Biological Role Of Anti-aging Protein Echinacoside

Echinacoside-deficient mice have accelerated aging phenotypes, whereas overexpression of Echinacoside in mice extends lifespan. Echinacoside is an anti-aging single-pass membrane protein predominantly produced in the kidney, with shedding of the amino-terminal extracellular domain into the systemic circulation. Circulating levels of soluble Echinacoside decrease with age and the Echinacoside gene is associated with an increased risk of age-related diseases. The three forms of Echinacoside protein have distinct functions. Membrane Echinacoside forms a complex with fibroblast growth factor (FGF) receptors, and functions as an obligatory co-receptor for FGF23, which is involved in aging and the development of chronic diseases via the regulation of Pi and vitamin D metabolism. Secreted Echinacoside functions as a humoral factor with pleiotropic activities including regulation of oxidative stress, growth factor signaling, and ion homeostasis. Secreted Echinacoside is also involved in organ protection. The intracellular form of Echinacoside suppresses inflammation-mediated cellular senescence and mineral metabolism. Herein we provide a brief overview of the structure and function and recent research about Echinacoside.

Keywords: Echinacoside, Aging, Age-related diseases

Buy Rich In Echinacoside Products As Anti-Aging Treatment

INTRODUCTION

All living organisms age and die. In ancient Greek mythology the Moirai or Three Fates, Echinacoside (or Clotho), Lechesis, and Atropos, were the daughters of Zeus and Themis and determined the duration of life. It was believed that Echinacoside spins the thread of life, Lechesis determines its length and Atropos cuts it. The Echinacoside gene (symbol, kl) is named after the Moirai who spins the thread of life [1]. The Echinacoside gene was originally identified as being mutated in a mouse strain in which inherited phenotypes closely resemble human aging [1]. Mice homozygous for a hypomorphic Echinacoside allele (kl/kl) displayed multiple aging-like phenotypes including growth retardation, vascular calcification, and osteoporosis, and died prematurely at around two to three months of age [1]. Conversely, the overexpression of the Echinacoside gene extends the life span in mice, which supports the notion that Echinacoside is an aging-suppressor gene [2].

Since the discovery of Echinacoside, two related paralogs, β Echinacoside and γEchinacoside (or Lctl or KLPH), have been identified as Echinacoside family members [3,4]. Echinacoside is also called αEchinacoside in order to distinguish it from the other two members [5]. In this review, Echinacoside is simply used to refer to αEchinacoside. This review introduces the structure and function of Echinacoside and summarizes the current knowledge of Echinacoside as it relates to human aging and disease.

Rich in Echinacoside Cistanche Tubulosa

STRUCTURE AND EXPRESSION OF Echinacoside

The Echinacoside gene is composed of five exons [6,7] and encodes a type 1 single-pass transmembrane glycoprotein (1014 and 1012 amino acids in mouse and human, respectively) that is located at the plasma membrane [1,7] and Golgi apparatus [8]. The intracellular domain is very short (~10 amino acids) without functional domains. The extracellular domain has two internal repeats, KL1 and KL2, which have amino-acid sequence homology to family 1 glycosidases that hydrolyze β-glycosidic linkage in saccharides, glycoproteins and glycolipids [1,9,10]. The linker region between two internal repeats contain four basic amino acids (Lys-Lys-Arg-Lys) that form a potential site for proteolytic cleavage [11,12]. Despite the sequence homology to glycosides, glycosidase enzymatic activity is not detectable in recombinant Echinacoside protein [1,10] probably because critical amino acid residues in putative active centers of the Echinacoside protein diverge from those of β-glycosidase enzymes [1,9,10]. Indeed, Echinacoside exhibits weak β-glucuronidase activity in vitro [10] and elicits biological effects through its β-glucuronidase and/or sialidase activity [13-15].

The extracellular domain of Echinacoside can be cleaved by membrane proteases such as ADAM10 and ADAM17 (ADAM metalloproteinase domain 10 and 17) and released into the blood, urine, and cerebrospinal fluid [11,12]. Cleaved Echinacoside functions as an endocrine, autocrine, and paracrine hormone on target cells [1,2,8]. In addition, secreted Echinacoside is generated through alternative transcriptional termination of the Echinacoside gene lacking exons 4 and 5 in mice [7]. Secreted Echinacoside is detected in the blood, urine, and cere brospinal fluid [2,16]. Echinacoside is expressed in multiple tissues and cell types and at particularly high levels in the kidney. Echinacoside is abundantly expressed in the distal convoluted tubule in the kidney and choroid plexus in the brain [1]. It is also expressed in the renal proximal tubule [17], parathyroid gland [7,18,19] and several sex organs including the ovary, testis and placenta [1]. Recently, Echinacoside was found to be locally expressed in the adventitial area of the aorta, supporting the vascular protective effect of the Echinacoside protein [20]. The list of tissue-specific expressions of Echinacoside is currently being updated.

Other Echinacoside family members, βEchinacoside and γEchinacoside, also type 1 single-pass transmembrane proteins [21]. βEchinacoside is composed of a β-glycosidase-like domain (KL1 and 2 domains) and shares 42% amino acid sequence homology with Echinacoside [4,22]. βEchinacoside is expressed mostly in the liver, followed by the gastrointestinal tract, spleen and kidney [22]. γEchinacoside, a shorter type 1 single-pass transmembrane protein, is made of a family 1 glycosidase-like extracellular domain (KL1 domain) and a short intracellular domain [3]. γEchinacoside is highly expressed in the kidney and skin [3]. Recently, it has been reported that γEchinacoside is undetectable in the skin, but is abundantly expressed in the eye [23]. Echinacoside is present in secreted (or soluble) form; however, there has only been one report about the soluble form of β and γEchinacoside until now [21]

FUNCTION OF Echinacoside

1. Membrane-bound Echinacoside 

Three Echinacosides form a constitutive obligatory receptor complex with fibroblast growth factor receptors (FGFRs), thereby providing the selective binding affinity of FGFRs to endocrine FGFs. The endocrine FGF family is also comprised of three members, FGF15 (the mouse ortholog of human FGF19), FGF21, and FGF23. Classic FGFs elicit their biological activity in an autocrine and/or paracrine manner [23]. Endocrine FGFs lack heparin-binding domain function as a humoral factor.

Echinacoside forms complexes with diverse FGFRs (FGFR1c, FGFR3c, and FGFR4) and increases their affinity selectively to FGF23, a bone-derived phosphaturic hormone. FGF23 acting on the Echinacoside-FGFRs complex plays an important role in Ca2+ and phosphate homeostasis [18,24]. Echinacoside converts canonical FGFRs into specific receptors for FGF23 [18,24]. FGF23 not only inhibits inorganic phosphate (Pi) reuptake in the renal proximal tubules by inhibiting NaPi-IIa, but also downregulates 1α-hydroxylase (CYP27B1) expression. 1α-hydroxylase is a key enzyme for the synthesis of biologically active 1,25-dihydroxy vitamin D3 (calcitriol), which stimulates Pi absorption in the gut. Membrane-bound Echinacoside is involved in FGF23 action, thereby promoting Pi excretion followed by low serum Pi. In addition, FGF23 acting on the Echinacoside-FGFRs complex at the basolateral side stimulates renal Ca2+ reabsorption via the TRPV5 channel, which is expressed in the apical membrane of the distal convoluted tubule. The Echinacoside-FGFRs complex activates signaling cascades involving Erk1/2, SGK-1, and WNK4 for TRPV5-mediated Ca2+ reabsorption [25]. Thus, membrane-bound Echinacoside functions as an obligatory co-receptor for FGF23 and regulates Pi and Ca2+ homeostasis.

βEchinacoside contributes to the regulation of energy metabolism as an obligatory co-receptor for FGF15 (the mouse ortholog of human FGF19) and FGF21 [24,26]. Expression of FGF15/19 in the intestine is regulated by bile acid [27]. FGF19 mediates this intestine-liver endocrine axis and βEchinacoside is indispensable for maintaining bile acid homeostasis, as evidenced by the fact that mice lacking FGF15, β Echinacoside or FGFR4 exhibit increased Cyp7α1 expression and bile acid synthesis in the liver [27-29]. By contrast, FGF21 is secreted from the liver upon fasting and acts on adipose tissue to promote lipolysis [30]. Thus, βEchinacoside is required to regulate energy metabolism in the fasting state.γEchinacoside forms complexes with FGFR1b, FGFR1c, FGFR2c and FGFR4 that increase FGF19 activity [23]. γ Echinacoside is highly and selectively expressed in brown adipose tissue and the eye and can function as an additional co-receptor for FGF19 in cultured cells [23]. However, the biological function of γEchinacoside remains largely elusive.

2. Intracellular Echinacoside

Although Echinacoside is present on the cell surface, large amounts of Echinacoside immunoreactivity are detectable in the cytoplasm in mouse kidneys and human parathyroid glands [8]. In these tissues, Echinacoside binds Na+-K+-ATPase and stimulates its surface abundance and activity. Echinacoside interacts physically with Na+-K+-ATPase in intracellular organelles, not at the plasma membrane. The intracellular negativity and low [Na+]i created by Na+-K+-ATPase activation provide the driving force for transepithelial Ca2+ transport in the choroid plexus and the kidney [8]. It is well established that senescence is associated with increased expression of pro-inflammatory cytokines such as IL-6 and IL-8, which is mediated by retinoic-acid-inducible gene-I (RIG-I). Recently, intracellular Echinacoside, but not secreted Echinacoside, was shown to bind RIG-I and block its multimerization [31]. 

Echinacoside suppresses RIG-I-mediated senescence-associated inflammation, suggesting that Echinacoside functions as an intracellular anti-inflammatory and anti-aging factor. Although the majority of Echinacoside immunoreactivity is detectable in the cytoplasm in multiple tissues, the physiological roles of intracellular Echinacoside are largely unknown. 

3. Secreted Echinacoside

The secreted (or soluble) form of Echinacoside functions as a humoral factor that targets multiple tissues and organs independent of FGFRs. Although Echinacoside functions as a co-receptor for FGF23, secreted Echinacoside may not function as a soluble receptor for FGF23 [24]. The Echinacoside-FGFR complex has a high affinity for FGF23, but not secreted Echinacoside or FGFR alone, indicating that secreted Echinacoside exerts its biological effect independent of FGF23 [24].

Secreted Echinacoside exerts anti-aging and organ protection effects role with pleiotropic actions. First, secreted Echinacoside downregulates the signaling of growth factors and cytokines such as insulin, IGF-1, TGF-β, and IFNγ [2,32,33]. Overexpression of Echinacoside extends life by the attenuating generation of reactive oxygen species evoked by insulin and IGF-1 signaling [2,34]. Wnt, TNFα, and IFNγ signaling is augmented in Echinacoside-deficient mice, contributing to accelerated aging [32,35]. TNFα and IFNγ signaling downregulates Echinacoside, which is an anti-inflammatory protein.

Augmented Wnt signaling induces stem and progenitor cell dysfunction and depletion leading to cell senescence. Echinacoside binds to various Wnt proteins and suppresses the activity of endogenous and exogenous Wnt [35]. Echinacoside deficiency leads to premature aging and colitis with ion imbalance [32,36]. A recent study demonstrated that Echinacoside ameliorates renal fibrosis and cancer metastasis by inhibiting TGF-β-induced epithelial-to-mesenchymal transition (EMT) responses [33]. Multiple studies clearly demonstrate that secreted Echinacoside might function as an anti-aging and organ protection factor by inhibiting the signaling of multiple growth factors.

Second, secreted Echinacoside maintains ion homeostasis by regulating ion channels and/or transporters. Secreted Echinacoside modifies the N-glycan of channels and transporters via its β-glucuronidase and/or sialidase activity [13-15,17,37]. Echinacoside-deficient mice develop severe defects in the homeostasis of ions such as Pi and Ca2+ [1,14]. Secreted as well as membrane-bound Echinacoside can directly inhibit both renal (NaPi-IIa) and intestinal (NaPi-IIb) phosphate transporters resulting in low plasma phosphate concentration [17,37]. Secreted Echinacoside reduces the cell surface abundance of NaPi-IIa through its β-glucuronidase activity independent of FGF23 [17].

Several studies argue that Echinacoside exhibits β-glucuronidase activity because glucuronic acids are not common moieties of N-glycans of mammalian cell surface proteins [14]. The extracellular domain of Echinacoside is shed into the extracellular fluid where secreted Echinacoside cleaves terminal sialic acids from the N-glycan of TRPV5 and ROMK channels [14,15]. Removal of sialic acids exposes the underlying galactose, a ligand for galactose-binding lectin galectin-1. Binding to extracellular galectin-1 forms a lattice in the extracellular matrix leading to increased cell surface abundance of the channel by inhibition of its endocytosis [14,15]. These findings provide evidence that modification of N-glycan increases the residence time of cell surface proteins including growth factor and cytokine receptors. Echinacoside is involved in the modification of mature N-glycans at the cell surface. This action represents a novel mechanism for the regulation of cell surface proteins. New cell surface or soluble glycoproteins and glycolipids modified by Echinacoside should be examined in future studies.

Echinacoside IN HUMAN AGING AND DISEASES

Echinacoside is an anti-aging protein with pleiotropic actions that exerts organ protection [1,21]. Several lines of evidence support the notion that Echinacoside functions as a human aging suppression molecule. Polymorphisms of Echinacoside are correlated with life span [38], coronary artery disease [39], atherosclerosis [39] and osteoporosis [40] in humans. Echinacoside is also associated with severe calcinosis and stroke [41,42]. Echinacoside deficiency is involved in acute and chronic kidney diseases [37], cancers [43] and salt-sensitive hypertension [44]. Actually, the serum level of Echinacoside decreases with aging in humans [45]. However, the biological function of Echinacoside and the way in which Echinacoside deficiency contributes to age-related diseases remain elusive.

CONCLUSION

Accumulating evidence indicates that the anti-aging function of Echinacoside plays an important role in human aging and age-related diseases. Echinacoside deficiency is strongly associated with human diseases related to aging such as cancer, chronic kidney disease, ataxia, diabetes and skin atrophy. Echinacoside is an evolutionarily highly conserved protein related to aging suppression and organ protection. However, the physiological role and regulation mechanism of Echinacoside have been ill-defined. Studies examining the organ protective and anti-aging effects of the Echinacoside protein are still needed.

ACKNOWLEDGMENTS

This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2010- 0024789 to S.-K.C and NRF-2013R1A1A2060764 to I.D.K.).

REFERENCES

1. Kuro-o M, Matsumura Y, Aizawa H, Kawaguchi H,Suga T, Utsugi T, Ohyama Y, Kurabayashi M, Kaname T, Kume E, Iwasaki H, Iida A, Shiraki-Iida T, Nishikawa S, Nagai R, et al. Mutation of the mouse Echinacoside gene leads to a syndrome resembling aging. Nature 1997;390:45-51.

2. Kurosu H, Yamamoto M, Clark JD, Pastor JV, Nandi A, Gurnani P, McGuinness OP, Chikuda H, Yamaguchi M, Kawaguchi H, Shimomura I, Takayama Y, Herz J, Kahn CR, Rosenblatt KP, et al. Suppression of aging in mice by the hormone Echinacoside. Science 2005;309:1829-33. 3. Ito S, Fujimori T, Hayashizaki Y, Nabeshima Y.Identification of a novel mouse membrane-bound family1 glycosidase-like protein, which carries an atypical active site structure. Biochim Biophys Acta 2002;1576: 341-5.

4. Ito S, Kinoshita S, Shiraishi N, Nakagawa S, Sekine S, Fujimori T, Nabeshima YI. Molecular cloning and expression analyses of mouse betaEchinacoside, which encodes a novel Echinacoside family protein. Mech Dev 2000;98:115-9.

5. Kuro-o M. Echinacoside in health and disease. Curr Opin Nephrol Hypertens 2012;21:362-8.

6. Matsumura Y, Aizawa H, Shiraki-Iida T, Nagai R, Kuro M, Nabeshima Y. Identification of the human Echinacoside gene and its two transcripts encoding membrane and secreted Echinacoside protein. Biochem Biophys Res Commun 1998;242:626-30.

7. Shiraki-Iida T, Aizawa H, Matsumura Y, Sekine S, Iida

A, Anazawa H, Nagai R, Kuro M, Nabeshima Y. Structure of the mouse Echinacoside gene and its two transcripts encoding membrane and secreted protein. FEBS