Elaine D. Por

Persistent Nociception Triggered by Nerve Growth Factor (NGF) Is Mediated by TRPV1 and Oxidative Mechanisms

Nerve growth factor (NGF) is elevated in certain chronic pain conditions and is a sufficient stimulus to cause lasting pain in humans, but the actual mechanisms underlying the persistent effects of NGF remain incompletely understood. We developed a rat model of NGF-induced persistent thermal hyperalgesia and mechanical allodynia to determine the role of transient receptor potential vanilloid 1 (TRPV1) and oxidative mechanisms in the persistent effects of NGF. Persistent thermal hypersensitivity and mechanical allodynia require de novo protein translation and are mediated by TRPV1 and oxidative mechanisms. By comparing effects after systemic (subcutaneous), spinal (intrathecal) or hindpaw (intraplantar) injections of test compounds, we determined that TRPV1 and oxidation mediate persistent thermal hypersensitivity via peripheral and spinal sites of action and mechanical allodynia via only a spinal site of action. Therefore, NGF-evoked thermal and mechanical allodynia are mediated by spatially distinct mechanisms. NGF treatment evoked sustained increases in peripheral and central TRPV1 activity, as demonstrated by increased capsaicin-evoked nocifensive responses, increased calcitonin gene-related peptide release from hindpaw skin biopsies, and increased capsaicin-evoked inward current and membrane expression of TRPV1 protein in dorsal root ganglia neurons. Finally, we showed that NGF treatment increased concentrations of linoleic and arachidonic-acid-derived oxidized TRPV1 agonists in spinal cord and skin biopsies. Furthermore, increases in oxidized TRPV1-active lipids were reduced by peripheral and spinal injections of compounds that completely blocked persistent nociception. Collectively, these data indicate that NGF evokes a persistent nociceptive state mediated by increased TRPV1 activity and oxidative mechanisms, including increased production of oxidized lipid TRPV1 agonists.

The cancer/testis antigen cage with oncogenic potential stimulates cell proliferation by up-regulating cyclins D1 and E in an AP-1- and E2F-dependent manner

A cancer/testis antigen, CAGE, is widely expressed in various cancer tissues and cancer cell lines but not in normal tissues except the testis. In the present study, ectopic expression of CAGE in fibroblast cells resulted in foci formation, suggesting its cell-transforming ability. Using stable HeLa transfectant clones with the tetracycline-inducible CAGE gene, we found that CAGE overexpression stimulated both anchorage-dependent and -independent cell growth in vitro and promoted tumor growth in a xenograft mouse model. Cell cycle analysis showed that CAGE augments the levels of cyclin D1 and E, thereby activating cyclin-associated cyclin-dependent kinases and subsequently accelerating the G1 to S progression. Moreover, increased cyclin D1 and E levels in CAGE-overexpressing cells were observed even in a growth arrested state, indicating a direct effect of CAGE on G1 cyclin expression. CAGE-induced expression of cyclins D1 and E was found to be mediated by AP-1 and E2F-1 transcription factors, and among the AP-1 members, c-Jun and JunD appeared to participate in CAGE-mediated up-regulation of cyclin D1. CAGE overexpression also enhanced retinoblastoma phosphorylation and subsequent E2F-1 nuclear translocation. In contrast, small interfering RNA-mediated knockdown of CAGE suppressed the expression of G1 cyclins, activation of AP-1 and E2F-1, and cell proliferation in both HeLa cervical cancer cells and Malme-3M melanoma cells. These results suggest that the cancer/testis antigen CAGE possesses oncogenic potential and promotes cell cycle progression by inducing AP-1- and E2F-dependent expression of cyclins D1 and E.

β-Arrestin-2 Desensitizes the Transient Receptor Potential Vanilloid 1 (TRPV1) Channel

Background: The TRPV1 receptor is an ionotropic receptor implicated in a variety of pain and inflammatory disorders. Results: β-Arrestin-2 scaffolds phosphodiesterase PDE4D5 to TRPV1 to regulate receptor phosphorylation and activity. Conclusion: β-Arrestin-2 functions as a scaffold protein to mediate TRPV1 desensitization in multiple cell models. Significance: Our findings presented herein provide compelling support for the contribution of β-arrestins as scaffolding proteins in the regulation of ligand-gated ion channels. Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel activated by multiple stimuli and is implicated in a variety of pain disorders. Dynamic sensitization of TRPV1 activity by A-kinase anchoring protein 150 demonstrates a critical role for scaffolding proteins in nociception, yet few studies have investigated scaffolding proteins capable of mediating receptor desensitization. In this study, we identify β-arrestin-2 as a scaffolding protein that regulates TRPV1 receptor activity. We report β-arrestin-2 association with TRPV1 in multiple cell models. Moreover, siRNA-mediated knockdown of β-arrestin-2 in primary cultures resulted in a significant increase in both initial and repeated responses to capsaicin. Electrophysiological analysis further revealed significant deficits in TRPV1 desensitization in primary cultures from β-arrestin-2 knock-out mice compared with wild type. In addition, we found that β-arrestin-2 scaffolding of phosphodiesterase PDE4D5 to the plasma membrane was required for TRPV1 desensitization. Importantly, inhibition of PDE4D5 activity reversed β-arrestin-2 desensitization of TRPV1. Together, these results identify a new endogenous scaffolding mechanism that regulates TRPV1 ligand binding and activation.

Activation of Mu Opioid Receptors Sensitizes Transient Receptor Potential Vanilloid Type 1 (TRPV1) via β-Arrestin-2-Mediated Cross-Talk

The transient receptor potential family V1 channel (TRPV1) is activated by multiple stimuli, including capsaicin, acid, endovanilloids, and heat (>42C). Post-translational modifications to TRPV1 result in dynamic changes to the sensitivity of receptor activation. We have previously demonstrated that β-arrestin2 actively participates in a scaffolding mechanism to inhibit TRPV1 phosphorylation, thereby reducing TRPV1 sensitivity. In this study, we evaluated the effect of β-arrestin2 sequestration by G-protein coupled receptors (GPCRs) on thermal and chemical activation of TRPV1. Here we report that activation of mu opioid receptor by either morphine or DAMGO results in β-arrestin2 recruitment to mu opioid receptor in sensory neurons, while activation by herkinorin does not. Furthermore, treatment of sensory neurons with morphine or DAMGO stimulates β-arrestin2 dissociation from TRPV1 and increased sensitivity of the receptor. Conversely, herkinorin treatment has no effect on TRPV1 sensitivity. Additional behavioral studies indicate that GPCR-driven β-arrestin2 sequestration plays an important peripheral role in the development of thermal sensitivity. Taken together, the reported data identify a novel cross-talk mechanism between GPCRs and TRPV1 that may contribute to multiple clinical conditions.

A-Kinase Anchoring Protein 150 Mediates Transient Receptor Potential Family V Type 1 Sensitivity to Phosphatidylinositol-4,5-Bisphosphate

A-kinase anchoring protein 150 (AKAP150) is a scaffolding protein that controls protein kinase A- and C-mediated phosphorylation of the transient receptor potential family V type 1 (TRPV1), dictating receptor response to nociceptive stimuli. The phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) anchors AKAP150 to the plasma membrane in naive conditions and also affects TRPV1 activity. In the present study, we sought to determine whether the effects of PIP2 on TRPV1 are mediated through AKAP150. In trigeminal neurons and CHO cells, the manipulation of cellular PIP2 led to significant changes in the association of AKAP150 and TRPV1. Following PIP2 degradation, increased TRPV1:AKAP150 coimmunoprecipitation was observed, resulting in increased receptor response to capsaicin treatment. Phospholipase C activation in neurons isolated from AKAP150−/− animals indicated that PIP2-mediated inhibition of TRPV1 in the whole-cell environment requires expression of the scaffolding protein. Furthermore, the addition of PIP2 to neurons isolated from AKAP150 wild-type mice reduced PKA sensitization of TRPV1 compared with isolated neurons from AKAP150−/− mice. These findings suggest that PIP2 degradation increases AKAP150 association with TRPV1 in the whole-cell environment, leading to sensitization of the receptor to nociceptive stimuli.

PP2B/calcineurin-mediated desensitization of TRPV1 does not require AKAP150

Activation of protein kinases and phosphatases at the plasma membrane often initiates agonist-dependent signalling events. In sensory neurons, AKAP150 (A-kinase-anchoring protein 150) orientates PKA (protein kinase A), PKC (protein kinase C) and the Ca2+/calmodulin-dependent PP2B (protein phosphatase 2B, also known as calcineurin) towards membrane-associated substrates. Recent evidence indicates that AKAP150-anchored PKA and PKC phosphorylate and sensitize the TRPV1 (transient receptor potential subfamily V type 1 channel, also known as the capsaicin receptor). In the present study, we explore the hypothesis that an AKAP150-associated pool of PP2B catalyses the dephosphorylation and desensitization of TRPV1. Biochemical, electrophysiological and cell-based experiments indicate that PP2B associates with AKAP150 and TRPV1 in cultured TG (trigeminal ganglia) neurons. Gene silencing of AKAP150 reduces basal phosphorylation of TRPV1. However, functional studies in neurons isolated from AKAP150-/- mice indicate that the anchoring protein is not required for pharmacological desensitization of TRPV1. Behavioural analysis of AKAP150-/- mice further support this notion, demonstrating that agonist-stimulated desensitization of TRPV1 is sensitive to PP2B inhibition and does not rely on AKAP150. These findings allow us to conclude that pharmacological desensitization of TRPV1 by PP2B may involve additional regulatory components.

Regulation of ROS Production and Vascular Function by Carbon Monoxide

Carbon monoxide (CO) is a gaseous molecule produced from heme by heme oxygenase (HO). CO interacts with reduced iron of heme-containing proteins, leading to its involvement in various cellular events via its production of mitochondrial reactive oxygen species (ROS). CO-mediated ROS production initiates intracellular signal events, which regulate the expression of adaptive genes implicated in oxidative stress and functions as signaling molecule for promoting vascular functions, including angiogenesis and mitochondrial biogenesis. Therefore, CO generated either by exogenous delivery or by HO activity can be fundamentally involved in regulating mitochondria-mediated redox cascades for adaptive gene expression and improving blood circulation (i.e., O2 delivery) via neovascularization, leading to the regulation of mitochondrial energy metabolism. This paper will highlight the biological effects of CO on ROS generation and cellular redox changes involved in mitochondrial metabolism and angiogenesis. Moreover, cellular mechanisms by which CO is exploited for disease prevention and therapeutic applications will also be discussed.

Phosphorylation regulates TRPV1 association with β-arrestin-2

Post-translational modifications in TRPV1 (transient receptor potential vanilloid 1) play a critical role in channel activity. Phosphorylation of serine/threonine residues within the N- and C-termini of TRPV1 are implicated in receptor sensitization and activation. Conversely, TRPV1 desensitization occurs via a calcium-dependent mechanism and leads to receptor de-phosphorylation. Importantly, we recently demonstrated that TRPV1 association with β-arrestin-2 is critical to receptor desensitization via its ability to scaffold the phosphodiesterase PDE4D5 to the receptor, regulating TRPV1 phosphorylation. In the present study, we demonstrate that phosphorylation of TRPV1 and β-arrestin-2 regulates this association at the membrane. Under serum-free media conditions, we observed a significant decrease in TRPV1 and β-arrestin-2 association in transfected CHO (Chinese-hamster ovary) cells. Pharmacological activation of the kinases PKA (protein kinase A) and PKC (protein kinase C) led to a robust increase in TRPV1 and β-arrestin-2 association, whereas inhibition of PKA and PKC decreased association. Previously, we identified potential PKA residues (Ser(116), Thr(370)) in the N-terminus of TRPV1 modulated by β-arrestin-2. In the present study we reveal that the phosphorylation status of Thr(370) dictates the β-arrestin-2 and TRPV1 association. Furthermore, we demonstrate that CK2 (casein kinase 2)-mediated phosphorylation of β-arrestin-2 at Thr(382) is critical for its association with TRPV1. Taken together, the findings of the present study suggest that phosphorylation controls the association of TRPV1 with β-arrestin-2.

Metallopeptidase inhibition potentiates bradykinin-induced hyperalgesia

&NA; The neuropeptide bradykinin (BK) sensitizes nociceptor activation following its release in response to inflammatory injury. Thereafter, the bioactivity of bradykinin is controlled by the enzymatic activities of circulating peptidases. One such enzyme, the metalloendopeptidase EC3.4.24.15 (EP24.15), is co‐expressed with bradykinin receptors in primary afferent neurons. In this study, using approaches encompassing pharmacology, biochemistry, cell biology, and behavioral animal models, we identified a crucial role for EP24.15 and the closely related EP24.16 in modulating bradykinin‐mediated hyperalgesia. Pharmacological analyses indicated that EP24.15 and EP24.16 inhibition significantly enhances bradykinin type‐2 receptor activation by bradykinin in primary trigeminal ganglia cultures. In addition, bradykinin‐induced sensitization of TRPV1 activation was increased in the presence of the EP24.15/16 inhibitor JA‐2. Furthermore, behavioral analyses illustrated a significant dose–response relationship between JA‐2 and bradykinin‐mediated thermal hyperalgesia. These results indicate an important physiological role for the metallopeptidases EP24.15 and EP24.16 in regulating bradykinin‐mediated sensitization of primary afferent nociceptors. Metalloendopeptidases EP24.15 and EP24.16 regulate inflammatory sensitization of primary afferent nociceptors by mediating bradykinin type‐2 receptor activation by bradykinin.

Secretion Profile of Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium During Wound Healing

Purpose The purpose of this study was to characterize the secretion profile of induced pluripotent stem cell-derived retinal pigment epithelium (iPS-RPE) during wound healing. iPS-RPE was used to develop an in vitro wound healing model. We hypothesized that iPS-RPE secretes cytokines and growth factors which act in an autocrine manner to promote migration and proliferation of cells during wound healing. Methods iPS-RPE was grown in transwells until fully confluent and pigmented. The monolayers were scratched to induce a wound. Levels of Ki-67, β-catenin, e-cadherin, n-cadherin, and S100A4 expression were analyzed by immunofluorescent labeling. Cell culture medium samples were collected from both the apical and basolateral sides of the transwells every 72 hours for 21 days. The medium samples were analyzed using multiplex ELISA to detect secreted growth factors and cytokines. The effects of conditioned medium on collagen gel contraction, cell proliferation, and migration were measured. Results iPS-RPE underwent epithelial-mesenchymal transition (EMT) during wound healing as indicated by the translocation of β-catenin to the nucleus, cadherin switch, and expression of S100A4. GRO, GM-CSF, MCP-1, IL-6, and IL-8 were secreted by both the control and the wounded cell cultures. VEGF, FGF-2, and TGFβ expression were detected at higher levels after wounding than those in control. The proteins were found to be secreted in a polarized manner. The conditioned medium from wounded monolayers promoted collagen gel contraction, as well as proliferation and migration of ARPE 19 cells. Conclusions These results indicate that after the monolayer is wounded, iPS-RPE secretes proteins into the culture medium that promote increased proliferation, contraction, and migration.