New insights into TRP channels

Abstract

An increasing number of studies have implicated that the activation of innate immune system and inflammatory mechanisms are of importance in the pathogenesis of numerous diseases. The innate immune system is present in almost all multicellular organisms in response to pathogens or tissue injury, which is performed via germ-line encoded pattern-recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs) or dangers-associated molecular patterns (DAMPs). Intracellular pathways linking immune and inflammatory response to ion channel expression and function have been recently identified. Among ion channels, transient receptor potential (TRP) channels are a major family of non-selective cation-permeable channels that function as polymodal cellular sensors involved in many physiological and pathological processes. In this review, we summarize current knowledge about classifications, functions, and interactions of TRP channels and PRRs, which may provide new insights into their roles in the pathogenesis of inflammatory diseases.

Introduction

The activation of innate immune system and inflammatory mechanisms are of importance in the pathogenesis of numerous diseases. The innate immune system is the first line of defense against pathogens and tissue injury, which is responsible for initiating immune responses to resolve infections and repair damaged tissues. Initiation of the innate immune response is triggered by recognition of pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs) by pathogen recognition receptors (PRRs).¹ Engagement of PRRs by PAMPs and DAMPs leads to a multitude of changes in the transcriptional and posttranslational cascades of innate immune cells that bring proinflammatory cytokines, chemokines, and growth factors into circulation.² Intracellular pathways linking immune and inflammatory response to ion channels have been identified.^3,4 Among ion channels, transient receptor potential (TRP) channels are a major family of non-selective cation-permeable channels, which are classified into 6 subfamilies on the basis of sequence homology and function as diverse cellular sensors.⁵ There are strong indications that TRP channels are involved in the pathogenesis of many inflammatory diseases.⁶ This brief review summarizes current evidence that relates to the interactions of PRRs and TRP channels, which may provide new insights into their roles and regulatory mechanisms.

Transient Receptor Potential Channels: Classification and Function

Transient receptor potential (TRP) channels are a family of non-selective cation-permeable channels that function as polymodal cellular sensors involved in many physiological processes.⁷ Mutations in genes encoding TRP channels have been demonstrated to be the cause of several inherited diseases that affect a variety of systems including the renal, skeletal, and nervous system.^4,6,8 As shown in Figure 1, all TRP channels are membrane proteins with 6 putative transmembrane segments (S1–S6) and a cation-permeable pore region between S5 and S6. Currently, 28 different mammalian TRP channels are identified and classified into 6 subfamilies on the basis of sequence homology: TRP canonical (TRPC; TRPC1–7), TRP vanilloid (TRPV; TRPV1–6), TRP melastatin (TRPM; TRPM1–8), TRP polycystin (TRPP; TRPP2, TRPP3, TRPP5), TRP mucolipin (TRPML; TRPML1–3), and TRP ankyrin (TRPA; TRPA1).^6,9 Different subfamilies of TRP channels display a variety of gating mechanism and cation selectivity,^10,11 which can be opened by direct ligand binding, G-protein coupled signaling, or membrane depolarization.^12,13 TRPC channels consist of the proteins closely related to the first identified member of TRP channels, the “canonical” Drosophila TRP, now named as TRPC1. Functionally, TRPC family has been implicated in a diverse set of diseases such as hypertension, vascular inflammation, cardiac hypertrophy, and progressive kidney failure. The mammalian TRPV channels are thermo- and chemosensitive channels which are composed of 6 members.¹⁴ TRPV1 is the best characterized member of TRPVs, which can be activated by a diverse range of stimuli, including membrane depolarization, noxious heat, vanilloid and endocannabinoid compounds, extracellular protons, and inflammatory mediators. It has been shown that TRPV1 plays a pivotal role in pain and neurogenic inflammation.¹⁵ TRPM channels exhibit highly variable permeability to Ca²⁺ and Mg²⁺, ranging from Ca²⁺ impermeable (TRPM4 and TRPM5) to highly Ca²⁺ and Mg²⁺ permeable (TRPM6 and TRPM7).^16,17 Although TRPMs have not yet been fully functionally characterized, TRPM1 appears to function as a tumor suppressor and TRPM3 is considered as a possible candidate gene involved in the etiology of amyotrophic lateral sclerosis (ALS).^18,19 The TRPML subfamily consists of 3 members, which are primarily intracellular proteins in cytosolic compartments^20,21 and characterized by a large E1 loop (between S1 and S2) with several N-glycosilation sites. All TRPMLs have short cytosolic tails (between 61 to 72 amino acids) with a palmitoylation site in a cysteine-rich region.^21,22 TRPP subfamily has 3 members, TRPP2, TRPP3, and TRPP5 (also named as PKD2, PKD2L1, and PKD2L2, respectively), mutations in Trpp2 and PKD1 lead to autosomal dominant polycystic kidney disease.²³ The only member of the TRPA subfamily TRPA1 has recently emerged as another potential therapeutic target in the treatment of chronic visceral inflammation and pain except for TRPV1. Meanwhile, the role of TRPA1 in gastrointestinal inflammatory disorders is becoming increasingly clear.²⁴ Although connections of TRP channels to a plethora of diseases are well documented, the regulatory mechanisms of TRP channels and their effects on particulars of Ca²⁺ homeostasis and cellular functions are not well understood. This review focuses on the aspects of interactions between TRP channels and PRRs in mediating the immune and inflammatory responses.

Figure 1. Classification and structural topology of transient receptor potential (TRP) channels. All TRP channels are membrane proteins with 6 putative transmembrane segments (S1–S6) and a cation-permeable pore region between S5 and S6. The cytoplasmic amino (N) and carboxy (C) termini are variable in length and contain different domains. Currently, 28 different mammalian TRP channels are identified and classified into 6 subfamilies on the basis of sequence homology: TRP canonical (TRPC; TRPC1–7), TRP vanilloid (TRPV; TRPV1–6), TRP melastatin (TRPM; TRPM1–8), TRP polycystin (TRPP; TRPP2, TRPP3, TRPP5), TRP mucolipin (TRPML; TRPML1–3), and TRP ankyrin (TRPA; TRPA1).

Innate Immune Receptors: Classification and Function

The innate immune response is initiated through the activation of PRRs by PAMPs or DAMPs. Currently, 4 major classes of PRRs have been identified consisting of transmembrane proteins such as the Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), as well as cytoplasmic proteins such as the Retinoic acid-inducible gene (RIG)-I-like receptors (RLRs) and NOD-like receptors (NLRs).²⁵ Among them, TLRs and NLRs are 2 major subfamilies of PRRs, which provide immediate responses against pathogenic invasion or tissue injury.^1,26,27 TLRs, the first identified PRRs, are type I transmembrane proteins with an extracellular or luminal binding domain composed of leucine-rich repeats (LRRs) and intracellular Toll–interleukin 1 (IL-1) receptor (TIR) domains required for downstream signal transductions.^28,29 TLRs are normally divided into 2 major subgroups based on their cellular localization and PAMP ligands.³⁰ TLR3, TLR7, TLR8, and TLR9 detect virus-derived ssRNA or dsRNA and are located in intracellular vesicles such as the endoplasmic reticulum (ER), endosomes and lysosomes. Conversely, TLR1, TLR2, TLR4, TLR5, and TLR6 are located on the cell surface and can sense various PAMPs, including lipopolysaccharide (LPS)4, peptidoglycan, bacterial flagellum, and fungal cell wall components.^31,32 As shown in Figure 2, upon activation, TLRs recruit a specific set of adaptor molecules that harbor TIR domain, such as MyD88 and TIR-domain-containing adaptor inducing IFN-β (TRIF), and initiate downstream signaling events, thereby lead to the secretion of proinflammatory mediators.³³ In addition, TLR signaling simultaneously induces maturation of dendritic cells (DCs), which is responsible for the induction of adaptive immunity.³⁰

Figure 2. Classification, activation and function of Toll-like receptors (TLRs) and NOD-like receptors (NLRs). TLRs are transmembrane proteins with an extracellular or luminal binding domain composed of leucine-rich repeats (LRRs) and intracellular Toll–interleukin 1 (IL-1) receptor (TIR) domains required for downstream signal transduction. NLRs share highly conserved structures consisting of (1) a centrally located, conservative nucleotide-binding oligomerization domain (NBD) that mediates self-oligomerization and is essential for the activation of NLRs, (2) a C-terminal leucine-rich repeat (LRR) domain that is involved in recognition of conserved microbial patterns or other ligands, and (3) a N-terminal effector domain, which is responsible for protein–protein interaction with adaptor molecules that result in signal transduction. Inflammasomes are a subfamily of NLR protein complexes that recognize a diverse set of inflammation-inducing stimuli and that control the production of important proinflammatory cytokines such as IL-1β and IL-18.

The NLR family of intracellular PRRs has recently been identified and plays a critical role in the control of inflammatory and immune responses through the modulation of different signaling pathways, including those dependent on NF-κB and caspase-1-mediated cleavage of IL-1β and IL-18.³⁴ NLR proteins share highly conserved structures^29,35and are divided into several subfamilies based on the nature of their N-terminal domains (Fig. 2).³⁶ NOD1 and NOD2 are 2 well-characterized members of noninflammasome NLRs, which mediate the activation of NF-κB and mitogen-activated protein kinases (MAPKs) in response to peptidoglycan-related molecules.³⁷ In dendritic cells, macrophages, and monocytes, activation of NODs leads mainly to the production of proinflammatory cytokines and expression of co-stimulatory molecules and adhesion molecules.³⁸ In addition to being present in inflammatory cells, studies from our laboratory and by others have found that NOD2 is also highly expressed in the kidney including renal proximal tubule epithelial cells³⁹ and glomerular cells⁶⁹ and cardiovascular system including vascular smooth muscle cells⁴⁰ and endothelial cells.⁴¹

Inflammasomes are a subfamily of NLR protein complexes that control the production of proinflammatory cytokines such as IL-1β and IL-18.⁴² Furthermore, they have been found to regulate other important aspects of inflammation and tissue repair such as pyroptosis, a form of cell death.⁴³ Among the NLRs, NLRP1, NLRP3, NLRP6, NLRP12, and IPAF have been identified to assemble into inflammasomes. The inflammasome is composed of the intracellular adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) and a “sensor NLR” that function as molecular scaffolds for the activation of caspase-1 to secrete IL-1β and IL-18.⁴⁴ The NLRP3 inflammasome is one of the most extensively studied NLR members and is activated by a wide range of signals of pathogenic, endogenous, and environmental origin.^45,46 In addition, NLRP3 is also alerted to the presence of endogenous danger signal molecules released during or indicative of tissue injury, such as extracellular ATP, hyaluronan, amyloid-β fibrils, and uric acid crystals.⁴⁷ Currently, 3 distinct mechanisms have been proposed to account for NLRP3 activation: potassium efflux,^48,49 the generation of reactive oxygen species (ROS),⁵⁰ and phagolysosomal destabilization.^42,43,51 Except for the critical role of NLRP3 in common autoimmune diseases, more recent studies have indicated that NLRP3 inflammasome contributes to the regulation of insulin signaling,⁵² and the pathogenesis of atherosclerosis,⁵¹ myocardial ischemia-reperfusion injury,⁵³ and neurodegeneration.^43,54 In this review, we describe recent findings of the interactions between PRRs and TRP channels.

TLR-Mediated Inflammatory Response is Associated with TRP Channel-Dependent Ca²⁺ Signaling

There is considerable evidence indicating that intracellular Ca²⁺ as a second messenger participates in TLR4-dependent signaling. However, how intracellular Ca²⁺ is increased in response to LPS and how they affect inflammatory response is poorly understood. An increasing number of studies have indicated that TLR-mediated immune response is associated with TRP channel-dependent Ca²⁺ signaling (Fig. 3A). Studies from Tauseef M et al. have identified a function of TRPC6 in endothelial cells showing that TRPC6-dependent Ca²⁺ signaling intersects with the TLR4 signaling pathway and hence contributes to LPS-induced lung endothelial permeability and NF-κB–dependent lung inflammation.⁵⁵ They found that LPS induces Ca²⁺ entry in endothelial cells in a TLR4-dependent manner and deletion of TRPC6 renders mice resistant to this endotoxin-induced barrier dysfunction and inflammation, and protects against sepsis-induced lethality. Furthermore, they showed that LPS induces the production of the second messenger DAG in endothelial cells, which directly activates TRPC6 in a TLR4-dependent manner. TRPC6-mediated Ca²⁺ entry in turn activates myosin light chain kinase (MYLK), a regulator of endothelial contractility, resulting in LPS/TLR4-mediated NF-κB activation and contributing to the mechanism of lung inflammation.⁵⁵ In addition to TRPC6, recent studies have also observed other TRP channels in mediating LPS-induced signaling pathways. A report has shown that LPS binds to receptors in trigeminal neurons and evoked a concentration-dependent increase in intracellular Ca²⁺ and inward currents. Furthermore, they found that LPS significantly sensitizes TRPV1 to capsaicin which is blocked by a selective TLR4 antagonist, indicating that LPS is capable of directly activating trigeminal neurons, and sensitizing TRPV1 via a TLR4-mediated mechanism.⁵⁶ Recent studies by Yamashiro et al. have shown that TRPV2, but not members of the TRPC or TRPM families, mediates intracellular Ca²⁺ mobilization, which is involved in LPS-induced TNF-α and IL-6 expression in NF-κB-dependent manner in RAW264 macrophage.⁵⁷ Because in these cells LPS acts through TLR4, it is tempting to speculate on a role of TRPV2 in modulating TLR4-dependent signaling in macrophages. Among TRPMs, TRPM4 has been demonstrated to be critically involved in LPS-induced endothelial cell death and inhibition of TRPM4 activity protects endothelium against LPS injury⁵⁸ suggesting that the effects of LPS are, at least in part, associated with TRPM4 signaling.

Figure 3. Summarized recent findings of the interactions between pattern-recognition receptors (PRRs) and transient receptor potential (TRP) channels. (A) TLR-mediated inflammatory response is associated with TRP channel-dependent Ca²⁺ signaling. (B) TRP channel-dependent Ca²⁺ signaling intersects with NLR signaling pathways.

TRP Channel-Dependent Ca²⁺ Signaling Intersects with NLR-Signaling Pathways

Although hyperhomocysteinemia (hHcys) has been recognized as an important independent risk factor in the progression of end-stage renal disease (ESRD) and in the development of cardiovascular complications related to ESRD,^59-61 the mechanisms triggering the pathogenic actions of hHcys are not yet fully understood. Recent studies from our laboratory investigated the contribution of NOD2 to the development of glomerulosclerosis in hHcys. Our results showed that NOD2 deficiency ameliorates renal injury in mice with hHcys. We further found that NOD2 induces TRPC6 expression and activity, leading to intracellular Ca²⁺ release, ultimately results in podocyte cytoskeleton rearrangement and apoptosis (Fig. 3B). This study for the first time establishes a previously unknown function of NOD2 for the regulation of TRPC6 channels, suggesting that TRPC6-dependent Ca²⁺ signaling is one of critical signal transduction pathways that links innate immunity mediator NOD2 to podocyte injury.⁶² In addition, TRPC6 has been linked to hypertension, cardiac hypertrophy, cardiac fibrosis, and neuronal degeneration. Therefore, pharmacological targeting of NOD2-mediated TRPC6 signaling pathways at multiple levels may help design a new approach to develop therapeutic strategies for prevention and the treatment of hHcys-associated end-organ damage.

On the contrary, TRPM2-mediated calcium mobilization can also induce NLRP3 inflammasome activation as shown in Figure 3B.⁶³ Although reactive oxygen species (ROS) have a central role in NLRP3 inflammasome activation, how ROS signal assembly of the NLRP3 inflammasome remains elusive. It has been shown that accumulation of ROS can induce a calcium influx via the TRPM2 channel.^64,65 TRPM2 is an oxidant-activated nonselective cation channel that is widely expressed in mammalian tissues and immune cells and plays a crucial role in innate immune regulation.⁶⁶ TRPM2-mediated calcium influx has been implicated in ROS-induced chemokine production in monocytes.⁶⁵ ROS stimulates intracellular ADP-ribose formation which, in turn, opens TRPM2 channels. These channels act as an endogenous redox sensor for mediating ROS-induced Ca²⁺ entry and the subsequent specific Ca²⁺-dependent cellular reactions such as endothelial hyper-permeability and apoptosis and inflammatory neutrophil infiltration.^64,65 Zhong et al. have identified liposomes as novel activators of the NLRP3 inflammasome and further demonstrated that liposome-induced inflammasome activation also requires mitochondrial ROS. Moreover, they found that stimulation with liposomes/crystals induces ROS-dependent calcium influx via the TRPM2 channel, macrophages deficient in TRPM2 drastically impair NLRP3 inflammasome activation and IL-1β secretion.⁶³ In addition, TRPM2 channel has been demonstrated to be required for innate immunity against Listeria monocytogenes, a known activator of the NLRP3 inflammasome.^67-69 Collectively, these results indicate that TRPM2 as a key factor that links oxidative stress to the NLRP3 inflammasome activation and targeting TRPM2 may be effective for the treatment of NLRP3 inflammasome-associated inflammatory disorders.

Concluding Remarks

Despite remarkable progress in TRP channels and PRRs, there are still numerous aspects of their activation that need to be understood. In particular, the importance of TRP channel and PRR interactions has just started to be elucidated. Therefore, understanding the interactions of the TRP channel and PRR pathways will provide new insights into their roles in the pathogeneses of inflammatory diseases which are involved in the changes in ion transport.

Glossary

Abbreviations:

ALS

amyotrophic lateral sclerosis

ASC

apoptosis-associated speck-like protein containing a CARD

BIR

baculovirus inhibition of apoptosis protein repeat

CARD

caspase-recruiting domain

CIITA

class II transactivator

CLRs

C-type lectin receptors

DAG

diacylglycerol

ER

endoplasmic reticulum

FIIND

function to find domain

Hcys

homocysteine

IRF

IFN regulatory factor

LPS

lipopolysaccharide

LRRs

leucine-rich repeats

LRRCT

leucine-rich repeat C-terminal

MAPK

mitogen-activated protein kinase

MDP

muramyl dipeptide

MKKs

MAPK kinases

MYD88

myeloid differentiation factor 88

MYLK

myosin light chain kinase

NAIP

NLR apoptosis inhibitory protein

NBD

nucleotide-binding oligomerization domain

NLRs

NOD-like receptors

NLRPs

NOD-like receptor pyrin domain containing

NODs

nucleotide binding and oligomerization domain-containing proteins

PYD

pyrin domain

RIG

Retinoic acid-inducible gene

RIP2

receptor-interacting protein 2

ROS

reactive oxygen species

TAK1

TGF-β-activated kinase 1

TBK1

TANK-binding kinase 1

TIR

Toll–interleukin 1 (IL-1) receptor

TIRAP

TIR domain–containing adapter protein

TLRs

Toll-like receptors

TRAF6

TNF receptor-associated factor 6

TRAM

TRIF-related adaptor molecule

TRIF

TIR-domain-containing adaptor inducing IFN-β

TRP

transient receptor potential

TRPA

TRP ankyrin

TRPC

TRP canonical

TRPM

TRP melastatin

TRPP

TRP polycystin

TRPPM

TRP mucolipin

TRPV

TRP vanilloid