Compartmentation and compartment-specific regulation of PDE5 by protein kinase G allows selective cGMP-mediated regulation of platelet functions

It is generally accepted that nitric oxide (NO) donors, such as sodium nitroprusside (SNP), or phosphodiesterase 5 (PDE5) inhibitors, including sildenafil, each impact human platelet function. Although a strong correlation exists between the actions of NO donors in platelets and their impact on cGMP, agents such as sildenafil act without increasing global intra-platelet cGMP levels. This study was undertaken to identify how PDE5 inhibitors might act without increasing cGMP. Our data identify PDE5 as an integral component of a protein kinase G1β (PKG1β)-containing signaling complex, reported previously to coordinate cGMP-mediated inhibition of inositol-1, 4, 5-trisphosphate receptor type 1 (IP₃R1)-mediated Ca²⁺-release. PKG1β and PDE5 did not interact in subcellular fractions devoid of IP₃R1 and were not recruited to IP₃R1-enriched membranes in response to cGMP-elevating agents. Activation of platelet PKG promoted phosphorylation and activation of the PDE5 fraction tethered to the IP₃R1-PKG complex, an effect not observed for the nontethered PDE5. Based on these findings, we elaborate a model in which PKG selectively activates PDE5 within a defined microdomain in platelets and propose that this mechanism allows spatial and temporal regulation of cGMP signaling in these cells. Recent reports indicate that sildenafil might prove useful in limiting in-stent thrombosis and the thrombotic events associated with the acute coronary syndromes (ACS), situations poorly regulated with currently available therapeutics. We submit that our findings may define a molecular mechanism by which PDE5 inhibition can differentially impact selected cellular functions of platelets, and perhaps of other cell types.     

Polyphosphoinositide Changes in Rabbit Platelets Stimulated with Platelet Activating Factor During the Formation of Platelet-fibrin Clots

Phosphoinositide metabolism in rabbit platelets prelabelled with [³²P]phosphate and [³H]inositol was stimulated by platelet activating factor (PAF, 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) with stirring at 200 rpm for 120 s in the presence of polymerising fibrin produced by the action of batroxobin (B. atrox) (also referred to by the proprietary name Reptilase) on fibrinogen. Under these conditions platelet-fibrin clots formed and retracted around the stirring bar. Phosphoinositides were extracted with chloroform: methanol: HC1. The role of the secretion of platelet granule contents in the phosphoinositide changes was examined by comparison of the effects of 1 nM PAF which did not cause secretion, with 50 nM PAF which caused extensive secretion. Stimulation of platelets with PAF in the presence of polymerising fibrin caused a greater decrease in the amount and labelling of extractable phosphatidylinositol 4,5-bisphosphate (PIP₂) than was observed with platelets stimulated in the presence of fibrinogen. With 1 nM PAF, the decrease (1.26 ± 0.11 nmol/10⁹ platelets) in amount of extractable PIP₂ when platelets were stimulated in the presence of polymerising fibrin compared with in the presence of fibrinogen was accounted for by an increase in the amount of phosphatidylinositol 4-phosphate (PIP). With 50 nM PAF, the decrease in amount of extractable PIP₂ (1.09±0.11 nmol/10⁹ platelets) was not accounted for by an increase in the amount of PIP; the decrease in the amount of [³H]inositol label in PIP₂ in platelets stimulated in the presence of polymerising fibrin was accounted for by the sum of the increases in PIP labelling and the label associated with interfacial protein from the lipid extractions. When fibrin polymerisation was blocked with glycyl-L-prolyl-L-arginyl-L-proline (GPRP), the large decrease in extractable PIP₂ and the increase in the association of label with the interfacial protein did not occur. Thus, both the formation of a fibrin network, and the changes that accompany the secretion of granule contents, are necessary for the association of the ³H-labelled material with interfacial protein. Blocking thromboxane A₂ formation had no effect on the changes in response to 50 nM PAF. Although PAF stimulated phospholipase C, resulting in increases in amount and ³²P-labelling of phosphatidic acid and ³H-labelling of inositol bisphosphate and inositol phosphate, the increases were similar in the presence of polymerising fibrin or fibrinogen. Thus, further stimulation of phospholipase C does not occur in association with clot formation. The specific radioactivities of labelling with [³H]inositol of the phosphoinositides in unstimulated platelets differed (PIP₂> phosphatidylinositol (PI) > PIP). Upon stimulation of the platelets with 1 nM PAF, the specific radioactivity of PIP rose above that of PI and toward that of PIP₂, indicating that the increase in PIP was due to degradation of PIP₂. Thus, the large decrease in extractable PIP₂ and increase in formation of PIP caused by the presence of polymerising fibrin appear to be due to increased degradation of PIP₂ to PIP.     

Thrombin-induced platelet aggregation,phosphoinositide metabolism and protein phosphorylation in NIDDM patients treated by diet,sulphonylurea or insulin

Summary We studied thrombin-induced metabolism of phosphoinositide, protein phosphorylation and platelet aggregation in platelets from 32 NIDDM patients and 12 control subjects. To clarify the effect of diet, sulphonylureas, or insulin treatment, the subjects were divided into three groups based on the type of treatment. Thrombin-induced platelet aggregation was measured with an aggregometer. Low-dose thrombin (0.25 U/ml)-stimulated platelet aggregation in diabetic patients was significantly increased compared with the control subjects. Platelet aggregation in the sulphonylurea and insulin groups was significantly lower than in the diet group. On the other hand, in platelets incubated with [³²P]orthophosphate, thrombin-induced incorporation of ³²P radioactivity into phosphatidic acid (PA) was significantly lower in the sulphonylurea and insulin groups than in the diet group. Thrombin-induced incorporation of [³²P] radioactivity into phosphatidylinositol (PIP) for 10 s was significantly higher in the sulphonylurea group than in the diet group. There were no differences in thrombin-induced 47 kDa protein phosphorylation between platelets from the diet, sulphonylurea, or insulin groups. These results suggest that sulphonylureas and insulin induce suppression of thrombin-induced activation of phospholipase C, which mediates hydrolysis of PIP and PIP₂ and production of PA, which leads to inhibition of platelet aggregation.Abbreviations NIDDM Non-insulin-dependent diabetes mellitus - IDDM insulin-dependent diabetes mellitus - PA phosphatidic acid - PI phosphatidylinositol - PIP phosphatidylinositol 4-monophosphate - PIP₂ phosphatidylinositol 4,5-bisphosphate - TPA 12-O-tetradecanoylphorbol-13-actate - IP₃ inositol 1,4,5-trisphophate - E/I expiration/inspiration - HPLTC high performance thin layer chromatography     

Phosphatidylinositol 4,5-bisphosphate (PIP2) stimulates the electrogenic Na/HCO3 cotransporter NBCe1-A expressed in Xenopus oocytes

Bicarbonate transporters are regulated by signaling molecules/ions such as protein kinases, ATP, and Ca²⁺. While phospholipids such as PIP₂ can stimulate Na-H exchanger activity, little is known about phospholipid regulation of bicarbonate transporters. We used the patch-clamp technique to study the function and regulation of heterologously expressed rat NBCe1-A in excised macropatches from Xenopus laevis oocytes. Exposing the cytosolic side of inside-out macropatches to a 5% CO₂/33 mM HCO₃⁻ solution elicited a mean inward current of 14 pA in 74% of macropatches attached to pipettes (−V_p = −60 mV) containing a low-Na⁺, nominally HCO₃⁻-free solution. The current was 80–90% smaller in the absence of Na⁺, approximately 75% smaller in the presence of 200 μM DIDS, and absent in macropatches from H₂O-injected oocytes. NBCe1-A currents exhibited time-dependent rundown that was inhibited by removing Mg²⁺ in the presence or absence of vanadate and F⁻ to reduce general phosphatase activity. Applying 5 or 10 μM PIP₂ (diC8) in the presence of HCO₃⁻ induced an inward current in 54% of macropatches from NBC-expressing, but not H₂O-injected oocytes. PIP₂-induced currents were HCO₃⁻-dependent and somewhat larger following more NBCe1-A rundown, 62% smaller in the absence of Na⁺, and 90% smaller in the presence of 200 μM DIDS. The polycation neomycin (250–500 μM) reduced the PIP₂-induced inward current by 69%; spermine (100 μM) reduced the current by 97%. Spermine, poly-D-lysine, and neomycin all reduced the baseline HCO₃⁻-induced inward currents by as much as 85%. In summary, PIP₂ stimulates NBCe1-A activity, and phosphoinositides are regulators of bicarbonate transporters.     

Activation of the atrial KACh channel by the βγ subunits of G proteins or intracellular Na+ ions depends on the presence of phosphatidylinositol phosphates

The βγ subunits of GTP-binding proteins (G_βγ) activate the muscarinic K⁺ channel (K_ACh) in heart by direct binding to both of its component subunits. K_ACh channels can also be gated by internal Na⁺ ions. Both activation mechanisms show dependence on hydrolysis of intracellular ATP. We report that phosphatidylinositol 4,5-bisphosphate (PIP₂) mimics the ATP effects and that depletion or block of PIP₂ retards the stimulatory effects of G_βγ subunits or Na⁺ ions on channel activity, effects that can be reversed by restoring PIP₂. Thus, regulation of K_ACh channel activity may be crucially dependent on PIP₂ and phosphatidylinositol signaling. These striking functional results are in agreement with in vitro biochemical studies on the PIP₂ requirement for G_βγ stimulation of G protein receptor kinase activity, thus implicating phosphatidylinositol phospholipids as a potential control point for G_βγ-mediated signal transduction.     

Coated platelets function in platelet-dependent fibrin formation via integrin αIIbβ3 and transglutaminase factor XIII

Coated platelets, formed by collagen and thrombin activation, have been characterized in different ways: i) by the formation of a protein coat of α-granular proteins; ii) by exposure of procoagulant phosphatidylserine; or iii) by high fibrinogen binding. Yet, their functional role has remained unclear. Here we used a novel transglutaminase probe, Rhod-A14, to identify a subpopulation of platelets with a cross-linked protein coat, and compared this with other platelet subpopulations using a panel of functional assays. Platelet stimulation with convulxin/thrombin resulted in initial integrin α_IIbβ₃ activation, the appearance of a platelet population with high fibrinogen binding, (independently of active integrins, but dependent on the presence of thrombin) followed by phosphatidylserine exposure and binding of coagulation factors Va and Xa. A subpopulation of phosphatidylserine-exposing platelets bound Rhod-A14 both in suspension and in thrombi generated on a collagen surface. In suspension, high fibrinogen and Rhod-A14 binding were antagonized by combined inhibition of transglutaminase activity and integrin α_IIbβ₃. Markedly, in thrombi from mice deficient in transglutaminase factor XIII, platelet-driven fibrin formation and Rhod-A14 binding were abolished by blockage of integrin α_IIbβ₃. Vice versa, star-like fibrin formation from platelets of a patient with deficiency in α_IIbβ₃ (Glanzmann thrombasthenia) was abolished upon blockage of transglutaminase activity. We conclude that coated platelets, with initial α_IIbβ₃ activation and high fibrinogen binding, form a subpopulation of phosphatidylserine-exposing platelets, and function in platelet-dependent star-like fibrin fiber formation via transglutaminase factor XIII and integrin α_IIbβ₃.     

Analysis of C5a-mediated chemotaxis by lentiviral delivery of small interfering RNA

Immune cells respond to chemotactic signals by means of G protein-coupled receptors. Attempts to elucidate the function of specific G protein family members in these responses is complicated by redundancy among the different G protein isoforms. We have used lentiviral-based RNA interference to eliminate expression of specific G protein subunits selectively in J774A.1 mouse macrophages. The chemotactic response to the complement factors C5a and C3a is ablated in cells lacking Gβ₂ but is unaffected in cells lacking Gβ₁, Gαi₂, or Gαi₃. Similarly, the C5a-mediated calcium response of single cells is either absent or significantly delayed and weakened by Gβ₂ knockdown. Assessment of Akt1 phosphorylation levels in response to C5a shows rapid and sustained phosphorylation in both wild-type cells and cells lacking Gβ₁. Cells lacking Gβ₂ retain the rapid response but cannot sustain phospho-Akt1 levels. The phenotype of cells lacking Gβ₂ can be reversed by overexpression of either human Gβ₂ or mouse Gβ₁. These data demonstrate the usefulness of lentiviral-based RNA interference in the systematic analysis of a signaling pathway, and they suggest that in J774A.1 cells, Gβ₂-derived Gβγ is the most effective mediator of chemotaxis to C5a.     

Platelet Gi protein Gαi2 is an essential mediator of thrombo-inflammatory organ damage in mice

Platelets are crucial for hemostasis and thrombosis and exacerbate tissue injury following ischemia and reperfusion. Important regulators of platelet function are G proteins controlled by seven transmembrane receptors. The G_i protein Gα_i2 mediates platelet activation in vitro, but its in vivo role in hemostasis, arterial thrombosis, and postischemic infarct progression remains to be determined. Here we show that mice lacking Gα_i2 exhibit prolonged tail-bleeding times and markedly impaired thrombus formation and stability in different models of arterial thrombosis. We thus generated mice selectively lacking Gα_i2 in megakaryocytes and platelets (Gnai2^fl/fl/PF4-Cre mice) and found bleeding defects comparable to those in global Gα_i2-deficient mice. To examine the impact of platelet Gα_i2 in postischemic thrombo-inflammatory infarct progression, Gnai2^fl/fl/PF4-Cre mice were subjected to experimental models of cerebral and myocardial ischemia/reperfusion injury. In the model of transient middle cerebral artery occlusion stroke Gnai2^fl/fl/PF4-Cre mice developed significantly smaller brain infarcts and fewer neurological deficits than littermate controls. Following myocardial ischemia, Gnai2^fl/fl/PF4-Cre mice showed dramatically reduced reperfusion injury which correlated with diminished formation of the ADP-dependent platelet neutrophil complex. In conclusion, our data provide definitive evidence that platelet Gα_i2 not only controls hemostatic and thrombotic responses but also is critical for the development of ischemia/reperfusion injury in vivo.Platelet activation at sites of vascular injury is essential for normal hemostasis but also is a major pathomechanism underlying acute ischemic disease states such as stroke or myocardial infarction, which represent leading causes of death and severe disability worldwide (1–3).Upon vascular injury, exposed extracellular matrix constituents of the damaged vessel wall allow initial adhesion of platelets, initiating intracellular signaling cascades that result in platelets’ firm adhesion and aggregation (2, 3). Activated platelets deliver diffusible local mediators, such as ADP or thromboxane A₂, to recruit and activate additional platelets into the growing thrombus. Hence, ADP potentiates the aggregatory effects of other stimuli such as thrombin and collagen and thereby contributes to stable thrombus formation. These mediators orchestrate platelet signaling by activating G protein-coupled receptors (GPCRs) (4). In particular, platelet activation by ADP is mediated by two GPCRs, P2Y₁, which couples to the heterotrimeric G protein G_q, and P2Y₁₂, which couples to G_i proteins (5). Deficiency of either P2Y₁ or P2Y₁₂ receptors leads to a reduced aggregation response following ADP stimulation, suggesting a complementary function of the two types of G proteins, G_q and G_i, in the induction of platelet activation (6–9).Only P2Y₁₂ receptors are therapeutically targeted by antagonists, which inhibit platelet aggregation in patients (10, 11). Correspondingly, mice lacking the P2Y₁₂ receptor exhibit a profound defect in platelet activation (7, 8) with prolonged bleeding times which correlate with impaired formation and stability of thrombi. However, the impact of this pathway on the progression of thrombo-inflammatory infarcts in the postischemic brain and heart is unknown. The P2Y₁₂ receptor is reported to signal selectively through the G protein G_i2, although biochemical reconstitution experiments suggest that it interacts with other G_i isoforms such as G_i3 (11–13). Platelet G_i2, however, may interact not only with P2Y₁₂ but also with additional GPCRs present in platelets. Regardless of these considerations, and unlike murine platelets deficient in P2Y₁₂ receptors, Gα_i2-deficient platelets show only a moderate inhibition of platelet aggregation in vitro, and the translation of this defect into the in vivo situation has not been reported thus far (14, 15).A different approach to study how Gα_i2 affects in vivo thrombotic activity of platelets came from a knockin mouse line in which regulator of G-protein signaling (RGS)-insensitive Gα_i2 (G184S) was expressed (16). However, the complexity and severity of the phenotype is likely to limit further studies on the progression of platelet-dependent thrombo-inflammatory infarcts (16).Here, we show that Gα_i3 only partially compensates for the loss of Gα_i2, revealing that platelet Gα_i2 plays a dual role in both thrombus formation in injured vessels and in progression of tissue damage after focal cerebral ischemia or myocardial infarction.     

Inositol 1,4,5-tris-phosphate activation of inositol tris-phosphate receptor Ca2+ channel by ligand tuning of Ca2+ inhibition

Inositol 1,4,5-tris-phosphate (IP₃) binding to its receptors (IP₃R) in the endoplasmic reticulum (ER) activates Ca²⁺ release from the ER lumen to the cytoplasm, generating complex cytoplasmic Ca²⁺ concentration signals including temporal oscillations and propagating waves. IP₃-mediated Ca²⁺ release is also controlled by cytoplasmic Ca²⁺ concentration with both positive and negative feedback. Single-channel properties of the IP₃R in its native ER membrane were investigated by patch clamp electrophysiology of isolated Xenopus oocyte nuclei to determine the dependencies of IP₃R on cytoplasmic Ca²⁺ and IP₃ concentrations under rigorously defined conditions. Instead of the expected narrow bell-shaped cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) response centered at ≈300 nM–1 μM, the open probability remained elevated (≈0.8) in the presence of saturating levels (10 μM) of IP₃, even as [Ca²⁺]_i was raised to high concentrations, displaying two distinct types of functional Ca²⁺ binding sites: activating sites with half-maximal activating [Ca²⁺]_i (K_act) of 210 nM and Hill coefficient (H_act) ≈2; and inhibitory sites with half-maximal inhibitory [Ca²⁺]_i (K_inh) of 54 μM and Hill coefficient (H_inh) ≈4. Lowering IP₃ concentration was without effect on Ca²⁺ activation parameters or H_inh, but decreased K_inh with a functional half-maximal activating IP₃ concentration (K_IP3) of 50 nM and Hill coefficient (H_IP3) of 4 for IP₃. These results demonstrate that Ca²⁺ is a true receptor agonist, whereas the sole function of IP₃ is to relieve Ca²⁺ inhibition of IP₃R. Allosteric tuning of Ca²⁺ inhibition by IP₃ enables the individual IP₃R Ca²⁺ channel to respond in a graded fashion, which has implications for localized and global cytoplasmic Ca²⁺ concentration signaling and quantal Ca²⁺ release.     

Absence of the β subunit (cchb1) of the skeletal muscle dihydropyridine receptor alters expression of the α1 subunit and eliminates excitation-contraction coupling

The multisubunit (α_1S, α₂/δ, β₁, and γ) skeletal muscle dihydropyridine receptor transduces transverse tubule membrane depolarization into release of Ca²⁺ from the sarcoplasmic reticulum, and also acts as an L-type Ca²⁺ channel. The α_1S subunit contains the voltage sensor and channel pore, the kinetics of which are modified by the other subunits. To determine the role of the β₁ subunit in channel activity and excitation-contraction coupling we have used gene targeting to inactivate the β₁ gene. β₁-null mice die at birth from asphyxia. Electrical stimulation of β₁-null muscle fails to induce twitches, however, contractures are induced by caffeine. In isolated β₁-null myotubes, action potentials are normal, but fail to elicit a Ca²⁺ transient. L-type Ca²⁺ current is decreased 10- to 20-fold in the β₁-null cells compared with littermate controls. Immunohistochemistry of cultured myotubes shows that not only is the β₁ subunit absent, but the amount of α_1S in the membrane also is undetectable. In contrast, the β₁ subunit is localized appropriately in dysgenic, mdg/mdg, (α_1S-null) cells. Therefore, the β₁ subunit may not only play an important role in the transport/insertion of the α_1S subunit into the membrane, but may be vital for the targeting of the muscle dihydropyridine receptor complex to the transverse tubule/sarcoplasmic reticulum junction.     

Cytoskeletal perturbation leads to platelet dysfunction and thrombocytopenia in variant forms of Glanzmann thrombasthenia

Several patients have been reported to have variant dominant forms of Glanzmann thrombasthenia, associated with macrothrombocytopenia and caused by gain-of-function mutations of ITGB3 or ITGA2B leading to reduced surface expression and constitutive activation of integrin α_IIbβ₃. The mechanisms leading to a bleeding phenotype of these patients have never been addressed. The aim of this study was to unravel the mechanism by which ITGB3 mutations causing activation of α_IIbβ₃ lead to platelet dysfunction and macrothrombocytopenia. Using platelets from two patients carrying the β₃ del647-686 mutation and Chinese hamster ovary cells expressing different α_IIbβ₃-activating mutations, we showed that reduced surface expression of α_IIbβ₃ is due to receptor internalization. Moreover, we demonstrated that permanent triggering of α_IIbβ₃-mediated outside-in signaling causes an impairment of cytoskeletal reorganization arresting actin turnover at the stage of polymerization. The induction of actin polymerization by jasplakinolide, a natural toxin that promotes actin nucleation and prevents depolymerization of stress fibers, in control platelets produced an impairment of platelet function similar to that of patients with variant forms of dominant Glanzmann thrombasthenia. del647-686β₃-transduced murine megakaryocytes generated proplatelets with a reduced number of large tips and asymmetric barbell-proplatelets, suggesting that impaired cytoskeletal rearrangement is the cause of macrothrombocytopenia. These data show that impaired cytoskeletal remodeling caused by a constitutively activated α_IIbβ₃ is the main effector of platelet dysfunction and macrothrombocytopenia, and thus of bleeding, in variant forms of dominant Glanzmann thrombasthenia.     

Dominant inheritance of a novel integrin β3 mutation associated with a hereditary macrothrombocytopenia and platelet dysfunction in two Italian families

Background

Defects of integrin α_IIbβ₃ are typical of Glanzmann’s thrombasthenia, an inherited autosomal recessive bleeding disorder characterized by the failure of platelets to aggregate in response to all physiological agonists, but with no abnormalities in the number or size of platelets. Although large heterogeneity has been described for Glanzmann’s thrombasthenia, no family has so far been described as having an autosomal dominant form of this disease.

Design and Methods

We describe two Italian families with moderate thrombocytopenia with large platelets, defective platelet function and moderate/severe mucocutaneous bleeding, transmitted as an autosomal dominant trait and associated with a novel integrin β₃-gene (ITGB3) mutation.

Results

The characteristics of our families are moderate macrothrombocytopenia and defective platelet function associated with a mild reduction of surface α_Ib β₃, impaired platelet aggregation to physiological agonists but not to ristocetin, normal clot retraction, reduced fibrinogen binding and expression of activated α_IIbβ₃ upon stimulation, normal platelet adhesion to immobilized fibrinogen but reduced platelet spreading and tyrosine phosphorylation, indicating defective α_IIbβ₃-mediated outside-in signaling. Molecular analysis revealed a novel mutation of ITGB3 that determines an in-frame deletion producing the loss of amino acids 647–686 of the βTD ectodomain of integrin β₃. Haplotype analysis indicated that the two families inherited the mutation from a common ancestral chromosome.

Conclusions

This novel autosomal dominant macrothrombocytopenia associated with platelet dysfunction raises interesting questions about the role of integrin β₃, and its βTD domain, in platelet formation and function.     

Targeted inactivation of αi2 or αi3 disrupts activation of the cardiac muscarinic K+ channel, IK+Ach, in intact cells

Cardiac muscarinic receptors activate an inwardly rectifying K⁺ channel, I_K^+Ach, via pertussis toxin (PT)-sensitive heterotrimeric G proteins (in heart G_i2, G_i3, or G_o). We have used embryonic stem cell (ES cell)-derived cardiocytes with targeted inactivations of specific PT-sensitive α subunits to determine which G proteins are required for receptor-mediated regulation of I_K^+Ach in intact cells. The muscarinic agonist carbachol increased I_K^+Ach activity in ES cell-derived cardiocytes from wild-type cells, in cells lacking α_o, and in cells lacking the PT-insensitive G protein α_q. In cells with targeted inactivation of α_i2 or α_i3, channel activation by both carbachol and adenosine was blocked. Carbachol-induced channel activation was restored in the α_i2- and α_i3-null cells by reexpressing the previously targeted gene and guanosine 5′-[γ-thio] triphosphate was able to fully activate I_K^+Ach in excised membranes patches from these mutants. In contrast, negative chronotropic responses to both carbachol and adenosine were preserved in cells lacking α_i2 or α_i3. Our results show that expression of two specific PT-sensitive α subunits (α_i2 and α_i3 but not α_o) is required for normal agonist-dependent activation of I_K^+Ach and suggest that both α_i2- and α_i3-containing heterotrimeric G proteins may be involved in the signaling process. Also the generation of negative chronotropic responses to muscarinic or adenosine receptor agonists do not require activation of I_K^+Ach or the expression of α_i2 or α_i3.     

Pharmacological inhibition of PI5P4Kα/β disrupts cell energy metabolism and selectively kills p53-null tumor cells

Most human cancer cells harbor loss-of-function mutations in the p53 tumor suppressor gene. Genetic experiments have shown that phosphatidylinositol 5-phosphate 4-kinase α and β (PI5P4Kα and PI5P4Kβ) are essential for the development of late-onset tumors in mice with germline p53 deletion, but the mechanism underlying this acquired dependence remains unclear. PI5P4K has been previously implicated in metabolic regulation. Here, we show that inhibition of PI5P4Kα/β kinase activity by a potent and selective small-molecule probe disrupts cell energy homeostasis, causing AMPK activation and mTORC1 inhibition in a variety of cell types. Feedback through the S6K/insulin receptor substrate (IRS) loop contributes to insulin hypersensitivity and enhanced PI3K signaling in terminally differentiated myotubes. Most significantly, the energy stress induced by PI5P4Kαβ inhibition is selectively toxic toward p53-null tumor cells. The chemical probe, and the structural basis for its exquisite specificity, provide a promising platform for further development, which may lead to a novel class of diabetes and cancer drugs.

There are two synthetic routes for phosphatidylinositol 4,5-bisphosphate, or PI(4,5)P₂, a versatile phospholipid with both structural and signaling functions in most eukaryotic cells (1 –3). The bulk of PI(4,5)P₂ is found at the inner leaflet of the plasma membrane and is synthesized from phosphatidylinositol 4-phosphate, or PI(4)P, by type 1 phosphatidylinositol phosphate kinase PI4P5K (4, 5). A smaller fraction of PI(4,5)P₂ is generated from the much rarer phosphatidylinositol 5-phosphate, or PI(5)P, through the activity of type 2 phosphatidylinositol phosphate kinase PI5P4K (6, 7). Although PI5P4K is as abundantly expressed as PI4P5K (8), its function is less well understood (9). It has been proposed that PI5P4K may play a role in suppressing PI(5)P, which is often elevated by stress (10, 11), or produce local pools of PI(4,5)P₂ at subcellular compartments such as Golgi and nucleus (12).Higher animals have three PI5P4K isoforms, α, β, and γ, which are encoded by three different genes, PIP4K2A, PIP4K2B, and PIP4K2C. The three isoforms differ, at least in vitro, significantly in enzymatic activity: PI5P4Kα is two orders of magnitude more active than PI5P4Kβ, while PI5P4K-γ has very little activity (13). PI5P4Ks are dimeric proteins (14), and the possibility that they can form heterodimers may have important functional implications, especially for the lesser active isoforms (15, 16). PI5P4Kβ is the only isoform that preferentially localizes to the nucleus (17).Genetic studies have implicated PI5P4Kβ in metabolic regulation (18, 19). Mice with both PIP4K2B genes inactivated manifest hypersensitivity to insulin stimulation (adult males are also leaner). Although this is consistent with the observation that PI(5)P levels, which can be manipulated by overexpressing PI5P4K or a bacterial phosphatase that robustly produces PI(5)P from PI(4,5)P₂, correlate positively with PI3K/Akt signaling, the underlying molecular mechanisms remain undefined (20). Both male and female PIP4K2B ^−/− mice are mildly growth retarded. Inactivation of the only PI5P4K isoform in Drosophila also produced small and developmentally delayed animals (21). These phenotypes may be related to suppressed TOR signaling (22, 23), but again, the underlying mechanism is unclear since TORC1 is downstream of, and positively regulated by, PI3K/Akt. Knocking out the enzymatically more active PI5P4Kα, in contrast, did not produce any overt metabolic or developmental phenotypes (19).Malignant transformation is associated with profound changes in cell metabolism (24, 25). Although metabolic reprograming generally benefits tumor cells by increasing energy and material supplies, it can also, counterintuitively, generate unique dependencies (26, 27). Loss of p53, a tumor suppressor that is mutated in most human cancers, has been shown to render cells more susceptible to nutrient stress (28, 29) and to the antidiabetic drug metformin (30, 31). Although TP53 ^−/− and PIP4K2B ^−/− mice are themselves viable, combining the two is embryonically lethal (19). Knocking out three copies of PI5P4K (PIP4K2A ^−/− PIP4K2B ^+/− ) greatly reduces tumor formation and cancer-related death in TP53 ^−/− animals (19). The synthetic lethal interaction between p53 and PI5P4Kα/β was thought to result from suppressed glycolysis and increased reactive oxygen species (19), although how the lipid kinases impact glucose metabolism remains uncertain.Given the interest in the physiological function of this alternative synthetic route for PI(4,5)P₂, and the potential of PI5P4K inactivation in treating type 2 diabetes and cancer, several attempts have been made to identify chemical probes that target various PI5P4K isoforms, which yielded compounds with micromolar affinity and unknown selectivity (32 –35). Here, we report the development of a class of PI5P4Kα/β inhibitors that have much improved potency and better-defined selectivity. Using the chemical probe, we show that transient inhibition of the lipid kinases alters cell energy metabolism and induces different responses in muscle and cancer cells.     

PIP5KI gamma is required for cardiovascular and neuronal development

All eukaryotic cells contain the phospholipid phosphatidylinositol 4, 5-bisphosphate (PIP2) that serves multiple roles in signal transduction cascades. Type I phosphatidylinositol-4-phosphate 5-kinase (PIP5KI) catalyzes the synthesis of PIP2 by phosphorylating phosphatidylinositol 4 phosphate. Although the classical isoforms of PIP5KI (designated as alpha, beta, and gamma) all generate the same phospholipid product, they have significantly dissimilar primary structures and expression levels in different tissues, and they appear to localize within different compartments within the cell. Therefore, it appears likely that PIP5KI isoforms have overlapping, but not identical, functions. Here we show that targeted disruption of PIP5KIgamma causes widespread developmental and cellular defects. PIP5KIgamma-null embryos have myocardial developmental defects associated with impaired intracellular junctions that lead to heart failure and extensive prenatal lethality at embryonic day 11.5 of development. Loss of PIP5KIgamma also results in neural tube closure defects that were associated with impaired PIP2 production, adhesion junction formation, and neuronal cell migration. These data, along with those of other PIP5KI isoforms, indicate that individual PIP5KI isoenzymes fulfill specific roles in embryonic development.     

Phosphatidylinositol-4,5-biphosphate-dependent rearrangement of TRPV4 cytosolic tails enables channel activation by physiological stimuli

Most transient receptor potential (TRP) channels are regulated by phosphatidylinositol-4,5-biphosphate (PIP₂), although the structural rearrangements occurring on PIP₂ binding are currently far from clear. Here we report that activation of the TRP vanilloid 4 (TRPV4) channel by hypotonic and heat stimuli requires PIP₂ binding to and rearrangement of the cytosolic tails. Neutralization of the positive charges within the sequence ¹²¹KRWRK¹²⁵, which resembles a phosphoinositide-binding site, rendered the channel unresponsive to hypotonicity and heat but responsive to 4α-phorbol 12,13-didecanoate, an agonist that binds directly to transmembrane domains. Similar channel response was obtained by depletion of PIP₂ from the plasma membrane with translocatable phosphatases in heterologous expression systems or by activation of phospholipase C in native ciliated epithelial cells. PIP₂ facilitated TRPV4 activation by the osmotransducing cytosolic messenger 5′-6’-epoxyeicosatrienoic acid and allowed channel activation by heat in inside-out patches. Protease protection assays demonstrated a PIP₂-binding site within the N-tail. The proximity of TRPV4 tails, analyzed by fluorescence resonance energy transfer, increased by depleting PIP₂ mutations in the phosphoinositide site or by coexpression with protein kinase C and casein kinase substrate in neurons 3 (PACSIN3), a regulatory molecule that binds TRPV4 N-tails and abrogates activation by cell swelling and heat. PACSIN3 lacking the Bin-Amphiphysin-Rvs (F-BAR) domain interacted with TRPV4 without affecting channel activation or tail rearrangement. Thus, mutations weakening the TRPV4–PIP₂ interacting site and conditions that deplete PIP₂ or restrict access of TRPV4 to PIP₂—in the case of PACSIN3—change tail conformation and negatively affect channel activation by hypotonicity and heat.     

Isolation and Structure of Two Prostaglandin Endoperoxides That Cause Platelet Aggregation

Incubation for a short time of arachidonic acid with the microsomal fraction of a homogenate of the vesicular gland of sheep in the presence of 1 mM p-mercuribenzoate followed by extraction and silicic acid chromatography yielded two prostaglandin endoperoxides. The structures of these compounds, i.e., 15-hydroperoxy-9α,11α-peroxidoprosta-5,13-dienoic acid (prostaglandin G₂) and 15-hydroxy-9α,11α-peroxidoprosta-5,13-dienoic acid (prostaglandin H₂), were assigned mainly by a number of chemical transformations into previously known prostaglandins. The new prostaglandins were 50-200 times (prostaglandin G₂) and 100-450 times (prostaglandin H₂) more active than prostaglandin E₂ on the superfused aorta strip. The half-life of the prostaglandin endoperoxides in aqueous medium (about 5 min) was significantly longer than that of “rabbit aorta-contracting substance” released from guinea pig lung, indicating that none of the prostaglandin endoperoxides is identical with this factor. Addition of 10-300 ng/ml of the endoperoxides to suspensions of washed human platelets resulted in rapid aggregation. Furthermore, platelet aggregation induced by thrombin was accompanied by release of material reducible by stannous chloride into prostaglandin F_2α, thus indicating the involvement of endogenous prostaglandin endoperoxides in platelet aggregation.     

Platelet biogenesis and functions require correct protein O-glycosylation

Platelets express a variety of membrane and secreted glycoproteins, but the importance of glycosylation to platelet functions is poorly understood. To explore the importance of O-glycosylation, we generated mice with a targeted deletion of Cosmc in murine endothelial/hematopoietic cells (EHC) (EHC Cosmc^−/y). X-linked Cosmc encodes an essential chaperone that regulates protein O-glycosylation. This targeted mutation resulted in lethal perinatal hemorrhage in the majority of mice, and the surviving mice displayed severely prolonged tail-bleeding times and macrothrombocytopenia. EHC Cosmc^−/y platelets exhibited a marked decrease in GPIb-IX-V function and agonist-mediated integrin αIIbβ3 activation, associated with loss of interactions with von Willebrand factor and fibrinogen, respectively. Significantly, three O-glycosylated glycoproteins, GPIbα, αIIb, and GPVI normally on platelet surfaces that play essential roles in platelet functions, were partially proteolyzed in EHC Cosmc^−/y platelets. These results demonstrate that extended O-glycans are required for normal biogenesis of the platelets as well as the expression and functions of their essential glycoproteins, and that variations in O-glycosylation may contribute to altered hemostasis.     

In Situ Tensile Deformation and Mechanical Properties of α Platelets TC21 Alloy

The present study was focused on the relationship between an α platelet microstructure and the properties of TC21 alloy, and the tensile deformation process was revealed by in situ observation. To obtain the α platelet microstructures, the samples were administered a solution treatment (1000 °C for 15 min) and then cooled to room temperature by different cooling methods (furnace cooling (FC), open-door furnace cooling (OFC), air cooling (AC), and water quench (WQ), corresponding to an increased cooling rate). It is found that α platelets become thinner and colonies become narrower with the increase in cooling rate. The formation of the platelet microstructure is based on the preferred Burgers orientation relationship of {110}_β//{0001}_α and <111>_β//<112¯0>_α. The α platelets orientation changes with the cooling rate. These differences in α platelets thickness and orientation result in the excellent ductility of the sample with thick platelets and the high strength of the samples with thin platelets. During the in situ tensile deformation process, the crack propagation path is deflected in the presence of grain boundaries, α platelets, and α colonies with different orientations. The fracture of the sample with thick α platelets shows better ductility compared to those with thin α platelets.     

From the Cover: A thyroid hormone receptor mutation that dissociates thyroid hormone regulation of gene expression in vivo

Resistance to thyroid hormone (RTH) is most often due to point mutations in the β-isoform of the thyroid hormone (TH) receptor (TR-β). The majority of mutations involve the ligand-binding domain, where they block TH binding and receptor function on both stimulatory and inhibitory TH response elements. In contrast, a few mutations in the ligand-binding domain are reported to maintain TH binding and yet cause RTH in certain tissues. We introduced one such naturally occurring human RTH mutation (R429Q) into the germline of mice at the TR-β locus. R429Q knock-in (KI) mice demonstrated elevated serum TH and inappropriately normal thyroid-stimulating hormone (TSH) levels, consistent with hypothalamic–pituitary RTH. In contrast, 3 hepatic genes positively regulated by TH (Dio1, Gpd1, and Thrsp) were increased in R429Q KI animals. Mice were then rendered hypothyroid, followed by graded T₃ replacement. Hypothyroid R429Q KI mice displayed elevated TSH subunit mRNA levels, and T₃ treatment failed to normally suppress these levels. T₃ treatment, however, stimulated pituitary Gh levels to a greater degree in R429Q KI than in control mice. Gsta, a hepatic gene negatively regulated by TH, was not suppressed in R429Q KI mice after T₃ treatment, but hepatic Dio1 and Thrsp mRNA levels increased in response to TH. Cardiac myosin heavy chain isoform gene expression also showed a specific defect in TH inhibition. In summary, the R429Q mutation is associated with selective impairment of TH-mediated gene repression, suggesting that the affected domain, necessary for TR homodimerization and corepressor binding, has a critical role in negative gene regulation by TH.