A novel BRET-Based GAP assay reveals phosphorylation-dependent regulation of the RAC-specific GTPase activating protein ARHGAP25.

ARHGAP25, a RAC-specific GTPase activating protein (GAP), is an essential regulator of phagocyte effector functions such as phagocytosis, superoxide production, and transendothelial migration. Furthermore, its complex role in tumor behavior has recently been recognized. We previously demonstrated that phosphorylation of serine 363 in ARHGAP25 regulates hematopoietic stem cells and progenitor cells in mouse bone marrow. However, the significance of other potential phosphorylation sites of ARHGAP25 remained unknown. Now, we developed a novel, real-time bioluminescence resonance energy transfer (BRET) assay to monitor the GAP activity of ARHGAP25 in vitro. Using this approach, we revealed that phosphorylation of S363 and S488, but not that of S379-380, controls ARHGAP25's RACGAP activity. On the other hand, we found in granulocyte-differentiated human PLB-985 cells that superoxide production and actin depolymerization are regulated by residues S363 and S379-380. The present data demonstrate the value of our BRET-GAP assay and show that different phosphorylation patterns regulate ARHGAP25's GAP activity and its effect on superoxide production and phagocytosis.

Mitochondrial cAMP and Ca2+ metabolism in adrenocortical cells.

The biological effects of physiological stimuli of adrenocortical glomerulosa cells are predominantly mediated by the Ca2+ and the cAMP signal transduction pathways. The complex interplay between these signalling systems fine-tunes aldosterone secretion. In addition to the well-known cytosolic interactions, a novel intramitochondrial Ca2+-cAMP interplay has been recently recognised. The cytosolic Ca2+ signal is rapidly transferred into the mitochondrial matrix where it activates Ca2+-sensitive dehydrogenases, thus enhancing the formation of NADPH, a cofactor of steroid synthesis. Quite a few cell types, including H295R adrenocortical cells, express the soluble adenylyl cyclase within the mitochondria and the elevation of mitochondrial [Ca2+] activates the enzyme, thus resulting in the Ca2+-dependent formation of cAMP within the mitochondrial matrix. On the other hand, mitochondrial cAMP (mt-cAMP) potentiates the transfer of cytosolic Ca2+ into the mitochondrial matrix. This cAMP-mediated positive feedback control of mitochondrial Ca2+ uptake may facilitate the rapid hormonal response to emergency situations since knockdown of soluble adenylyl cyclase attenuates aldosterone production whereas overexpression of the enzyme facilitates steroidogenesis in vitro. Moreover, the mitochondrial Ca2+-mt-cAMP-Ca2+ uptake feedback loop is not a unique feature of adrenocortical cells; a similar signalling system has been described in HeLa cells as well.

Mitochondrial cAMP exerts positive feedback on mitochondrial Ca2+ uptake via the recruitment of Epac1.

We have previously demonstrated in H295R adrenocortical cells that the Ca2+-dependent production of mitochondrial cAMP (mt-cAMP) by the matrix soluble adenylyl cyclase (sAC; encoded by ADCY10) is associated with enhanced aldosterone production. Here, we examined whether mitochondrial sAC and mt-cAMP fine tune mitochondrial Ca2+ metabolism to support steroidogenesis. Reduction of mt-cAMP formation resulted in decelerated mitochondrial Ca2+ accumulation in intact cells during K+-induced Ca2+ signalling and also in permeabilized cells exposed to elevated perimitochondrial [Ca2+]. By contrast, treatment with the membrane-permeable cAMP analogue 8-Br-cAMP, inhibition of phosphodiesterase 2 and overexpression of sAC in the mitochondrial matrix all intensified Ca2+ uptake into the organelle. Identical mt-cAMP dependence of mitochondrial Ca2+ uptake was also observed in HeLa cells. Importantly, the enhancing effect of mt-cAMP on Ca2+ uptake was independent from both the mitochondrial membrane potential and Ca2+ efflux, but was reduced by Epac1 (also known as RAPGEF3) blockade both in intact and in permeabilized cells. Finally, overexpression of sAC in the mitochondrial matrix potentiated aldosterone production implying that the observed positive feedback mechanism of mt-cAMP on mitochondrial Ca2+ accumulation may have a role in the rapid initiation of steroidogenesis.This article has an associated First Person interview with the first author of the paper.

Developmental Role of Macrophage Cannabinoid-1 Receptor Signaling in Type 2 Diabetes.

Islet inflammation promotes ß-cell loss and type 2 diabetes (T2D), a process replicated in Zucker Diabetic Fatty (ZDF) rats in which ß-cell loss has been linked to cannabinoid-1 receptor (CB1R)-induced proinflammatory signaling in macrophages infiltrating pancreatic islets. Here, we analyzed CB1R signaling in macrophages and its developmental role in T2D. ZDF rats with global deletion of CB1R are protected from ß-cell loss, hyperglycemia, and nephropathy that are present in ZDF littermates. Adoptive transfer of CB1R-/- bone marrow to ZDF rats also prevents ß-cell loss and hyperglycemia but not nephropathy. ZDF islets contain elevated levels of CB1R, interleukin-1ß, tumor necrosis factor-α, the chemokine CCL2, and interferon regulatory factor-5 (IRF5), a marker of inflammatory macrophage polarization. In primary cultured rodent and human macrophages, CB1R activation increased Irf5 expression, whereas knockdown of Irf5 blunted CB1R-induced secretion of inflammatory cytokines without affecting CCL2 expression, which was p38MAPKα dependent. Macrophage-specific in vivo knockdown of Irf5 protected ZDF rats from ß-cell loss and hyperglycemia. Thus, IRF5 is a crucial downstream mediator of diabetogenic CB1R signaling in macrophages and a potential therapeutic target.

Hybrid inhibitor of peripheral cannabinoid-1 receptors and inducible nitric oxide synthase mitigates liver fibrosis.

Liver fibrosis, a consequence of chronic liver injury and a way station to cirrhosis and hepatocellular carcinoma, lacks effective treatment. Endocannabinoids acting via cannabinoid-1 receptors (CB1R) induce profibrotic gene expression and promote pathologies that predispose to liver fibrosis. CB1R antagonists produce opposite effects, but their therapeutic development was halted due to neuropsychiatric side effects. Inducible nitric oxide synthase (iNOS) also promotes liver fibrosis and its underlying pathologies, but iNOS inhibitors tested to date showed limited therapeutic efficacy in inflammatory diseases. Here, we introduce a peripherally restricted, orally bioavailable CB1R antagonist, which accumulates in liver to release an iNOS inhibitory leaving group. In mouse models of fibrosis induced by CCl4 or bile duct ligation, the hybrid CB1R/iNOS antagonist surpassed the antifibrotic efficacy of the CB1R antagonist rimonabant or the iNOS inhibitor 1400W, without inducing anxiety-like behaviors or CB1R occupancy in the CNS. The hybrid inhibitor also targeted CB1R-independent, iNOS-mediated profibrotic pathways, including increased PDGF, Nlrp3/Asc3, and integrin αvß6 signaling, as judged by its ability to inhibit these pathways in cnr1-/- but not in nos2-/- mice. Additionally, it was able to slow fibrosis progression and to attenuate established fibrosis. Thus, dual-target peripheral CB1R/iNOS antagonists have therapeutic potential in liver fibrosis.

Extramitochondrial OPA1 and adrenocortical function.

We have previously described that silencing of the mitochondrial protein OPA1 enhances mitochondrial Ca(2+) signaling and aldosterone production in H295R adrenocortical cells. Since extramitochondrial OPA1 (emOPA1) was reported to facilitate cAMP-induced lipolysis, we hypothesized that emOPA1, via the enhanced hydrolysis of cholesterol esters, augments aldosterone production in H295R cells. A few OPA1 immunopositive spots were detected in ∼40% of the cells. In cell fractionation studies OPA1/COX IV (mitochondrial marker) ratio in the post-mitochondrial fractions was an order of magnitude higher than that in the mitochondrial fraction. The ratio of long to short OPA1 isoforms was lower in post-mitochondrial than in mitochondrial fractions. Knockdown of OPA1 failed to reduce db-cAMP-induced phosphorylation of hormone-sensitive lipase (HSL), Ca(2+) signaling and aldosterone secretion. In conclusion, OPA1 could be detected in the post-mitochondrial fractions, nevertheless, OPA1 did not interfere with the cAMP - PKA - HSL mediated activation of aldosterone secretion.

Activation of the Nlrp3 inflammasome in infiltrating macrophages by endocannabinoids mediates beta cell loss in type 2 diabetes.

Type 2 diabetes mellitus (T2DM) progresses from compensated insulin resistance to beta cell failure resulting in uncompensated hyperglycemia, a process replicated in the Zucker diabetic fatty (ZDF) rat. The Nlrp3 inflammasome has been implicated in obesity-induced insulin resistance and beta cell failure. Endocannabinoids contribute to insulin resistance through activation of peripheral CB1 receptors (CB1Rs) and also promote beta cell failure. Here we show that beta cell failure in adult ZDF rats is not associated with CB1R signaling in beta cells, but rather in M1 macrophages infiltrating into pancreatic islets, and that this leads to activation of the Nlrp3-ASC inflammasome in the macrophages. These effects are replicated in vitro by incubating wild-type human or rodent macrophages, but not macrophages from CB1R-deficient (Cnr1(-/-)) or Nlrp3(-/-) mice, with the endocannabinoid anandamide. Peripheral CB1R blockade, in vivo depletion of macrophages or macrophage-specific knockdown of CB1R reverses or prevents these changes and restores normoglycemia and glucose-induced insulin secretion. These findings implicate endocannabinoids and inflammasome activation in beta cell failure and identify macrophage-expressed CB1R as a therapeutic target in T2DM.

Mitochondrial Ca2+ uptake is inhibited by a concerted action of p38 MAPK and protein kinase D.

Angiotensin II elicits cytosolic Ca2+ signal that is transferred into the mitochondria. Previously we found in H295R cells that this signal transfer is enhanced by both the inhibition of p38 MAPK and a novel isoform of PKC [G. Szanda, P. Koncz, A. Rajki, A. Spät, Participation of p38 MAPK and a novel-type protein kinase C in the control of mitochondrial Ca2+ uptake, Cell Calcium 43 (2008) 250-259]. Now we report that simultaneous activation of these protein kinases (by TNFalpha and PMA+an inhibitor of the conventional PKC isoforms, respectively) attenuates the transfer of cytosolic Ca2+ signal, elicited by depolarisation or store-operated Ca2+ influx, into the mitochondria. The Ca2+ uptake enhancing effect of the p38 MAPK inhibitor SB202190 is due to the inhibition of p38 MAPK and not to a direct mitochondrial action. Protein kinases reduce mitochondrial [Ca2+] by inhibiting the uptake mechanism. The threshold of mitochondrial Ca2+ uptake may depend on the activity of p38 MAPK. The silencing of protein kinase D (PKD) also results in enhanced transfer of Ca2+ signal from the cytosol into the mitochondria. Our data indicate that Ca2+ mobilising agonists, through the simultaneous activation of p38 MAPK, a novel PKC isoform and PKD, exert a negative feed-forward action on mitochondrial Ca2+ uptake, thus reducing the risk of Ca2+ overload.

Effect of cytosolic Mg2+ on mitochondrial Ca2+ signaling.

Cytosolic Ca2+ signals are followed by mitochondrial Ca2+ uptake, which, in turn, modifies several biological processes. Mg2+ is known to inhibit Ca2+ uptake by isolated mitochondria, but its significance in intact cells has not been elucidated. In HEK293T cells, activation of purinergic receptors with extracellular ATP caused cytosolic Ca2+ signals associated with parallel changes in cytosolic [Mg2+]. Neither signals were affected by omitting bivalent cations from the extracellular medium. The effect of store-operated Ca2+ influx on cytosolic Mg2+ concentration ([Mg2+]c) was negligible. Uncaged Ca2+ displaced Mg2+ from cytosolic binding sites, but for an equivalent Ca2+ signal, the change in [Mg2+] was significantly smaller than that measured after adding extracellular ATP. Inositol 1,4,5-trisphosphate mobilized Ca2+ and Mg2+ from internal stores in permeabilized cells. The increase of [Mg2+] in the range that occurred in ATP-stimulated cells inhibited mitochondrial Ca2+ uptake in permeabilized cells without affecting mitochondrial Ca2+ efflux. Therefore, the Mg2+ signal generated by Ca2+ mobilizing agonists may attenuate mitochondrial Ca2+ uptake.

Participation of p38 MAPK and a novel-type protein kinase C in the control of mitochondrial Ca2+ uptake.

Angiotensin II elicits cytosolic and mitochondrial Ca2+ signal in H295R adrenocortical cells. We found that Ca2+ uptake rate and peak values in small mitochondrial regions both depend on the colocalization of these mitochondrial regions with GFP-marked endoplasmic reticular (ER) vesicles. The dependence of the Ca2+ response on this colocalization is abolished by SB202190 and PD169316, inhibitors of p38 MAPK, as well as by transfection with siRNA against p38 MAPK mRNA. The same manoeuvres result in an increased ratio of global mitochondrial to global cytosolic Ca2+ response, indicating that inhibition of p38 MAPK is followed by enhanced mitochondrial Ca2+ uptake. alpha-Toxin and TNFalpha, agents which similarly to angiotensin II increase the phosphorylation of p38, failed to affect mitochondrial Ca2+ uptake, indicating that activation of p38 MAPK is necessary but not sufficient for the inhibition of Ca2+ uptake. Bisindolylmaleimide, an inhibitor of the conventional and novel-type protein kinase C isoforms also evokes enhanced mitochondrial Ca2+ uptake, whereas Gö6976 that inhibits the conventional isoforms only failed to exert any effect. These data show that angiotensin II attenuates Ca2+ uptake predominantly into mitochondria that do not colocalize with ER, by a mechanism involving p38 MAPK and a novel-type PKC.

Mitochondrial Ca2+ uptake with and without the formation of high-Ca2+ microdomains.

The mitochondrial Ca(2+) uniporter has low affinity for Ca(2+), therefore it has been assumed that submicromolar Ca(2+) signals cannot induce mitochondrial Ca(2+) uptake. The close apposition of the plasma membrane or the endoplamic reticulum (ER) to the mitochondria and the limited Ca(2+) diffusion in the cytoplasm result in the formation of perimitochondrial high-Ca(2+) microdomains (HCMDs) capable of activating mitochondrial Ca(2+) uptake. The possibility of mitochondrial Ca(2+) uptake at low submicromolar [Ca(2+)](c) has not yet been generally accepted. Earlier we found in permeabilized glomerulosa, luteal and pancreatic beta cells that [Ca(2+)](m) increased when [Ca(2+)](c) was raised from 60 nM to less than 200 nM. Here we report data obtained from H295R (adrenocortical) cells transfected with ER-targeted GFP. Cytoplasmic Ca(2+) response to angiotensin II was different in mitochondrion-rich and mitochondrion-free domains. The mitochondrial Ca(2+) response to angiotensin II correlated with GFP fluorescence indicating the vicinity of ER. When the cells were exposed to K(+) (inducing Ca(2+) influx), no correlation was found between the mitochondrial Ca(2+) signal and the vicinity of the plasma membrane or the ER. The results presented here provide evidence that mitochondrial Ca(2+) uptake may occur both with and without the formation of HCMDs within the same cell.

Prostaglandin F2alpha potentiates the calcium dependent activation of mitochondrial metabolism in luteal cells.

Cytoplasmic Ca2+ signals are transferred to the mitochondria and activate the Krebs cycle. We have compared the efficiency of this process for two Ca2+ mobilising agonists, PGF2alpha and ATP (acting at metabotropic P2 receptors) in rat luteal cells. [Ca2+]c, [Ca2+]m and mitochondrial NAD(P)H were monitored by means of microspectrofluorimetry and confocal microscopy. While both agonists caused similar elevations of [Ca2+]c, changes in NAD(P)H were larger in response to PGF2alpha than to ATP. PGF2alpha more effectively increased NAD(P)H level also in mouse luteal cells. PGF2alpha caused a faster rate of rise of NAD(P)H fluorescence than ATP when reoxidation was prevented with rotenone, suggesting a faster rate of NAD(P)+ reduction. The NAD(P)H response to both agonists was dependent on the mobilisation of stored Ca2+. We found no difference in the efficacy of transmission of the [Ca2+]c signal to mitochondria in response to PGF2alpha and ATP. Raising [Ca2+]c with ionomycin increased the NAD(P)H signal, which was further raised by PGF2alpha but not by ATP. These data suggest that PGF2alpha potentiates the Ca2+-induced stimulation of mitochondrial metabolism by a Ca2+-independent mechanism and shows that agonists may modulate mitochondrial function differentially through a novel process beyond the simple transfer of Ca2+ from ER to mitochondria.