cAMP guided his way: a life for G protein-mediated signal transduction and molecular pharmacology-tribute to Karl H. Jakobs.

Karl H. Jakobs, former editor-in-chief of Naunyn-Schmiedeberg's Archives of Pharmacology and renowned molecular pharmacologist, passed away in April 2018. In this article, his scientific achievements regarding G protein-mediated signal transduction and regulation of canonical pathways are summarized. Particularly, the discovery of inhibitory G proteins for adenylyl cyclase, methods for the analysis of receptor-G protein interactions, GTP supply by nucleoside diphosphate kinases, mechanisms in phospholipase C and phospholipase D activity regulation, as well as the development of the concept of sphingosine-1-phosphate as extra- and intracellular messenger will presented. His seminal scientific and methodological contributions are put in a general and timely perspective to display and honor his outstanding input to the current knowledge in molecular pharmacology.

Sphingosine kinase 2 is a negative regulator of inflammatory macrophage activation.

Sphingosine kinases (SPHK) generate the sphingolipid sphingosine-1-phosphate, which, among other functions, is a potent regulator of inflammation. While SPHK1 produces S1P to promote inflammatory signaling, the role of SPHK2 is unclear due to divergent findings in studies utilizing gene depletion versus inhibition of catalytic activity. We sought to clarify how SPHK2 affects inflammatory signaling in human macrophages, which are main regulators of inflammation. SPHK2 expression and activity were rapidly decreased within 6 h upon stimulating primary human macrophages with lipopolysaccharide (LPS), but was upregulated after 24 h. At 24 h following LPS stimulation, targeting SPHK2 with the inhibitor ABC294640, a specific siRNA or by using Sphk2-/- mouse peritoneal macrophages increased inflammatory cytokine production. Downregulation of SPHK2 in primary human macrophages within 6 h of LPS treatment was blocked by inhibiting autophagy. SPHK2 overexpression or inhibiting autophagy 6 h after human macrophage activation with LPS suppressed inflammatory cytokine release. Mechanistically, SPHK2 suppressed LPS-triggered NF-κB activation independent of its catalytic activity and prevented increased mitochondrial ROS formation downstream of LPS. In conclusion, SPHK2 is an anti-inflammatory protein in human macrophages that is inversely coupled to inflammatory cytokine production. This needs consideration when targeting SPHK2 with specific inhibitors.

Activation of Adenylyl Cyclase Causes Stimulation of Adenosine Receptors.

BACKGROUND/AIMS: Signaling of Gs protein-coupled receptors (GsPCRs) is accomplished by stimulation of adenylyl cyclase, causing an increase of the intracellular cAMP concentration, activation of the intracellular cAMP effectors protein kinase A (PKA) and Epac, and an efflux of cAMP, the function of which is still unclear. METHODS: Activation of adenylyl cyclase by GsPCR agonists or cholera toxin was monitored by measurement of the intracellular cAMP concentration by ELISA, anti-phospho-PKA substrate motif phosphorylation by immunoblotting, and an Epac-FRET assay in the presence and absence of adenosine receptor antagonists or ecto-nucleotide phosphodiesterase/pyrophosphatase2 (eNPP2) inhibitors. The production of AMP from cAMP by recombinant eNPP2 was measured by HPLC. Extracellular adenosine was determined by LC-MS/MS, extracellular ATP by luciferase and LC-MS/MS. The expression of eNPP isoenzymes 1-3 was examined by RT-PCR. The expression of multidrug resistance protein 4 was suppressed by siRNA. RESULTS: Here we show that the activation of GsPCRs and the GsPCRs-independent activation of Gs proteins and adenylyl cyclase by cholera toxin induce stimulation of cell surface adenosine receptors (A2A or A2B adenosine receptors). In PC12 cells stimulation of adenylyl cyclase by GsPCR or cholera toxin caused activation of A2A adenosine receptors by an autocrine signaling pathway involving cAMP efflux through multidrug resistance protein 4 and hydrolysis of released cAMP to AMP by eNPP2. In contrast, in PC3 cells cholera toxin- and GsPCR-induced stimulation of adenylyl cyclase resulted in the activation of A2B adenosine receptors. CONCLUSION: Our findings show that stimulation of adenylyl cyclase causes a remarkable activation of cell surface adenosine receptors.

Long noncoding RNA LISPR1 is required for S1P signaling and endothelial cell function.

Sphingosine-1-Phosphate (S1P) is a potent signaling lipid. The effects of S1P are mediated by the five S1P receptors (S1PR). In the endothelium S1PR1 is the predominant receptor and thus S1PR1 abundance limits S1P signaling. Recently, lncRNAs were identified as a novel class of molecules regulating gene expression. Interestingly, the lncRNA NONHSAT004848 (LISPR1, Long intergenic noncoding RNA antisense to S1PR1), is closely positioned to the S1P1 receptors gene and in part shares its promoter region. We hypothesize that LISPR1 controls endothelial S1PR1 expression and thus S1P-induced signaling in endothelial cells. In vitro transcription and translation as well as coding potential assessment showed that LISPR1 is indeed noncoding. LISPR1 was localized in both cytoplasm and nucleus and harbored a PolyA tail at the 3'end. In human umbilical vein endothelial cells, as well as human lung tissue, qRT-PCR and RNA-Seq revealed high expression of LISPR1. S1PR1 and LISPR1 were downregulated in human pulmonary diseases such as COPD. LISPR1 but also S1PR1 were induced by inflammation, shear stress and statins. Knockdown of LISPR1 attenuated endothelial S1P-induced migration and spheroid outgrowth of endothelial cells. LISPR1 knockdown decreased S1PR1 expression, which was paralleled by an increase of the binding of the transcriptional repressor ZNF354C to the S1PR1 promoter and a reduction of the recruitment of RNA Polymerase II to the S1PR1 5'end. This resulted in attenuated S1PR1 expression and attenuated S1P downstream signaling. Collectively, the disease relevant lncRNA LISPR1 acts as a novel regulatory unit important for S1PR1 expression and endothelial cell function.

Mitochondrial fragmentation in human macrophages attenuates palmitate-induced inflammatory responses.

Macrophages in adipose tissue contribute to inflammation and the development of insulin resistance in obesity. Exposure of macrophages to saturated fatty acids alters cell metabolism and activates pro-inflammatory signaling. How fatty acids influence macrophage mitochondrial dynamics is unclear. We investigated the mechanism of palmitate-induced mitochondrial fragmentation and its impact on inflammatory responses in primary human macrophages. Fatty acids, such as palmitate, caused mitochondrial fragmentation in human macrophages. Increased mitochondrial fragmentation was also observed in peritoneal macrophages from hyperlipidemic apolipoprotein E knockout mice. Fatty acid-induced mitochondrial fragmentation was independent of the fatty acid chain saturation and required dynamin-related protein 1 (DRP1). Mechanistically, mitochondrial fragmentation was regulated by incorporation of palmitate into mitochondrial phospholipids and their precursors. Palmitate-induced endoplasmic reticulum stress and loss of mitochondrial membrane potential did not contribute to mitochondrial fragmentation. Macrophages treated with palmitate maintained intact mitochondrial respiration and ATP levels. Pharmacological or genetic inhibition of DRP1 enhanced palmitate-induced mitochondrial ROS production, c-Jun phosphorylation, and inflammatory cytokine expression. Our results indicate that mitochondrial fragmentation is a protective mechanism attenuating inflammatory responses induced by palmitate in human macrophages.

The activation of RhoC in vascular endothelial cells is required for the S1P receptor type 2-induced inhibition of angiogenesis.

Sphingosine-1-phosphate (S1P) is a multifunctional phospholipid inducing a variety of cellular responses in endothelial cells (EC). S1P responses are mediated by five G protein coupled receptors of which three types (S1P1R-S1P3R) have been described to be of importance in vascular endothelial cells (EC). Whereas the S1P1R regulates endothelial barrier function by coupling to Gαi and the monomeric GTPase Rac1, the signaling pathways involved in the S1P-induced regulation of angiogenesis are ill defined. We therefore studied the sprouting of human umbilical vein EC (HUVEC) in vitro and analyzed the activation of the RhoGTPases RhoA and RhoC. Physiological relevant concentrations of S1P (100-300nM) induce a moderate activation of RhoA and RhoC. Inhibition or siRNA-mediated depletion of the S1P2R preferentially decreased the activation of RhoC. Both manipulations caused an increase of sprouting in a spheroid based in vitro sprouting assay. Interestingly, a similar increase in sprouting was detected after effective siRNA-mediated knockdown of RhoC. In contrast, the depletion of RhoA had no influence on sprouting. Furthermore, suppression of the activity of G proteins of the Gα12/13 subfamily by adenoviral overexpression of the regulator of G protein signaling domain of LSC as well as siRNA-mediated knockdown of the Rho specific guanine nucleotide exchange factor leukemia associated RhoGEF (LARG) inhibited the S1P-induced activation of RhoC and concomitantly increased sprouting of HUVEC with similar efficacy. We conclude that the angiogenic sprouting of EC is suppressed via the S1P2R subtype. Thus, the increase in basal sprouting can be attributed to blocking of the inhibitory action of autocrine S1P stimulating the S1P2R. This inhibitory pathway involves the activation of RhoC via Gα12/13 and LARG, while the simultaneously occurring activation of RhoA is apparently dispensable here.

Glucocorticoids protect renal mesangial cells from apoptosis by increasing cellular sphingosine-1-phosphate.

Neutral ceramidase (NCDase) and sphingosine kinases (SphKs) are key enzymes regulating cellular sphingosine-1-phosphate (S1P) levels. In this study we found that stress factor-induced apoptosis of rat renal mesangial cells was significantly reduced by dexamethasone treatment. Concomitantly, dexamethasone increased cellular S1P levels, suggesting an activation of sphingolipid-metabolizing enzymes. The cell-protective effect of glucocorticoids was reversed by a SphK inhibitor, was completely absent in SphK1-deficient cells, and was associated with upregulated mRNA and protein expression of NCDase and SphK1. Additionally, in vivo experiments in mice showed that dexamethasone also upregulated SphK1 mRNA and activity, and NCDase protein expression in the kidney. Fragments (2285, 1724, and 1126 bp) of the rat NCDase promoter linked to a luciferase reporter were transfected into rat kidney fibroblasts and mesangial cells. There was enhanced NCDase promoter activity upon glucocorticoids treatment that was abolished by the glucocorticoid receptor antagonist RU-486. Single and double mutations of the two putative glucocorticoid response element sites within the promoter reduced the dexamethasone effect, suggesting that both glucocorticoid response elements are functionally active and required for induction. Our study shows that glucocorticoids exert a protective effect on stress-induced mesangial cell apoptosis in vitro and in vivo by upregulating NCDase and SphK1 expression and activity, resulting in enhanced levels of the protective lipid second messenger S1P.