Epac-1/Rap-1 signaling pathway orchestrates the reno-therapeutic effect of ticagrelor against renal ischemia/reperfusion model.

Despite the renal expression of P2Y12, the purinergic receptor for adenosine diphosphate, few data are available to discuss the renotherapeutic potential of ticagrelor, one of its reversible blockers. Indeed, the tonic inhibitory effect of this receptor has been linked to the activation of exchange protein activated by cyclic adenosine monophosphate-1 (Epac-1) protein through the cyclic adenosine monophosphate cascade. Epac-1 is considered a crossroad protein, where its activation has been documented to manage renal injury models. Hence, the current study aimed to investigate the possible therapeutic effectiveness of ticagrelor, against renal ischemia/reperfusion (I/R) model with emphasis on the involvement of Epac-1 signaling pathway using R-CE3F4, a selective Epac-1 blocker. Accordingly, rats were randomized into four groups; viz., sham-operated, renal I/R, I/R post-treated with ticagrelor for 3 days, and ticagrelor + R-CE3F4. Treatment with ticagrelor ameliorated the I/R-mediated structural alterations and improved renal function manifested by the reduction in serum BUN and creatinine. On the molecular level, ticagrelor enhanced renal Epac-1 mRNA expression, Rap-1 activation (Rap-1-GTP) and SOCS-3 level. On the contrary, it inhibited the protein expression of JAK-2/STAT-3 hub, TNF-α and MDA contents, as well as caspase-3 activity. Additionally, ticagrelor enhanced the protein expression/content of AKT/Nrf-2/HO-1 axis. All these beneficial effects were obviously antagonized upon using R-CE3F4. In conclusion, ticagrelor reno-therapeutic effect is partly mediated through modulating the Epac-1/Rap-1-GTP, AKT/Nrf-2/HO-1 and JAK-2/STAT-3/SOCS-3 trajectories, pathways that integrate to afford novel explanations to its anti-inflammatory, anti-oxidant, and anti-apoptotic potentials.

Epac1 Is Crucial for Maintenance of Endothelial Barrier Function through A Mechanism Partly Independent of Rac1.

Epac1 (exchange protein activated by cAMP) stabilizes the endothelial barrier, but detailed studies are limited by the side effects of pharmacological Epac1 modulators and transient transfections. Here, we compare the key properties of barriers between endothelial cells derived from wild-type (WT) and Epac1-knockout (KO) mice myocardium. We found that KO cell layers, unlike WT layers, had low and cAMP-insensitive trans-endothelial resistance (TER). They also had fragmented VE-cadherin staining despite having augmented cAMP levels and increased protein expression of Rap1, Rac1, RhoA, and VE-cadherin. The simultaneous direct activation of Rac1 and RhoA by CN04 compensated Epac1 loss, since TER was increased. In KO-cells, inhibition of Rac1 activity had no additional effect on TER, suggesting that other mechanisms compensate the inhibition of the Rac1 function to preserve barrier properties. In summary, Epac1 is crucial for baseline and cAMP-mediated barrier stabilization through mechanisms that are at least partially independent of Rac1.

The ADP receptor P2RY12 regulates osteoclast function and pathologic bone remodeling.

The adenosine diphosphate (ADP) receptor P2RY12 (purinergic receptor P2Y, G protein coupled, 12) plays a critical role in platelet aggregation, and P2RY12 inhibitors are used clinically to prevent cardiac and cerebral thrombotic events. Extracellular ADP has also been shown to increase osteoclast (OC) activity, but the role of P2RY12 in OC biology is unknown. Here, we examined the role of mouse P2RY12 in OC function. Mice lacking P2ry12 had decreased OC activity and were partially protected from age-associated bone loss. P2ry12-/- OCs exhibited intact differentiation markers, but diminished resorptive function. Extracellular ADP enhanced OC adhesion and resorptive activity of WT, but not P2ry12-/-, OCs. In platelets, ADP stimulation of P2RY12 resulted in GTPase Ras-related protein (RAP1) activation and subsequent αIIbß3 integrin activation. Likewise, we found that ADP stimulation induced RAP1 activation in WT and integrin ß3 gene knockout (Itgb3-/-) OCs, but its effects were substantially blunted in P2ry12-/- OCs. In vivo, P2ry12-/- mice were partially protected from pathologic bone loss associated with serum transfer arthritis, tumor growth in bone, and ovariectomy-induced osteoporosis: all conditions associated with increased extracellular ADP. Finally, mice treated with the clinical inhibitor of P2RY12, clopidogrel, were protected from pathologic osteolysis. These results demonstrate that P2RY12 is the primary ADP receptor in OCs and suggest that P2RY12 inhibition is a potential therapeutic target for pathologic bone loss.

A comparison between the effects of hydrophobic and hydrophilic statins on osteoclast function in vitro and ovariectomy-induced bone loss in vivo.

Statins potently inhibit 3-hydroxy-3-methylglutaryl-coenzyme A reductase, blocking downstream biosynthesis of isoprenoid lipids and causing inhibition of protein prenylation. Prenylated signaling molecules are essential for osteoclast function, consistent with our previous observation that mevastatin can inhibit osteoclast activity in vitro. Several reports suggest that statins may also have an anabolic effect on bone and stimulate osteoblast differentiation. This study sought to determine the effects of both hydrophobic and hydrophilic statins, particularly rosuvastatin (RSV), on osteoclast function in vitro and in vivo. All statins tested (RSV, pravastatin [PRA], cerivastatin [CER], and simvastatin [SIM]) caused accumulation of unprenylated Rap-1A in rabbit osteoclast-like cells and J774 macrophages in vitro and inhibited osteoclast-mediated resorption. The order of potency for inhibiting prenylation in vitro (at concentrations of 0.01-50 muM) was CER>SIM>RSV>PRA. The most potent hydrophilic statin (CER, 0.05 and 0.3 mg/kg) inhibited prenylation in rabbit osteoclasts 24 hours after a single subcutaneous (s.c.) injection more effectively than the most potent hydrophobic statin (RSV, 20 mg/kg). However, in a mouse model of osteoporosis, s.c. 0.05 mg/kg/day CER and 2 or 20 mg/kg/day RSV for 3 weeks only mildly prevented loss of cortical and trabecular bone induced by ovariectomy. No increase in bone formation rate was observed with statin treatment, suggesting that this effect was due to inhibition of osteoclast-mediated resorption rather than increased bone formation.

Zoledronate has an antitumor effect and induces actin rearrangement in dexamethasone-resistant myeloma cells.

New strategies are needed to overcome the resistance of multiple myeloma (MM) to dexamethasone (Dex). Several recent in vitro studies demonstrated the antitumor effect of nitrogen-containing amino-bisphosphonates (N-BPs) in various tumor cell lines. Inhibition of the prenylation of small G proteins is assumed to be one of the principal mechanisms by which N-BPs exert their effects. There have been few reports on N-BP treatment of MM cells that are resistant to Dex. Additionally, it is not known how small G proteins are altered in N-BP-treated MM cells. In this study, we evaluated the effect of the most potent N-BP, zoledronate (ZOL), on a Dex-resistant human MM cell subline (Dex-R) that we established from the well-documented RPMI8226 cell line. ZOL reduced the viability and induced apoptosis of Dex-R cells. Some of the ZOL-treated RPMI8226 cells and ZOL-treated Dex-R cells were elongated; however, elongated cells were not seen among the Dex-treated RPMI8226 cells. Furthermore, we found that portions of the small G proteins, Rho and Rap1A, were unprenylated in the ZOL-treated MM cells. Geranylgeraniol reduced the above-mentioned ZOL-induced effects. These findings suggest that ZOL may be beneficial for the treatment of Dex-resistant MM by suppressing the processing of RhoA and Rap1A.

Thrombopoietin promotes beta1-integrin-mediated adhesion in hematopoietic cells via the small GTPase Rapl.

OBJECTIVE: The interactions between cells and extracellular matrices in the bone marrow microenvironment are critical for normal hematopoiesis, controlling cell survival, proliferation, differentiation, and motility. A number of hematopoietic growth factors and cytokines can mediate these interactions by changing expression and/or activity of specific integrins, or by changing cell shape. Thrombopoietin (TPO) has previously been shown to stimulate adhesion. in certain hematopoietic cell types, although the exact mechanisms by which adhesion is promoted remain elusive. MATERIALS AND METHODS: The role of TPO in hematopoietic cell adhesion was determined with fibronectin adhesion and binding assays, flow cytometry, and immunocytochemistry using the hematopoietic cell line UT-7/TPO and bone marrow-derived primary mouse megakaryocytes. The role of Rapl in TPO-mediated adhesion was determined using a RaplGAP overexpressing UT-7/TPO cell line, in which Rapl could not be activated. RESULTS: We found that TPO promoted hematopoietic cell adhesion by causing cytoskeletal reorganization and not by increasing integrin expression, localization, or affinity, as previously hypothesized. Through studies using the UT-7/TPO-RaplGAP cell line, we found that TPO-mediated cell shape change occurred via activation of Rapl. CONCLUSIONS: These data demonstrate an important role for TPO in mediating interactions in the bone marrow microenvironment and make a significant contribution to our understanding of how TPO may affect hematopoiesis.

Synthesis and biological activity of isoprenoid bisphosphonates.

Bisphosphonates have been used in the clinic to treat osteoporosis and to reduce bone resorption and the accompanying pathological bone fractures that attend a number of malignancies including multiple myeloma and cancers of the prostate, breast, and lung. There is also evidence that some bisphosphonates have direct anticancer activity. Expansion of the current class of bisphosphonates may lead to compounds that more selectively and potently target these cancers through inhibition of the mevalonate pathway. To this end, a set of dialkyl bisphosphonates bearing isoprenoid chains of varying lengths has been synthesized. Some of these compounds were found to have biological activity on post-translational processing of the oncogenic small GTPases, Ras and Rap1a, in human-derived K562 leukemia cells. Most importantly, these compounds impair protein geranylgeranylation and not protein farnesylation.

Expression, activation, and subcellular localization of the Rap1 GTPase in cord blood-derived human megakaryocytes.

The expression of the small GTPase Rap1 in human megakaryocytes (MKs) differentiated from cord blood (CB)-derived progenitors was investigated. High levels of Rap1 were detected in the majority of mature megakaryocytes independently of days of culture, while a very low percentage of immature megakaryocytes was found to express a small amount of the protein. Rap1 was predominantly detected on internal alpha-granule but not on the plasma membrane. By contrast, CD41 was clearly present on the peripheral plasma membrane, although it also displayed an intracellular localization similar to that of Rap1. Upon thrombin stimulation, both Rap1 and CD41 translocated to the periphery of the cell. At the opposite, RhoA GTPase and glycoprotein Ibalpha were predominantly located at the plasma membrane and did not undergo relocation upon thrombin stimulation. Thrombin induced a dose- and time-dependent activation of Rap1 in mature megakaryocytes. By using a confocal microscopy approach with a specific probe, active Rap1 was detected exclusively at the peripheral plasma membrane. These results demonstrate that expression of Rap1 occurs during maturation rather than differentiation of megakaryocytes from cord blood progenitor cells. Moreover, we demonstrate that thrombin-activated Rap1 is exclusively localized at the peripheral plasma membrane.

Identification of biphenylcarboxylic acid derivatives as a novel class of bone resorption inhibitors.

UNLABELLED: A novel class of biphenylcarboxylic acid derivatives are described that inhibit osteoclastic bone resorption in vitro by promoting osteoclast apoptosis and that prevent ovariectomy-induced bone loss in vivo. The compounds act by a novel mechanism that seems to be distinct from existing antiresorptive drugs. INTRODUCTION: Many common bone diseases such as osteoporosis, Paget's disease, and cancer-associated bone disease are characterized by excessive bone loss caused by increased osteoclastic activity. Successful treatment of these diseases is based on osteoclast inhibition. The osteoclast inhibitory drugs that are currently available fall into relatively few mechanistic classes, indicating the need to identify novel antiresorptives. Here we describe a series of biphenylcarboxylic acid derivatives that have potent inhibitory effects on osteoclastic bone resorption in vitro and on ovariectomy-induced bone loss in vivo. MATERIALS AND METHODS: Compounds were tested for inhibitory effects on bone resorption in vitro using mouse osteoblast-bone marrow co-cultures, isolated rabbit osteoclasts, and mouse osteoclasts generated from bone marrow. Some experiments were also performed on human osteoclasts generated from peripheral blood mononuclear cells. We also investigated the effects of specific compounds on ovariectomy-induced bone loss in vivo in mice. RESULTS: One of the most potent compounds identified was the butanediol ester of biphenyl carboxylic acid (ABD056), which inhibited osteoclast formation in mouse osteoblast-bone marrow co-cultures by 50% (IC50) at a concentration of 26 microM and in macrophage-colony stimulating factor (M-CSF)- and RANKL-stimulated mouse bone marrow cultures with an IC50 of 8 microM. Mechanistic studies showed that ABD056 caused osteoclast apoptosis and inhibited TNFalpha-induced NF-kappaB activation. No inhibitory effects on osteoblast growth or differentiation were observed at concentrations of up to 100 microM. When administered to mice at doses of 5 and 10 mg/kg/day, ABD056 prevented ovariectomy-induced bone loss. CONCLUSIONS: Butanediol biphenylcarboxylic acid derivatives represent a new class of antiresorptive drug that might be of therapeutic value in the prevention and treatment of diseases characterized by osteoclast activation such as osteoporosis, cancer-associated bone disease, and Paget's disease of bone.

The neurofibromatosis 1 gene product neurofibromin regulates pituitary adenylate cyclase-activating polypeptide-mediated signaling in astrocytes.

Individuals with the neurofibromatosis 1 (NF1)-inherited tumor predisposition syndrome develop low-grade astrocytomas. The NF1 tumor suppressor gene product neurofibromin exhibits GTPase-activating activity (GAP) toward RAS, such that loss of neurofibromin expression leads to high levels of activated RAS and increased cell proliferation. Previous work has demonstrated that Nf1 inactivation in astrocytes leads to increased cell proliferation in vitro and in vivo, accompanied by increased RAS pathway activation. Studies on Nf1 mutant Drosophila have suggested that neurofibromin might also regulate cAMP signaling. Because intracellular cAMP levels have profound effects on astrocyte growth control, we sought to determine the contribution of neurofibromin to astrocyte cAMP regulation. In this report, we demonstrate that Nf1 inactivation in astrocytes results in reduced cAMP generation in response to PACAP and attenuated calcium influx and Rap1 activation. Based on the differential effects of forskolin and dibutyryl-cAMP on Nf1-/- astrocytes, neurofibromin likely functions at the level of adenylyl cyclase activation. Last, the reintroduction of a fragment of neurofibromin containing residues sufficient for restoring RAS-GAP function in Nf1-/- cells resulted in only partial restoration of neurofibromin-mediated cAMP regulation. These results demonstrate that neurofibromin positively influences cAMP generation and activation of cAMP growth regulatory targets in astrocytes and expands the role of the NF1 gene in astrocyte growth regulation.

The G(s)-coupled adenosine A(2B) receptor recruits divergent pathways to regulate ERK1/2 and p38.

Adenosine A(2B) receptors have been suggested to influence cell differentiation and proliferation. Human adenosine A(2B) receptors expressed in Chinese hamster ovary cells mediate phosphorylation and activation of the extracellular signal-regulated kinase (ERK1/2). Already low concentrations of agonists such as 5'-N-ethylcarboxamidoadenosine (NECA) are effective. Phosphorylation of the stress-activated protein kinase p38 was also potently induced by NECA (EC(50) 18.5 nM). These NECA-induced effects were mimicked by forskolin and 8-Br-cAMP. Inhibition of cAMP-dependent protein kinase (PKA) using H89 (N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide)) blocked phosphorylation of the cAMP response element-binding protein (CREB) and p38, but did not decrease NECA-induced ERK1/2 phosphorylation. NECA activated the small GTPase Rap1, and this was also not blocked by H89. Inhibition of phosphatidylinositol-3'-kinase (PI3K) by wortmannin inhibited adenosine A(2B) receptor-mediated ERK1/2 phosphorylation and activation of Rap1, without affecting CREB and p38 phosphorylation. A(2B) receptor-stimulated protein kinase B phosphorylation was sensitive to wortmannin, but not to H89. Thus, stimulation of adenosine A(2B) receptors activates both ERK1/2 and p38 via cAMP, but the downstream pathways are markedly different. ERK1/2 activation was dependent on PI3K but not on PKA. p38 activation by NECA was instead independent of PI3K but required cAMP and PKA. The potent activation of both MAPKs suggests a physiological role.

Galpha and Gbeta gamma require distinct Src-dependent pathways to activate Rap1 and Ras.

The Src tyrosine kinase is necessary for activation of extracellular signal-regulated kinases (ERKs) by the beta-adrenergic receptor agonist, isoproterenol. In this study, we examined the role of Src in the stimulation of two small G proteins, Ras and Rap1, that have been implicated in isoproterenol's signaling to ERKs. We demonstrate that the activation of isoproterenol of both Rap1 and Ras requires Src. In HEK293 cells, isoproterenol activates Rap1, stimulates Rap1 association with B-Raf, and activates ERKs, all via PKA. In contrast, the activation by isoproterenol of Ras requires Gbetagamma subunits, is independent of PKA, and results in the phosphoinositol 3-kinase-dependent activation of AKT. Interestingly, beta-adrenergic stimulation of both Rap1 and ERKs, but not Ras and AKT, can be blocked by a Src mutant (SrcS17A) that is incapable of being phosphorylated and activated by PKA. Furthermore, a Src mutant (SrcS17D), which mimics PKA phosphorylation at serine 17, stimulates Rap1 activation, Rap1/B-Raf association, and ERK activation but does not stimulate Ras or AKT. These data suggest that Rap1 activation, but not that of Ras, is mediated through the direct phosphorylation of Src by PKA. We propose that the beta(2)-adrenergic receptor activates Src via two independent mechanisms to mediate distinct signaling pathways, one through Galpha(s) to Rap1 and ERKs and the other through Gbetagamma to Ras and AKT.