgen regulation and cell cycle progression. [123,124] Both subunits are organized in four domains (Figure 4). The b1-subunit consists of a N-terminal heme-binding domain which is closely related to a family of heme-containing and NO-and oxygen-binding enzymes and therefore is referred to as the H-NOX domain. ...
The vasodilatory properties of nitric oxide (NO) have been utilized in pharmacotherapy for more than 130 years. Still today, NO-donor drugs are important in the management of cardiovascular diseases. However, inhaled NO or drugs releasing NO and organic nitrates are associated with noteworthy therapeutic shortcomings, including resistance to NO in some disease states, the development of tolerance during long-term treatment, and nonspecific effects, such as post-translational modification of proteins. The beneficial actions of NO are mediated by stimulation of soluble guanylate cyclase (sGC), a heme-containing enzyme which produces the intracellular signaling molecule cyclic guanosine monophosphate (cGMP). Recently, two classes of compounds have been discovered that amplify the function of sGC in a NO-independent manner, the so-called sGC stimulators and sGC activators. The most advanced drug, the sGC stimulator riociguat, has successfully undergone Phase III clinical trials for different forms of pulmonary hypertension.
... Recent findings indicate that other nuclear effects of NO altering the cell cycle can occur. A mechanism involved in the regulation of the cell cycle is the direct interaction of NO-sensitive GC with chromosomes during mitosis (26). More recently, NO has been shown to modulate chromatin folding in human endothelial cells through class II histone deacetylases (27). ...
Nitric oxide (NO) has been suggested to be a pathophysiological modulator of cell proliferation, cell cycle arrest and apoptosis. In this context, opposite effects can be exerted by NO under diverse conditions. Indeed, several studies have indicated that low relative concentrations of NO seem to favour cell proliferation and anti-apoptotic responses whereas higher levels of NO favour pathways inducing cell cycle arrest, mitochondria respiration, senescence or apoptosis. Here, we report the NO effects on both promotion and inhibition of cell proliferation with particular concern on cardiovascular disease, diabetes and stem cells. Moreover, we focus on molecular mechanisms of action involved in the control of cell cycle progression, which include both cGMP-dependent and -independent pathways. This growing field may lead to broad and novel targeted therapies against cardiovascular diseases especially during concomitant type 2 diabetes, as well as novel bioimaging NO-based diagnostic tools.
... The finding that the a 1 /b 1-heterodimer showed the expected diffuse cytosolic localization, while the C-a 1 /b 1-heterodimer was also present in the nucleus, is interesting in the context of reports suggesting a role for the b 1-subunit in the nucleus: Baltrons and colleagues have demonstrated that the b 1-subunit translocates to the nucleus for proteosomal degradation in rat astrocytes after treatment with bacterial endotoxin[19]. In a subsequent report the b 1-subunit was shown to be peripherally associated to chromosomes during mitosis and to play a role in chromatin condensation and cell cycle progression in rat C6 glioma cells[20]. The mechanism by which the b 1-subunit enters the nucleus is unknown since the protein lacks a recognizable nuclear localization signal[19]. ...
Nitric oxide sensitive guanylyl cyclase (NOsGC) is a heterodimeric enzyme formed by an α- and a β₁-subunit. A splice variant (C-α₁) of the α₁-subunit, lacking at least the first 236 amino acids has been described by Sharina et al. 2008 and has been shown to be expressed in differentiating human embryonic cells. Wagner et al. 2005 have shown that the amino acids 61-128 of the α₁-subunit are mandatory for quantitative heterodimerization implying that the C-α₁-splice variant should lose its capacity to dimerize quantitatively.
In the current study we demonstrate preserved quantitative dimerization of the C-α₁-splice by co-purification with the β₁-subunit. In addition we used fluorescence resonance energy transfer (FRET) based on fluorescence lifetime imaging (FLIM) using fusion proteins of the β₁-subunit and the α₁-subunit or the C-α₁ variant with ECFP or EYFP. Analysis of the respective combinations in HEK-293 cells showed that the fluorescence lifetime was significantly shorter (≈0.3 ns) for α₁/β₁ and C-α₁/β₁ than the negative control. In addition we show that lack of the amino-terminus in the α₁ splice variant directs it to a more oxidized subcellular compartment.
We conclude that the amino-terminus of the α₁-subunit is dispensable for dimerization in-vivo and ex-vivo, but influences the subcellular trafficking.
... Its expression has been detected in all studied tissues, albeit at varying levels and with different subunit compositions [8]. Although sGC was originally purified from a cytosolic fraction, it was subsequently found associated with cellular membranes in a variety of cell types [9][10][11][12][13][14][15]. Each sGC subunit has two isoforms (α1/α2 and β1/β2) encoded by a total of 4 separate genes. ...
Soluble guanylyl cyclase (sGC) is a key protein in the nitric oxide (NO)/-cGMP signaling pathway. sGC activity is involved in a number of important physiological processes including smooth muscle relaxation, neurotransmission and platelet aggregation and adhesion. Regulation of sGC expression and activity emerges as a crucial factor in control of sGC function in normal and pathological conditions. Recently accumulated evidence strongly indicates that the regulation of sGC expression is a complex process modulated on several levels including transcription, post-transcriptional regulation, translation and protein stability. Presently our understanding of mechanisms governing regulation of sGC expression remains very limited and awaits systematic investigation. Among other ways, the expression of sGC subunits is modulated at the levels of mRNA abundance and transcript diversity. In this review we summarize available information on different mechanisms (including transcriptional activation, mRNA stability and alternative splicing) involved in the modulation of mRNA levels of sGC subunits in response to various environmental clues. We also summarize and cross-reference the information on human sGC splice forms available in the literature and in genomic databases. This review highlights the fact that the study of the biological role and regulation of sGC splicing will bring new insights to our understanding of NO/cGMP biology.
... NO protects the myocardium against I/R injury through the following mechanisms: (1) involvment of NO-sensitive guanylyl cyclase [58,59], (2) mediated activation of the second messenger cGMP [60] (3) inhibition of Ca 2+ influx [61], (4) opening of sarcolemmal K ATP channels [62] (5) activation of mitochondrial K ATP channel activity [63], (6) termination of ONOO-induced lipid radical chain propagation [64], and (7) stimulation of cyclooxygenase-2(COX-2) [65]. ...
Nitric oxide (NO) is produced in almost all tissues and it exerts a variety of biological actions under both physiological and pathological conditions. It is synthesized by three distinct enzymes: endothelial (eNOS), neuronal (nNOS) and inducible (iNOS) nitricoxide synthases. NO is a cardioprotective mediator in powerful cardioprotective processes such as pre- and post-conditioning ischemia;they operate largely in a NO-dependent manner. However, the activity of different NOSs isoforms as well as, the bioavailability of NO can be affected by a variety of disease conditions (in particular diabetes) and pathological situations associated with significantly elevated levels of the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α). These adversely affect NO-signaling, as well as the efficacy and safety of treatments with NO or NO-containing agents.
... Thus, the reduction in protein oxidative stress, apoptotic cell death and neuronal degeneration observed in zaprinast-treated animals most probably results from decreased activation of microglia/macrophages. Available data indicate that rat brain microglia do not express functional NO-GC and are thus unable to form cGMP in response to NO (Teunissen et al. 2000;van Staveren et al. 2005;Pifarre et al. 2009). However, rat brain microglia in culture has been reported to express mRNA for NPR-A and NPR-B (Moriyama et al. 2006) and expression of PDE1B has been shown to increase during monocyte to macrophage differentiation and to play a major role in regulating cGMP levels in these cells (Bender and Beavo 2006b). ...
J. Neurochem. (2010) 112 , 807–817.
Abstract
Recent evidence obtained in cultured glial cells indicates that cGMP‐mediated pathways regulate cytoskeleton dynamics, glial fibrillary acidic protein expression and motility in astrocytes, as well as inflammatory gene expression in microglia, suggesting a role in the regulation of the glial reactive phenotype. The aim of this work was to examine if cGMP regulates the glial inflammatory response in vivo following CNS damage caused by a focal cryolesion onto the cortex in rats. Results show that treatment with the cGMP phosphodiesterase inhibitor zaprinast (10 mg/kg i.p.) 2 h before and 24 and 48 h after the lesion results 3 days post‐lesion in notably enhanced astrogliosis manifested by increased glial fibrillary acidic protein immunoreactivity and protein levels around the lesion. In contrast, zaprinast decreased the number of round/ameboid lectin‐positive cells and the expression of the activated microglia/macrophage markers Iba‐1 and CD11b indicating decreased recruitment and activation of these cells. This altered inflammatory response is accompanied by a decrease in protein oxidative stress, apoptotic cell death and neuronal degeneration. In addition, zaprinast enhanced angiogenesis in the lesioned cortex probably as a result of vascular endothelial growth factor expression in reactive astrocytes. These results suggest that regulation of the glial inflammatory response may contribute to the reported neuroprotective effects of cGMP‐phosphodiesterase inhibitors in brain injury.
Nitric oxide (NO), a signaling molecule in the cardiovascular system, has been receiving increasing attention since Furchgott, Ignarro, and Murad were awarded the Nobel Prize in Physiology and Medicine for the discovery in 1998. Soluble guanylate cyclase (sGC), as an NO receptor, is a key metalloprotein in mediating NO signaling transduction. sGC is activated by NO to catalyze the conversion of guanosine 5′-triphosphate (GTP) to cyclic guanylate monophosphate (cGMP). The dysfunction of NO signaling results in many pathological disorders, including several cardiovascular diseases, such as arterial hypertension, pulmonary hypertension, heart failure and so on. Significant advances in its structure, function, mechanism, and physiological and pathological roles have been made throughout the past 15 years. We herein review the progress of sGC on structural, functional investigations, as well as the proposed activation/deactivation mechanism. The heme-dependent sGC stimulators and heme-independent sGC activators have also been summarized briefly.
Die gefäßerweiternden Eigenschaften von Stickstoffmonoxid (NO) werden in der Pharmakotherapie bereits seit mehr als 130 Jahren genutzt, und auch heute noch spielen NO-Donatoren eine bedeutende Rolle bei der Behandlung von Herzkreislauferkrankungen. Inhaliertes NO oder organische Nitrate und NO-freisetzende Medikamente sind jedoch mit erheblichen therapeutischen Nachteilen behaftet, beispielsweise einer Toleranzentwicklung bei Langzeitbehandlung und unspezifischen Effekten wie der posttranslationalen Modifikation von Proteinen. Die positiven Wirkungen von NO werden durch die Stimulation der löslichen Guanylatcyclase (sGC) vermittelt, einem Häm-haltigen Enzym, welches das intrazelluläre Signalmolekül cyclisches Guanosinmonophosphat (cGMP) bildet. Vor kurzem wurden zwei Substanzklassen entdeckt, die die Wirkung der sGC NO-unabhängig verstärken, die sogenannten sGC-Stimulatoren und sGC-Aktivatoren. Der erste Vertreter dieser neuen Medikamentenklassen, der sGC-Stimulator Riociguat, zeigte überzeugende Wirksamkeit in klinischen Phase-III-Studien bei unterschiedlichen Formen des Lungenhochdrucks.
Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and generates the second messenger cyclic GMP (cGMP). Recently, purified sGC α1β1 has been shown to additionally generate the cyclic pyrimidine nucleotides cCMP and cUMP. However, since cyclic pyrimidine nucleotide formation occurred only the presence of Mn(2+) but not Mg(2+), the physiological relevance of these in vitro findings remained unclear. Therefore, we studied cyclic nucleotide formation in intact cells. We observed NO-dependent cCMP- and cUMP formation in intact HEK293 cells overexpressing sGC α1β1 and in RFL-6 rat fibroblasts endogenously expressing sGC, using HPLC-tandem mass spectrometry. The identity of cCMP and cUMP was confirmed by HPLC-time-of-flight mass spectrometry. Our data indicate that cCMP and cUMP play second messenger roles and that Mn(2+) is a physiological sGC cofactor.
Neuronal nitric-oxide synthase (nNOS) has various splicing variants and different subcellular localizations. nNOS can be found
also in the nucleus; however, its exact role in this compartment is still not completely defined. In this report, we demonstrate
that the PDZ domain allows the recruitment of nNOS to nuclei, thus favoring local NO production, nuclear protein S-nitrosylation, and induction of mitochondrial biogenesis. In particular, overexpression of PDZ-containing nNOS (nNOSα) increases
S-nitrosylated CREB with consequent augmented binding on cAMP response element consensus sequence on peroxisome proliferator-activated
receptor γ co-activator (PGC)-1α promoter. The resulting PGC-1α induction is accompanied by the expression of mitochondrial
genes (e.g., TFAM, MtCO1) and increased mitochondrial mass. Importantly, full active nNOS lacking PDZ domain (nNOSβ) does not localize
in nuclei and fails in inducing the expression of PGC-1α. Moreover, we substantiate that the mitochondrial biogenesis normally
accompanying myogenesis is associated with nuclear translocation of nNOS. We demonstrate that α-Syntrophin, which resides
in nuclei of myocytes, functions as the upstream mediator of nuclear nNOS translocation and nNOS-dependent mitochondrial biogenesis.
Overall, our results indicate that altered nNOS splicing and nuclear localization could be contributing factors in human muscular
diseases associated with mitochondrial impairment.
Soluble guanylate cyclase (sGC) is a critical heme-containing enzyme involved in NO signaling. The dimerization of sGC subunits is necessary for its bioactivity and its mechanism is a striking and an indistinct issue. The roles of heme domain cysteines of the sGC on the dimerization and heme binding were investigated herein. The site-directed mutations of three conserved cysteines (C78A, C122A and C174S) were studied systematically and the three mutants were characterized by gel filtration analysis, UV–vis spectroscopy and heme transfer examination. Cys78 was involved in heme binding but not referred to the dimerization, while Cys174 was demonstrated to be involved in the homodimerization. These results provide new insights into the cysteine-related dimerization regulation of sGC.
Human soluble guanylate cyclase (hsGC), a NO sensor/NO receptor of a heterodimeric hemoprotein, plays a critical role in the NO-sGC-cGMP signaling pathway, and also reveals a novel nitrite reductase activity. This indicates that hsGC could activate itself by catalytic reduction of nitrite to NO instead of receiving NO from nitric oxide synthase (NOS), which provides valuable insight into the physiological function of the homodimeric hsGC.
The aim of this work was to study the effect of glucagon-like peptide-2 (GLP-2) on the cyclic guanosine monophosphate (cGMP) signalling pathway and whether insulin or epidermal growth factor (EGF) might modulate the effects of GLP-2. GLP-2 produced a dose-dependent decrease in intracellular sodium nitroprusside-induced cGMP production. However, insulin induced an increase in the levels of cGMP that was dose-dependently decreased by the addition of GLP-2. By contrast, EGF induced a decrease in cGMP production, which was further reduced by the addition of GLP-2. To assess whether variations in cGMP production might be related with changes in some component of soluble guanylyl cyclase (sGC), the expression of the α1, α2, and β1 subunits were determined by Western blot analysis. At 1 h, GLP-2 produced a decrease in the expression of both α1 and β1 in the cytosolic fraction, but at 24 h only β1was reduced. As expected, insulin induced an increase in the expression of both subunits after 1 h of incubation; this was decreased by the addition of GLP-2. Likewise, incubation with EGF for 24 h produced a decrease in the expression of both subunits that was maximal when GLP-2 was added. In addition, incubation with insulin for 1 h produced an increase in the expression of the α2 subunit, which was reduced by the addition of GLP-2. These results suggest that GLP-2 inhibits cGMP production by decreasing the cellular content of at least one subunit of the heterodimeric active form of the sGC, independently of the presence of insulin or EFG. This may open new insights into the actions of this neuropeptide.
The activation of cAMP-response element binding protein (CREB)-dependent gene expression seems a crucial step in the molecular cascade that mediates the formation of long-lasting memories. This view is based both on correlative evidence and on functional assays that demonstrate, through loss- and gain-of-function experiments, the impact of CREB manipulation on memory performance. Mechanistically, CREB's role in memory is thought to be a consequence of its participation in long-term forms of synaptic plasticity. Recent studies demonstrate that CREB, in addition to synaptic plasticity, also modulates the intrinsic excitability of the neuron. This discovery reveals new intriguing connections between intrinsic and synaptic plasticity and is likely to have a significant impact on our understanding of the role of CREB in memory formation.
We previously showed that treatment with bacterial lipopolysaccharide (LPS) or pro-inflammatory cytokines decreases NO-sensitive guanylyl cyclase (GC(NO)) activity in astrocytes by decreasing the half-life of the obligate GC(NO) beta1 subunit in a NO-independent but transcription- and translation-dependent process. Here we show that LPS-induced beta1 degradation requires proteasome activity and is independent of NFkappaB activation or beta1 interaction with HSP90. Immunocytochemistry and confocal microscopy analysis revealed that LPS promotes colocalization of the predominantly soluble beta1 protein with ubiquitin and the 20S proteasome in nuclear aggregates that present characteristics of clastosomes, nuclear bodies involved in proteolysis via the ubiquitin-proteasome system. Proteasome and protein synthesis inhibitors prevented LPS-induced clastosome assembly and nuclear colocalization of beta1 with ubiquitin and 20S proteasome, strongly supporting a role for these transient nuclear structures in GC(NO) down-regulation during neuroinflammation.
Cyclic GMP accumulation induced by noradrenaline in astrocyte-enriched primary cultures from rat cerebrum involves synthesis of NO, as evidenced by the competitive inhibition exerted by the NO synthase inhibitor NG-monomethyl-L-arginine (IC50 = 3 microM). Furthermore, the noradrenaline effect was potently inhibited by haemoglobin (IC50 = 25 nM) and potentiated by superoxide dismutase, indicating that NO synthesis and cyclic GMP formation may occur in different subsets of astrocytes. Investigation of the receptors implicated by using selective adrenoceptor agonists and antagonists indicates that about 75% of the NO-dependent noradrenaline response is mediated by alpha 1-adrenoceptors and the rest by beta-adrenoceptors, with no evidence for potentiating effects between the two receptor types. This noradrenaline effect appears to require Ca2+ entry, since it is strongly dependent on extracellular Ca2+ but is not affected by conditions that will abolish intracellular Ca2+ mobilization (incubation with neomycin or pretreatment with carbachol). Inhibition by pretreatment with pertussis toxin is in agreement with involvement of the alpha 1A-adrenoceptor subtype in this Ca(2+)-dependent effect. However, implication of an unknown alpha 1-adrenoceptor subtype cannot be disregarded, because a similar inhibition is exerted by the presumably selective alpha 1B- and alpha 1C-adrenoceptor blocking agent chloroethylclonidine. Treatment of the cultures with the protein kinase C activator phorbol 12-myristate 13-acetate inhibits to a great extent the noradrenaline-induced cyclic GMP formation.
Soluble guanylyl cyclase (sGC), the target enzyme of the signalling molecule NO, contains one prosthetic haem group and consists of an alpha and a beta subunit. So far, only the alpha1beta1 heterodimer has been shown to exist in different cells and tissues, and most biochemical studies of sGC have been performed with the alpha1 beta1 heterodimer. Here we demonstrate for the first time the natural occurrence of the alpha2 subunit on the protein level. The alpha2 subunit co-precipitated with the beta1 subunit from human placenta, showing the existence of the alpha2 beta1 isoform in vivo. The new enzyme was expressed in and purified from cells from the Spodoptera frugiperda ovary cell line Sf 9. Spectral analysis showed that the alpha2 beta1 heterodimer contains a prosthetic haem group revealing the same characteristics as the haem in the alpha1 beta1 form. The kinetic properties of both isoforms and sensitivity towards NO were indistinguishable. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of sGC, abolished NO-stimulated activity of both heterodimers. The new NO-independent activator, 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), increased the maximal NO-stimulated activity of the new isoform, caused a leftward-shift in the NO concentration-response curve and turned CO into an effective activator, as it did for the alpha1 beta1 heterodimer (200-fold activation). In summary, the differences in primary structure of both alpha subunits are contrasted by their functional similarity. Further studies will be needed to elucidate the physiological purpose of the new isoform.
Soluble guanylyl cyclase (sGC) is an α/β-heterodimeric hemoprotein that, upon interaction with the intercellular messenger
molecule NO, generates cGMP. Although the related family of particulate guanylyl cyclases (pGCs) forms active homodimeric
complexes, it is not known whether homodimerization of sGC subunits occurs. We report here the expression in Sf9 cells of
glutathione S-transferase-tagged recombinant human sGCα1 and β1 subunits, applying a novel and rapid purification method based on GSH-Sepharose
affinity chromatography. Surprisingly, in intact Sf9 cells, both homodimeric GSTα/α and GSTβ/β complexes were formed that
were catalytically inactive. Upon coexpression of the respective complementary subunits, GSTα/β or GSTβ/α heterodimers were
preferentially formed, whereas homodimers were still detectable. When subunits were mixed after expression, e.g.GSTβ and β or GSTα and β, no dimerization was observed. In conclusion, our data suggest the previously unrecognized possibility
of a physiological equilibrium between homo- and heterodimeric sGC complexes.
Protein kinase A (PKA) and the nuclear A-kinase-anchoring protein AKAP95 have previously been shown to localize in separate compartments in interphase but associate at mitosis. We demonstrate here a role for the mitotic AKAP95-PKA complex. In HeLa cells, AKAP95 is associated with the nuclear matrix in interphase and redistributes mostly into a chromatin fraction at mitosis. In a cytosolic extract derived from mitotic cells, AKAP95 recruits the RIIalpha regulatory subunit of PKA onto chromatin. Intranuclear immunoblocking of AKAP95 inhibits chromosome condensation at mitosis and in mitotic extract in a PKA-independent manner. Immunodepletion of AKAP95 from the extract or immunoblocking of AKAP95 at metaphase induces premature chromatin decondensation. Condensation is restored in vitro by a recombinant AKAP95 fragment comprising the 306-carboxy-terminal amino acids of the protein. Maintenance of condensed chromatin requires PKA binding to chromatin-associated AKAP95 and cAMP signaling through PKA. Chromatin-associated AKAP95 interacts with Eg7, the human homologue of Xenopus pEg7, a component of the 13S condensin complex. Moreover, immunoblocking nuclear AKAP95 inhibits the recruitment of Eg7 to chromatin in vitro. We propose that AKAP95 is a multivalent molecule that in addition to anchoring a cAMP/PKA-signaling complex onto chromosomes, plays a role in regulating chromosome structure at mitosis.
Nitric oxide (NO) remains the only firmly established endogenous modulator of soluble guanylyl cyclase (sGC) activity, but physiological, structural, and biochemical evidence now suggests that in vivo regulation of sGC involves direct interaction with other factors. We searched for such endogenous modulators in human umbilical vein endothelial cells and COS-7 cells. The cytosolic fraction of both cell types stimulated the activity of semipurified sGC severalfold in the absence or presence of a saturating concentration of NO. The cytosolic factor was sensitive to proteinase K and destroyed by boiling, suggesting that it contains a protein component. Size exclusion chromatography revealed peaks of activity between 40 and 70 kDa. The sGC-activating effect was further purified by ion exchange chromatography. In the presence of the benzylindazole YC-1 or NO, the partially purified factor synergistically activated sGC, suggesting that this factor had a mode of activation different from that of YC-1 or NO. Four candidate activators were identified from the final purification step by matrix-assisted laser desorption ionization mass spectrometry analysis. Using an sGC affinity matrix, one of them, the molecular chaperone Hsp70, was shown to directly interact with sGC. This interaction was further confirmed by co-immunoprecipitation in lung tissues and by co-localization in smooth muscle cells. sGC and Hsp70 co-localized at the plasma membrane, supporting the idea that sGC can be translocated to the membrane. Hsp70 co-purifies with the sGC-activating effect, and immunodepletion of Hsp70 from COS-7 cytosol coincided with a marked attenuation of the sGC-activating effect, yet the effect was not rescued by the addition of pure Hsp70. Thus, Hsp70 is a novel sGC-interacting protein that is responsible for the sGC-activating effect, probably in association with other factors or after covalent modification.
The 90-kDa heat shock protein (hsp90) regulates the stability and function of many client proteins, including members of the NO-cGMP signaling pathway. Soluble guanylyl cyclase (sGC), an NO receptor, was recently reported to be an hsp90-interacting partner. In the present study, we show that hsp90 binds to both subunits of the most common sGC form (alpha(1)beta(1)) when these are expressed individually but only interacts with beta(1) in the heterodimeric form of the enzyme. Characterization of the region of hsp90 required to bind each subunit in immunoprecipitation experiments revealed that residues 310 to 456 of hsp90 interact with the sGC subunits. The region of beta(1) responsible for binding to hsp90beta was mapped using in vitro binding assays and immunoprecipitation experiments and was found to lie in the regulatory domain. The physiological importance of the hsp90/sGC interaction was investigated by treating rat smooth muscle cells with the hsp90 inhibitors radicicol and geldanamycin (GA) and determining both sGC activity and protein levels. Long-term (24 or 48 h) inhibition of hsp90 resulted in a strong decrease of both alpha(1) and beta(1) protein levels and sGC activity. Moreover, incubation of smooth muscle cells with the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) blocked the GA-induced down-regulation of sGC. We conclude that the N-terminal region of the beta(1) subunit mediates binding of the heterodimeric form of sGC to hsp90 and that this interaction involves the M domain of hsp90. Hsp90 binding to sGC regulates the pool of active enzymes by affecting the protein levels of the two subunits.
In addition to genetic information, mitotic chromosomes transmit essential components for nuclear assembly and function in a new cell cycle. A specialized chromosome domain, called the perichromosomal layer, perichromosomal sheath, chromosomal coat, or chromosome surface domain, contains proteins required for a variety of cellular processes, including the synthesis of messenger RNA, assembly of ribosomes, repair of DNA double-strand breaks, telomere maintenance, and apoptosis regulation. The layer also contains many proteins of unknown function and is a major target in autoimmune disease. Perichromosomal proteins are found along the entire length of chromosomes, excluding centromeres, where sister chromatids are paired and spindle microtubules attach. Targeting of proteins to the perichromosomal layer occurs primarily during prophase, and they generally remain associated until telophase. During interphase, perichromosomal proteins localize to nucleoli, the nuclear envelope, nucleoplasm, heterochromatin, centromeres, telomeres, and/or the cytoplasm. It has been suggested that the perichromosomal layer may contribute to chromosome structure, as several of the associated proteins have functions in chromatin remodeling during interphase. We review the identified proteins associated with this chromosome domain and briefly discuss their known functions during interphase and mitosis.
Nitric oxide (NO) is a relative newcomer to pharmacology, as the paper which initiated the field was published only 25 years ago. Nevertheless its impact is such that to date more than 31,000 papers have been published with NO in the title and more than 65,000 refer to it in some way. The identification of NO with endothelium-derived relaxing factor and the discovery of its synthesis from L-arginine led to the realisation that the L-arginine: NO pathway is widespread and plays a variety of physiological roles. These include the maintenance of vascular tone, neurotransmitter function in both the central and peripheral nervous systems, and mediation of cellular defence. In addition, NO interacts with mitochondrial systems to regulate cell respiration and to augment the generation of reactive oxygen species, thus triggering mechanisms of cell survival or death. This review will focus on the role of NO in the cardiovascular system where, in addition to maintaining a vasodilator tone, it inhibits platelet aggregation and adhesion and modulates smooth muscle cell proliferation. NO has been implicated in a number of cardiovascular diseases and virtually every risk factor for these appears to be associated with a reduction in endothelial generation of NO. Reduced basal NO synthesis or action leads to vasoconstriction, elevated blood pressure and thrombus formation. By contrast, overproduction of NO leads to vasodilatation, hypotension, vascular leakage, and disruption of cell metabolism. Appropriate pharmacological or molecular biological manipulation of the generation of NO will doubtless prove beneficial in such conditions.
British Journal of Pharmacology (2006) 147, S193–S201. doi:10.1038/sj.bjp.0706458
Stimulation of freshly isolated rat hepatocytes with lysophosphatidic acid (LPA) resulted in LPA1 receptor-mediated and nitricoxide-dependent up-regulation of the immediate early genes iNOS (inducible nitric-oxide synthase (NOS)) and mPGES-1 (microsomal prostaglandin E synthase-1). Because LPA is a ligand for both cell surface and intracellular receptor sites and a potent endothelial NOS (eNOS) activator, we hypothesized that NO derived from activated nuclearized eNOS might participate in gene regulation. Herein we show, by confocal microscopy performed on porcine cerebral endothelial cells expressing native LPA1-receptor and eNOS and on HTC4 rat hepatoma cells co-transfected with recombinant human LPA1-receptor and fused eNOS-GFP cDNA, a dynamic eNOS translocation from peripheral to nuclear regions upon stimulation with LPA. Nuclear localization of eNOS and its downstream effector, soluble guanylate cyclase, were demonstrated in situ in rat liver specimens by immunogold labeling using specific antibodies. Stimulation of this nuclear fraction with LPA and the NO donor sodium nitroprusside resulted, respectively, in increased production of nitrite (and eNOS phosphorylation) and cGMP; these separate responses were also correspondingly blocked by NOS inhibitor L-NAME and soluble guanylate cyclase inhibitor ODQ. In addition, sodium nitroprusside evoked a sequential increase in nuclear Ca2+ transients, activation of p42 MAPK, NF-kappaB binding to DNA consensus sequence, and dependent iNOS RNA. This study describes a hitherto unrecognized molecular mechanism by which nuclear eNOS through ensuing NO modulates nuclear calcium homeostasis involved in gene transcription-associated events. Moreover, our findings strongly support the concept of the nucleus as an autonomous signaling compartment.
Soluble guanylyl cyclase (sGC) is a cGMP-generating enzyme carrying a heme prosthetic group that functions as a nitric oxide (NO) sensor. sGC is present in most cells types, including the vascular endothelium, where its biological functions remain largely unexplored. Herein, we have investigated the role of sGC in angiogenesis and angiogenesis-related properties of endothelial cells (EC). Initially, we determined that sGC was present and enzymatically active in the chicken chorioallantoic membrane (CAM) during the days of maximal angiogenesis. In the CAM, inhibition of endogenous sGC inhibited neovascularization, whereas activation promoted neovessel formation. Using zebrafish as a model for vascular development, we did not detect any effect on vasculogenesis upon sGC blockade, but we did observe an abnormal angiogenic response involving the cranial and intersegmental vessels, as well as the posterior cardinal vein. In vitro, pharmacological activation of sGC or adenovirus-mediated sGC gene transfer promoted EC proliferation and migration, whereas sGC inhibition blocked tube-like network formation. In addition, sGC inhibition blocked the migratory response to vascular EC growth factor. Cells infected with sGC-expressing adenoviruses exhibited increased extracellular signal-regulated kinase 1/2 and p38 MAPK activation that was sensitive to sGC inhibition by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, suggesting that these mitogen-activated protein kinases are downstream effectors of sGC in EC. A functional role for p38 in cGMP-stimulated migration was demonstrated using SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole]; pharmacological inhibition of p38 attenuated BAY 41-2272 [5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine] and sGC overexpression-induced EC mobilization. We conclude that sGC activation promotes the expression of angiogenesis-related properties by EC and that sGC might represent a novel target to modulate neovessel formation.
The growth and progression of prostate cancer are dependent on androgens and androgen receptor (AR), which act by modulating gene expression. Utilizing a gene microarray approach, we have identified the alpha1-subunit gene of soluble guanylyl cyclase (sGC) as a novel androgen-regulated gene. A heterodimeric cytoplasmic protein composed of one alpha and one beta subunit, sGC mediates the widespread cellular effects of nitric oxide (NO). We report here that, in prostate cancer cells, androgens stimulate the expression of sGCalpha1. A cloned human sGCalpha1 promoter is activated by androgen in an AR-dependent manner, suggesting that sGCalpha1 may be a direct AR target gene. Disruption of sGCalpha1 expression severely compromises the growth of both androgen-dependent and androgen-independent AR-positive prostate cancer cells. Overexpression of sGCalpha1 alone is sufficient for stimulating prostate cancer cell proliferation. Interestingly, the major growth effect of sGCalpha1 is independent of NO and cyclic guanosine monophosphate, a major mediator of the sGC enzyme. These data strongly suggest that sGCalpha1 acts in prostate cancer via a novel pathway that does not depend on sGCbeta1. Tissue studies show that sGCalpha1 expression is significantly elevated in advanced prostate cancer. Thus, sGCalpha1 may be an important mediator of the procarcinogenic effects of androgens.
Double strand break (DSB) recognition is the first step in the DSB damage response and involves activation of ataxia telangiectasia-mutated (ATM) and phosphorylation of targets such as p53 to trigger cell cycle arrest, DNA repair, or apoptosis. It was reported that activation of ATM- and Rad3-related (ATR) kinase by DSBs also occurs in an ATM-dependent manner. On the other hand, Ku70/80 is known to participate at a later time point in the DSB response, recruiting DNA-PKcs to facilitate non-homologous end joining. Because Ku70/80 has a high affinity for broken DNA ends and is abundant in nuclei, we examined their possible involvement in other aspects of the DSB damage response, particularly in modulating the activity of ATM and other phosphatidylinositol (PI) 3-related kinases during DSB recognition. We thus analyzed p53(Ser18) phosphorylation in irradiated Ku-deficient cells and observed persistent phosphorylation in these cells relative to wild type cells. ATM or ATR inhibition revealed that this phosphorylation is mainly mediated by ATM-dependent ATR activity at 2 h post-ionizing radiation in wild type cells, whereas in Ku-deficient cells, this occurs mainly through direct ATM activity, with a secondary contribution from ATR via a novel ATM-independent mechanism. Using ATM/Ku70 double-null cell lines, which we generated, we confirmed that ATM-independent ATR activity contributed to persistent phosphorylation of p53(Ser18) in Ku-deficient cells at 12 h post-ionizing radiation. In summary, we discovered a novel role for Ku70/80 in modulating ATM-dependent ATR activation during DSB damage response and demonstrated that these proteins confer a protective effect against ATM-independent ATR activation at later stages of the DSB damage response.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
beta-Catenin nuclear import has been found to be independent of classical nuclear localization signal (NLS) nuclear import factors. Here, we test the hypothesis that beta-catenin interacts directly with nuclear pore proteins to mediate its own transport. We show that beta-catenin, unlike importin-beta, does not interact detectably with Phe/Gly(FG)-repeat-rich nuclear pore proteins or nucleoporins (Nups). Moreover, unlike NLS-containing proteins, beta-catenin nuclear import is not inhibited by wheat germ agglutinin (WGA) or excess importin-beta. These results suggest beta-catenin nuclear translocation does not involve direct interactions with FG-Nups. However, beta-catenin has two regions that can target it to the nucleus, and its import is cold sensitive, indicating that beta-catenin nuclear import is still an active process. Transport is blocked by a soluble form of the C-cadherin cytoplasmic domain, suggesting that masking of the nuclear targeting signal may be a mechanism of regulating beta-catenin subcellular localization.
NO-sensitive guanylyl cyclase (GC) acts as the effector molecule for NO and therefore plays a key role in the NO/cGMP signalling cascade. Besides the long known GC isoform (α1β1), another heterodimer (α2β1) has recently been identified to be associated with PSD-95 in brain.Here, we report on the tissue distribution of all known guanylyl cyclase subunits to elucidate the isoform content in different tissues of the mouse. The guanylyl cyclase subunit levels were assessed with quantitative real-time PCR, and the most important results were verified in Western blots. We demonstrate the major occurrence of the α2β1 heterodimer in brain, find a significant amount in lung and lower amounts in all other tissues tested. In brain, the levels of the α2β1 and α1β1 isoforms were comparable; in all other tissues, the α1β1 heterodimer was the predominating isoform. The highest guanylyl cyclase content was found in lung; here the GC amounted to approximately twice as much as in brain.In sum, the major occurrence of the α2β1 heterodimer suggests a special role in synaptic transmission; whether this isoform outside the brain also occurs in neuronal networks has to be addressed in future studies.
Nitric oxide is a new type of signalling molecule in both the central and peripheral nervous systems. It does not behave like a conventional neurotransmitter; instead, it may be best described as an inter-cellular second messenger. Nitric oxide is formed from the amino acid, L-arginine, and probably exerts many of its actions by stimulating soluble guanylate cyclase, hence causing an accumulation of cyclic GMP in target cells. The L-arginine-nitric oxide-cyclic GMP pathway is expressed widely throughout the central nervous system and it is becoming implicated in an increasing number of phenomena, both physiological and pathological.
In immunohistochemical studies of rat liver tissue slices and purified nuclei, adenosine 3':5'-cyclic monophosphate (cAMP) and guanosine 3':5'-cyclic monophosphate (cGMP) immunofluorescence on the nuclear membrane are sequentially increased after glucagon administration. An explanation for the increased cGMP immunofluorescence was sought in experiments in which guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2]activity of hepatic subcellular fractions was determined. The results showed that a nuclear guanylate cyclase exists which can be distinguished from the soluble and crude particulate guanylate cyclases. The activity of the nuclear enzyme was increased by 35% in nuclei isolated from rats 30 min after glucagon injection, the time at which maximal nuclear membrane cGMP immunofluorescence is observed. Because glucagon altered both cAMP location and levels prior to the observed changes in nuclear cGMP metabolism, the hypothesis that cAMP acted as the second messenger was tested. In vitro incubation of nuclei isolated from control rats with 10(-5) M cAMP produced a 25% increase in nuclear guanylate cyclase activity. With nuclei isolated from glucagon-treated rats, no significant increase in enzyme activity was observed; this indicates that maximal stimulation of nuclear guanylate cyclase by cAMP occurred at levels that are obtained in vivo after glucagon administration. These findings suggest that hepatic nuclear cGMP content may be regulated by a specific organelle guanylate cyclase and that cAMP may be one of the determinants of this enzyme's activity.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
A complex structure, visible by electron microscopy, surrounds each chromosome during mitosis. The organization of this structure is distinct from that of the chromosomes and the cytoplasm. It forms a perichromosomal layer that can be isolated together with the chromosomes. This layer covers the chromosomes except in centromeric regions. The perichromosomal layer includes nuclear and nucleolar proteins as well as ribonucleoproteins (RNPs). The list of proteins and RNAs identified includes nuclear matrix proteins (perichromin, peripherin), nucleolar proteins (perichro-monucleolin, Ki-67 antigen, B23 protein, fibrillarin, p103, p52), ribosomal proteins (S1) and snRNAs (U3 RNAs). Only limited information is available about how and when the perichromosomal layer is formed. During early prophase, the proteins extend from the nucleoli towards the periphery of the nucleus. Thin cordon-like structures reach the nuclear envelope delimiting areas in which chromosomes condense. At telophase, the proteins are associated with the part of the chromosomes remaining condensed and accumulate in newly formed nucleoli in regions where chromatin is already decondensed. The perichromosomal layer contains several different classes of proteins and RNPs and it has been attributed various roles: (1) in chromosome organization, (2) as a barrier around the chromosomes, (3) involvement in compartmentation of the cells in prophase and telophase and (4) a binding site for chromosomal passenger proteins necessary to the early process of nuclear assembly.
Signal transduction with the diatomic radical nitric oxide (NO) is involved in a number of important physiological processes, including smooth muscle relaxation and neurotransmission. Soluble guanylate cyclase (sGC), a heterodimeric enzyme that converts guanosine triphosphate to cyclic guanosine monophosphate, is a critical component of this signaling pathway. sGC is a hemoprotein; it is through the specific interaction of NO with the sGC heme that sGC is activated. Over the last decade, much has been learned about the unique heme environment of sGC and its interaction with ligands like NO and carbon monoxide. This review will focus on the role of sGC in signaling, its relationship to the other nucleotide cyclases, and on what is known about sGC genetics, heme environment and catalysis. The latest understanding in regard to sGC will be incorporated to build a model of sGC structure, activation, catalytic mechanism and deactivation.
Guanylyl cyclases (GC) catalyze the formation of the intracellular signal molecule cyclic GMP from GTP. For some years it has been known that the heme-containing soluble guanylyl cyclase (sGC) is stimulated by NO and NO-containing compounds. The sGC enzyme consists of two subunits (alpha(1) and beta(1)). In the present study, the alpha(1) and beta(1)-subunits were identified in the guinea pig cochlea at the electron microscopic level using a post-embedding immuno-labeling procedure. Ultrathin sections of LR White embedded specimens were incubated with various concentrations of two rabbit polyclonal antibodies to the alpha(1)- and beta(1)-subunit, respectively. The immunoreactivity was visualized by a gold-labeled secondary antibody in an energy-filtering transmission electron microscope (EFTEM). Marked immunoreactivity for both antibodies was found in the inner and outer hair cells, with numerous gold particles at the border of the cuticular plates, associated with the cell nuclei or attached to electron-dense parts of the cytoplasm. In the pillar cells and apical Deiters cells, soluble guanylyl cyclase immunoreactivity was located at the rim of the cuticular plates and between the microtubuli bundles. Together with the recently identified nitric oxide synthase isoforms [Eur. Arch. Otorhinolaryngol. 254 (1997) 396; Eur. Arch. Otorhinolaryngol. 255 (1998) 483], the soluble guanylyl cyclase may be involved in signalling processes in the organ of Corti.
Whole brain spheroids provide a suitable model to study neurodevelopment. In the literature a role for the nitric oxide (NO)-cyclic guanosine 3',5'-monophosphate (cGMP) signalling pathway during development has frequently been suggested. In this study we investigated whether functional cGMP pathways were present in differentiated spheroids. In 3-week-old spheroids soluble guanylate cyclase was stimulated with N-methyl D-aspartic acid or sodium nitroprusside (NO donor). The results showed that the NO synthase-cGMP pathway is present in the culture system. Soluble guanylate cyclase-dependent cGMP formation was found in NO synthase containing neurons, in neurons of the GABAergic, glutamatergic and cholinergic system, and in astroglia and oligodendroglia. Activation of particulate guanylate cyclase by atrial natriuretic peptide also triggered an increase in cGMP production. Particulate guanylate cyclase was found in astroglia and in microglia as well as in glutamic acid decarboxylase and calbindin containing structures and neuronal NO synthase containing neurons. Chronic inhibition of NO synthase during culture development had no effect on soluble or particulate guanylate cyclase functioning. Similarly, inhibition of soluble guanylate cyclase during culture development did not have any effect on NO synthase and particulate guanylate cyclase functioning. It is concluded that NO synthase and both soluble and particulate guanylate cyclase are present in whole brain spheroid cultures and that their activity can be influenced by several stimuli. The spheroid culture system constitutes a suitable model to study the NO-cGMP pathway during brain development in mammals.
Despite the widespread use of nitric oxide as a signalling molecule in the central nervous system, the molecular makeup of its receptor, soluble guanylyl cyclase (sGC), therein is poorly understood. Accordingly, RT-PCR and in situ hybridization were used to identify sGC subunits expressed in rat brain. In addition to the expected mRNA for alpha 1 and beta1 subunits, message for the beta 2 subunit was detected in the cerebellum at all developmental stages investigated (1--150 days postnatum). The use of degenerate primers allowed the identification of mRNA coding for the rat alpha 2 subunit, which was also expressed at every age studied. All but beta 2 were detected by in situ hybridization in the brains of both 8-day-old and adult rats. The distribution patterns indicated that in some areas, e.g. caudate-putamen and nucleus accumbens, sGC probably exists mainly as the alpha 1 beta 1 heterodimer. In others, e.g. hippocampus and olfactory bulb, alpha 2 beta 1 is likely to be dominant. In the cerebellum, alpha 1 and beta 1 message was strong in the Purkinje cell layer but was not confined to Purkinje cells: smaller cells, presumed to be the Bergmann glia, were also labelled. In contrast, alpha 2 mRNA was concentrated in cerebellar granule cells. Western blotting indicated an excess of alpha 1 over beta 1 protein in the cerebellum, the reverse of what was found in the lung. It is concluded that, in molecular terms, sGC is likely to be more complex and exhibit more regional variation in the brain than previously thought. The functional consequences of this heterogeneity require investigation.
Figure 1. NO is functionally active in human normal melanocytes and nonmetastatic melanoma cells. Intracellular cGMP levels (□) following incubation with SIN-1 (1 mM) and the melanin content (▪) in cultured human melanocytes (A) and in melanoma cell lines (nonmetastatic: 530, 1F6; sporadically metastatic: M14, Mel57; highly metastatic: MV3, BLM) (B) were determined as described in Materials and Methods. Results (pmol cGMP) are normalized to protein content (mg) and are expressed as percent induction compared with the unstimulated control melanocytes (100%). Data present means ±SEM of four replicates of four independent experiments. Values of melanin content are means ±SEM of triplicates of three independent experiments. All values presented in (A) are significantly different from controls. * p <0.05 vs control; ** p <0.05 vs unpigmented BLM.
The signaling molecule nitric oxide (NO) exerts most of its effects by the stimulation of the NO-sensitive guanylyl cyclase. Two isoforms of the NO receptor molecule exist: the ubiquitously occurring alpha(1)beta(1) and the alpha(2)beta(1) with a more limited distribution. As the isoforms are functionally indistinguishable, the physiological relevance of these isoforms remained unclear. The neuronal NO synthase has been reported to be associated with PSD-95. Here, we demonstrate the interaction of the so far unnoticed alpha(2)beta(1) isoform with PSD-95 in rat brain as shown by coprecipitation. The interaction is mediated by the alpha(2) C-terminal peptide and the third PDZ domain of PSD-95. As a consequence of the PSD-95 interaction, the so far considered "soluble" alpha(2)beta(1) isoform is recruited to the membrane fraction of synaptosomes, whereas the alpha(1)beta(1) isoform is found in the cytosol. Our results establish the alpha(1)beta(1) as the cytosolic and the alpha(2)beta(1) as the membrane-associated NO-sensitive guanylyl cyclase and suggest the alpha(2)beta(1) isoform as the sensor for the NO formed by the PSD-95-associated neuronal NO synthase.
Nitric oxide (NO) is a ubiquitous, cell-permeable intercellular messenger. The current concept assumes that NO diffuses freely through the plasma membrane into the cytoplasm of a target cell, where it activates its cytosolic receptor enzyme, soluble guanylyl cyclase (sGC). Recent evidence, however, suggests that cellular membranes are not only the predominant site of calcium-dependent NO synthesis, but also the site of its distribution and binding. Here we extend this concept to NO signalling to show that active sGC is partially associated with the plasma membrane in a state of enhanced NO sensitivity. After cellular activation, sGC further translocates to the membrane fraction in human platelets and associates with the NO-synthase-containing caveolar fraction in rat lung endothelial cells, in a manner that is dependent on the concentration of intracellular calcium. Our data suggest that the entire NO signalling pathway is more spatially confined than previously assumed and that sGC dynamically translocates to the plasma membrane, where it is sensitized to NO.
NO-sensitive guanylyl cyclase (GC) acts as the effector molecule for NO and therefore plays a key role in the NO/cGMP signalling cascade. Besides the long known GC isoform (alpha(1)beta(1)), another heterodimer (alpha(2)beta(1)) has recently been identified to be associated with PSD-95 in brain.Here, we report on the tissue distribution of all known guanylyl cyclase subunits to elucidate the isoform content in different tissues of the mouse. The guanylyl cyclase subunit levels were assessed with quantitative real-time PCR, and the most important results were verified in Western blots. We demonstrate the major occurrence of the alpha(2)beta(1) heterodimer in brain, find a significant amount in lung and lower amounts in all other tissues tested. In brain, the levels of the alpha(2)beta(1) and alpha(1)beta(1) isoforms were comparable; in all other tissues, the alpha(1)beta(1) heterodimer was the predominating isoform. The highest guanylyl cyclase content was found in lung; here the GC amounted to approximately twice as much as in brain. In sum, the major occurrence of the alpha(2)beta(1) heterodimer suggests a special role in synaptic transmission; whether this isoform outside the brain also occurs in neuronal networks has to be addressed in future studies.
In retrospect, basic research in the fields of NO and cyclic GMP during the past two decades appears to have followed a logical course beginning with the findings that NO and cyclic GMP are vascular smooth muscle relaxants, that nitroglycerin relaxes smooth muscle by metabolism to NO, progressing to the discovery that mammalian cells synthesize NO, and finally the revelation that NO is a neurotransmitter mediating vasodilation in specialized vascular beds. A great deal of basic and clinical research on the physiological and pathophysiological roles of NO in cardiovascular function has been conducted since the discovery that EDRF is NO. The new knowledge on NO should enable investigators in this field to develop novel and more effective therapeutic strategies for the prevention, diagnosis and treatment of numerous cardiovascular disorders. Since NO elicits a protective and beneficial action in many disease states, novel NO donor drugs for clinical use should prove to be very effective drugs for the treatment of essential hypertension, stroke, coronary artery disease, vascular complications of diabetes, impotency and other disorders involving the vascular system.
Microglia can be isolated with high purity but low yield by shaking off loosely adherent cells from mixed glial cultures. Here we describe a new technique for isolating microglia with an average yield close to 2,000,000 microglial cells/mouse pup, more than five times higher than that of the shaking method. Confluent mixed glial cultures are subjected to mild trypsinization (0.05-0.12%) in the presence of 0.2-0.5 mM EDTA and 0.5-0.8 mM Ca2+. This results in the detachment of an intact layer of cells containing virtually all the astrocytes, leaving undisturbed a population of firmly attached cells identified as >98% microglia. These almost pure microglial preparations can be kept in culture for weeks and show proliferation and phagocytosis. Treatment with macrophage colony-stimulating factor and lipopolysaccharide, alone or in the presence of interferon gamma, induces typical microglial responses in terms of proliferation, morphological changes, nuclear factor-kappaB translocation, NO, and tumor necrosis alpha release and phagocytosis. This method allows for the preparation of highly enriched mouse or rat microglial cultures with ease and reproducibility. Because of its high yield, it can be especially convenient when high amounts of microglial protein/mRNA are required or in cases in which the starting material is limited, such as microglial cultures from transgenic animals.
Since 1922 when Wu proposed the use of the Folin phenol reagent for
the measurement of proteins (l), a number of modified analytical pro-
cedures ut.ilizing this reagent have been reported for the determination
of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and
in insulin (10).
Although the reagent would seem to be recommended by its great sen-
sitivity and the simplicity of procedure possible with its use, it has not
found great favor for general biochemical purposes.
In the belief that this reagent, nevertheless, has considerable merit for
certain application, but that its peculiarities and limitations need to be
understood for its fullest exploitation, it has been studied with regard t.o
effects of variations in pH, time of reaction, and concentration of react-
ants, permissible levels of reagents commonly used in handling proteins,
and interfering subst.ances. Procedures are described for measuring pro-
tein in solution or after precipitation wit,h acids or other agents, and for
the determination of as little as 0.2 y of protein.
Dopamine receptors exhibit tissue- and cell type-specific expression that is modulated during development, aging and in diseases such as Parkinson's. The molecular mechanisms regulating expression of dopamine receptors are not well understood, in part due to the lack of a model cell line that not only expresses endogenous dopamine receptors but also has the requisite regulatory mechanisms. Here, we demonstrate that the CAD catecholaminergic cell line expresses D1, D2, D3 and D5 dopamine receptor subtypes and associated signaling proteins. CAD cell differentiation induced by serum withdrawal increases the levels of D1 receptor mRNA by transcriptional up-regulation. This increase is also mimicked by the neurotrophin NT3. Interestingly, the increase of D1 receptor mRNA does not result in increased levels of D1 receptor protein in differentiated CAD cells. Furthermore, while the D1 receptor protein is expressed in differentiated CAD cells, it loses its ability to activate adenylyl cyclase. We demonstrate that the post-transcriptional regulation is not due to decreased D1 receptor mRNA stability or generation of a truncated D1 receptor mRNA, and that the down-regulation of D1 receptor function in differentiated CAD cells is mediated by post-translational mechanisms that decrease cell surface receptor expression by altering receptor processing and trafficking.
Soluble guanylyl cylase (sGC) has been identified for being a receptor for the gaseous transmitters nitric oxide and carbon monoxide. Currently four subunits alpha1, alpha2, beta1, and beta2 have been characterized. Heterodimers of alpha and beta-subunits as well as homodimers of the beta2-subunit are known to constitute functional sGC which use GTP to form cGMP a potent signal molecule in a multitude of second messenger cascades. Since NO-cGMP signaling plays a pivotal role in neuronal development we analyzed the maturational expression pattern of the newly characterized alpha2-subunit of sGC within the brain of Wistar rats by means of RNase protection assay and immunohistochemistry. alpha2-subunit mRNA as well as immunoreactive alpha2-protein increased during postnatal cerebral development. Topographical analysis revealed a selective high expression of the alpha2-subunit in the choroid plexus and within developing sensory systems involving the olfactory and somatosensory system of the forebrain as well as parts of the auditory and visual system within the hindbrain. In cultured cortical neurons the alpha2-subunit was localized to the cell membrane, especially along neuronal processes. During the first 11 days of postnatal development several cerebral regions showed a distinct expression of the alpha2-subunit which was not paralleled by the alpha1/beta1-subunits especially within the developing thalamo-cortical circuitries of the somatosensory system. However, at later developmental stages all three subunits became more homogenously distributed among most cerebral regions, indicating that functional alpha1/beta1 and alpha2/beta1 heterodimers of sGC could be formed. Our findings indicate that the alpha2-subunit is an essential developmentally regulated constituent of cerebral sensory systems during maturation. In addition the alpha2-subunit may serve other functions than forming a functional heterodimer of sGC during the early phases of sensory pathway refinement.
By the formation of the second messenger cGMP, NO-sensitive guanylyl cyclase (GC) plays a key role within the NO/cGMP signaling cascade which participates in vascular regulation and neurotransmission. The enzyme contains a prosthetic heme group that acts as the acceptor site for NO. High affinity binding of NO to the heme moiety leads to an up to 200-fold activation of the enzyme. Unexpectedly, NO dissociates with a half-life of a few seconds which appears fast enough to account for the deactivation of the enzyme in biological systems. YC-1 and its analogs act as NO sensitizers and led to the discovery of a novel pharmacologically and conceivably physiologically relevant regulatory principle of the enzyme. The two isoforms of the heterodimeric enzyme (alpha1beta1, alpha2beta1) are known that are functionally indistinguishable. The alpha2beta1-isoform mainly occurs in brain whereas the alpha1beta1-enzyme shows a broader distribution and represents the predominantly expressed form of NO-sensitive GC. Until recently, the enzyme has been thought to occur in the cytosol. However, latest evidence suggests that the alpha2-subunit mediates the membrane association of the alpha2beta1-isoform via interaction with a PDZ domain of the post-synaptic scaffold protein PSD-95. Binding to PSD-95 locates this isoform in close proximity to the NO-generating synthases thereby enabling the NO sensor to respond to locally elevated NO concentrations. In sum, the two known isoforms may stand for the neuronal and vascular form of NO-sensitive GC reflecting a possible association to the neuronal and endothelial NO-synthase, respectively.
Double-stranded RNA (dsRNA) is an important regulator of gene expression in many eukaryotes. It triggers different types of gene silencing that are collectively referred to as RNA silencing or RNA interference. A key step in known silencing pathways is the processing of dsRNAs into short RNA duplexes of characteristic size and structure. These short dsRNAs guide RNA silencing by specific and distinct mechanisms. Many components of the RNA silencing machinery still need to be identified and characterized, but a more complete understanding of the process is imminent.
In Alzheimer's disease (AD) brains increased NO synthase (NOS) expression is found in reactive astrocytes surrounding amyloid plaques. We have recently shown that treatment with beta-amyloid peptides or IL-1beta down-regulates NO-sensitive soluble guanylyl cyclase (sGC) in cultured astrocytes and in adult rat brain. In this work, we have examined sGC activity and expression in postmortem brain tissue of AD patients and matched controls. No significant alteration was observed in basal or NO-stimulated sGC activity, nor in sGC beta1 and alpha1 subunit levels in cortical extracts of AD brains. Immunohistochemistry showed intense and widespread labeling of sGC beta1 in cortical and hippocampal neurons and white matter fibrillar astrocytes, while grey matter astrocytes were faintly stained. In AD, expression of sGC in neurons and fibrillar astrocytes is not altered but is markedly reduced in reactive astrocytes surrounding amyloid plaques. Immunostaining for sGC beta1 was also lacking in reactive astrocytes in cortex and subcortical white matter in Creutzfeldt-Jakob disease brains and in subacute and chronic plaques in multiple sclerosis (MS) brains. Thus, induction of astrocyte reactivity is associated with decreased capacity to generate cGMP in response to NO both in vitro and in vivo. This effect may be related to the development of the astroglial inflammatory response.
Endogenous nitric oxide (NO), and possibly NO-releasing drugs, can both inhibit and promote vascular proliferative disorders, such as atherosclerosis and restenosis. The cell types and signaling pathways that mediate these opposing effects are controversial. It is widely assumed that the NO-mediated synthesis of the second messenger cGMP and the activation of cGMP-dependent protein kinase type I (cGKI) inhibits the proliferation of vascular smooth muscle cells and, thus, vascular remodeling. However, recent data from transgenic mouse models challenge this view. Here, we propose that cGMP signaling through cGKI might promote vasculoproliferative processes and their clinical complications. This new concept has important implications for the use of cGMP-elevating drugs in humans and might help to identify novel therapeutic strategies for vascular proliferative diseases.
Although the importance of the cyclic GMP (cGMP) signaling pathway in cardiac myocytes is well established, little is known about its regulation. Ca2+-dependent translocation of nitric oxide (NO) sensitive guanylyl cyclase (GCNO) to the cell membrane has been recently proposed to play a role. The aim of this study was to determine the possible functional relevance of GCNO bound to the cardiomyocyte membrane.
Cytosolic and particulate fractions of adult rat cardiomyocytes were isolated and blotted, and their GCNO activity was assayed in parallel experiments.
In untreated cardiomyocytes, approximately 30% of beta1-and alpha1-subunits of GCNO and a similar proportion of GCNO activity were found in the particulate fraction. The dependence of GCNO activity on pH, Ca2+, GTP and NO donor concentrations was similar in particulate and cytosolic fractions. Treatment of cardiomyocytes with the ionophore A23187 caused GCNO to translocate to the sarcolemma, increased GCNO activity in this fraction, and potentiated NO-mediated cGMP synthesis. These effects appeared to be mediated by Ca2+-dependent changes on the phosphorylation status of GCNO, since they were enhanced by the non-selective inhibitor staurosporine and by the selective inhibitor of Ca2+/calmodulin-dependent protein kinase KN-93. The effect of drugs increasing intracellular Ca2+ on cGMP synthesis was clearly correlated with their effects on membrane-associated GCNO activity but not with their effects on cytosol-associated GCNO.
These results are the first evidence that 1) GCNO is associated with the cell membrane in cardiomyocytes, 2) the regulation of membrane-associated GCNO differs from that of cytosolic GCNO, and 3) membrane association may have a crucial role in determining the response of cells to NO.
Nitric oxide (NO) performs multiple physiological roles as a biological signaling molecule. The role of NO and cGMP signaling in embryonic stem (ES) cell-derived cardiomyocytes (CM) has been investigated but many questions remain. In this study, we examined the expression of the NO signaling pathway components nitric oxide synthase (NOS-1, 2, 3), soluble guanylyl cyclase (sGCalpha(1) and beta(1)) and protein kinase G (PKG) genes and sGC activity in murine ES cells subjected to differentiation by embryoid body (EB) formation. We found that in undifferentiated ES cells, NOS-1, NOS-3, and sGCbeta(1) were detected while NOS-2, sGCalpha(1), and PKG were very low or undetectable. When ES cells were subjected to differentiation, NOS-1 abruptly decreased within one day, NOS-2 mRNA became detectable after several days, and NOS-3 increased after 7-10 days. Levels of sGCalpha(1), sGCbeta(1), and PKG all increased gradually over a several day time course of differentiation in EB outgrowths. Analysis of sGC activity in cell lysates derived from undifferentiated ES cells revealed that NO could not stimulate cGMP. However, lysates from differentiated EB outgrowths produced abundant cGMP levels after NO stimulation. Purification of ES-cell derived CM revealed that mRNA expression of all the NOS isoforms was very low to absent while sGCalpha(1) and beta(1) subunit mRNAs were abundant and sGC-mediated cGMP production was apparent in this population of cells. These data suggest that cGMP-mediated NO signaling may play a minor role, if any, in undifferentiated ES cells but could be involved in the early differentiation events or physiological processes of ES cells or ES cell-derived lineages.
Despite the recognition that the NO-cGMP signaling pathway is involved in so many physiological and pathological events, a clear understanding of many of the functions of this signaling pathway remains elusive. Because of its pleiotropic and often transient actions, its modulation for therapeutic purposes in multiple pathological states is a complex issue. Recent work that combines the areas of developmental and stem cell biology and NO-cGMP signaling in various models may help us elucidate some of these functions and even discover novel actions for this signaling paradigm. This review will discuss some of the recent work in these areas, with additional focus on the nitric oxide receptor, soluble guanylyl cyclase.
Nitric oxide (NO*) has been proposed to be a physiological modulator of cell proliferation, able to promote in most cases cell cycle arrest. In this review I explore the molecular basis of this mechanism of action. The modulatory action of NO* on the intracellular concentration of cGMP and the machinery directly involved in the control of cell cycle progression, including the expression and activity of diverse cyclins and cyclin-dependent kinases, their physiological inhibitors, and the master transcriptional regulator retinoblastoma protein, will be discussed. The role of NO* in proliferation mediated by tyrosine kinase receptors such as the epidermal growth factor receptor and downstream signalling pathways will also be considered. Finally, the involvement of NO* in proliferative processes relevant for normal development will be outlined.
Mitotic chromosome structure has been the cell biology equivalent of a 'riddle, wrapped in a mystery, inside an enigma'. Observations that genetic knockout or knockdown of condensin subunits or topoisomerase II cause only minimal perturbation in overall chromosome condensation, together with analysis of early stages of chromosome condensation and effects produced by histone H1 depletion, suggest a need to reconsider textbook models of mitotic chromosome condensation and organization.
Nitric oxide (NO) exerts most of its physiological effects through activation of a predominantly soluble guanylyl cyclase (sGC). In mammalian cells sGC exists as a heterodimer of alpha and beta subunits. Currently, four subunits (alpha1, alpha2, beta1, and beta2) have been characterized. We used in situ hybridization with subunit-specific 33P-labeled oligonucleotide probes to compare the anatomical distribution of sGC subunit mRNAs in rat and monkey brains. Message for all subunits except beta2 was detected in both species. The sGC subunit showing the highest expression and widest distribution was beta1. High expression for all subunits was found in basal ganglia, olfactory system, hippocampus, cortex, and cerebellum. Minor species differences in the relative distribution of alpha subunits were observed. In general, the alpha1 message was more prominent in monkey brain and the alpha2 message in rat brain. This was more evident in limbic areas and cerebellar cortex. Some differences were also observed in subunit laminar distribution in cerebral cortex. The limited species differences in sGC subunit distribution suggest that in primates, as occurs in rodents, the NO-cGMP signaling pathway will be involved in important brain functions such as memory formation, sensory processing, and behavior.
The signaling molecule nitric oxide (NO), first described as endothelium-derived relaxing factor (EDRF), acts as physiological activator of NO-sensitive guanylyl cyclase (NO-GC) in the cardiovascular, gastrointestinal, and nervous systems. Besides NO-GC, other NO targets have been proposed; however, their particular contribution still remains unclear. Here, we generated mice deficient for the β1 subunit of NO-GC, which resulted in complete loss of the enzyme. GC-KO mice have a life span of 3–4 weeks but then die because of intestinal dysmotility; however, they can be rescued by feeding them a fiber-free diet. Apparently, NO-GC is absolutely vital for the maintenance of normal peristalsis of the gut. GC-KO mice show a pronounced increase in blood pressure, underlining the importance of NO in the regulation of smooth muscle tone in vivo. The lack of an NO effect on aortic relaxation and platelet aggregation confirms NO-GC as the only NO target regulating these two functions, excluding cGMP-independent mechanisms. Our knockout model completely disrupts the NO/cGMP signaling cascade and provides evidence for the unique role of NO-GC as NO receptor.
• cardiovascular
• knockout mice
• cGMP
• platelet aggregation
• smooth muscle relaxation
We previously showed that treatment with bacterial lipopolysaccharide (LPS) or pro-inflammatory cytokines decreases NO-sensitive guanylyl cyclase (GC(NO)) activity in astrocytes by decreasing the half-life of the obligate GC(NO) beta1 subunit in a NO-independent but transcription- and translation-dependent process. Here we show that LPS-induced beta1 degradation requires proteasome activity and is independent of NFkappaB activation or beta1 interaction with HSP90. Immunocytochemistry and confocal microscopy analysis revealed that LPS promotes colocalization of the predominantly soluble beta1 protein with ubiquitin and the 20S proteasome in nuclear aggregates that present characteristics of clastosomes, nuclear bodies involved in proteolysis via the ubiquitin-proteasome system. Proteasome and protein synthesis inhibitors prevented LPS-induced clastosome assembly and nuclear colocalization of beta1 with ubiquitin and 20S proteasome, strongly supporting a role for these transient nuclear structures in GC(NO) down-regulation during neuroinflammation.