Miguel Perez-Aso

Activation of adenosine A2A receptor reduces osteoclast formation via PKA- and ERK1/2-mediated suppression of NFκB nuclear translocation

We previously reported that adenosine, acting at adenosine A2A receptors (A2AR), inhibits osteoclast (OC) differentiation in vitro (A2AR activation OC formation reduces by half) and in vivo. For a better understanding how adenosine A2AR stimulation regulates OC differentiation, we dissected the signalling pathways involved in A2AR signalling.

Different β-adrenoceptor subtypes coupling to cAMP or NO/cGMP pathways: implications in the relaxant response of rat conductance and resistance vessels

To analyse the relative contribution of β1‐, β2‐ and β3‐adrenoceptors (Adrb) to vasodilatation in conductance and resistance vessels, assessing the role of cAMP and/or NO/cGMP signalling pathways.

Pharmacological blockade of adenosine A2A receptors diminishes scarring

Adenosine A2A receptor (A2AR) stimulation promotes wound healing and is required for the development of fibrosis in murine models of scleroderma and cirrhosis. Nonetheless, the role of A2AR in the formation of scars following skin trauma has not been explored. Here, we examined the effect of pharmacological blockade of A2AR, with the selective adenosine A2AR‐antagonist ZM241385 (2.5 mg/ml), in a murine model of scarring that mimics human scarring. We found that application of the selective adenosine A2AR antagonist ZM241385 decreased scar size and enhanced the tensile strength of the scar. Within the scar itself, collagen alignment and composition (marked reduction in collagen 3), but not periostin, biglycan, or fibronectin accumulation, was improved by application of ZM241385. Moreover, A2AR blockade reduced the number of myofibroblasts and angiogenesis but not macrophage infiltration in the scar. Taken together, our work strongly suggests that pharmacological A2AR blockade can be used to diminish scarring while improving the collagen composition and tensile strength of the healed wound.—Perez‐Aso, M., Chiriboga, L., Cronstein, B. N. Pharmacological blockade of adenosine A2A receptors diminishes scarring. FASEB J. 26, 4254–4263 (2012). www.fasebj.org

Adenosine 2A receptor promotes collagen production by human fibroblasts via pathways involving cyclic AMP and AKT but independent of Smad2/3

Activation of adenosine A2A receptor (A2AR) promotes fibrosis and collagen synthesis. However, the underlying mechanism is still unclear, not least because cAMP, its principal effector, has been found to inhibit TGFβ1‐induced collagen synthesis. Here, we show that in primary normal human dermal fibroblasts, A2AR stimulation with CGS21680 elicits a modest cAMP increase (150±12% of control; EC50 54.8 nM), which stimulates collagen1 (Col1) and collagen3 (Col3), but maximal cAMP resulting from direct activation of adenylyl cyclase by forskolin (15,689±7038% of control; EC50 360.7 nM) inhibits Col1 and increases Col3. Similar to Col1 expression, fibroblast proliferation increased following physiological cAMP increases by CGS21680 but was inhibited by cAMP increases beyond the physiological range by forskolin. The A2AR‐mediated increase of Col1 and Col3 was mediated by AKT, while Col3, but not Col1, expression was dependent on p38 and repressed by ERK. TGFβ1 induced phosphorylation of Smad2/3 and increased Col3 expression, which was prevented by Smad3 depletion. In contrast, CGS21680 did not activate Smad2/3, and Smad2/3 knockdown did not prevent CGS21680‐induced Col1 or Col3 increases. Our results indicate that cAMP is a concentration‐dependent switch for collagen production via noncanonical, AKT‐dependent, Smad2/3‐independent signaling. These observations explain the paradoxical effects of cAMP on collagen expression.—Perez‐Aso, M., Fernandez, P., Mediero, A., Chan, E. S., and Cronstein, B. N. Adenosine 2A receptor promotes collagen production by human fibroblasts via pathways involving cyclic AMP and AKT but independent of Smad2/3. FASEB J. 28, 802–812 (2014). www.fasebj.org

Direct or indirect stimulation of adenosine A2A receptors enhances bone regeneration as well as bone morphogenetic protein-2

Promoting bone regeneration and repair of bone defects is a need that has not been well met to date. We have previously found that adenosine, acting via receptors (A2AR) promotes wound healing and inhibits inflammatory osteolysis and hypothesized that A2AR might be a novel target to promote bone regeneration. Therefore, we determined whether direct A2AR stimulation or increasing endogenous adenosine concentrations via purine transport blockade with dipyridamole regulates bone formation. We determined whether coverage of a 3 mm trephine defect in a mouse skull with a collagen scaffold soaked in saline, bone morphogenetic protein‐2 (BMP‐2; 200 ng), 1 μM CGS21680 (A2AR agonist, EC50 = 160 nM), or 1 μM dipyridamole (EC50 = 32 nM) promoted bone regeneration. Microcomputed tomography examination demonstrated that CGS21680 and dipyridamole markedly enhanced bone regeneration as well as BMP‐2 8 wk after surgery (60 ± 2%, 79 ± 2%, and 75 ± 1% bone regeneration, respectively, vs. 32 ± 2% in control, P < 0.001). Blockade by a selective A2AR antagonist (ZM241385, 1 μM) or deletion of A2AR abrogated the effect of CGS21680 and dipyridamole on bone regeneration. Both CGS21680 and dipyridamole treatment increased alkaline phosphatase‐positive osteoblasts and diminished tartrate resistance acid phosphatase‐positive osteoclasts in the defects. In vivo imaging with a fluorescent dye for new bone formation revealed a strong fluorescent signal in treated animals that was equivalent to BMP‐2. In conclusion, stimulation of A2AR by specific agonists or by increasing endogenous adenosine levels stimulates new bone formation as well as BMP‐2 and represents a novel approach to stimulating bone regeneration.—Mediero, A., Wilder, T., Perez‐Aso, M., Cronstein, B. N. Direct or indirect stimulation of adenosine A2A receptors enhances bone regeneration as well as bone morphogenetic protein‐2. FASEB J. 29, 1577‐1590 (2015). www.fasebj.org

Adenosine A2A receptors promote collagen production by a Fli1- and CTGF-mediated mechanism

IntroductionAdenosine, acting through the A2A receptor, promotes tissue matrix production in the skin and the liver and induces the development of dermal fibrosis and cirrhosis in murine models. Since expression of A2A receptors is increased in scleroderma fibroblasts, we examined the mechanisms by which the A2A receptor produces its fibrogenic effects.MethodsThe effects of A2A receptor ligation on the expression of the transcription factor, Fli1, a constitutive repressor for the synthesis of matrix proteins, such as collagen, is studied in dermal fibroblasts. Fli1 is also known to repress the transcription of CTGF/CCN2, and the effects of A2A receptor stimulation on CTGF and TGF-β1 expression are also examined.ResultsA2A receptor occupancy suppresses the expression of Fli1 by dermal fibroblasts. A2A receptor activation induces the secretion of CTGF by dermal fibroblasts, and neutralization of CTGF abrogates the A2A receptor-mediated enhancement of collagen type I production. A2AR activation, however, resulted in a decrease in TGF-β1 protein release.ConclusionsOur results suggest that Fli1 and CTGF are important mediators of the fibrogenic actions of adenosine and the use of small molecules such as adenosine A2A receptor antagonists may be useful in the therapy of dermal fibrosis in diseases such as scleroderma.

Apremilast, a novel phosphodiesterase 4 (PDE4) inhibitor, regulates inflammation through multiple cAMP downstream effectors

IntroductionThis work was undertaken to delineate intracellular signaling pathways for the PDE4 inhibitor apremilast and to examine interactions between apremilast, methotrexate and adenosine A2A receptors (A2AR).MethodsAfter apremilast and LPS incubation, intracellular cAMP, TNF-α, IL-10, IL-6 and IL-1α were measured in the Raw264.7 monocytic murine cell line. PKA, Epac1/2 (signaling intermediates for cAMP) and A2AR knockdowns were performed by shRNA transfection and interactions with A2AR and A2BR, as well as with methotrexate were tested in vitro and in the murine air pouch model. Statistical differences were determined using one or two-way ANOVA or Student’s t test. The alpha nominal level was set at 0.05 in all cases. A P value of < 0.05 was considered significant.ResultsIn vitro, apremilast increased intracellular cAMP and inhibited TNF-α release (IC50=104nM) and the specific A2AR-agonist CGS21680 (1μM) increased apremilast potency (IC50=25nM). In this cell line, apremilast increased IL-10 production. PKA, Epac1 and Epac2 knockdowns prevented TNF-α inhibition and IL-10 stimulation by apremilast. In the murine air pouch model, both apremilast and MTX significantly inhibited leukocyte infiltration, while apremilast, but not MTX, significantly inhibited TNF-α release. The addition of MTX (1 mg/kg) to apremilast (5 mg/kg) yielded no more inhibition of leukocyte infiltration or TNF-α release than with apremilast alone.ConclusionsThe immunoregulatory effects of apremilast appear to be mediated by cAMP through the downstream effectors PKA, Epac1, and Epac2. A2AR agonism potentiated TNF-α inhibition by apremilast, consistent with the cAMP-elevating effects of that receptor. Because the A2AR is also involved in the anti-inflammatory effects of MTX, the mechanism of action of both drugs involves cAMP-dependent pathways and is therefore partially overlapping in nature.

The three α1-adrenoceptor subtypes show different spatio-temporal mechanisms of internalization and ERK1/2 phosphorylation.

We analyzed the kinetic and spatial patterns characterizing activation of the MAP kinases ERK 1 and 2 (ERK1/2) by the three α1-adrenoceptor (α1-AR) subtypes in HEK293 cells and the contribution of two different pathways to ERK1/2 phosphorylation: protein kinase C (PKC)-dependent ERK1/2 activation and internalization-dependent ERK1/2 activation. The different pathways of phenylephrine induced ERK phosphorylation were determined by western blot, using the PKC inhibitor Ro 31-8425, the receptor internalization inhibitor concanavalin A and the siRNA targeting β-arrestin 2. Receptor internalization properties were studied using CypHer5 technology and VSV-G epitope-tagged receptors. Activation of α1A- and α1B-ARs by phenylephrine elicited rapid ERK1/2 phosphorylation that was directed to the nucleus and inhibited by Ro 31-8425. Concomitant with phenylephrine induced receptor internalization α1A-AR, but not α1B-AR, produced a maintained and PKC-independent ERK phosphorylation, which was restricted to the cytosol and inhibited by β-arrestin 2 knockdown or concanavalin A treatment. α1D-AR displayed constitutive ERK phosphorylation, which was reduced by incubation with prazosin or the selective α1D antagonist BMY7378. Following activation by phenylephrine, α1D-AR elicited rapid, transient ERK1/2 phosphorylation that was restricted to the cytosol and not inhibited by Ro 31-8425. Internalization of the α1D-AR subtype was not observed via CypHer5 technology. The three α1-AR subtypes present different spatio-temporal patterns of receptor internalization, and only α1A-AR stimulation translates to a late, sustained ERK1/2 phosphorylation that is restricted to the cytosol and dependent on β-arrestin 2 mediated internalization.

Adenosine A2A Receptor and TNF-α Regulate the Circadian Machinery of the Human Monocytic THP-1 Cells

Morning stiffness and increased symptoms of inflammatory arthritis are among the most common manifestations of rheumatoid arthritis (RA). Tumor necrosis alpha (TNF-α), an important mediator of inflammation in RA, regulates the circadian expression of clock proteins, and adenosine A2A receptors (A2AR) mediate many of the anti-inflammatory and antirheumatic actions of methotrexate, the cornerstone drug in the treatment of RA. We found that A2AR activation and TNF-α activated the clock core loop of the human monocytic THP-1 cell line. We further observed that interleukin (IL)-10, but not IL-12, mRNA expression fluctuates in a circadian fashion and that TNF-α and A2AR stimulation combined increased IL-10 expression. Interestingly, TNF-α, but not CGS21680, dramatically inhibited IL-12 mRNA expression. The demonstration that A2AR and TNF-α regulate the intrinsic circadian clock in immune cells provides an explanation for both the pathologic changes in circadian rhythms in RA and for the adverse circadian effects of methotrexate, such as fatigue.

Activation of EPAC1/2 is essential for osteoclast formation by modulating NFκB nuclear translocation and actin cytoskeleton rearrangements

Bisphosphonates inhibit osteoclast differentiation/function via inhibition of Rap1A isoprenylation. As Rap1 is the effector of exchange protein directly activated by cAMP (EPAC) proteins, we determined the role of EPAC in osteoclast differentiation. We examined osteoclast differentiation as the number of primary murine/human bone‐marrow precursors that differentiated into multinucleated TRAP‐positive cells in the presence of EPAC‐selective stimulus (8‐pCTP‐2‘‐O‐Me‐cAMP, 100 μM; 8‐pCTP‐2‘‐O‐Me‐cAMP‐AM, 1 μM) or inhibitor brefeldin A (BFA), ESI‐05, and ESI‐09 (10 μM each). Rap1 activity was assessed, and signaling events, as well as differentiation in EPAC1/2‐knockdown RAW264.7 cells, were studied. Direct EPAC1/2 stimulation significantly increased osteoclast differentiation, whereas EPAC1/2 inhibition diminished differentiation (113±6%, P<0.05, and 42±10%, P<0.001, of basal, respectively). Rap1 activation was maximal 15 min after RANKL stimulation (147±9% of basal, P< 0.001), whereas silencing of EPAC1/2 diminished activated Rap1 (43±13 and 20±15% of control, P<0.001) and NFκB nuclear translocation. TRAP‐staining revealed no osteoclast differentiation in EPAC1/2‐KO cells. Cathepsin K, NFATc1, and osteopontin mRNA expression decreased in EPAC1/2‐KO cells when compared to control. RhoA, cdc42, Rac1, and FAK were activated in an EPAC1/2‐dependent manner, and there was diminished cytoskeletal assembly in EPAC1/2‐KO cells. In summary, EPAC1 and EPAC2 are critical signaling intermediates in osteoclast differentiation that permit RANKL‐stimulated NFκB nuclear translocation and actin rearrangements. Targeting this signaling intermediate may diminish bone destruction in inflammatory arthritis.—Mediero, A., Perez‐Aso, M., Cronstein, B. N., Activation of EPAC1/2 is essential for osteoclast formation by modulating NFκB nuclear translocation and actin cytoskeleton rearrangements. FASEB J. 28, 4901–4913 (2014). www.fasebj.org