An overview of carbonic anhydrases and membrane channels of synoviocytes in inflamed joints

Abstract

The highly aggressive fibroblast-like synoviocytes (FLSs) are inflammatory mediators involved in synovial joint destruction. Membrane channels and transporters are essential components of the cell migration apparatus and are involved in various cellular functions. Although evidence is emerging that cell migration is a physiological/pathological process, the mechanism of highly dynamic synoviocytes linked to the membrane channels and carbonic anhydrases (CAs) in inflamed joints is only partially understood. In this review, topics covered will give a brief overview of CAs and the membrane channels of synoviocytes. We have also systematically focused on the role of FLS channels and transporters under various conditions, including rheumatoid arthritis (RA), to understand the pathophysiology of the migration of synoviocytes as inflammatory mediators in joints.

1. Introduction

Rheumatoid arthritis (RA) is a common inflammatory autoimmune disease that induces diarthrodial joint inflammation¹. The fibroblast-like synoviocytes (FLSs), located in the synovium, mediate synovial joint destruction by releasing metalloproteinases (MMPs) and secreting cytokines, including interleukin (IL)-6, IL-Iβ, IL-8, and tumour necrosis factor (TNF)-α in RA^2–6. Immune cells including macrophages, T cells, B cells, mast cells, and etc. are activated in RA and play crucial roles to secrete various cytokines and mediate inflammation of joint⁷. Moreover, TGF-β and platelet-derived growth factor (PDGF) levels were elevated in the RA synovial fluids^8,9. The inflamed synovium activates local FLS and induces the invasion of FLS¹⁰. Figure 1 represents the inflammatory mediators including immune cells and cytokines in pathogenesis of RA.

Figure 1.

Inflammatory mediators in pathogenesis of RA. Synovial fluid interacts with inflammatory cytokines and immune cells, which have role in inflammation and joint destruction in RA synovium. Inflammation and hyperplasia of FLS involve in pannus formation of joint.

Cellular migration is not only an important physiological process related to wound healing, immune defence, and angiogenesis but also affects pathological processes, including tumour metastases and arthritis¹¹. Moreover, the maintenance of a polarised state is the basis for cell migration¹². Although the morphological polarisation of neuronal cells and epithelial cells is more critical than that of migrating cells, several mechanisms of the polarisation among classical polarised cells, such as neuronal and epithelial cells, and migrating cells follow similar principles. FLS are highly dynamic, and the chemotactic ability of the inflamed synovial fluid provides direction to the migration, invasion, and inflammatory/proliferative signalling events of pannus formation in the joint. To our knowledge, the membrane channels of FLS in migration have not been elucidated systematically. There is potential evidence for the involvement of carbonic anhydrases (CAs) and membrane channels in FLS migration in inflamed joints and has been discussed in the following sections.

2. Features of FLS

Normal synovial tissue is divided into two functional layers: a surface layer and sublining layer¹³. The surface layer is in direct contact with the intra-articular cavity, 1–2 layers deep consisting of two predominating cell types: macrophage-like synoviocytes (type A, MLS) derived from bone marrow, and FLS descended from the mesenchymal stem cells (type B)^10,13. Those two cell types are essential for maintaining joint homeostasis. The FLS is relatively richer than MLS and displays a variety of surface adhesion molecules, such as ICAM-1, CD90, and matrix proteins to help in the anchoring with extracellular matrix^14,15. The FLS contains endocrine and sensory functions and also possesses the epithelium-like nature¹⁶. While the typical feature of FLS is synovial hyperplasia during the inflammation, this review is designed to understand the dynamic role of FLS in the pathogenesis of RA and its associated membrane channels.

3. Carbonic anhydrases

Carbonic anhydrases are zinc metalloenzymes. The physiological role of CAs is related to membrane transporters and will be discussed in brief. CAs catalyse the reversible reactions of CO₂ and water to produce H⁺ and HCO₃^–. They play a prominent role in the transport of CO₂ and protons across biological membranes, such as intercellular, intracellular, and extracellular spaces and are involved in diverse physiological functions, including pH regulation, fluids, and enzymes secretion and bone resorption^17–19. Several CA isozymes are expressed in mammals²⁰. Architecturally, CAs include cytosolic isoenzymes (CA I, CA II, CA III, CA VII, and CA VIII), membrane-associated isozymes (CA IV, CA IX, CA XII, and CA XIV), mitochondrial isoenzymes (CA VA and CA VB), and secreted CA isoenzyme (CA VI)²¹. CAs produce HCO₃^–, which fuels the HCO₃^– transporter²². The Na⁺/HCO₃^– cotransporter, NBC1 cooperates with CA II and CA IV to modulate the intracellular pH²³. High H⁺ concentration is found in the synovial fluid of RA patients, indicating that acidic pH reflects the pathophysiology of inflammation²⁴. CA and HCO₃^–-modulating transporters contribute to the modulation of synovial pH. The intensity of the inflammation process and ache-related symptoms in RA-affected patients inversely correlate with the tissue pH values^24,25. Tissue acidosis was found to be unfavourable for the progression of both antibody-mediated and cellular immunity processes²⁶. Although evidence indicates the importance of pH regulation of synovial tissues in various conditions, the precise role of the pH regulatory mechanism and its related transporters needs to be clarified. Beyond pH regulation, substantial evidence has indicated that CAs are also involved in bone resorption, hypoxia, and autoantibody formation. Expression of CA I was increased in the synovium of spondylitis and may accelerate calcification and bone resorption²⁷. The overexpression of CA IX and XII, associated with tumour hypoxia, was revealed in the inflamed synovium^28,29. Enhanced oxidative stress of erythrocytes in RA has addressed its correlation with CA II autoantibody formation³⁰.

Despite these differences in the role of CA, the regulatory role of CA in transporter machinery involves cell migration coordinated with cell adhesion molecules and ion transporters. The function of the CAs was to acidify the extracellular environment, thereby reducing cell adhesion and consequently increasing invasion and migration of tumour cells³¹. Especially, CA IX and CA XII were enhanced by hypoxic condition in tumour cells³². Hypoxia-inducible factor (HIF) affected the migration, cellular pH, and cell survival associated with tumour growth³³. The CA IX has been linked to cell–cell connections in the cell membrane, controlled by E-cadherin³⁴. It also regulated the cell migration by inhibiting E-cadherin associated with cell adhesion and interacting with the bicarbonate transporter, anion exchanger 2, in the leading edge regions in SiHa cells³¹. Bicarbonate transporters not only controlled the pH of the cells but also affected cell migration³⁵. The deficiency of SLC4A4 (NBCe1), an electrogenic Na⁺/HCO₃^– co-transporter, was influenced by cell migration by interfering with the intracellular pH regulatory mechanism in MDA-MB 231 breast cancer cells³⁶. However, information on CAs on the RA FLS remains unclear. The verification of regulatory and migration role of CAs in FLS will provide a new scope for synovial physiology.

4. Membrane ion channels of FLS

4.1. Aquaporins

The aquaporins (AQPs) are water or small molecule-transporting channel proteins across the plasma membranes of various human tissues and cell types³⁷. Thirteen types of AQPs (AQP0–AQP12) from mammalian tissues have been cloned and sequenced³⁸. The AQPs are classified into two groups: water selective channel (orthodox AQPs) and water, glycerol, nitrate (AQP6), and urea channel (aquaglyceroporins; AQP 3, AQP7, and AQP9)³⁹. The permeability of AQPs is dependent on osmotic and hydrostatic gradients and pH values. Several investigations have shown the involvement of AQPs in cartilage damage in joint diseases like RA and osteoarthritis (OA). AQP1 is distributed in the articular cartilage and the synovium⁴⁰. AQP1 is also expressed in chondrocytes and synoviocytes of RA patients⁴¹. Up-regulated AQP1 found in the inflamed synovial tissues of RA patients might play a potential pathological role in hydrarthrosis and joint swelling⁴². Acetazolamide, AQP1 inhibitor, was decreased AQP1 protein level via inhibition of NF-κB activation and subsequent reduction of hind pow swelling in adjuvant-induced arthritis rats, suggesting that attenuation of AQP1 mediates anti-arthritis effect⁴². It is well-known that AQP4 possesses high water permeability than that of AQP1⁴³ and its role in the nervous system has been studied⁴⁴. AQP4 is over-activated in rat articular chondrocytes and high homologues of AQP4 between rat and human⁴⁵; however, the pathological role of AQP4 in RA is still unclear. AQP9 was strongly induced upon treatment with TNF-α in FLS and was also expressed in the RA and OA synovial tissues⁴¹. Although the pathological roles of AQP in the synovial tissues remain to be elucidated, experimental evidence has revealed that AQPs are involved in the pathogenesis of hydrarthrosis and synovitis (Table 1).

Table 1.

AQPs in FLS.

AQP	Mechanism	Species	Ref.
AQP1	Hydrarthrosis and joint swelling Inhibiting NF-κB pathway by AQP1 inhibitor	Adjuvant-injected arthritis rats	42
AQP4	Over-activated AQP4 in articular chondrocytes	Articular chondrocytes, adjuvant-injected arthritis rats	45
AQP9	Hydrarthrosis	HepG2, FLS from OA and RA patients	41

4.2. K⁺ channels

Ca²⁺-activated potassium channel K_Ca1.1 (known as BK, Maxi-K, Slo1, or KCNMA1) is the only member of the K_Ca1.1 potassium channel family⁴⁶. The K_Ca1.1 channel consists of α-subunits and β-subunits comprising of four different isoforms (β₁, β₂, β₃, and β₄)⁴⁷. The K_Ca1.1 was a major K⁺ channel expressed in FLS plasma membrane in RA⁴⁸. Blocking the K_Ca1.1 channel in RA FLS by inhibiting the α-subunit interrupted Ca²⁺ homeostasis; the proliferation, migration, and the invasiveness of cells; and the cytokines and chemokines⁴⁸. The K⁺ channels in the plasma membrane of cells play a critical role in regulating β₁ integrins by influencing Ca²⁺ homeostasis⁴⁹. The FLS cells express a variety of integrins, α₄, α₅, α₆, and β₁ isotype⁵⁰. Blocking of K_Ca1.1 channel interrupted Ca²⁺ homeostasis, thus affecting integrin expression⁴⁹. Enhanced integrin ligation increased cytokine signalling and growth factor expression, thus leading to the expression of matrix MMPs⁵⁰. Blocking of K_Ca1.1 activity or its expression reduced the FLS proliferation and expression of pro-MMP2 and attenuated the subsequent FLS invasion. On the contrary, activated K_Ca1.1 or overexpression of the channel enhanced the invasiveness of FLS⁵¹. Regulation of K_Ca1.1 of FLS also affected the proliferation and migration of CCR7^– effector memory T cells, another major cell type implicated in the progression of RA⁵².

4.3. Acid-sensing ion channels

Acid-sensing ion channels (ASICs) mediate tissue acidosis by pH changes are known as voltage-insensitive, ligand-gated cation channels with protons^53,54. The ASICs are associated with inflammatory pain, and especially ASIC1 and ASIC3 contribute to the musculoskeletal pain⁵⁵. The ASIC3 is expressed in the sensory neurons that innervate the synovial joints by increasing the intracellular Ca²⁺ levels upon sensing a decrease of pH in the inflamed joint^56,57. Synovial inflammation and inflammatory cytokine levels were increased that led to joint destruction in ASIC3–/– mice⁵⁵. FLS were activated with the decrease in pH; the acidic environment increased the intracellular Ca²⁺ levels by ASIC3⁵⁷. Activation of FLS in acidic pH mediates the accumulation of inflammatory cytokines. In addition, activation of ASIC3 by acidic pH evokes Ca²⁺ signalling, which lead to the apoptosis of FLS by phosphorylation of the MAP kinase ERK in synovial inflammation; thus, it could be a blockade of synovial proliferation⁵⁸. Activation of ASIC3 can be a therapeutic strategy for reducing inflammatory FLS level and subsequent disease progression in an inflamed joint.

4.4. Ca²⁺ signalling of FLS

Intracellular Ca²⁺ plays crucial roles in various physiological processes, including the flow of nerve impulses, muscle contraction, cell division, and hormone secretion⁵⁹. Enhanced Ca²⁺-activated phosphatase calcineurin activity and Ca²⁺ release by proinflammatory cytokine were observed in RA FLS, suggesting that dysregulated Ca²⁺ signalling involved in the pathogenesis of chronic arthritis⁶⁰. In addition, synovial fluid of patients with RA contains ATP⁶¹ and FLS expressed P2X7 receptor and functionally involved in ATP-dependent Ca²⁺ release and subsequently mediated IL-6 release⁶². Generally, the cytosol is surrounded by two major Ca²⁺ sources; the intracellular Ca²⁺ stores including sarco/endoplasmic reticulum (SR/ER), nucleus, golgi, and mitochondria and the extracellular media⁶³. The Ca²⁺ is released from intracellular stores or enters into the cells through the plasma membrane⁶⁴. The Ca²⁺ homeostasis is maintained by two types of membrane ATPase, the SR/ER Ca²⁺-ATPase (SERCA) and plasma membrane Ca²⁺-ATPase (PMCA). These pumps are involved in reduction of cytosolic Ca²⁺, from cytosol to intracellular Ca²⁺ stores by the SERCA and to the extracellular space by the PMCA⁶⁵. Na⁺/Ca²⁺ exchangers are also known to have a critical role in Ca²⁺ removing mechanism with Na⁺ regulation^66,67. The ER also contains inositol-1,4,5-trisphosphate (IP₃) receptors (IP₃Rs) and ryanodine receptors (RyRs), which provide conduits for the rapid release of Ca²⁺⁶⁸. The agonist stimulation such as receptor activation leads to the generation of IP₃, which releases to the cytosol and binds to the intracellular membranes to release Ca²⁺ from the intracellular stores of Ca²⁺⁶⁹. Although the Ca²⁺ signalling and its signalling proteins have been well established, the network of Ca²⁺ signalling in FLS needs to be clarified more extensively.

4.5. TRP channels

Although there is relatively low evidence in Ca²⁺ signalling network in FLS, studies of transient receptor potential (TRP) channels have been performed in various reports. The TRP channels have been known to be nonselective cation channels and play a critical role in inflammatory pain of arthritis^70,71. FLS express the TRP family proteins, including TRPC (TRPC-canonical) 1, TRPC5, TRPA (TRP-ankyrin) 1, TRPV (TRPV-vanilloid) 1, TRPV2, TRPV4, TRPM (TRPM-melastatin) 7, and TRPM8^72–76. We will discuss FLS-related TRP channel activation and will provide information on the following section. The detailed mechanism is summarised in Figure 2 and Table 2.

Figure 2.

Activators of TRP channels in RA FLS. The activators of FLS-associated TRP channels are summarised. The mechanism of action is represented in Table 2. Activation of TRP channels increases intracellular Ca²⁺ level and is involved in various functions including the reduced MMP secretion, joint destruction, enhancement of pain, and apoptosis of inflamed FLS. TRPV: transient receptor potential vanilloid channels; TRPC: transient receptor potential canonical channels; TRPM: transient receptor potential melastatin channels; TRPA: transient receptor potential ankyrin channels.

Table 2.

TRP channels in FLS.

TRPs	Mechanism	Species	Ref.
TRPC1/TRPC5	Reduced MMP secretion and joint inflammation	Human FLS, mouse joint tissue	76,77
TRPV1	Promoted inflammation and joint destruction	SW982 human synovial cells	88
TRPV2	Reduced expression of the MMP2 and MMP3 proteins	FLS from DA (severe and erosive arthritis)	90
TRPV4	Reduced IL-8 production	FLS with RA and without RA, MH7A	89
TRPA1	Increased pain-related response	Human FLS, ddY mice	75
TRPM3	Decreased hyaluronan secretion	HIG-82 cells (FLS cell-line), joint tissue of RA patients	94
TRPM7	Activated ER stress, increased apoptosis of FLS	FLS from RA	99

4.5.1. TRPC

The TRPC1 and TRPC5 were expressed in secretory FLS^76,77. The expression of oxidoreductase thioredoxin, a well-known oxidative stress marker, was increased in RA synovial fluids to counteract oxidative stress^78–80. The thioredoxin was considered as a costimulatory component with cytokines in FLS⁸¹ and can be secreted⁸². Extracellular reduced form of thioredoxin enhanced the activities of TRPC1 and TRPC5 channels as new extracellular targets⁷⁷. Inhibition of these channels by antibodies enhanced MMP secretion and suppressed the thioredoxin-mediated inhibitory effect on secretion⁷⁷. More recently, the study of TRPC5 KO mice and inhibition of TRPC5 channels by antagonist addressed the enhanced inflamed joint and hyperalgesia⁷⁶, suggesting that functional modulation of TRPC 1/5 could be considered as therapeutic targets for RA.

4.5.2. TRPA

The TRPA1 is a cold-sensitive and Ca²⁺-permeable nonselective cation channel and plays an essential role in inflammation and pain⁸³. For the evidence of TRPA1 expression in FLS, mRNA of TRPA1 has been detected in SW982 human synoviocytes⁷⁴. Diphenyleneiodonium (DPI) as a TRPA1 activator induced Ca²⁺ signal in TRPA1-expressing FLS and pain response in ddY mice⁷⁵. More recently, it has been reported that proinflammatory FLS can be attenuated by TRPA1 activation. TNF-stimulated FLS enhanced protein level of TRPA1 and subsequent stimulation of TRPA1 enhanced the necrosis⁸⁴.

4.5.3. TRPV

The TRPV channels sense heat, protons, lipids, and osmolarity^85,86. The RA and OA patients possess pain linked to TRPV1⁷³. Capsaicin, an agonist of TRPV1, increased IL-6 mRNA and protein levels by promoting pro-inflammatory cytokines⁸⁷. Activation of TRPV1 enhanced mRNA level and protein level of IL-6 in FLS from RA and OA patient and application of TRPV1 antagonist could be therapeutic strategy to modulate nociception from arthritis⁷³. Activated FLS-mediated TNF-α secretion enhanced the expression of TRPV1 and TRPV4 in SW982 human synoviocytes⁸⁸. Especially, 4α-phorbol-12,13-didecanoate (4α-PDD), a selective TRPV4 agonist and hypotonic stimulation induced an increased intracellular Ca²⁺ level and decreased IL-8 secretion in RA⁸⁹. The enhanced TRPV2 expression was associated with invasion of FLS from rats using gene profile technique⁷². However, functional activation of TRPV2 by specific TRPV2 agonists, O1821 and LER13, dramatically reduced IL-1β-mediated expression of the MMP2 and MMP3 proteins in FLS and reduced the severity of disease and genetic deletion of TRPV2 enhanced the invasiveness of FLS⁹⁰. Expression of TRPV2 involves in invasion mechanism and further functional stimulation of TRPV2 attenuates the invasiveness, suggesting that regulation of TRPV2 can be also novel therapeutic strategy of RA such as TRPC and TRPA channels.

4.5.4. TRPM

The Ca²⁺ entry through TRPM3 is involved in cell survival, death, growth, and differentiation⁹¹. Hyaluronan, known as the major component of the extracellular matrix, was increased in RA patients⁹². The increased secretion of hyaluronan from RA FLS was reduced by TRPM3 activator pregnenolone sulphate, activating TRPM3-mediated Ca²⁺ entry^93,94. The TRPM7 mediates a variety of functions, such as cell cycle, migration differentiation, and regulation of Ca²⁺ homeostasis and it is correlated with the oxidative stress-induced cell injury^95–97. It has been proposed that hypoxia and low glucose also lead to ER stress in RA joints⁹⁸. Inhibition of TRPM7 by Gd³⁺ and 2-aminoethoxydiphenyl borate (2-APB) induced RA FLS apoptosis by activating ER stress⁹⁹.

5. Future perspectives

We limited our review to the most relevant channels related to cell migration in RA FLS. Evidence related to cell migration by water and ion channels addressed the housekeeping functions. The migration or invasion is the major feature of cancer cell. The major consequences between the cancer cells and inflamed FLS are hypoxia and acidic circumstances¹⁰⁰. The cancer cells employ a circuit of ion transporters and enzymes to avoid the detrimental consequences of hypoxic and acidic tumour microenvironment. Alterations of CA IX and CA XII are associated with various cancers and considered oncogenic factors¹⁰¹. The overexpression of CA IX and XII in the inflamed synovium^28,29 provides the several similarities of pathology between inflamed synovium and cancer (Figure 3). Such similarities between microenvironments including hypoxia, acidic pH, and enhanced CA IX and XII can be speculated, as RA FLS would share the migration mechanism with cancer cells. Thus, therapeutic options in cancer therapy can be expanded and exploited for the RA model.

Figure 3.

Potential function of CAs in inflamed synovium. CA I was overexpressed in the synovium of the patients with ankylosing spondylitis²⁷. The overexpression of CA IX and XII was revealed in the inflamed synovium. Although determination of CA isoenzymes on the RA FLS remains unclear, there are several similarities between inflamed synovium and cancer. CA: carbonic anhydrase; pH_e: extracellular pH.

Currently, experimental evidence for the involvement of CAs and FLS membrane channels in RA is limited. The physiological and pathological roles of ion channels and transporters in dynamic FLS migration have not yet been studied in detail. Here, we have summarised the studies on membrane channels and regulatory enzymes of RA-FLS with an aim to understand their migrated state. However, many questions regarding RA-FLS still need to be clarified. What are the exact molecular mechanisms by which ion transporter affects the FLS migration apparatus? What are the exact components of synovial fluid that mediate the FLS dynamics? What are the components affecting the differential expression of CAs and membrane channels in FLS? What is the combined mechanism of CAs as regulatory enzymes? Several membrane channels and transporters show tissue-specific expression. Thus, unravelling the mechanisms by which ion channels and transporters are positioned in and modulate the migration of activated FLS will be a rewarding pursuit for the coming years. The motivation of channel physiologists is also needed to develop potential therapeutics to counter the critical pathophysiological involvement of FLS migration in joints in RA.

Funding Statement

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean Government (MSIT; [NRF-2019R1F1A1046785]) and by the grant from the Gachon University Gil Medical Center Project [FRD 2018–07].

Authors’ contributions

JHH and MJJ conceptualized and designed the study. MJJ prepared and revised the manuscript critically for intellectual content. JHH approved the final version of the manuscript.

Disclosure statement

The authors declare that they have no conflicts of interest with the contents of this article.