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2 Role of Transglutaminase 2 (TG2) in Oxidative Stress-Related Diseases

2 Role of Transglutaminase 2 (TG2) in Oxidative Stress-Related Diseases

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E.M. Jeong and I.-G. Kim

Fig. 14.2 Role of TG2 in the development of oxidative stress-associated diseases. TG2 regulation

by oxidative stress is involved in the pathogeneses of various diseases as described in Table 14.1.

ROS reactive oxygen species, ER endoplasmic reticulum, AT2 receptor angiotensin II receptor

type 2, ECM extracellular matrix, DM diabetes mellitus

Moreover, a causal role of TG2 has been verified in various animal models using

TG2-deficient mice.

These findings indicate that TG2 is a downstream effector of the redox signaling

pathway, even though the exact molecular mechanism by which TG2 is activated

has not been thoroughly elucidated. In the following sections, we evaluate the body

of literature describing the regulation of TG2 by oxidative stimuli, especially

focusing on TG2 regulation by transcriptional controls (Sect. 14.3), posttranslational modifications of TG2 (Sect. 14.4), and regulation by cellular mediators (Sect. 14.5). A full understanding of ROS-mediated TG2 activation could help

to design targeted therapeutics to prevent ROS-associated diseases.


ROS-Sensitive Transcription Factors Involved

in the Regulation of TG2 Promoter Activity

14.3.1 Nuclear Factor κ-Light-Chain-Enhancer of Activated

B Cells (NF-κB)

NF-κB is a transcription factor that plays a pivotal role in inflammation, immunity,

and cancer biology (Perkins 2007). NF-κB is activated via signaling pathways

under diverse oxidative stress conditions. The mechanisms of oxidative stressdependent NF-κB activation are depicted in Fig. 14.3. All pathways are dependent

on IκBα phosphorylation by three kinds of kinases: tyrosine kinase, casein kinase II


Regulation of Transglutaminase 2 by Oxidative Stress


Fig. 14.3 Transcriptional regulation of TG2 via oxidative stress signaling pathways. (1) NF-κB;

NF-κB is activated via three different signaling pathways in response to oxidative stress (Pinkas

et al. 2007). H2O2 or hypoxia induces phosphorylation at Tyr42 of IκBα. UV activates CK2, which

phosphorylates at the carboxyl terminal PEST domain of IκBα. Finally, genotoxic stress activates

ATM, a primary DNA damage sensor kinase that phosphorylates NEMO, leading to IKK activation and subsequent IκBα phosphorylation at Ser32 and Ser36. Phosphorylated IκBα proteins are

degraded via the ubiquitin proteasomal pathway, favoring NF-κB activation. TG2 transcription has

been reported to be activated by all oxidative stimuli described above (Jeong et al. 2009).

However, until now, only that genotoxic stress/ATM/IKK/NF-κB signal pathway was proven to

be involved in TG2 expression (Ai et al. 2012). (2) HIF-1; TG2 promoter is occupied by HIF-1

complex that consists of HIF-1α and HIF-1β subunits, resulting in its transcription activation in

response to hypoxia (Jang et al. 2010). HIF-1β is constitutively expressed, whereas HIF-1α is

hydroxylated by PHD and ubiquitinated by pVHL under normoxic conditions, resulting in its

destruction. PHD is not active in the absence of O2 and is inactivated by ROS generated from

mitochondrial complex III under low O2 conditions, favoring HIF-1 complex formation and

initiation of target gene transcription (Sabharwal and Schumacker 2014). However, it has not

been reported yet whether ROS are involved in HIF-1α-dependent TG2 expression under hypoxic

conditions. CK2 casein kinase 2, ATM ataxia telangiectasia mutated, NEMO NF-κB essential

modulator, IKK IκB kinase, pVHL Von Hippel–Lindau tumor suppressor, PEST a peptide

sequence rich in proline (P), glutamic acid (E), serine (S), and threonine (T ), HIF-1 hypoxiainducible factor-1, SOD1 superoxide dismutase 1, PHD prolyl hydroxylase

(CK2), and IκB kinase (IKK), each responding to different oxidative stimuli

(Perkins 2007). Genotoxic stress induced by ionizing radiation (IR) or some cancer

drugs activates ataxia telangiectasia mutated (ATM) kinase that phosphorylates

NF-κB essential modulator (NEMO) and forms an active IκB kinase (IKK)

complex to phosphorylate IκBα (Wu et al. 2006). UV induces CK2-dependent


E.M. Jeong and I.-G. Kim

phosphorylation of IκBα in its peptide sequence rich in proline, glutamic acid,

serine, and threonine (PEST) domain (Perkins and Gilmore 2006). H2O2 and

hypoxia promote phosphorylation of IκBα at tyrosine 42 residue, leading to its

degradation or separation from NF-κB (Perkins and Gilmore 2006). In all the above

cases, phosphorylated IκBα is eventually destroyed, thus resulting in NF-κB


The TG2 mRNA level increases in response to cytokines like TNFα and IL-6 via

the NF-κB-dependent signaling pathway (Kuncio et al. 1998; Suto et al. 1993). The

NF-κB signaling pathway seems to be also involved in TG2 expression induced by

various oxidative stimuli such as H2O2, UV, and doxorubicin (Jeong et al. 2009;

Mehta 1994). Actually, TG2 transcription increases via the ATM-dependent NF-κB

signaling pathway under genotoxic stress conditions (Ai et al. 2012) (Fig. 14.3). A

member of the NF-κB family, p65, binds to two functional sites located at -2121

and -1361 relative to the translation start site of TG2 promoter in human breast

cancer cells (Ai et al. 2012), indicating that TG2 is a bona fide target gene of


NF-κB-dependent expression of TG2 may be crucial to the pathogenesis of

oxidative stress-related diseases such as chemo- and radio-resistant cancer (Cao

et al. 2008; Herman et al. 2006), pathogenic inflammatory responses, and fibrosis

(Falasca et al. 2008; Oh et al. 2011). Interestingly, TG2 is known to activate NF-κB

signaling through its crosslinking activity for IκBα or PPARγ (Lee et al. 2004;

Maiuri et al. 2008). This implies that there might exist a positive feedback loop

generated by NF-κB and TG2, causing constitutive NF-κB activation. On the other

hand, a TG2-ablated mouse shows no obvious phenotype (De Laurenzi and Melino

2001), indicating that TG2 may be a responder to oxidative stress in a variety of

pathological contexts. Thus, TG2 can be considered as an effective druggable target

for NF-κB-related diseases (Kim 2006; Verma and Mehta 2007).

14.3.2 Hypoxia-Inducible Factor-1 (HIF-1)

HIF-1 is an oxygen-sensitive heterodimeric transcription factor comprising HIF-1α

and HIF-1β. Although HIF-1β is constitutively expressed, HIF-1α is only stabilized

at the protein level under hypoxic conditions. Subsequently, HIF-1α binds to the

promoter region of target genes and activates their transcription. In the presence of

oxygen, HIF prolyl hydroxylases (PHD) catalyzes the hydroxylation of HIF-1α at

proline residues. The hydroxylated HIF-1α is targeted by a component of the

ubiquitin E3 ligase complex, von-Hippel–Lindau-tumor-suppressor (pVHL) and

subsequently destroyed by the proteasome (Schofield and Ratcliffe 2004). Under

hypoxic conditions, PHD is inactive and HIF-1α destruction pathway is suppressed,

leading to the formation of transcriptionally active HIF-1 complex.

ROS are crucially involved in the regulation of HIF-1α protein stability under

hypoxic conditions (Fig. 14.3). Hyperoxia is generally regarded as an inducer of

ROS, which are generated by incomplete reduction of oxygen during respiration in


Regulation of Transglutaminase 2 by Oxidative Stress


the mitochondrial matrix. Paradoxically, a low oxygen concentration also produces

superoxide anions from the outer ubiquinone-binding site of the mitochondrial

complex III into the intermembrane space. This is probably due to the extended

presence of the semiquinone radical in complex III (Sabharwal and Schumacker

2014) (Fig. 14.3). Hypoxia-induced ROS inactivate PHD and subsequently increase

the HIF-1α expression level, allowing HIF-1 complex to activate the target gene


TG2 expression is induced by hypoxia in a HIF-1α-dependent manner in various

cancer cells (Fig. 14.3) (Jang et al. 2010). TG2 promoter contains three potential

hypoxia response elements (HRE), which are conserved sequences found in the

promoter regions of hypoxia-inducible genes. Among them, in response to hypoxia,

the HIF-1 complex binds to only one functional HRE located at -371/-368 relative

to the translation start site of TG2 promoter in U373MG human glioblastoma cells

(Jang et al. 2010). Thus, TG2 is an actual target gene of HIF-1.

Hypoxia-responsive TG2 might play a crucial role in tumor cell survival,

invasion, and chemo- and radio-resistance (Jang et al. 2010). In addition, according

to recent reports, HIF-1α-dependent expression of TG2 is associated with hypoxiainduced smooth muscle cell proliferation in the pulmonary artery in a pulmonary

hypertension model (Penumatsa et al. 2014). Since hypoxia plays a central role in

numerous pathophysiologies, it is suspected that HIF-1α-dependent expression of

TG2 might also be involved in other cellular responses to hypoxia such as metabolic adaptation and angiogenesis (Basso and Ratan 2013; Wang et al. 2013).


Post-Translational Modifications (PTMs) of TG2

in Response to Oxidative Stress

14.4.1 Vicinal Disulfides

Studies have reported that TG2 is regulated by several PTMs including vicinal

disulfide formation, SUMOylation, and ubiquitination through the ROS-mediated

signaling pathway (Fig. 14.4). Of the PTMs, oxidation of cysteine residues in TG2

is considered to be a crucial regulatory mechanism for its activation because human

TG2 has 20 cysteines in the amino acid sequence and even the active site has

cysteine (Cys277). The sulfhydryl group (-SH) of cysteine is highly reactive to ROS

or RNS, forming oxidative modifications such as disulfide bridges, S-glutathionylation, S-nitrosylation, S-sulfenylation, S-sulfinylation, S-sulfonylation, Scysteinylation, and S-sulfhydration (Grek et al. 2013). Thus, it often acts as a sensor

in redox-sensitive proteins (Green and Paget 2004).

Previously, Fork and colleagues showed that purified guinea pig liver transglutaminase, TG2, was inactivated by treatment with various thiol oxidants or

alkylating agents (Chung and Folk 1970; Connellan and Folk 1969). On the other

hand, oxidized TG2 was reactivated in vitro by treatment of thiol-based reducing


E.M. Jeong and I.-G. Kim

Fig. 14.4 ROS induce post-translational modifications of TG2. (1) Intramolecular disulfides;

oxidative stress induces the formation of vicinal disulfide bond (Cys230-Cys370) in TG2, which

facilitates another vicinal disulfide (Cys370-Cys371) formation (Bogeski et al. 2011). TG2 with

vicinal disulfides is inactive, but can be reduced and activated by thioredoxin, which may be

secreted from IFN-γ-stimulated monocytes or possibly the celiac intestinal mucosa (Jin

et al. 2011). S-Glutathionylation at Cys230 of TG2 is suspected to be a transient modification to

promote vicinal disulfide formation (Bogeski et al. 2011). (2) S-Nitrosylation; under nitrosative

stress, TG2 can be modified by S-nitrosylation at up to 15 cysteine residues, resulting in its

inactivation in vitro and in vivo (Lai et al. 2001; Santhanam et al. 2010). (3) Ubiquitination; a

high level of ROS induces intracellular calcium overload that promotes TG2 degradation via the

ubiquitin proteasome pathway (Jeong et al. 2009). (4) SUMOylation; CFTRΔF508-induced ROS

induce TG2 interaction with PIASy, a SUMO E3 ligase. Consequently, TG2 is modified by

SUMO-1, which increases TG2 protein level via inhibition of ubiquitin proteasomal degradation,

favoring TG2 activation (Luciani et al. 2009). ROS reactive oxygen species, RNS reactive nitrogen

species, [Ca2+]i intracellular calcium concentration, GSH reduced glutathione, GSSG oxidized

glutathione, IFN-γ interferon gamma, PTM post-translational modification, PIASy protein inhibitor of activated STAT y, SUMO-1 small ubiquitin-like modifier-1

reagents such as dithiothreitol (DTT), whereas alkylated TG2 was not. The active

site Cys277 was not involved in the oxidation process. They suggested that TG2

might be inactivated through the formation of an intramolecular disulfide bridge

between certain regulatory cysteine residues.

TG2 conformation changes substantially upon activation. TG2 exhibits an open

form during calcium-mediated activation, however, it presents a closed inactive

form that is induced by binding to the nucleotide (see Chap. 1). Recently, an

intramolecular disulfide bond between Cys370 and Cys371 was found by solving

crystal structures of the open form of TG2 in complex with a reactive gluten peptide

mimetic inhibitor (Pinkas et al. 2007). Meanwhile, the x-ray crystal structures of

GTP or ATP-bound TG2, which have closed forms, revealed another vicinal

disulfide between Cys230 and Cys370 (Han et al. 2010; Jang et al. 2014). However,

the vicinal disulfides did not exist in the crystal structures of the GDP-bound form

(Liu et al. 2002). It seems that Cys370-Cys371 stabilizes the open structure of TG2

and Cys230-Cys370 stabilizes the nucleoside triphosphate binding closed conformation of TG2. Notably, residues of Cys230, Cys370, and Cys371 are proximate to each

other in the crystal structures of both the open and closed forms of TG2

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