Adriana Rodrigues

Melanocortin 5 receptor activates ERK1/2 through a PI3K-regulated signaling mechanism

Melanocortin 5 receptor (MC5R) is a G protein coupled receptor (GPCR) with high affinity for alpha-melanocyte-stimulating hormone (alpha-MSH). To unravel some of the downstream cell-signaling pathways activated by this receptor, HEK293 cells were transiently and stably transfected with a vector encoding green fluorescent protein (GFP)-tagged MC5R. In these cells the receptor was correctly addressed to the cell surface and was functional, as shown by the MC5R-induced formation of intracellular cAMP. In fact, the MC5R agonist alpha-MSH induced a 10- or 16-fold increase (transient or stable cells, respectively) above the cAMP levels found in unstimulated cells. Moreover, in cells stably expressing MC5R-GFP, alpha-MSH promoted ERK1/2 phosphorylation in a dose-dependent manner (EC50=7.3 nM) with the maximal effect occurring after 5 min of agonist incubation. The signaling pathway conveyed through ERK1/2 is not linked to cAMP, since the phosphorylation of these kinases is unchanged by the inhibition of adenylyl cyclase. Also, ERK1/2 activation is not significantly affected by protein kinase A (PKA), protein kinase C (PKC) and protein kinase B or Akt (Akt/PKB) specific inhibitors. However, alpha-MSH-induced ERK1/2 activation is abolished by the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002. Altogether, these findings demonstrate that MC5R signals through a PI3K-regulated Akt-independent pathway leading to downstream activation of ERK1/2. The involvement of these MAPK suggests that MC5R could be implicated in cellular proliferation or differentiation mechanisms.

Intracellular signaling mechanisms of the melanocortin receptors: current state of the art

The melanocortin system is composed by the agonists adrenocorticotropic hormone and α, β and γ-melanocyte-stimulating hormone, and two naturally occurring antagonists, agouti and agouti-related protein. These ligands act by interaction with a family of five melanocortin receptors (MCRs), assisted by MCRs accessory proteins (MRAPs). MCRs stimulation activates different signaling pathways that mediate a diverse array of physiological processes, including pigmentation, energy metabolism, inflammation and exocrine secretion. This review focuses on the regulatory mechanisms of MCRs signaling, highlighting the differences among the five receptors. MCRs signal through G-dependent and independent mechanisms and their functional coupling to agonists at the cell surface is regulated by interacting proteins, namely MRAPs and β-arrestins. The knowledge of the distinct modulation pattern of MCRs signaling and function may be helpful for the future design of novel drugs able to combine specificity, safety and effectiveness in the course of their therapeutic use.

Melanocortin 5 receptor signaling and internalization: Role of MAPK/ERK pathway and β-arrestins 1/2

The Melanocortin 5 receptor (MC5R) is a G-protein coupled receptor (GPCR) that exhibits high affinity for α-MSH. Here we present evidence for MC5R-GFP internalization and subsequent recycling to cell surface, in α-MSH-stimulated HeLa cells. This melanocortin induces a biphasic activation of ERK1/2 with an early peak at 15min, a G(i)-protein driven, β-arrestins 1/2 independent process, and a late sustained activation that is regulated by β-arrestins 1/2. ERK1/2 lead to downstream phosphorylation of 90-kDa ribosomal S6 kinases (p90RSK) and mitogen- and stress-activated protein kinase 1 (MSK1). Only a small fraction (10%) of phosphorylated p90RSK and ERK1/2 translocates to the nucleus inducing c-Fos expression. α-MSH also activates CREB through cAMP/PKA pathway. In 3T3-L1 adipocytes, where MC5R is endogenously expressed, α-MSH also induces phosphorylation and cytosolic retention of the same signaling molecules. These findings provide new evidence on the signaling mechanisms underlying MC5R biological response to α-MSH.

Real-Time PCR Study of Ang1, Ang2, Tie-2, VEGF, and KDR Expression in Human Erectile Tissue during Aging

INTRODUCTION Aging is a recognized risk factor for erectile dysfunction (ED), contributing independently to vascular damage of penile tissue. Vascular maintenance depends on angiogenic balance in tissues. Vascular endothelial growth factor (VEGF) is a modulator of endothelial cells functions, after engagement to specific receptor kinase domain region (KDR). Other factors, such as angiopoietins, cross talk with VEGF, modulating its effects. Angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) compete for binding to Tie-2 and, while Ang1 promotes vascular stabilization, Ang2 acts as a partial agonist or antagonist of Ang1 signaling, depending on VEGF bioavailability. AIMS To quantify the expression of Ang1, Ang2, Tie-2, VEGF, and KDR by real-time polymerase chain reaction (PCR) in human corpus cavernosum (CC) from young and aged healthy individuals. METHODS   Human CC fragments were obtained from organ donors without known risk factors to ED and divided in two groups: young (16-35 years) and aged (59-74 years). RNA was extracted and converted to cDNA. Real-time PCR reactions employed appropriate primers. KDR, Tie-2, Akt, and phospho-Akt protein levels were also assessed by Western blotting (WB). Computer-assisted evaluation of vascular areas was performed. MAIN OUTCOME MEASURES Study of angiopoietins-Tie-2 and VEGF-KDR systems in human CC during aging by real-time PCR and WB. The ratios Ang1/Tie-2 and VEGF/KDR and Akt levels were also determined. RESULTS Real-time PCR results showed a sixfold significant reduction in the Ang1/Tie-2 ratio during aging. Ang2, VEGF, and KDR expression results were highly variable. Nevertheless, the ratio VEGF/KDR was significantly higher in the aged individuals. Akt and phospho-Akt levels were similar in both groups. Immunohistological evaluation revealed a significant decrease in vascular areas and endothelial surface in CC with aging, despite no differences found in vessel number. CONCLUSIONS The obtained results suggest an aging-associated downregulation of angiopoietins/Tie-2 system and an apparent compensatory upregulation of the VEGF/KDR system.

Effects of Aging and Cardiovascular Disease Risk Factors on the Expression of Sirtuins in the Human Corpus Cavernosum

INTRODUCTION Sirtuin (SIRT)1 was recently identified in human corpus cavernosum (CC). We hypothesized that other sirtuins could also be expressed in the CC. Expression of these enzymes in tissues is affected by aging, the main independent risk factor for erectile dysfunction besides other cardiovascular disease risk factors (CVDRF), such as diabetes or obesity. AIM The aim of this study was to characterize the expression of SIRT1-3 and SIRT5-7 in human CC relatively to age and CVDRF. METHODS Samples of CC collected from patients submitted to programmed surgeries or organ donors were divided in three groups according to age and presence of CVDRF. Expression of SIRT1-3 and SIRT5-7 mRNAs was assessed by real-time polymerase chain reaction. Cellular localization and semi-quantification of sirtuins proteins were performed by immunofluorescence and Western blotting (WB), respectively. Nuclear factor kappa B (NFkB)-p65, inducible (iNOS) and endothelial nitric oxide synthase (eNOS) levels were also assayed by WB. MAIN OUTCOME MEASURES The main outcome measure was to characterize the expression of SIRT1-3 and SIRT5-7 in human CC. RESULTS SIRT1-3 and SIRT5-7 mRNAs were detected in all individuals, without statistical differences among groups, excepting SIRT7 that decreased four times in aged groups relatively to young (P = 0.013). WB analysis demonstrated that aged individuals with CVDRF presented higher levels of SIRT7 protein relatively to young (P = 0.0495) and lower levels of SIRT3 protein relatively to healthy aged (P = 0.0077). Expression of NFkB-p65 and iNOS were higher in aged than in young individuals (P = 0.0185; P = 0.004, respectively). No differences in other sirtuins or total eNOS were seen among groups although phospho eNOS Ser(1177) levels decreased in groups of aged men relatively to young (P = 0.0043; P = 0.0099). CONCLUSIONS Our results demonstrate for the first time expression of SIRT2-3 and SIRT5-7 in the human CC. Aged individuals with CVDRF presented an increase in SIRT7 protein levels and a decrease in mitochondrial SIRT3. This finding suggests that CVDRF induces the loss of antioxidant defense mechanisms leading to endothelial injury.

Effect of Aging and Cardiovascular Risk Factors on Receptor Tie1 Expression in Human Erectile Tissue

INTRODUCTION Erectile dysfunction is highly prevalent in patients with advanced age or cardiovascular disease risk factors (CVDRFs). These conditions interfere on expression of vascular growth factors and respective receptors causing disturbance in endothelial function. AIM This study aims to assess the effect of aging and CVDRF on the expression of tyrosine kinase with immunoglobulin-like and EGF-like domains (Tie) 1 in human corpus cavernosum (CC). METHODS CC fragments obtained from programmed surgeries or organ donors were divided into three groups: young, healthy aged, and aged with CVDRF. Angiopoietin (Ang) 1, Ang2, Tie1, and Tie2 mRNA and protein levels were assessed by real-time polymerase chain reaction and Western blotting, respectively. Dual-immunolabeling of Tie1 with specific markers of endothelium and smooth muscle and Ang1 and Ang2 was performed. MAIN OUTCOME MEASURES To characterize the expression of Tie1 in human CC and elucidate its potential inhibitory effect in Ang-Tie2 system. RESULTS Analysis of mRNAs demonstrated a decrease in Tie1 expression in CVDRF individuals compared with aged or young healthy individuals. No variation for Tie2, Ang1, or Ang2 expression was observed among the studied groups. In all analyzed CC fragments, a 125 kDa band, Tie1, was detected. This protein presented a significant age-related decrease, specially in individuals with CVDRF. Immunofluorescence study revealed Tie1 expression in the endothelium of samples of all experimental groups. CONCLUSIONS Employing different methodological approaches, we show for the first time that Tie1 is expressed in human CC endothelium, and its level of expression diminishes in aged individuals, particularly those with CVDRF. This finding reinforces the view that delivery of Ang1 to the CC of erectile dysfunction affected CVDRF patients is able to activate a beneficial Tie2 response.

Characterization of TGF-β expression and signaling profile in the adipose tissue of rats fed with high-fat and energy-restricted diets

Transforming growth factor beta (TGF-β) plays an important role in the pathogenesis of obesity, influencing the release of inflammation mediators and promoting remodeling and collagen deposition in the adipose tissue (AT). In this context, this work aims to elucidate whether TGF-β and Smad-dependent or Smad-independent signaling pathways contribute to regional differentiation of AT in high-fat diet (HFD) and energy-restricted (ER) rat models. For this, TGF-β, TGF-β receptors I and II, PAI-1 and GLUT4 mRNA levels were quantified by real-time PCR, and western blotting assays allowed the semiquantification of TGF-β and proteins from Smad3, ERK1/2 and Akt signaling pathways in subcutaneous and visceral (epididymal, retroperitoneal and mesenteric) fat depots from control, HFD and ER-treated rats. HFD was associated to increased levels of TGF-β and PAI-1 mRNA in epididymal and retroperitoneal visceral fat depots, while ER diet induced a reduction of TGF-β mRNA levels in mesenteric, but surprisingly an increase in retroperitoneal fat. Regarding the different signaling pathways, contrarily to what was found for Smad3, activation of ERK1/2 and Akt in response to HFD was detected in all the visceral but not in subcutaneous fat depots. ER-treated rats presented a more heterogeneous signaling response, as well as decreased TGF-β receptors expression, in the different visceral fat depots. In conclusion, subcutaneous and visceral AT respond differently to distinct diet patterns regarding TGF-β expression and activated signaling pathways. Furthermore, the present study points that visceral AT should not be understood as a homogeneous entity since that response also varied in the different fat depots.

Structural insights on melanocortin 5 receptor targeting to cell surface

The melanocortin 5 receptor (MC5R) is a G-protein coupled receptor (GPCR) with a typical seventransmembrane-domain structure. It is assumed that all GPCRs undergo several post-transcriptional modifications during synthesis and folding until finally target to the cell membrane. The available information about the specific domains responsible for the correct targeting of the melanocortin receptors to the cell surface is scarce. Regarding MC5R, it was shown that the first 20 aminoacids of the human receptor can be deleted without affecting the ligand binding affinity, but further deletions of the N terminus resulted in total loss of binding. These results were not directly correlated with the cell surface expression levels of the receptor. Thus, we aim to define the specific MC5R domains important to its correct trafficking and signaling, and preliminary results are here presented.

Cell surface targeting of the Melanocortin 5 Receptor (MC5R) requires serine-rich terminal motifs

The Melanocortin 5 Receptor (MC5R) is a cell surface receptor that belongs to the class of G-protein coupled receptors (GPCRs), which comprises an intracellular carboxylic domain, seven transmembrane helices and an extracellular amino terminal. Over the last few years, MC5R has been implicated in the regulation of lipid metabolism in exocrine glands, muscle and even in adipose tissue and its function is quite dependent on its correct cell membrane targeting. In this context, the purpose of this work was to study the role of MC5R N-terminus in the receptor trafficking from the endoplasmic reticulum (ER) through the Golgi complex to the plasma membrane. Analysis of N-terminal deleted forms of MC5R revealed that the first 21 amino acids contain the information responsible for the receptor cell surface expression and the removal of further amino acids interfere with the receptor synthesis. In this setting, several mutant forms of the receptor were created by site directed mutagenesis of the MC5R first 21 amino acids and their presence at the plasma membrane was assessed. We have found that two small motifs, constituted by residues Ser4/Ser5 and Ser17/Glu18, are clearly involved in the correct targeting of MC5R to the cell surface. Fluorescence microscopy analysis has revealed that MC5R constructs with mutations in those residues are mainly retained at the ER/Golgi complex. Furthermore, the homodimerization ability of the receptor is maintained in these mutant forms, suggesting that other mechanisms are involved in the regulation of the anterograde transport of MC5R by those N-terminal domains.

Peripherally administered melanocortins induce mice fat browning and prevent obesity

Background/objectivesThe browning of white adipose tissue (WAT) has been in the spotlight during the last years, becoming an attractive approach to combat obesity. Melanocortin neuropeptides, such as α-melanocyte-stimulating hormone (α-MSH), are well-known regulators of appetite at the central nervous system, but its role in adipocyte metabolism is poorly elucidated. This study sought to verify if α-MSH can induce transdifferentiation of white to brown/beige adipocytes and to determine whether it can ameliorate the obesity phenotype.MethodsThe browning effect of α-MSH was determined in isolated adipocytes using the 3T3-L1 cell line and in inguinal subcutaneous adipose tissue (ingWAT) of diet-induced obese (DIO) mice by quantifying the expression of browning hallmark genes, oxygen consumption, and mitochondrial biogenesis. α-MSH protection from diet-induced obesity was evaluated by analyzing mice body weight, fat mass, and lipid and glucose serum profiles.ResultsHere, we report that α-MSH activates a thermogenic gene program and increases the mitochondrial respiratory rate in 3T3-L1 adipocytes and ingWAT of DIO mice. Without affecting food intake, peripheral administration of α-MSH decreases body weight and ingWAT mass, promoting a significant rise in the number of smaller adipocytes, whereas it lowered the larger ones. Additionally, there was an increase in the mass of brown adipose tissue. Browning activation occurs concomitantly with improvement on serum lipid profile, insulin resistance, and glucose homeostasis.ConclusionsThis study highlights the anti-obesity properties of melanocortins by promoting ingWAT browning and provides new perspectives for future designing of more effective therapeutic strategies.