Jean-Yves Tano

The Constitutive Function of Native TRPC3 Channels Modulates Vascular Cell Adhesion Molecule-1 Expression in Coronary Endothelial Cells Through Nuclear Factor κB Signaling

Rationale: Upregulation of endothelial vascular cell adhesion molecule (VCAM)-1 and the subsequent increase in monocyte recruitment constitute critical events in atherogenesis. We have recently shown that in human coronary artery endothelial cells (HCAECs) regulated expression of VCAM-1 depends, to a significant extent, on expression and function of the Ca2+-permeable channel transient receptor potential canonical (TRPC)3, regardless of the ability of the stimulatory signal to induce regulated Ca2+ influx, leading to the hypothesis that TRPC3 constitutive, rather than regulated function, contributes to the underlying signaling mechanism. Objective: The present studies addressed this important question and gathered mechanistic insight on the signaling coupling constitutive TRPC3 function to VCAM-1 expression. Methods and Results: In HCAECs, maneuvers that prevent Ca2+ influx or knockdown of TRPC3 markedly reduced tumor necrosis factor (TNF)&agr;-induced VCAM-1 and monocyte adhesion. TNF&agr; also induced TRPC3 expression and TRPC3-mediated constitutive cation influx and currents. Stable (HEK293 cells) or transient (HCAECs) overexpression of TRPC3 enhanced TNF&agr;-induced VCAM-1 compared to wild-type cells. I&kgr;B&agr; phosphorylation/degradation was reduced by TRPC3 knockdown and increased by channel overexpression. Inhibition of calmodulin completely prevented nuclear factor &kgr;B activation, whereas blocking calmodulin-dependent kinases or NADPH oxidases rendered partial inhibition. Conclusions: Our findings indicate that in HCAECs expression of VCAM-1 and monocyte adhesion depend, to a significant extent, on TRPC3 constitutive function through a signaling mechanism that requires constitutive TRPC3-mediated Ca2+ influx for proper activation of nuclear factor &kgr;B, presumably through Ca2+-dependent activation of the calmodulin/calmodulin-dependent kinase axis.

Impairment of survival signaling and efferocytosis in TRPC3-deficient macrophages.

We have recently shown that in macrophages proper operation of the survival pathways phosphatidylinositol-3-kinase (PI3K)/AKT and nuclear factor kappa B (NFkB) has an obligatory requirement for constitutive, non-regulated Ca(2+) influx. In the present work we examined if Transient Receptor Potential Canonical 3 (TRPC3), a member of the TRPC family of Ca(2+)-permeable cation channels, contributes to the constitutive Ca(2+) influx that supports macrophage survival. We used bone marrow-derived macrophages obtained from TRPC3(-/-) mice to determine the activation status of survival signaling pathways, apoptosis and their efferocytic properties. Treatment of TRPC3(+/+) macrophages with the pro-apoptotic cytokine TNFα induced time-dependent phosphorylation of IκBα, AKT and BAD, and this was drastically reduced in TRPC3(-/-) macrophages. Compared to TRPC3(+/+) cells TRPC3(-/-) macrophages exhibited reduced constitutive cation influx, increased apoptosis and impaired efferocytosis. The present findings suggest that macrophage TRPC3, presumably through its constitutive function, contributes to survival signaling and efferocytic properties.

Bone marrow deficiency of TRPC3 channel reduces early lesion burden and necrotic core of advanced plaques in a mouse model of atherosclerosis.

AIMS Macrophage apoptosis plays a determinant role in progression of atherosclerotic lesions. An important goal in atherosclerosis research is to identify new components of macrophage apoptosis that can eventually be exploited as molecular targets in strategies aimed at manipulating macrophage function in the lesion. In the previous work from our laboratory, we have shown that transient receptor potential canonical 3 (TRPC3) channel is an obligatory component of survival mechanisms in human and murine macrophages and that TRPC3-deficient non-polarized bone marrow-derived macrophages exhibit increased apoptosis, suggesting that in vivo TRPC3 might influence lesion development. In the present work, we used a bone marrow transplantation strategy as a first approach to examine the impact of macrophage deficiency of TRPC3 on early and advanced atherosclerotic lesions of Apoe(-/-) mice. METHODS AND RESULTS After 3 weeks of high-fat diet, lesions in mice transplanted with bone marrow from Trpc3(-/-) donors were smaller and with reduced cellularity than controls. Advanced lesions from these mice exhibited reduced necrotic core, less apoptotic macrophages, and increased collagen content and cap thickness. In vitro, TRPC3-deficient macrophages polarized to the M1 phenotype showed reduced apoptosis, whereas both M1 and M2 macrophages had increased efferocytic capacity. CONCLUSIONS Bone marrow deficiency of TRPC3 has a dual beneficial effect on lesion progression by reducing cellularity at early stages and necrosis in the advanced plaques. Our findings represent the first evidence for a role of a member of the TRPC family of cation channels in mechanisms associated with atherosclerosis.

Requirement for non-regulated, constitutive calcium influx in macrophage survival signaling

The phosphatidylinositol-3-kinase (PI3K)/AKT axis and the Nuclear Factor kappa B (NFκB) pathway play critical roles in macrophage survival. In cells other than macrophages proper operation of those two pathways requires Ca²(+) influx into the cell, but if that is the case in macrophages remains unexplored. In the present work we used THP-1-derived macrophages and a pharmacological approach to examine for the first time the role of constitutive, non-regulated Ca²(+) influx in PI3K/AKT and NFκB signaling. Blocking constitutive function of Ca²(+)-permeable channels with the organic channel blocker SKF96365 completely prevented phosphorylation of IκBα, AKT and its downstream target BAD in TNFα-treated macrophages. A similar effect was observed upon treating macrophages with the calmodulin (CAM) inhibitor W-7 or the calmodulin-dependent kinase II (CAMKII) inhibitor KN-62. In addition, pre-treating macrophages with SKF96365 significantly enhanced TNFα-induced apoptosis. Our findings suggest that in THP-1-derived macrophages survival signaling depends, to a significant extent, on constitutive Ca²(+) influx presumably through a mechanism that involves the CAM/CAMKII axis as a coupling component between constitutive Ca²(+) influx and activation of survival signaling.

On the potential role of source and species of diacylglycerol in phospholipase-dependent regulation of TRPC3 channels

Members of the Transient Receptor Potential Canonical (TRPC) family of channel forming proteins are among the most important Ca2+-permeable cation channels in non-excitable cells. Physiologically, TRPC channels are activated downstream receptor-dependent stimulation of phospholipases, either by store-operated or non-store operated mechanisms. TRPC3, a member of the TRPC3/6/7 subfamily, has been largely studied mostly due to its ability to function in one or the other modes, depending on cell type and expression conditions. The role of TRPC3 as a non-store operated channel has been attributed to its ability to respond to diacylglycerol (DAG) either exogenously applied or endogenously produced following activation of receptor-stimulated phospholipases. Despite the vast amount of information accumulated on this topic, some critical aspects related to phospholipase-dependent DAG-mediated regulation of TRPC3 remain unclear and/or unexplored. Among these, the source and species of native DAG, modulation by different DAG-generating phospholipases and protein kinase C-dependent inhibition of TRPC3 in its native environment are just few examples. The present essay is intended to compile existing knowledge on the nature of phospholipase-derived DAGs, their biophysical properties and current evidence on phospholipase-dependent regulation of TRPC3, to speculate on potential scenarios that may eventually provide answers to some of the above questions.

Macrophage function in atherosclerosis: potential roles of TRP channels.

Cation channels of the Transient Receptor Potential Canonical (TRPC) group, which belong to the larger TRP superfamily of channel proteins, are critical players in cardiovascular disease. Recent studies underscored a role of TRPC3 in macrophage survival and efferocytosis, two critical events in atherosclerosis lesion development. Also, other members of the TRP channel superfamily are found expressed in monocytes/macrophages, where they participate in processes that might be of significance to atherogenesis. These observations set a framework for future studies aimed at defining the ultimate functions not only of TRPC3, but probably other TRP channels, in macrophage biology. The purpose of this manuscript is to provide a timely revision of existing evidence on the role of members of the TRP channel superfamily, in particular TRPCs, in macrophages and discuss it in the context of the macrophage’s function in atherogenesis.

Reduced endoplasmic reticulum stress-induced apoptosis and impaired unfolded protein response in TRPC3-deficient M1 macrophages

Endoplasmic reticulum (ER) stress is a prominent mechanism of macrophage apoptosis in advanced atherosclerotic lesions. Recent studies from our laboratory showed that advanced atherosclerotic plaques in Apoe(-/-) mice with bone marrow deficiency of the calcium-permeable channel Transient Receptor Potential Canonical 3 (TRPC3) are characterized by reduced areas of necrosis and fewer apoptotic macrophages than animals transplanted with Trpc3(+/+) bone marrow. In vitro, proinflammatory M1 but not anti-inflammatory M2 macrophages derived from Trpc3(-/-)Apoe(-/-) animals exhibited reduced ER stress-induced apoptosis. However, whether this was due to a specific effect of TRPC3 deficiency on macrophage ER stress signaling remained to be determined. In the present work we used polarized macrophages derived from mice with macrophage-specific deficiency of TRPC3 to examine the expression level of ER stress markers and the activation status of some typical mediators of macrophage apoptosis. We found that the reduced susceptibility of TRPC3-deficient M1 macrophages to ER stress-induced apoptosis correlates with an impaired unfolded protein response (UPR), reduced mitochondrion-dependent apoptosis, and reduced activation of the proapoptotic molecules calmodulin-dependent protein kinase II and signal transducer and activator of transcription 1. Notably, none of these pathways was altered in TRPC3-deficient M2 macrophages. These findings show for the first time an obligatory requirement for a member of the TRPC family of cation channels in ER stress-induced apoptosis in macrophages, underscoring a rather selective role of the TRPC3 channel on mechanisms related to the UPR signaling in M1 macrophages.

Evidence for Operation of Nicotinic and Muscarinic Acetylcholine Receptor-Dependent Survival Pathways in Human Coronary Artery Endothelial Cells

Nicotinic acetylcholine receptors (nAChRs) have recently emerged as critical players in modulation of endothelial function. In particular, studies on endothelial cells from different vascular beds have shown anti‐apoptotic actions of nicotinic stimulation, but whether there is actually activation of survival signaling downstream nAChR function has not been explored. In the present work we used human coronary artery endothelial cells (HCAECs) and a pharmacological approach to examine the impact of cholinergic stimulation on survival signaling pathways. Our findings show that cholinergic receptors promote activation of three typical survival routes: the phosphatidyl‐inositol‐3‐kinase (PI3K)/AKT axis, activated downstream muscarinic and nAChRs; the JAK2/STAT3 axis, activated downstream nAChR; and ERK1/2 MAP kinases, activated by both muscarinic acetylcholine receptor (mAChR) and nAChR. Based on their sensitivity to α‐bungarotoxin, nicotinic regulation of JAK2/STAT3 and ERK1/2 occurs downstream α7‐nAChRs. The present findings suggest that in HCAECs the two cholinergic receptors may act concertedly to induce an efficient survival response of coronary cells when exposed to pro‐apoptotic stimuli. J. Cell. Biochem. 112: 1978–1984, 2011.

INVOLVEMENT OF CALMODULIN AND CALMODULIN KINASE II IN TUMOR NECROSIS FACTOR ALPHA-INDUCED SURVIVAL OF BONE MARROW DERIVED MACROPHAGES

We previously showed that survival signaling in TNFα-treated, human THP1-derived macrophages (TDMs) has an obligatory requirement for constitutive Ca(2+) influx through a mechanism involving calmodulin/calmodulin kinase II (CAM/CAMKII). We also demonstrated that such requirement also applies to the protective actions of TNFα in murine bone marrow-derived macrophages (BMDMs) and that TRPC3 channels mediate constitutive Ca(2+) influx. Using a pharmacological approach we here examined if in BMDMs, similarly to TDMs, TNFα-induced survival signaling also involves CAM/CAMKII. In BMDMs, TNFα induced rapid activation of the survival pathways NFκB, AKT and p38MAPK. All these routes were activated in a PI3K-dependent fashion. Activation of AKT and NFκB, but not that of p38MAPK, was abrogated by the CAM inhibitor W7, while KN-62, a CAMKII inhibitor, prevented activation of AKT and p38MAPK but not that of NFκB. Inhibition of CAM or CAMKII completely prevented the protective actions of TNFα. Our observations indicate that in BMDMs CAM and CAMKII have differential contributions to the components of TNFα-dependent survival signaling and underscore a complex interplay among canonical survival routes. These findings set a signaling framework to understand how constitutive Ca(2+) influx couples to macrophage survival in BMDMs.

Molecular and Cellular Aspects of Atherosclerosis: Emerging Roles of TRPC Channels

Recruitment of circulating monocytes to activated areas of the endothelium and their migration to the subintimal inflammatory foci represents one of the earliest events in atherogenesis (Linton and Fazio 2003; Hansson 2005). Importantly, monocyte recruitment can be recognized throughout all lesional stages including advanced lesions, where plaque infiltration and neovascularization occur. Indeed, available experimental evidence supports the notion that in advanced stages monocyte infiltration contributes to plaque instability and rupture (Virmani, Burke et al. 2006). Monocyte recruitment to the subendothelial milieu implies a sequence of events that begin with monocyte rolling along and tethering to the endothelial surface, firm adhesion and activation, and ultimately migration to the subintima. At the molecular level, the entire sequence entails interaction of integrins on the monocyte surface with cell adhesion molecules (CAMs) expressed on the endothelial cell. Monocyte rolling and tethering is mainly mediated by CAMs from the selectin group (v.g., E-selectin) while firm adhesion and migration are mostly mediated by CAMs from the immunoglobulin (Ig) superfamily, such as intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Compelling evidence accumulated over the last decade has clearly established that VCAM-1 (CD106) has a prominent role in mediating attachment and migration of monocytes (the Cluster of Differentiation nomenclature (Zola, Swart et al. 2005) is given for reference, but “VCAM-1” will be used throughout the text).