Cannabinoid receptor interacting protein suppresses agonist-driven CB1 receptor internalization and regulates receptor replenishment in an agonist-biased manner

Cannabinoid receptor interacting protein 1a (CRIP1a) is a CB1 receptor (CB1R) distal C-terminus-associated protein that modulates CB1R signaling via G proteins, and CB1R down-regulation but not desensitization (Blume et al. [2015] Cell Signal., 27, 716-726; Smith et al. [2015] Mol. Pharmacol., 87, 747-765). In this study, we determined the involvement of CRIP1a in CB1R plasma membrane trafficking. To follow the effects of agonists and antagonists on cell surface CB(1)Rs, we utilized the genetically homogeneous cloned neuronal cell line N18TG2, which endogenously expresses both CB1R and CRIP1a, and exhibits a well-characterized endocannabinoid signaling system. We developed stable CRIP1a-over-expressing and CRIP1a-siRNA-silenced knockdown clones to investigate gene dose effects of CRIP1a on CB1R plasma membrane expression. Results indicate that CP55940 or WIN55212-2 (10 nM, 5 min) reduced cell surface CB1R by a dynamin-and clathrin-dependent process, and this was attenuated by CRIP1a over-expression. CP55940-mediated cell surface CB1R loss was followed by a cycloheximide-sensitive recovery of surface receptors (30120 min), suggesting the requirement for new protein synthesis. In contrast, WIN55212-2-mediated cell surface CB(1)Rs recovered only in CRIP1a knockdown cells. Changes in CRIP1a expression levels did not affect a transient rimonabant (10 nM)mediated increase in cell surface CB(1)Rs, which is postulated to be as a result of rimonabant effects on \u27non-agonist-driven\u27 internalization. These studies demonstrate a novel role for CRIP1a in agonist-driven CB1R cell surface regulation postulated to occur by two mechanisms: 1) attenuating internalization that is agonist-mediated, but not that in the absence of exogenous agonists, and 2) biased agonist-dependent trafficking of de novo synthesized receptor to the cell surface

The association of N-palmitoylethanolamine with the FAAH inhibitor URB597 impairs melanoma growth through a supra-additive action

<p>Abstract</p> <p>Background</p> <p>The incidence of melanoma is considerably increasing worldwide. Frequent failing of classical treatments led to development of novel therapeutic strategies aiming at managing advanced forms of this skin cancer. Additionally, the implication of the endocannabinoid system in malignancy is actively investigated.</p> <p>Methods</p> <p>We investigated the cytotoxicity of endocannabinoids and their hydrolysis inhibitors on the murine B16 melanoma cell line using a MTT test. Enzyme and receptor expression was measured by RT-PCR and enzymatic degradation of endocannabinoids using radiolabeled substrates. Cell death was assessed by Annexin-V/Propidium iodine staining. Tumors were induced in C57BL/6 mice by s.c. flank injection of B16 melanoma cells. Mice were injected i.p. for six days with vehicle or treatment, and tumor size was measured each day and weighted at the end of the treatment. Haematoxylin-Eosin staining and TUNEL assay were performed to quantify necrosis and apoptosis in the tumor and endocannabinoid levels were quantified by HPLC-MS. Tube formation assay and CD31 immunostaining were used to evaluate the antiangiogenic effects of the treatments.</p> <p>Results</p> <p>The <it>N</it>-arachidonoylethanolamine (anandamide, AEA), 2-arachidonoylglycerol and <it>N</it>- palmitoylethanolamine (PEA) reduced viability of B16 cells. The association of PEA with the fatty acid amide hydrolase (FAAH) inhibitor URB597 considerably reduced cell viability consequently to an inhibition of PEA hydrolysis and an increase of PEA levels. The increase of cell death observed with this combination of molecules was confirmed in vivo where only co-treatment with both PEA and URB597 led to decreased melanoma progression. The antiproliferative action of the treatment was associated with an elevation of PEA levels and larger necrotic regions in the tumor.</p> <p>Conclusions</p> <p>This study suggests the interest of targeting the endocannabinoid system in the management of skin cancer and underlines the advantage of associating endocannabinoids with enzymatic hydrolysis inhibitors. This may contribute to the improvement of long-term palliation or cure of melanoma.</p

The association of hyperuricemia and immediate postpartum hypertension in women without a diagnosis of chronic hypertension

Our objective was to determine if elevated uric acid (UA) is associated with postpartum hypertension (PP HTN) in women without chronic hypertension. This is a secondary analysis of a randomized trial. We compared those with elevated UA to those with normal UA. Logistic regression was conducted to determine the association of elevated UA with PP HTN. Five hundred and fifty-six women met criteria. An UA level ≥ 5.2 mg/dL was associated with immediate PP HTN (adjusted odds ratio 2.44, 95% CI 1.63–3.64). The association was stronger among overweight and obese women. We conclude that hyperuricemia is associated with PP HTN, especially among obese women

The association of hyperuricemia and immediate postpartum hypertension in women without a diagnosis of chronic hypertension

Our objective was to determine if elevated uric acid (UA) is associated with postpartum hypertension (PP HTN) in women without chronic hypertension. This is a secondary analysis of a randomized trial. We compared those with elevated UA to those with normal UA. Logistic regression was conducted to determine the association of elevated UA with PP HTN. Five hundred and fifty-six women met criteria. An UA level ≥ 5.2 mg/dL was associated with immediate PP HTN (adjusted odds ratio 2.44, 95% CI 1.63–3.64). The association was stronger among overweight and obese women. We conclude that hyperuricemia is associated with PP HTN, especially among obese women

Maternal human telomerase reverse transcriptase variants are associated with preterm labor and preterm premature rupture of membranes

<div><p>Objective</p><p>Premature aging and short telomere lengths of fetal tissues are associated with spontaneous preterm labor (PTL) and preterm premature rupture of membranes (pPROM). Maintenance of telomere length is performed by the enzyme telomerase. Human telomerase reverse transcriptase (hTERT) is a subunit of telomerase, and its dysfunction affects telomere shortening. This study assessed whether maternal or fetal genetic variations in the hTERT gene are associated with PTL or pPROM.</p><p>Methods</p><p>A case (PTL or pPROM) control (term birth) genetic association study was conducted in 654 non-Hispanic white mothers (438 term, 162 PTL, 54 pPROM) and 502 non-Hispanic white newborns (346 term, 116 PTB, 40 pPROM). Maternal and fetal DNA samples were genotyped for 23 single nucleotide polymorphisms (SNPs) within the hTERT gene. Allele frequencies were compared between cases and controls, stratified by PTL and pPROM. Maternal and fetal data were analyzed separately.</p><p>Results</p><p>Allelic differences in one SNP of hTERT (rs2853690) were significantly associated with both PTL (adjusted OR 2.24, 95%CI 1.64–3.06, p = 2.32e-05) and with pPROM (adjusted OR 7.54, 95%CI 3.96–14.33, p = 2.39e-07) in maternal DNA. There was no significant association between the hTERT SNPs analyzed and PTL or pPROM in the fetal samples.</p><p>Conclusion</p><p>hTERT polymorphisms in fetal DNA do not associate with PTL or pPROM risk; however, maternal genetic variations in hTERT may play a contributory role in risk of PTL and PPROM.</p></div

Logistic regression results for fetal single locus allele frequencies among cases and controls and association with preterm labor.

<p>Logistic regression results for fetal single locus allele frequencies among cases and controls and association with preterm labor.</p

hTERT SNPs that were genotyped for maternal and fetal analyses and those that were excluded from analysis.

<p>hTERT SNPs that were genotyped for maternal and fetal analyses and those that were excluded from analysis.</p

Baseline characteristics of fetal cases and controls.

<p>Baseline characteristics of fetal cases and controls.</p

Logistic regression results for maternal single locus allele frequencies among cases and controls and association with preterm labor.

<p>Logistic regression results for maternal single locus allele frequencies among cases and controls and association with preterm labor.</p

Logistic regression results for fetal single locus allele frequencies among cases and controls and association with preterm premature rupture of membranes.

<p>Logistic regression results for fetal single locus allele frequencies among cases and controls and association with preterm premature rupture of membranes.</p