*2.2. Signal Transduction of Na,K-ATPase by CTS*

It is noteworthy that low-dose ouabain treatment activates the specific signaling without affecting the intracellular ion concentrations [21]. Mounting evidence has shown that Na,K-ATPase is engaged in the cardiac glycoside-mediated signaling cascades [22]. In the context of the signal-transducing functions of Na,K-ATPase, this pump is capable of communicating with signaling molecules by ligand-induced conformational changes within the signalosome [10]. Ouabain binding to the Na,K-ATPase in caveolae activates not only inositol triphosphate receptor (IP3R)-mediated Ca2+ release from S/ER but also Src kinase-mediated signal cascades [8]. CTS-induced conformational changes occurring within the caveolar sodium pump α1 subunit, which is a negative regulator of Src, allow the activation of Src [9].

Currently, Na,K-ATPase complex with Src is regarded as a unique receptor for CTS [9]. Activated Src triggers the execution of downstream signaling cascades, such as epidermal growth factor receptor (EGFR) transactivation-mediated Ras/Raf/mitogen-activated protein kinase (MAPK) kinase (MEK)/extracellular signal-regulated kinase (ERK) 1/2 pathways [23], inducing gene activation [10]. Src is known to be a key player in the Na,K-ATPase-mediated inter-receptor crosstalk with EGFR [24]. Activated as well are phosphoinositide 3-kinase (PI3K) [25] and EGFR-transactivated generation of reactive oxygen species (ROS), and the latter explains the clinical role of Na,K-ATPase as an oxidant amplifier contributing to the progression of cardiac fibrosis [9]. In addition, Src activates the phospholipase C (PLC) pathway [26] that also contributes to the IP3-induced Ca2+ release [10]. The signaling pathways of the sodium transporter are assumed to be implicated in hypertension, renal disease, diabetes, metabolic disease, and cardiovascular diseases. When there are alterations in the signaling pathways, signaling receptor function would change, causing hypertension, cardiac hypertrophy, tissue fibrosis, or cancer [9].

#### *2.3. Ouabain-Induced Hypertension and Related Diseases*

Ouabain, a well-known positive ionotropic agent in the heart, is also an endogenous adrenal cortical hormone because it can be synthesized in the adrenal glands [27]. It is known that the infusion of ouabain for a prolonged period in rodents [4–6], elevation of endogenous ouabain [28], and ouabain-like factors [29] in the circulation are associated with the development of hypertension. The elevation of endogenous ouabain also facilitates the proliferation of myocardial and smooth muscle cells [27]. Circulating endogenous ouabain inhibits the activity of the ouabain-sensitive Na,K-ATPase α2 subunit [4] and inhibition of the sodium pump by cardiac glycosides, such as ouabain, is associated with the pathogenesis of hypertension [17], cataract [18], and diabetic diseases [16], through the elevation of the intracellular Ca2+ concentrations.

#### **3. TCTP, as an Intracellular Na,K-ATPase Suppressor**

TCTP, alternatively referred to as fortilin, p23, and histamine-releasing factor (HRF), is a highly conserved multifunctional 172-amino-acid protein that is under a high degree of regulation both at transcriptional and translational levels [30]. TCTP plays fundamental roles in the regulation of cell cycle progression, apoptosis, autophagy, survival, stress responses, growth and development, and cytokine-like activities, among others [30]. Given the multiplicity of roles it plays, dysregulation of TCTP can lead to cardiovascular and metabolic diseases including systemic and pulmonary arterial hypertension [31], atherosclerosis, diabetes, carcinogenesis, and allergic and inflammatory disorders [32].

Our research group has identified TCTP as a cytoplasmic repressor for Na,K-ATPase [33]. In contrast to ouabain that binds to the outer part of the sodium pump, TCTP interacts with the cytosolic domain of Na,K-ATPase [33]. Searching for the cytoplasmic molecules that are potentially associated with the Na,K-ATPase α subunit, we screened a rat skeletal muscle DNA library using a yeast two-hybrid system. We found that the third large cytoplasmic domain (CD3) of Na,K-ATPase α1 and α2 isoforms interacts with the rat TCTP [33].

While studying the interaction of TCTP with the sodium pump in HeLa cells, we discovered the inhibitory effect of TCTP on Na,K-ATPase activity. Interaction of rat TCTP with the sodium pump does not affect the mRNA and protein levels of the Na,K-ATPase α subunit, indicating a relatively short-term regulation by TCTP [33]. TCTP inhibited the sodium pump activity in a dose-dependent manner [33]. In addition, the C-terminal region (102–172 residues) of rat TCTP was the moiety involved in the inhibition of pump activity of Na,K-ATPase [33]. These observations suggest that TCTP is possibly playing a promotive role in hypertension and raise the question of whether TCTP elevates cytosolic Ca2+ in vivo [33]. Interaction of the sodium pump with TCTP is now generally accepted to have important roles not only in the biological functions and signaling but also in the pathophysiology of human diseases [34–36].

It has been shown that TCTP plays either protective or causative roles in certain diseases in some conditions, but it is clear that the dysregulation of TCTP contributes to the development of various pathophysiological processes [37], as shown in several genetically altered mouse models (Table 1). Conventional or tissue/cell-specific TCTP-overexpressing transgenic mice (TCTP-TG) were used to study the gain-of-function of TCTP. Conversely, heterozygous TCTP-deficient mice (TCTP+/−), in which TCTP gene is deficient causing a loss of function, were studied because of embryonic lethality of homozygous TCTP knockout mice (TCTP−/−) [38]. This review focuses on the roles of TCTP in hypertension and related diseases, including atherosclerosis, cataracts, and other metabolic disorders that have been studied in genetically engineered animal models.


**Table 1.** Genetically modified mouse models that exhibit elevated or reduced expressions of TCTP and its phenotypes and pathophysiological significances in vivo.

↑ indicates increase; ↓ indicates decrease.

#### **4. Pathophysiology of Hypertension in TCTP-TG**

Animal models have been used for conducting etiological studies of genetic hypertension. These include spontaneous hypertensive rats (SHRs), Dahl salt-sensitive rats, and transgenic models generated by overexpression of particular genes [53]. To address the questions raised regarding the pathophysiological consequences of TCTP-mediated Na,K-ATPase inhibition in vivo, we constructed transgenic mice overexpressing TCTP (TCTP-TG). It was established in either C57BL/6×CBA hybrid or C57BL/6N inbred backgrounds using pCAGGS-TCTP cDNA construct consisting of cytomegalovirus enhancer (CMV-IE) and chicken β-actin promotor (Figure 1A).

**Figure 1.** Generation of TCTP-TG and TCTP-TG/ApoE KO mice. (**A**) Schematic representation of the gene construct used for TCTP transgenic overexpression in mice. The TCTP transgene of pCAGGS-TCTP cDNA containing CMV-IE enhancer and chicken β-actin promoter constructed in the transgenic expression vector pCAGGS was used to generate the transgenic mice. (**B**) Strategy of crossbreeding for generation of nTG/ApoE KO and TCTP-TG/ApoE KO mice exhibiting identical genetic backgrounds.

Their phenotypic characteristics are partly affected by the genetic background of TCTP-TG because, for example, cataractogenesis was profound in C57BL/6N background mice [41] while both types of transgenic mice show hypertensive properties regardless of their genetic backgrounds [39,41,44]. Because of the decreased fertility of inbred offspring, TCTP-TG was generated in C57BL/6×CBA hybrid strains in the majority of our investigations. For the study regarding atherogenesis, TCTP-TG was subjected to crossbreeding to generate apolipoprotein (ApoE)-deficient TCTP-TG (ApoE-KO/TCTP-TG) with a hybrid background (Figure 1B).

#### *4.1. Systemic Arterial Hypertension by Na,K-ATPase Inhibition in TCTP-TG*

Both C57BL/6×CBA and C57BL/6N mice overexpressing TCTP showed phenotypes of hypertension and hybrid mice were used in the study. The gender-related dimorphism regarding BP was observed, also reported in heterozygous female TGR(mRen2)27 transgenic rats [54], possibly due to the differences in hormonal changes or vascular responsiveness between female and male TCTP-TG mice occurring with ageing [39]. At approximately 6 weeks after birth, systemic arterial hypertension developed in both male and female TCTP-TG but after 19–20 weeks female TCTP-TG did not exhibit a significant difference in BP compared with non-transgenic (nTG) mice [39]. Therefore, heterozygous male TCTP-TG mice were studied for further investigations.

As with arterial BP elevation, an increase in left ventricular systolic pressure (LVSP) was found in TCTP-TG mice [39]. The thickening of the left ventricular posterior walls was observed in 15- to 16-month-old male TG mice, possibly caused by the sustained elevation of BP, but TCTP-TG did not exhibit the significant cardiac dysfunction [39]. TCTP-TG showed the characteristics of hypertension: first, the alteration of vascular functions that exhibit enhanced contractility of aortic rings devoid of endothelial layer, and secondly, augmented sensitivity to vasoconstrictor stimuli such as norepinephrine (NE) and serotonin (5-HT). Moreover, vascular relaxation reactivity following sodium nitroprusside (SNP) treatment after NE-induced contraction was decreased, indicating the reduced sensitivity to vasodilators [39]. Impaired vasodilatory responses accompanied by the hypercontractile profile in the vasculature isolated from TCTP-TG mice aberrantly elevated the BP [39].

As expected, vascular smooth muscle cells (VSMCs) isolated from TCTP-TG showed suppressed activity of Na,K-ATPase α1 and α2 isoforms that are the predominant Na,K-ATPase in mouse aorta, confirming the TCTP-induced sodium pump inhibition in VSMCs [39]. The expression of α1 in VSMCs from TCTP-TG (TG-VSMCs) was elevated, whereas that of α2 did not show any significant changes [39]. Because transient TCTP overexpression in HeLa cells did not affect the Na,K-ATPase expression, as observed in our earlier studies [33], elevated α1 expression in TCTP-TG mice suggests a potential compensatory mechanism due to the prolonged pump inhibition in vivo [39].

Then, we confirmed that inhibition of Na,K-ATPase activity in TG-VSMCs induces augmented intracellular Ca2+ mobilization, possibly through the reverse mode of NCX. Upon 5-HT stimuli, TG-VSMCs exhibited elevated Ca2+ transients that were mobilized from the ER [39]. Of interest, ouabain treatment at a concentration that blocks almost all the activity of Na,K-ATPase did not further enhance the 5-HT-evoked Ca2+ transients in TG-VSMCs whereas that of non-transgenic mice was elevated by ouabain, compared with untreated cells [39]. This phenomenon can be explained by the fact that ouabain may have negligible impact on TCTP-TG whose homeostasis has already been established by TCTP-induced Na,K-ATPase inhibition [39]. Likewise, VSMCs of TCTP-TG showed increased sensitivity to vasoconstrictor, elevated levels of Ca2+ in the resting state, and increased capacity for intracellular Ca2+ mobilization, and ultimately enhanced contraction by stimuli, accompanied by reduction in vasorelaxant-induced relaxation [39].

Taking these observations together, we delineated the pathophysiology of TCTPinduced hypertension using TCTP-TG, as depicted in Figure 2. Repression of Na,K-ATPase by TCTP overexpression results in the rise of cytosolic Na+ level, and subsequent elevation of cytosolic Ca2+ mobilization and Ca2+ storage, possibly through the reversion action of NCX, ultimately inducing the hyper-contractility of VSMC in TCTP-TG. Clearly, based on the fact that high blood pressure implies the dysfunctions in cardiac and/or arterial parameters [55], non-significant cardiac malfunctions indicate that TCTP-TG-induced hypertension is mediated by vascular malfunctions, as shown by elevated contractility and reduced relaxation in VSMC from TCTP-TG mice. This phenomenon is similar to that of ouabain treatment-induced hypertension exhibiting augmented vascular reactivity to vasopressors in renal artery of rats [56], confirming that TCTP can be viewed as an endogenous ouabain-like entity. Based on the observation that TCTP+/<sup>−</sup> and wild-type mice did not show a significant difference in systolic BP [45], overexpression of TCTP might be essential in inducing arterial systemic hypertension in vivo.

**Figure 2.** TCTP overexpression-induced consequences in transgenic TCTP-overexpressing (TCTP-TG) mice. Transgenic overexpression of TCTP led to the Na,K-ATPase inhibition, which results in the development of hypertension through elevated cytosolic Ca2+ levels and vascular contractility and tone. Such an increase in Ca2+ can also occur in the lens epithelial cells of TCTP-TG, at this time, inducing the activation of proteolytic enzyme and lens swelling, all of which affect the cataractogenic processes. RhoA/Rho kinase activation and resultant MYPT-1 phosphorylation in TCTP-TG appears to partly contribute to the increased sensitivity of myofibrils upon constrictor stimuli, thereby increasing the contractility of VSMC in TCTP-TG (↑ indicates increase).

#### *4.2. Upregulation of RhoA/Rho Kinase Signaling in TCTP-TG*

The contractility of smooth muscle cells is largely determined by the phosphorylation status of the regulatory myosin light chain (MLC) whose activity is under regulation by myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) [57]. Vasoconstrictors like Ang II binding to G-protein coupled receptors (GPCR) activates Gq/11, which in turn activates phospholipase C (PLC)-β and elevates Ca2+ concentration, thereby stimulating MLCK activity and induces G12/13 to activate RhoA/Rho kinase and, in turn, inhibits MLCP. A myosin binding subunit, the myosin phosphatase target unit (MYPT-1) of MLCP, is one of the most important substrates of Rho kinase [57]. Both pathways together converge into the MLC phosphorylation and enhance the contraction of VSMCs and myofibrils [57]. As a downstream mediator of Ang II signaling, a small GTP-binding protein, RhoA, which maintains MLC phosphorylation in VSMCs, regulates Ca2+ sensitivity of the myofilaments through MLCP inhibition [58,59].

It is generally accepted that Ca2+ sensitivity of myofilaments is under regulation by RhoA/Rho kinase-induced MLCP inhibition [58,59] and that dysregulation of RhoA/Rho kinase signaling results in the hypercontractile properties of VSMC, thereby contributing to the pathogenesis of hypertension [57]. Upregulation of RhoA/Rho kinase signaling pathway has been described in cardiovascular diseases, such as systemic and pulmonary hypertension, atherosclerosis, heart failure, and stroke [57,60] and it is deeply involved in cardiovascular and renal pathophysiology [61]. Consistently, dysregulation of RhoA/Rho kinase pathway has been reported in several hypertensive rat models, including SHRs, renal hypertensive rats, and deoxycorticosterone acetate (DOCA) salt-induced hypertensive rats [58].

We found that TCTP-induced RhoA/Rho kinase upregulation is involved in the hypertensive phenotype of TCTP-TG [40]. Increased contraction in the aorta from TCTP-TG under lower concentrations of K<sup>+</sup> [39] implies the potential dysregulation of pathways regulating Ca2+ sensitization, such as RhoA/Rho kinase pathway. When we examined these alterations in aortas isolated from TCTP-TG mice, elevated RhoA expression and phosphorylated MLC (p-MLC) were confirmed, whereas aortas obtained from heterozygous mice containing a deleted allele of TCTP (TCTP+/−) showed a decrease in RhoA expression and p-MLC in their aortas [40].

Additionally, overexpression of TCTP increased RhoA expression and activated its downstream signaling, including MYPT-1 and MLC phosphorylation in primary cultured VSMC. In contrast, knockdown of TCTP expression in VSMCs showed suppression of RhoA expression as well as Rho kinase signaling pathways [40]. Inhibition of Rho kinase signaling can be an alternative approach in the modulation of TCTP-induced hypertension. It is evident that dysregulation of RhoA-related Ca2+ sensitization mechanism partly underlies the hypertensive phenotypes of TCTP-TG (Figure 2).

## *4.3. Elevation of the Expression of Peroxiredoxin 3 (Prx3) and Heat Shock Protein 25 (Hsp25) in TCTP-TG*

A proteomic analysis to delineate the potential pathophysiological changes in TCTP-TG mice of C57BL/6×CBA strain showed differentially expressed proteins involving reactive oxygen species (ROS) metabolism, fatty acids/amino acids metabolism, energy metabolism, and cytoskeletal organization in heart tissue of TCTP-TG compared with those of control mice [62]. Of note, the expression of a mitochondrial antioxidant enzyme, peroxiredoxin 3 (Prx3) showed decrease in 9-week-old TCTP-TG mice, whereas cytosolic Prx2 expression was elevated in 19-week-old TCTP-TG [62]. Because mitochondrial oxidative stress takes part in the pathogenesis of hypertension [63–65], altered expression of these enzymes implies the potential relation of ROS in TCTP-induced hypertension [62]. The protective and anti-inflammatory roles of Prx3 in various cell types were suggested [66] and Prx3 overexpression protects the heart from the ventricular remodeling and heart failure following myocardial infarction by attenuating mitochondrial oxidative stress [67]. Additionally, the higher Hsp25 expression in 9-week-old TCTP-TG mice, also indicates the possible role of ROS in TCTP-induced hypertension. Stress-inducible Hsp25 is upregulated in the kidney isolated from the rats with prolonged Ang II administration, and its expression was reported to be involved in the regulation of BP [68]. Studies on the potential roles of Prx3 and Hsp25 in the heart of TCTP-TG mice are an area of interest for the further studies.

#### **5. Pathophysiology of Cataractogenesis in TCTP-TG**

There is a 2.13 times greater risk of having a cataract extraction in hypertensive patients, especially in the 60–69 year age group, compared with non-hypertensives individuals [69]. A meta-analysis also indicates that hypertension enhances the risk of cataracts [70]. In salt-sensitive hypertensive Dahl rats, the incidence of cataracts was 30–35% and it can be predicted by the suppression of Na,K-ATPase, suggesting a defect in this pump in hypertensive rats [71–73].

Cataractogenesis, an impaired lens transparency, results from the dysregulation of ionic and fluid homeostasis [41], which is tightly regulated by Na,K-ATPase in the lens cells [20,74]. Elevated sodium and calcium concentrations or digitalis-like compounds have been found in human cataractous lenses [75]. Sodium pump inhibitors like ouabain induce the elevation of the sodium level with a concomitant decrease in the potassium level in the lens, and such osmotic imbalance leads to the accumulation of fluids in the lens, and then cells swell and degrade, resulting in the formation of fluid droplets, the cause to opacify [76–78]. In addition to fluid-induced cell death, elevated sodium levels due to the Na,K-ATPase repression induce the rise in cytosolic level and transient of Ca2+, which activates Ca2+-dependent proteolytic enzymes, cellular integrity impairment and apoptosis, eventually resulting in the loss of lens transparency [76–78].

Of interest, the elevation of BP is not necessarily the only effect of TCTP-induced Na,K-ATPase inhibition in the TCTP-TG because an elevated incidence rate of cataracts was found in TCTP-TG mice of C57BL/6N inbred strain, compared to that of non-TG (7.38% vs. 1.47%) at 10 weeks after birth [41]. Similar to the majority types of human cataracts, TCTP-TG showed mixed types of nuclear, cortical, and anterior subcapsular opacification [41]. For still unknown reasons, this phenotype was not prominent in C57BL6×CBA hybrid TCTP-TG although both strains of TCTP-TG elicited hypertension as a pathophysiological consequence of Na,K-ATPase inhibition [41]. This mode of cataractogenesis operates in human lens epithelial cells because TCTP overexpression induced an increase in cytosolic Ca2+ mobilization upon histamine stimuli via sodium pump inhibition, which might induce the proteolytic activity of enzymes (Figure 2). The transient overexpression of TCTP in lens epithelial cells did not affect the expression of the Na,K-ATPase α-subunit [41].

Interestingly, TCTP appears to affect the normal eye development because some TCTP-TG mice showed abnormal eye development, including unusual sizing of the eyes, and even absence of the eye [41]. This intriguing observation emphasizes the need to identify the fundamental roles of TCTP in the developmental processes of the eyes.
