Sphingosine Kinases at the Intersection of Pro-Inflammatory LPS and Anti-Inflammatory Endocannabinoid Signaling in BV2 Mouse Microglia Cells.

Microglia, the resident immune cells of the central nervous system, play important roles in brain homeostasis as well as in neuroinflammation, neurodegeneration, neurovascular diseases, and traumatic brain injury. In this context, components of the endocannabinoid (eCB) system have been shown to shift microglia towards an anti-inflammatory activation state. Instead, much less is known about the functional role of the sphingosine kinase (SphK)/sphingosine-1-phosphate (S1P) system in microglia biology. In the present study, we addressed potential crosstalk of the eCB and the S1P systems in BV2 mouse microglia cells challenged with lipopolysaccharide (LPS). We show that URB597, the selective inhibitor of fatty acid amide hydrolase (FAAH)-the main degradative enzyme of the eCB anandamide-prevented LPS-induced production of tumor necrosis factor-α (TNFα) and interleukin-1ß (IL-1ß), and caused the accumulation of anandamide itself and eCB-like molecules such as oleic acid and cis-vaccenic acid ethanolamide, palmitoylethanolamide, and docosahexaenoyl ethanolamide. Furthermore, treatment with JWH133, a selective agonist of the eCB-binding cannabinoid 2 (CB2) receptor, mimicked the anti-inflammatory effects of URB597. Interestingly, LPS induced transcription of both SphK1 and SphK2, and the selective inhibitors of SphK1 (SLP7111228) and SphK2 (SLM6031434) strongly reduced LPS-induced TNFα and IL-1ß production. Thus, the two SphKs were pro-inflammatory in BV2 cells in a non-redundant manner. Most importantly, the inhibition of FAAH by URB597, as well as the activation of CB2 by JWH133, prevented LPS-stimulated transcription of SphK1 and SphK2. These results present SphK1 and SphK2 at the intersection of pro-inflammatory LPS and anti-inflammatory eCB signaling, and suggest the further development of inhibitors of FAAH or SphKs for the treatment of neuroinflammatory diseases.

Rare Phytocannabinoids Exert Anti-Inflammatory Effects on Human Keratinocytes via the Endocannabinoid System and MAPK Signaling Pathway.

Increasing evidence supports the therapeutic potential of rare cannabis-derived phytocannabinoids (pCBs) in skin disorders such as atopic dermatitis, psoriasis, pruritus, and acne. However, the molecular mechanisms of the biological action of these pCBs remain poorly investigated. In this study, an experimental model of inflamed human keratinocytes (HaCaT cells) was set up by using lipopolysaccharide (LPS) in order to investigate the anti-inflammatory effects of the rare pCBs cannabigerol (CBG), cannabichromene (CBC), Δ9-tetrahydrocannabivarin (THCV) and cannabigerolic acid (CBGA). To this aim, pro-inflammatory interleukins (IL)-1ß, IL-8, IL-12, IL-31, tumor necrosis factor (TNF-ß) and anti-inflammatory IL-10 levels were measured through ELISA quantification. In addition, IL-12 and IL-31 levels were measured after treatment of HaCaT cells with THCV and CBGA in the presence of selected modulators of endocannabinoid (eCB) signaling. In the latter cells, the activation of 17 distinct proteins along the mitogen-activated protein kinase (MAPK) pathway was also investigated via Human Phosphorylation Array. Our results demonstrate that rare pCBs significantly blocked inflammation by reducing the release of all pro-inflammatory ILs tested, except for TNF-ß. Moreover, the reduction of IL-31 expression by THCV and CBGA was significantly reverted by blocking the eCB-binding TRPV1 receptor and by inhibiting the eCB-hydrolase MAGL. Remarkably, THCV and CBGA modulated the expression of the phosphorylated forms (and hence of the activity) of the MAPK-related proteins GSK3ß, MEK1, MKK6 and CREB also by engaging eCB hydrolases MAGL and FAAH. Taken together, the ability of rare pCBs to exert an anti-inflammatory effect in human keratinocytes through modifications of eCB and MAPK signaling opens new perspectives for the treatment of inflammation-related skin pathologies.

The TRPV1 Receptor Is Up-Regulated by Sphingosine 1-Phosphate and Is Implicated in the Anandamide-Dependent Regulation of Mitochondrial Activity in C2C12 Myoblasts.

The sphingosine 1-phosphate (S1P) and endocannabinoid (ECS) systems comprehend bioactive lipids widely involved in the regulation of similar biological processes. Interactions between S1P and ECS have not been so far investigated in skeletal muscle, where both systems are active. Here, we used murine C2C12 myoblasts to investigate the effects of S1P on ECS elements by qRT-PCR, Western blotting and UHPLC-MS. In addition, the modulation of the mitochondrial membrane potential (ΔΨm), by JC-1 and Mitotracker Red CMX-Ros fluorescent dyes, as well as levels of protein controlling mitochondrial function, along with the oxygen consumption were assessed, by Western blotting and respirometry, respectively, after cell treatment with methanandamide (mAEA) and in the presence of S1P or antagonists to endocannabinoid-binding receptors. S1P induced a significant increase in TRPV1 expression both at mRNA and protein level, while it reduced the protein content of CB2. A dose-dependent effect of mAEA on ΔΨm, mediated by TRPV1, was evidenced; in particular, low doses were responsible for increased ΔΨm, whereas a high dose negatively modulated ΔΨm and cell survival. Moreover, mAEA-induced hyperpolarization was counteracted by S1P. These findings open new dimension to S1P and endocannabinoids cross-talk in skeletal muscle, identifying TRPV1 as a pivotal target.

Effects of Rare Phytocannabinoids on the Endocannabinoid System of Human Keratinocytes.

The decriminalization and legalization of cannabis has paved the way for investigations into the potential of the use of phytocannabinoids (pCBs) as natural therapeutics for the treatment of human diseases. This growing interest has recently focused on rare (less abundant) pCBs that are non-psychotropic compounds, such as cannabigerol (CBG), cannabichromene (CBC), Δ9-tetrahydrocannabivarin (THCV) and cannabigerolic acid (CBGA). Notably, pCBs can act via the endocannabinoid system (ECS), which is involved in the regulation of key pathophysiological processes, and also in the skin. In this study, we used human keratinocytes (HaCaT cells) as an in vitro model that expresses all major ECS elements in order to systematically investigate the effects of CBG, CBC, THCV and CBGA. To this end, we analyzed the gene and protein expression of ECS components (receptors: CB1, CB2, GPR55, TRPV1 and PPARα/γ/δ; enzymes: NAPE-PLD, FAAH, DAGLα/ß and MAGL) using qRT-PCR and Western blotting, along with assessments of their functionality using radioligand binding and activity assays. In addition, we quantified the content of endocannabinoid(-like) compounds (AEA, 2-AG, PEA, etc.) using UHPLC-MS/MS. Our results demonstrated that rare pCBs modulate the gene and protein expression of distinct ECS elements differently, as well as the content of endocannabinoid(-like) compounds. Notably, they all increased CB1/2 binding, TRPV1 channel stimulation and FAAH and MAGL catalytic activity. These unprecedented observations should be considered when exploring the therapeutic potential of cannabis extracts for the treatment of human skin diseases.

Probing Vulnerability of the gp41 C-Terminal Heptad Repeat as Target for Miniprotein HIV Inhibitors.

One of the therapeutic strategies in HIV neutralization is blocking membrane fusion. In this process, tight interaction between the N-terminal and C-terminal heptad-repeat (NHR and CHR) regions of gp41 is essential to promote membranes apposition and merging. We have previously developed single-chain proteins (named covNHR) that accurately mimic the complete gp41 NHR region in its trimeric conformation. They tightly bind CHR-derived peptides and show a potent and broad HIV inhibitory activity in vitro. However, the extremely high binding affinity (sub-picomolar) is not in consonance with their inhibitory activity (nanomolar), likely due to partial or temporal accessibility of their target in the virus. Here, we have designed and characterized two single-chain covNHR miniproteins each encompassing one of the two halves of the NHR region and containing two of the four sub-pockets of the NHR crevice. The two miniproteins fold as trimeric helical bundles as expected but while the C-terminal covNHR (covNHR-C) miniprotein is highly stable, the N-terminal counterpart (covNHR-N) shows only marginal stability that could be improved by engineering an internal disulfide bond. Both miniproteins bind their respective complementary CHR peptides with moderate (micromolar) affinity. Moreover, the covNHR-N miniproteins can access their target in the context of trimeric native envelope proteins and show significant inhibitory activity for several HIV pseudoviruses. In contrast, covNHR-C cannot bind its target sequence and neither inhibits HIV, indicating a higher vulnerability of C-terminal part of CHR. These results may guide the development of novel HIV inhibitors targeting the gp41 CHR region.

Structural and Thermodynamic Analysis of HIV-1 Fusion Inhibition Using Small gp41 Mimetic Proteins.

Development of effective inhibitors of the fusion between HIV-1 and the host cell membrane mediated by gp41 continues to be a grand challenge due to an incomplete understanding of the molecular and mechanistic details of the fusion process. We previously developed single-chain, chimeric proteins (named covNHR) that accurately mimic the N-heptad repeat (NHR) region of gp41 in a highly stable coiled-coil conformation. These molecules bind strongly to peptides derived from the gp41 C-heptad repeat (CHR) and are potent and broad HIV-1 inhibitors. Here, we investigated two covNHR variants differing in two mutations, V10E and Q123R (equivalent to V38E and Q40R in gp41 sequence) that reproduce the effect of HIV-1 mutations associated with resistance to fusion inhibitors, such as T20 (enfuvirtide). A detailed calorimetric analysis of the binding between the covNHR proteins and CHR peptides (C34 and T20) reveals drastic changes in affinity due to the mutations as a result of local changes in interactions at the site of T20 resistance. The crystallographic structure of the covNHR:C34 complex shows a virtually identical CHR-NHR binding interface to that of the post-fusion structure of gp41 and underlines an important role of buried interfacial water molecules in binding affinity and in development of resistance against CHR peptides. Despite the great difference in affinity, both covNHR variants demonstrate strong inhibitory activity for a wide variety of HIV-1 strains. These properties support the high potential of these covNHR proteins as new potent HIV-1 inhibitors. Our results may guide future inhibition approaches.