A plasma membrane-associated form of the androgen receptor enhances nuclear androgen signaling in osteoblasts and prostate cancer cells.

Androgen binding to the androgen receptor (AR) in the cytoplasm induces the AR to translocate to the nucleus, where it regulates the expression of target genes. Here, we found that androgens rapidly activated a plasma membrane-associated signaling node that enhanced nuclear AR functions. In murine primary osteoblasts, dihydrotestosterone (DHT) binding to a membrane-associated form of AR stimulated plasma membrane-associated protein kinase G type 2 (PKG2), leading to the activation of multiple kinases, including ERK. Phosphorylation of AR at Ser515 by ERK increased the nuclear accumulation and binding of AR to the promoter of Ctnnb1, which encodes the transcription factor ß-catenin. In male mouse osteoblasts and human prostate cancer cells, DHT induced the expression of Ctnnb1 and CTNN1B, respectively, as well as ß-catenin target genes, stimulating the proliferation, survival, and differentiation of osteoblasts and the proliferation of prostate cancer cells in a PKG2-dependent fashion. Because ß-catenin is a master regulator of skeletal homeostasis, these results explain the reported male-specific osteoporotic phenotype of mice lacking PKG2 in osteoblasts and imply that PKG2-dependent AR signaling is essential for maintaining bone mass in vivo. Our results suggest that widely used pharmacological PKG activators, such as sildenafil, could be beneficial for male and estrogen-deficient female patients with osteoporosis but detrimental in patients with prostate cancer.

The vitamin B12 analog cobinamide ameliorates azide toxicity in cells, Drosophila melanogaster, and mice.

CONTEXT: The azide anion (N3-) is highly toxic. It exists most commonly as sodium azide, which is used widely and is readily available, raising the potential for occupational incidents and use as a weapon of mass destruction. Azide-poisoned patients present with vomiting, seizures, hypotension, metabolic acidosis, and coma; death can occur. No specific azide antidote exists, with treatment being solely supportive. Azide inhibits mitochondrial cytochrome c oxidase and is likely oxidized to nitric oxide in vivo. Cytochrome c oxidase inhibition depletes intracellular adenosine triphosphate and increases oxidative stress, while increased nitric oxide causes hypotension and exacerbates oxidative damage. Here, we tested whether the cobalamin (vitamin B12) analog cobinamide, a strong and versatile antioxidant that also neutralizes nitric oxide, can reverse azide toxicity in mammalian cells, Drosophila melanogaster, and mice. RESULTS: We found cobinamide bound azide with a moderate affinity (Ka 2.87 × 105 M-1). Yet, cobinamide improved growth, increased intracellular adenosine triphosphate, and reduced apoptosis and malondialdehyde, a marker of oxidative stress, in azide-exposed cells. Cobinamide rescued Drosophila melanogaster and mice from lethal exposure to azide and was more effective than hydroxocobalamin. Azide likely generated nitric oxide in the mice, as evidenced by increased serum nitrite and nitrate, and reduced blood pressure and peripheral body temperature in the animals; the reduced temperature was likely due to reflex vasoconstriction in response to the hypotension. Cobinamide improved recovery of both blood pressure and body temperature. CONCLUSION: We conclude cobinamide likely acted by neutralizing both oxidative stress and nitric oxide, and that it should be given further consideration as an azide antidote.

Metabolic interaction between amino acid deprivation and cisplatin synergistically reduces phosphoribosyl-pyrophosphate and augments cisplatin cytotoxicity.

Cisplatin is a mainstay of cancer chemotherapy. It forms DNA adducts, thereby activating poly(ADP-ribose) polymerases (PARPs) to initiate DNA repair. The PARP substrate NAD+ is synthesized from 5-phosphoribose-1-pyrophosphate (PRPP), and we found that treating cells for 6 h with cisplatin reduced intracellular PRPP availability. The decrease in PRPP was likely from (1) increased PRPP consumption, because cisplatin increased protein PARylation and PARP1 shRNA knock-down returned PRPP towards normal, and (2) decreased intracellular phosphate, which down-regulated PRPP synthetase activity. Depriving cells of a single essential amino acid decreased PRPP synthetase activity with a half-life of ~ 8 h, and combining cisplatin and amino acid deprivation synergistically reduced intracellular PRPP. PRPP is a rate-limiting substrate for purine nucleotide synthesis, and cisplatin inhibited de novo purine synthesis and DNA synthesis, with amino acid deprivation augmenting cisplatin's effects. Amino acid deprivation enhanced cisplatin's cytotoxicity, increasing cellular apoptosis and DNA strand breaks in vitro, and intermittent deprivation of lysine combined with a sub-therapeutic dose of cisplatin inhibited growth of ectopic hepatomas in mice. Augmentation of cisplatin's biochemical and cytotoxic effects by amino acid deprivation suggest that intermittent deprivation of an essential amino acid could allow dose reduction of cisplatin; this could reduce the drug's side effects, and allow its use in cisplatin-resistant tumors.

Protein kinase G1 regulates bone regeneration and rescues diabetic fracture healing.

Bone fractures are a major cause of morbidity and mortality, particularly in patients with diabetes, who have a high incidence of fractures and exhibit poor fracture healing. Coordinated expression of osteoblast-derived vascular endothelial growth factor (VEGF) and bone morphogenic proteins (BMPs) is essential for fracture repair. The NO/cGMP/protein kinase G (PKG) signaling pathway mediates osteoblast responses to estrogens and mechanical stimulation, but the pathway's role in bone regeneration is unknown. Here, we used a mouse cortical-defect model to simulate bone fractures and studied osteoblast-specific PKG1-knockout and diabetic mice. The knockout mice had normal bone microarchitecture but after injury exhibited poor bone regeneration, with decreased osteoblasts, collagen deposition, and microvessels in the bone defect area. Primary osteoblasts and tibiae from the knockout mice expressed low amounts of Vegfa and Bmp2/4 mRNAs, and PKG1 was required for cGMP-stimulated expression of these genes. Diabetic mice also demonstrated low Vegfa and Bmp2/4 expression in bone and impaired bone regeneration after injury; notably, the cGMP-elevating agent cinaciguat restored Vegfa and BMP2/4 expression and full bone healing. We conclude that PKG1 is a key orchestrator of VEGF and BMP signaling during bone regeneration and propose pharmacological PKG activation as a novel therapeutic approach to enhance fracture healing.

Aortic pathology from protein kinase G activation is prevented by an antioxidant vitamin B12 analog.

People heterozygous for an activating mutation in protein kinase G1 (PRKG1, p.Arg177Gln) develop thoracic aortic aneurysms and dissections (TAAD) as young adults. Here we report that mice heterozygous for the mutation have a three-fold increase in basal protein kinase G (PKG) activity, and develop age-dependent aortic dilation. Prkg1R177Q/+ aortas show increased smooth muscle cell apoptosis, elastin fiber breaks, and oxidative stress compared to aortas from wild type littermates. Transverse aortic constriction (TAC)-to increase wall stress in the ascending aorta-induces severe aortic pathology and mortality from aortic rupture in young mutant mice. The free radical-neutralizing vitamin B12-analog cobinamide completely prevents age-related aortic wall degeneration, and the unrelated anti-oxidant N-acetylcysteine ameliorates TAC-induced pathology. Thus, increased basal PKG activity induces oxidative stress in the aorta, raising concern about the widespread clinical use of PKG-activating drugs. Cobinamide could be a treatment for aortic aneurysms where oxidative stress contributes to the disease, including Marfan syndrome.

Protein Kinase G Activation Reverses Oxidative Stress and Restores Osteoblast Function and Bone Formation in Male Mice With Type 1 Diabetes.

Bone loss and fractures are underrecognized complications of type 1 diabetes and are primarily due to impaired bone formation by osteoblasts. The mechanisms leading to osteoblast dysfunction in diabetes are incompletely understood, but insulin deficiency, poor glycemic control, and hyperglycemia-induced oxidative stress likely contribute. Here we show that insulin promotes osteoblast proliferation and survival via the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) signal transduction pathway and that PKG stimulation of Akt provides a positive feedback loop. In osteoblasts exposed to high glucose, NO/cGMP/PKG signaling was reduced due in part to the addition of O-linked N-acetylglucosamine to NO synthase-3, oxidative inhibition of guanylate cyclase activity, and suppression of PKG transcription. Cinaciguat-an NO-independent activator of oxidized guanylate cyclase-increased cGMP synthesis under diabetic conditions and restored proliferation, differentiation, and survival of osteoblasts. Cinaciguat increased trabecular and cortical bone in mice with type 1 diabetes by improving bone formation and osteocyte survival. In bones from diabetic mice and in osteoblasts exposed to high glucose, cinaciguat reduced oxidative stress via PKG-dependent induction of antioxidant genes and downregulation of excess NADPH oxidase-4-dependent H2O2 production. These results suggest that cGMP-elevating agents could be used as an adjunct treatment for diabetes-associated osteoporosis.

The activity of cGMP-dependent protein kinase Iα is not directly regulated by oxidation-induced disulfide formation at cysteine 43.

The type I cGMP-dependent protein kinases (PKGs) are key regulators of smooth muscle tone, cardiac hypertrophy, and other physiological processes. The two isoforms PKGIα and PKGIß are thought to have unique functions because of their tissue-specific expression, different cGMP affinities, and isoform-specific protein-protein interactions. Recently, a non-canonical pathway of PKGIα activation has been proposed, in which PKGIα is activated in a cGMP-independent fashion via oxidation of Cys43, resulting in disulfide formation within the PKGIα N-terminal dimerization domain. A "redox-dead" knock-in mouse containing a C43S mutation exhibits phenotypes consistent with decreased PKGIα signaling, but the detailed mechanism of oxidation-induced PKGIα activation is unknown. Therefore, we examined oxidation-induced activation of PKGIα, and in contrast to previous findings, we observed that disulfide formation at Cys43 does not directly activate PKGIα in vitro or in intact cells. In transfected cells, phosphorylation of Ras homolog gene family member A (RhoA) and vasodilator-stimulated phosphoprotein was increased in response to 8-CPT-cGMP treatment, but not when disulfide formation in PKGIα was induced by H2O2 Using purified enzymes, we found that the Cys43 oxidation had no effect on basal kinase activity or Km and Vmax values; however, PKGIα containing the C43S mutation was less responsive to cGMP-induced activation. This reduction in cGMP affinity may in part explain the PKGIα loss-of-function phenotype of the C43S knock-in mouse. In conclusion, disulfide formation at Cys43 does not directly activate PKGIα, and the C43S-mutant PKGIα has a higher Ka for cGMP. Our results highlight that mutant enzymes should be carefully biochemically characterized before making in vivo inferences.

A Novel, Direct NO Donor Regulates Osteoblast and Osteoclast Functions and Increases Bone Mass in Ovariectomized Mice.

Most US Food and Drug Administration (FDA)-approved treatments for osteoporosis target osteoclastic bone resorption. Only PTH derivatives improve bone formation, but they have drawbacks, and novel bone-anabolic agents are needed. Nitrates, which generate NO, improved BMD in estrogen-deficient rats and may improve bone formation markers and BMD in postmenopausal women. However, nitrates are limited by induction of oxidative stress and development of tolerance, and may increase cardiovascular mortality after long-term use. Here we studied nitrosyl-cobinamide (NO-Cbi), a novel, direct NO-releasing agent, in a mouse model of estrogen deficiency-induced osteoporosis. In murine primary osteoblasts, NO-Cbi increased intracellular cGMP, Wnt/ß-catenin signaling, proliferation, and osteoblastic gene expression, and protected cells from apoptosis. Correspondingly, in intact and ovariectomized (OVX) female C57Bl/6 mice, NO-Cbi increased serum cGMP concentrations, bone formation, and osteoblastic gene expression, and in OVX mice, it prevented osteocyte apoptosis. NO-Cbi reduced osteoclasts in intact mice and prevented the known increase in osteoclasts in OVX mice, partially through a reduction in the RANKL/osteoprotegerin gene expression ratio, which regulates osteoclast differentiation, and partially through direct inhibition of osteoclast differentiation, observed in vitro in the presence of excess RANKL. The positive NO effects in osteoblasts were mediated by cGMP/protein kinase G (PKG), but some of the osteoclast-inhibitory effects appeared to be cGMP-independent. NO-Cbi increased trabecular bone mass in both intact and OVX mice, consistent with its in vitro effects on osteoblasts and osteoclasts. NO-Cbi is a novel direct NO-releasing agent that, in contrast to nitrates, does not generate oxygen radicals, and combines anabolic and antiresorptive effects in bone, making it an excellent candidate for treating osteoporosis. © 2016 American Society for Bone and Mineral Research.

Nitric oxide as a mediator of estrogen effects in osteocytes.

Postmenopausal osteoporosis due to estrogen deficiency is a major health problem, and available therapies rely largely on the inhibition of bone resorption, because estrogen replacement is associated with risks. Estrogen promotes bone health in large part by increasing osteocyte survival, but the molecular mechanisms involved are only partly understood. We showed that estradiol stimulates nitric oxide (NO) production in osteocytes, leading to increased cGMP synthesis and activation of cGMP-dependent protein kinases (PKGs). Moreover, we found that 17ß-estradiol protects osteocytes against apoptosis via the NO/cGMP signaling pathway: type II PKG mediates estradiol-induced activation of the prosurvival kinases Erk and Akt, whereas type I PKG contributes to prosurvival signaling by directly phosphorylating and inactivating the cell death protein BAD. Preclinical data support an important role of NO in bone biology, and clinical trials suggest that NO donors may prevent bone loss in postmenopausal women. Our data provide novel insights into estrogen signaling through the NO/cGMP/PKG pathway and a rationale for using NO donors and other cGMP-elevating agents for treating postmenopausal osteoporosis.

Akt phosphorylation and regulation of transketolase is a nodal point for amino acid control of purine synthesis.

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway integrates environmental clues to regulate cell growth and survival. We showed previously that depriving cells of a single essential amino acid rapidly and reversibly arrests purine synthesis. Here we demonstrate that amino acids via mammalian target of rapamycin 2 and IκB kinase regulate Akt activity and Akt association and phosphorylation of transketolase (TKT), a key enzyme of the nonoxidative pentose phosphate pathway (PPP). Akt phosphorylates TKT on Thr382, markedly enhancing enzyme activity and increasing carbon flow through the nonoxidative PPP, thereby increasing purine synthesis. Mice fed a lysine-deficient diet for 2 days show decreased Akt activity, TKT activity, and purine synthesis in multiple organs. These results provide a mechanism whereby Akt coordinates amino acid availability with glucose utilization, purine synthesis, and RNA and DNA synthesis.

Nitrosyl-cobinamide (NO-Cbi), a new nitric oxide donor, improves wound healing through cGMP/cGMP-dependent protein kinase.

Nitric oxide (NO) donors have been shown to improve wound healing, but the mechanism is not well defined. Here we show that the novel NO donor nitrosyl-cobinamide (NO-Cbi) improved in vitro wound healing in several cell types, including an established line of lung epithelial cells and primary human lung fibroblasts. On a molar basis, NO-Cbi was more effective than two other NO donors, with the effective NO-Cbi concentration ranging from 3 to 10µM, depending on the cell type. Improved wound healing was secondary to increased cell migration and not cell proliferation. The wound healing effect of NO-Cbi was mediated by cGMP, mainly through cGMP-dependent protein kinase type I (PKGI), as determined using pharmacological inhibitors and activators, and siRNAs targeting PKG type I and II. Moreover, we found that Src and ERK were two downstream mediators of NO-Cbi's effect. We conclude that NO-Cbi is a potent inducer of cell migration and wound closure, acting via cGMP, PKG, Src, and extracellular signal regulated kinase (ERK).

Rho isoform-specific interaction with IQGAP1 promotes breast cancer cell proliferation and migration.

We performed a proteomics screen for Rho isoform-specific binding proteins to clarify the tumor-promoting effects of RhoA and C that contrast with the tumor-suppressive effects of RhoB. We found that the IQ-motif-containing GTPase-activating protein IQGAP1 interacts directly with GTP-bound, prenylated RhoA and RhoC, but not with RhoB. Co-immunoprecipitation of IQGAP1 with endogenous RhoA/C was enhanced when RhoA/C were activated by epidermal growth factor (EGF) or transfection of a constitutively active guanine nucleotide exchange factor (GEF). Overexpression of IQGAP1 increased GTP-loading of RhoA/C, while siRNA-mediated depletion of IQGAP1 prevented endogenous RhoA/C activation by growth factors. IQGAP1 knockdown also reduced the amount of GTP bound to GTPase-deficient RhoA/C mutants, suggesting that IQGAP enhances Rho activation by GEF(s) or stabilizes Rho-GTP. IQGAP1 depletion in MDA-MB-231 breast cancer cells blocked EGF- and RhoA-induced stimulation of DNA synthesis. Infecting cells with adenovirus encoding constitutively active RhoA(L63) and measuring absolute amounts of RhoA-GTP in infected cells demonstrated that the lack of RhoA(L63)-induced DNA synthesis in IQGAP1-depleted cells was not due to reduced GTP-bound RhoA. These data suggested that IQGAP1 functions downstream of RhoA. Overexpression of IQGAP1 in MDA-MB-231 cells increased DNA synthesis irrespective of siRNA-mediated RhoA knockdown. Breast cancer cell motility was increased by expressing a constitutively-active RhoC(V14) mutant or overexpressing IQGAP1. EGF- or RhoC-induced migration required IQGAP1, but IQGAP1-stimulated migration independently of RhoC, placing IQGAP1 downstream of RhoC. We conclude that IQGAP1 acts both upstream of RhoA/C, regulating their activation state, and downstream of RhoA/C, mediating their effects on breast cancer cell proliferation and migration, respectively.

Protein kinase G and focal adhesion kinase converge on Src/Akt/ß-catenin signaling module in osteoblast mechanotransduction.

Mechanical loading of bone induces interstitial fluid flow, leading to fluid shear stress (FSS) of osteoblasts. FSS rapidly increases the intracellular calcium concentration ([Ca(2+)]) and nitric oxide (NO) synthesis in osteoblasts and activates the protein kinase Akt. Activated Akt stimulates osteoblast proliferation and survival, but the mechanism(s) leading to Akt activation is not well defined. Using pharmacological and genetic approaches in primary human and mouse osteoblasts and mouse MC3T3 osteoblast-like cells, we found that Akt activation by FSS occurred through two parallel pathways; one required calcium stimulation of NO synthase and NO/cGMP/protein kinase G II-dependent activation of Src, and the other required calcium activation of FAK and Src, independent of NO. Both pathways cooperated to increase PI3K-dependent Akt phosphorylation and were necessary for FSS to induce nuclear translocation of ß-catenin, c-fos, and cox-2 gene expression and osteoblast proliferation. These data explain how mechanical stimulation of osteoblasts leads to increased signaling through a growth regulatory pathway essential for maintaining skeletal integrity.

Pro-survival effects of 17ß-estradiol on osteocytes are mediated by nitric oxide/cGMP via differential actions of cGMP-dependent protein kinases I and II.

Estrogens promote bone health in part by increasing osteocyte survival, an effect that requires activation of the protein kinases Akt and ERK1/2, but the molecular mechanisms involved are only partly understood. Because estrogens increase nitric oxide (NO) synthesis and NO can have anti-apoptotic effects, we examined the role of NO/cGMP signaling in estrogen regulation of osteocyte survival. Etoposide-induced death of MLO-Y4 osteocyte-like cells, assessed by trypan blue staining, caspase-3 cleavage, and TUNEL assays, was completely prevented when cells were pre-treated with 17ß-estradiol. This protective effect was mimicked when cells were pre-treated with a membrane-permeable cGMP analog and blocked by pharmacological inhibitors of NO synthase, soluble guanylate cyclase, or cGMP-dependent protein kinases (PKGs), supporting a requirement for NO/cGMP/PKG signaling downstream of 17ß-estradiol. siRNA-mediated knockdown and viral reconstitution of individual PKG isoforms demonstrated that the anti-apoptotic effects of estradiol and cGMP were mediated by PKG Iα and PKG II. Akt and ERK1/2 activation by 17ß-estradiol required PKG II, and cGMP mimicked the effects of estradiol on Akt and ERK, including induction of ERK nuclear translocation. cGMP induced BAD phosphorylation on several sites, and experiments with phosphorylation-deficient BAD mutants demonstrated that the anti-apoptotic effects of cGMP and 17ß-estradiol required BAD phosphorylation on Ser(136) and Ser(155); these sites were targeted by Akt and PKG I, respectively, and regulate BAD interaction with Bcl-2. In conclusion, 17ß-estradiol protects osteocytes against apoptosis by activating the NO/cGMP/PKG cascade; PKG II is required for estradiol-induced activation of ERK and Akt, and PKG Iα contributes to pro-survival signaling by directly phosphorylating BAD.

The combination of cobinamide and sulfanegen is highly effective in mouse models of cyanide poisoning.

CONTEXT: Cyanide is a component of smoke in residential and industrial fires, and accidental exposure to cyanide occurs in a variety of industries. Moreover, cyanide has the potential to be used by terrorists, particularly in a closed space such as an airport or train station. Current therapies for cyanide poisoning must be given by intravenous administration, limiting their use in treating mass casualties. OBJECTIVE: We are developing two new cyanide antidotes--cobinamide, a vitamin B(12) analog, and sulfanegen, a 3-mercaptopyruvate prodrug. Both drugs can be given by intramuscular administration, and therefore could be used to treat a large number of people quickly. We now asked if the two drugs would have an augmented effect when combined. MATERIALS AND METHODS: We used a non-lethal and two different lethal models of cyanide poisoning in mice. The non-lethal model assesses neurologic recovery by quantitatively evaluating the innate righting reflex time of a mouse. The two lethal models are a cyanide injection and a cyanide inhalation model. RESULTS: We found that the two drugs are at least additive when used together in both the non-lethal and lethal models: at doses where all animals died with either drug alone, the combination yielded 80 and 40% survival in the injection and inhalation models, respectively. Similarly, drug doses that yielded 40% survival with either drug alone, yielded 80 and 100% survival in the injection and inhalation models, respectively. As part of the inhalation model, we developed a new paradigm in which animals are exposed to cyanide gas, injected intramuscularly with an antidote, and then re-exposed to cyanide gas. This simulates cyanide exposure of a large number of people in a closed space, because people would remain exposed to cyanide, even after receiving an antidote. CONCLUSION: The combination of cobinamide and sulfanegen shows great promise as a new approach to treating cyanide poisoning.

Cyclic GMP and protein kinase G control a Src-containing mechanosome in osteoblasts.

Mechanical stimulation is crucial for bone growth and remodeling, and fluid shear stress promotes anabolic responses in osteoblasts through multiple second messengers, including nitric oxide (NO). NO triggers production of cyclic guanosine 3',5'-monophosphate (cGMP), which in turn activates protein kinase G (PKG). We found that the NO-cGMP-PKG signaling pathway activates Src in mechanically stimulated osteoblasts to initiate a proliferative response. PKGII was necessary for Src activation, a process that also required the interaction of Src with ß3 integrins and dephosphorylation of Src by a complex containing the phosphatases SHP-1 (Src homology 2 domain-containing tyrosine phosphatase 1) and SHP-2. PKGII directly phosphorylated and stimulated SHP-1 activity, and fluid shear stress triggered the recruitment of PKGII, Src, SHP-1, and SHP-2 to a mechanosome containing ß3 integrins. PKGII-null mice showed defective Src and ERK (extracellular signal-regulated kinase) signaling in osteoblasts and decreased ERK-dependent gene expression in bone. Our findings reveal a convergence of NO-cGMP-PKG and integrin signaling and establish a previously unknown mechanism of Src activation. These results support the use of PKG-activating drugs to mimic the anabolic effects of mechanical stimulation of bone in the treatment of osteoporosis.

A DNA polymerase-{alpha}{middle dot}primase cofactor with homology to replication protein A-32 regulates DNA replication in mammalian cells.

alpha-Accessory factor (AAF) stimulates the activity of DNA polymerase-alpha.primase, the only enzyme known to initiate DNA replication in eukaryotic cells ( Goulian, M., Heard, C. J., and Grimm, S. L. (1990) J. Biol. Chem. 265, 13221-13230 ). We purified the AAF heterodimer composed of 44- and 132-kDa subunits from cultured cells and identified full-length cDNA clones using amino acid sequences from internal peptides. AAF-132 demonstrated no homologies to known proteins; AAF-44, however, is evolutionarily related to the 32-kDa subunit of replication protein A (RPA-32) and contains an oligonucleotide/oligosaccharide-binding (OB) fold domain similar to the OB fold domains of RPA involved in single-stranded DNA binding. Epitope-tagged versions of AAF-44 and -132 formed a complex in intact cells, and purified recombinant AAF-44 bound to single-stranded DNA and stimulated DNA primase activity only in the presence of AAF-132. Mutations in conserved residues within the OB fold of AAF-44 reduced DNA binding activity of the AAF-44.AAF-132 complex. Immunofluorescence staining of AAF-44 and AAF-132 in S phase-enriched HeLa cells demonstrated punctate nuclear staining, and AAF co-localized with proliferating cell nuclear antigen, a marker for replication foci containing DNA polymerase-alpha.primase and RPA. Small interfering RNA-mediated depletion of AAF-44 in tumor cell lines inhibited [methyl-(3)H]thymidine uptake into DNA but did not affect cell viability. We conclude that AAF shares structural and functional similarities with RPA-32 and regulates DNA replication, consistent with its ability to increase polymerase-alpha.primase template affinity and stimulate both DNA primase and polymerase-alpha activities in vitro.

The phosphatidylinositol 3-kinase/akt cassette regulates purine nucleotide synthesis.

The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is highly conserved throughout evolution and regulates cell size and survival and cell cycle progression. It regulates the latter by stimulating procession through G(1) and the G(1)/S phase transition. Entry into S phase requires an abundant supply of purine nucleotides, but the effect of the PI3K/Akt pathway on purine synthesis has not been studied. We now show that the PI3K/Akt cassette regulates both de novo and salvage purine nucleotide synthesis in insulin-responsive mouse mesenchymal cells. We found that serum and insulin stimulated de novo purine synthesis in serum-starved cells largely through PI3K/Akt signaling, and pharmacologic and genetic inhibition of PI3K/Akt reduced de novo synthesis by 75% in logarithmically growing cells. PI3K/Akt regulated early steps of de novo synthesis by modulating phosphoribosylpyrophosphate production by the non-oxidative pentose phosphate pathway and late steps by modulating activity of the bifunctional enzyme aminoimidazole-carboxamide ribonucleotide transformylase IMP cyclohydrolase, an enzyme not previously known to be regulated. The effects of PI3K/Akt on purine nucleotide salvage were likely through regulating phosphoribosylpyrophosphate availability. These studies define a new mechanism whereby the PI3K/Akt cassette functions as a master regulator of cellular metabolism and a key player in oncogenesis.

Cyanide produced by human isolates of Pseudomonas aeruginosa contributes to lethality in Drosophila melanogaster.

Some Pseudomonas aeruginosa strains are cyanogenic, and cyanide may contribute to the bacterium's virulence. Using human isolates of P. aeruginosa, we have shown that Drosophila melanogaster suspended above cyanogenic strains become motionless and develop bradycardia and that flies injected with cyanogenic bacterial strains die more rapidly than those injected with noncyanogenic strains. Flies exposed to cyanogenic strains had high cyanide and low adenosine triphosphate (ATP) concentrations in body extracts, and treatment with a cyanide antidote equalized survival of flies injected with cyanogenic and noncyanogenic strains. P. aeruginosa PAO1 strain with a mutation in the hydrogen cyanide synthase gene cluster was much less toxic to flies than the parental cyanogenic strain or 2 knock-in strains. Transgenic flies overexpressing rhodanese, which detoxifies cyanide by converting it to thiocyanate, were resistant to cyanide and the increased virulence of cyanogenic strains. We conclude that D. melanogaster is a good model for studying cyanide produced by P. aeruginosa.

Effects of lovastatin on Rho isoform expression, activity, and association with guanine nucleotide dissociation inhibitors.

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (EC1.1.1.88) inhibitors (statins) reduce cholesterol synthesis and prevent cardiovascular disease; they can also inhibit prenylation of Ras and Rho proteins, and have anti-neoplastic effects. Rho proteins cycle between an active, GTP-bound, and an inactive, GDP-bound form, and Rho prenylation is important for Rho's interaction with upstream regulators and downstream effectors, but the effects of statins on Rho signaling are incompletely understood. We found that the HMG-CoA reductase inhibitor lovastatin markedly induced the expression of RhoA, B, and C in human erythroleukemia (HEL) cells. The drug increased RhoA and C only in their unprenylated forms, but it increased both prenylated and unprenylated RhoB and did not significantly affect N- and K-Ras prenylation, suggesting that it inhibited geranyl-geranylation more efficiently than farnesylation. Quantitative analysis of nucleotides bound to Rho demonstrated a 3.7-fold increase in Rho-GTP and a similar increase in Rho-GDP in lovastatin-treated cells, leaving the fraction of Rho in the active, GTP-bound form constant at 5.8%. Lovastatin reduced Rho association with Rho guanine dissociation inhibitor (RhoGDI)-alpha and -beta, and prenylation-deficient Rho mutants did not associate with RhoGDI. siRNA inhibition of RhoGDIalpha expression increased Rho-GTP, suggesting that decreased Rho/RhoGDIalpha association explained an increase in unprenylated Rho-GTP in lovastatin-treated cells. Unprenylated Rho A, B, and C were partly functional in activating serum response element-dependent transcription. In conclusion, we quantified effects of lovastatin on RhoA, B, and C isoforms, and provide a molecular mechanism whereby statins cause accumulation of unprenylated Rho-GTP.