Soluble αKlotho downregulates Orai1-mediated store-operated Ca2+ entry via PI3K-dependent signaling

αKlotho is a type 1 transmembrane anti-aging protein. αKlotho-deficient mice have premature aging phenotypes and an imbalance of ion homeostasis including Ca2+ and phosphate. Soluble αKlotho is known to regulate multiple ion channels and growth factor-mediated phosphoinositide-3-kinase (PI3K) signaling. Store-operated Ca2+ entry (SOCE) mediated by pore-forming subunit Orai1 and ER Ca2+ sensor STIM1 is a ubiquitous Ca2+ influx mechanism and has been implicated in multiple diseases. However, it is currently unknown whether soluble αKlotho regulates Orai1-mediated SOCE via PI3K-dependent signaling. Among the Klotho family, αKlotho downregulates SOCE while βKlotho or γKlotho does not affect SOCE. Soluble αKlotho suppresses serum-stimulated SOCE and Ca2+ release-activated Ca2+ (CRAC) channel currents. Serum increases the cell-surface abundance of Orai1 via stimulating vesicular exocytosis of the channel. The serum-stimulated SOCE and cell-surface abundance of Orai1 are inhibited by the preincubation of αKlotho protein or PI3K inhibitors. Moreover, the inhibition of SOCE and cell-surface abundance of Orai1 by pretreatment of brefeldin A or tetanus toxin or PI3K inhibitors prevents further inhibition by αKlotho. Functionally, we further show that soluble αKlotho ameliorates serum-stimulated SOCE and cell migration in breast and lung cancer cells. These results demonstrate that soluble αKlotho downregulates SOCE by inhibiting PI3K-driven vesicular exocytosis of the Orai1 channel and contributes to the suppression of SOCE-mediated tumor cell migration.


Introduction
Klotho is an aging-suppressor gene that encodes type 1 transmembrane glycoprotein called αKlotho [22,23]. Klotho-deficient (kl/kl) mice show accelerated aging phenotypes with a severe imbalance of ion homeostasis including Ca 2+ and phosphate (P i ) [22,24,36]. The Klotho family comprises three members: αKlotho (encoded by the αKlotho gene; also known as KL), βKlotho (encoded by the βKlotho gene; also known as KLB), and γKlotho (encoded by Lctl gene; also known as KLG) [19,20]. αKlotho has at least two functional modes including full-length membrane-bound form and soluble form. The membranous form of αKlotho binds to multiple fibroblast growth factor (FGF) receptors that function as an obligatory coreceptor for FGF23 to regulate P i and Ca 2+ homeostasis [7,24,36]. The extracellular domain of αKlotho is cleaved off and released into blood, urine, and cerebrospinal fluid to function as paracrine and/or endocrine Ji-Hee Kim and Eun Young Park contributed equally and thus share the first authorship. hormone [19,23]. This soluble form of αKlotho exerts aging suppression and organ protection with pleiotropic action including regulation of ion channels and growth factor signaling [19][20][21].
The ubiquitous second messenger Ca 2+ regulates various cellular behaviors. Store-operated Ca 2+ entry (SOCE) is vital for the maintenance of endoplasmic reticulum (ER) Ca 2+ stores at precise levels for signaling in both nonexcitable and excitable tissues to regulate a variety of cellular functions [31,32]. The molecular components of SOCE are Orai1 and STIM1 (stromal interaction molecule 1), a pore-forming subunit, and an ER Ca 2+ sensor, respectively. STIM1 is oligomerized and translocated to the plasma membrane during ER Ca 2+ depletion that thereby triggers Ca 2+ entry via Orai1, a Ca 2+ -selective channel at the plasma membrane [31,32]. SOCE is a downstream effector of growth factor signaling. The explicit mechanism of Orai1 activation by PI3K-driven growth factor signaling in physiological conditions remains elusive. Moreover, soluble αKlotho suppresses aging and protects multiple disease progression by regulating growth factor signaling [19,23]. The mechanism linking αKlotho and SOCE by growth factor signaling has not yet been identified. Here, we examined the mechanism by which soluble αKlotho regulates Orai1-mediated SOCE by growth factor stimulation and its functional implications.  [11,24,30]. Orai1 (mCherry-3xFlag-Orai1) and STIM1 (YFP-STIM1) plasmids were kindly provided from Drs. Joseph Yuan (University of North Texas, USA).
All DNA plasmids were transfected by using X-tremeGENE HP DNA transfection reagent Ⓡ (Roche, Mannheim, Germany) following the manufacturer's instructions. Experiments were conducted 48 h after transfection. For knockdown by siRNA, oligonucleotides were transfected into MDA-MB231 and H1693 cells with DharmaFect (cat no. T-2001-03, Horizon Discovery Ltd., Cambridge, UK) following the manufacturer's instructions. Cells were trypsinized, and re-seeded on the poly-lysine coated coverglasses after 48 h for live-cell Ca 2+ imaging or on the 6-well plate for in vitro wound-healing assay. Experiments were conducted after 24 h re-seeding the cells.

Real-time quantitative PCR analysis
Purified total RNA was extracted from the trypsinized pellets of HEK293FT cells through Hybrid-RTM total RNA purification kit (cat. n. 305-101, GeneAll, Seoul, South Korea) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized from 1 μg of total RNA by using a ReverTraAce® qPCR RT Master Mix with gDNA Remover (cat. n. FSQ-301, Toyobo, Osaka, Japan). The mRNA abundance was analyzed by real-time quantitative PCR with SYBR Green (cat. n. 204143, Qiagen, Germantown, MD, USA) using the following sequencespecific human primers: For the analysis of each gene expression, the experiments were performed in triplicate in a real-time PCR system (7900HT, Thermo Fisher Scientific). Data were analyzed following the 2 -ΔΔCt method with 18S as the reference gene.

Confocal microscopy
For immunofluorescence staining, HEK293 cells with an inducible mCherry-STIM1-T2A-Orai1-eGFP were grown on poly-L-lysine-coated coverslips. eGFP-Orai1 and mCherry-STIM1 protein were induced after 12~24 h tetracycline (5 μM) treatment [34] and were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. GFP and mCherry fluorescent images were obtained using a laser scanning confocal microscope (Zeiss, LSM 800, Jena, Germany) with Airyscan. Super-resolution image of the Orai1 expression on the plasma membrane and Airyscan image processing was acquired using the ZEN 2.3 software.
In vitro wound-healing assay A wound-healing assay was conducted as described previously [14,17]. Briefly, MDA-MB231 and H1693 cells were plated at 1 × 10 7 per well in a 6-well plate until grown to confluence. The cells were incubated with recombinant αKlotho protein (1 nM) in only 1% penicillin contained RPMI1640 media for 30 min and then exchanged with complete media with or without αKlotho protein. To distinguish cell migration from proliferation, all wound-healing assays were performed in the presence of anti-tumor drug mitomycin C (M4287, Sigma-Aldrich, a final concentration of 0.1 μg/ml) to prevent proliferation. The image was captured by a microscope after 24 h of drug treatment (time 0, initial time point). The migrated cells were counted using an ImageJ 1.48 (NIH, USA).

Data analysis and statistics
Results are presented as mean ± SEM. Statistical analysis was performed using a two-tailed unpaired Student's t test and one-way ANOVA followed by Tukey's multiple comparison tests by the GraphPad Prism Software (version 5.0, GraphPad Software, San Diego, CA, USA). p values less than 0.05 and 0.01 were considered significant for single and multiple comparisons, respectively. All experiments were repeated independently 3-4 times with similar results.
There are at least two types of functional αKlotho, membranous and soluble form [12]. We next examined which functional mode of αKlotho effectively regulates endogenous SOCE in HEK293FT cells. We found that both membranous and soluble αKlotho downregulate SOCE (Fig. 1e-f). The soluble form of αKlotho is more potent to suppress SOCE. Of note, overexpression of both membranous and secreted forms of αKlotho did not affect the expression of endogenous Orai1 and STIM1 in HEK293 cells (Fig. 1g). Together, soluble αKlotho is critical for SOCE regulation.
Soluble αKlotho downregulates serum-stimulated SOCE and CRAC current Soluble αKlotho has pleiotropic cellular function including regulation of ion channels and growth factor signaling [12,13,19]. Here, we examined whether soluble αKlotho regulates serum-stimulated SOCE and Ca 2+ release-activated Ca 2+ (CRAC) channel current in HEK293FT cells. Endogenous SOCE was significantly increased in the application of serum compared with that in serum-deprived conditions, and this stimulation was attenuated by pretreatment with recombinant αKlotho protein ( Fig. 2a and b).
Orai1 is a principal pore subunit of the CRAC channel [33]. Next, CRAC channel current density was measured in HEK293FT cells overexpressing Orai1 and STIM1 by ruptured whole-cell patch-clamp recording. ER Ca 2+ depletion evoked inward currents under dialyzed whole-cell configuration (Fig. 2c). Current-voltage (I-V) relationship curves showed characteristic inward rectifying CRAC currents (Fig.  2d). Soluble αKlotho reduced Orai1 current density and SOCE but had no apparent effects on the general properties of whole-cell currents (Fig. 2c-e). These results support that soluble αKlotho downregulates serum-stimulated SOCE and Orai1 currents.

Serum increases the cell-surface abundance of Orai1 via stimulating its exocytosis
We examined the time course of SOCE stimulation by serum treatment. The stimulation of endogenous SOCE by serum was detected after 10 min incubation and reached a maximal effect at 1 h (Fig. 3a). We and others reported that serum growth factors promote transient translocation of TRPC5 and TRPC6 channels to the plasma membrane [3,16,42]. Similarly, serum treatment promoted the relocalization of GFP-tagged Orai1 to the plasma membrane (Fig. 3b). Moreover, biotinylation assay showed that incubation with serum increased the steady-state surface abundance of Orai1 but not in the total cell lysates (Fig.  3c). Assessment of the cell-surface abundance of Orai1 was confirmed by no detection of intracellular protein at a biotinylated fraction (Fig. 3c). The growth factor stimulates a cell-surface abundance of TRPC channels via their SNARE-dependent vesicular exocytosis [9,16,43]. Thus, we examined whether a similar mechanism may involve the upregulation of Orai1 by serum. Brefeldin A (BFA) or tetanus toxin (TeNT) disrupt vesicular exocytosis.
Serum-stimulated cell-surface abundance of Orai1 was blunted by preincubation with BFA or TeNT (Fig. 3c and  d), indicating that steady-state vesicular exocytosis of Orai1 occurs in the presence of serum.  [16,42]. We explored whether a similar mechanism may contribute to the suppression of Orai1 and SOCE. We next measured the effects of αKlotho on a cell-surface abundance of Orai1 using biotinylation assay. Preincubation of soluble αKlotho prevented steady-state and serum-stimulated surface abundance of Orai1 ( Fig. 4a and b), which supports the notion that αKlotho reduces the cell-surface abundance of Orai1. These findings of plasma membrane expression of Orai1 were confirmed by Ca 2+ imaging showing that SOCE was inhibited by BFA or TeNT (Fig. 4c-f). The reduction in the cell-surface abundance of Orai1 by soluble αKlotho may result from decreased exocytosis and/or increased endocytosis of the channel. Moreover, inhibition of vesicular exocytosis of the channel by BFA or TeNT decreased SOCE and prevented further inhibition by soluble αKlotho (Fig. 4c-f). These results indicate that αKlotho reduces SOCE via downregulating vesicular exocytosis of the Orai1 channel. αKlotho inhibits SOCE and cell-surface abundance of Orai1 via PI3K-dependent pathway Activation of the PI3K-Akt pathway by serum growth factors increases the plasma membrane abundance of TRPC channels by stimulating their exocytosis [3,16,42]. Soluble αKlotho inhibits increased cell-surface abundance of TRPC3 and TRPC6 by inhibiting the PI3K-dependent pathway [9,16,42]. We next examined whether αKlotho suppresses SOCE and cell-surface abundance of Orai1 by inhibiting serumstimulated PI3K signaling. Inhibition of PI3K by preincubation with its blockers, wortmannin (WMN) or LY294002, reduced Akt phosphorylation (Fig. 5a-d). Accordingly, αKlotho also reduced serum-stimulated Akt phosphorylation ( Fig. 5e and f). Blockade of PI3K by preincubation with WMN or LY294002 inhibited serumstimulated cell-surface abundance of Orai1 ( Fig. 5g and h). Moreover, inhibition of PI3K by WMN or LY294002 abrogated SOCE and prevented further αKlotho-induced inhibition ( Fig. 5i and j). Collectively, these results support that soluble αKlotho suppresses SOCE via inhibiting PI3Kdependent exocytosis of the Orai1 channel.
αKlotho ameliorates serum-stimulated SOCE and migration in breast and lung cancer cells Orai1-mediated SOCE is critical for tumor cell migration and metastasis [14,44]. We previously reported that soluble αKlotho inhibits the migration of clear cell renal cell carcinoma via suppressing the IGF-1-stimulated PI3K pathway [15]. Therefore, we explored whether αKlotho inhibits Orai1-mediated SOCE and migration in breast and lung cancer cells. Orai1 was a primary molecular component of SOCE in both breast and lung cancer cells, MDA-MB231 and H1693, respectively (Fig. 6a-d). Moreover, both tumor cell migrations were also blunted by silencing ORAI1 (Fig.  6e and f). Consistent with results in HEK293FT cells, SOCE was significantly upregulated by application of serum compared with that by serum deprivation, and the stimulation was attenuated by incubation of αKlotho protein in  (Fig. 6g-j). Of note, inhibition of PI3K by WMN abrogated SOCE and prevented further αKlotho-mediated inhibition ( Fig. 6k and l) in both tumor cells supporting the notion that soluble αKlotho suppresses SOCE via inhibiting PI3K-dependent pathway. Accordingly, αKlotho also inhibited serumstimulated tumor cell migration (Fig. 6m and n).

Discussion
SOCE is essential for the maintenance of ER Ca 2+ stores at a precise level for cellular signaling and functions [31,32]. Disturbed SOCE-mediated Ca 2+ signaling and homeostasis of Ca 2+ store have been implicated in the pathogenesis of multiple diseases [31]. Major downstream signaling effectors of growth factor receptors are PLCγ and PI3K-Akt pathways. The cellular mechanism of Orai1 activation can be mediated by serum and/or growth factors triggering PLCγ activation, IP 3 generation, and Ca 2+ release from the ER store. Depletion of ER Ca 2+ store oligomerizes STIM1 to open the Orai1 channel at the plasma membrane [28]. PI3K-Akt pathway signaling contributes to the stimulation of exocytosis of multiple channels such as TRPC5 and TRPC6 [3,9,16,42]. The underlying mechanism of Orai1 regulation by PI3K-derived growth factor signaling remains unsolved. Our data demonstrate that activation of the PI3K-dependent signaling pathway by serum increases the cell-surface abundance of Orai1 via enhancing forward trafficking of the channel to the plasma membrane. These findings support that a similar mechanism may contribute to the downregulation of Orai1-mediated SOCE. Notably, PI3K inhibitors have pleiotropic effects. Therefore, the underlying mechanism by downstream effectors of PI3K to regulate the cellsurface expression of Orai1 awaits future study.
The aging process is closely related to altered growth factor signaling and ion imbalance including Ca 2+ and P i The lack of β-actin detection in the membrane fraction was used as a control for biotinylation. Surface and lysate denote biotinylated fraction and total cellular protein, respectively. d Densitometry of the surface abundance of Orai1 in panel c. **p < 0.01, data were analyzed by one-way ANOVA in a and d [18,19]. The membrane-bound form of αKlotho and βKlotho forms a binary complex with FGFRs, which serves as the physiological receptors for FGF23 and FGF19/21, respectively [7,[19][20][21]. We found that membr ano us α Klot ho bu t not β Kloth o or γ K l o t h o downregulates SOCE. Membranous αKlotho associated with FGF receptors functions as a coreceptor for FGF23 signaling to regulate P i [7,24,36]. Soluble αKlotho also regulates multiple ion channels [13]. Our data demonstrate that both types of αKlotho can downregulate SOCE. Soluble αKlotho is more potent to downregulate SOCE, supporting that soluble αKlotho is critical for Orai1-mediated SOCE.
Soluble αKlotho can up-or downregulate multiple channels via a distinct mechanism. αKlotho positively regulates several TRPV (TRPV2, 5, and 6) and K + channels (ROMK, Kv1.3, KCNQ1/KCNE1, and hERG channels) through increasing cell-surface abundance of the channels by modification of their N-glycan through sialidase or β-glucuronidase activity of αKlotho [1,2,6,26,27,29]. Modifying N-glycans of the channel by αKlotho delays its endocytosis resulting in increased cell-surface abundance [4,27]. Conversely, αKlotho negatively regulates multiple TRPC channels with a distinct mechanism. αKlotho directly binds to the VEGFR2/ TRPC1 complex to promote their cointernalization [25]. On the other hand, αKlotho downregulates the cellsurface abundance of TRPC6 and TRPC3 via inhibiting their PI3K-dependent exocytosis [16,42]. In the present study, αKlotho reduces the cell-surface abundance of Orai1 by inhibiting the serum-stimulated PI3K-dependent pathway. This supports the notion that the growth factor- Recently, the underlying mechanism of αKlotho on the downregulation of TRPC6 by growth factor-mediated PI3K signaling is unraveled [9,41]. Soluble αKlotho specifically targets α2-3-sialyllactose of monosialogangliosides highly enriched in the lipid raft and particularly downregulates lipid raft-dependent PI3K-Akt signaling to suppress TRPC6 [9,40,41]. Orai1 is localized in the lipid raft and binds directly to caveolin-1 and cholesterol [10,45,46]. At a steady-state, Orai1 continuously recycles between the endosome and the plasma membrane [45,46]. In the present study, we show that soluble αKlotho suppresses Orai1 surface abundance via inhibiting PI3K-dependent exocytosis of the channel. Hence, future studies will explore the mechanism that specific lipid raft-dependent PI3K/Akt signaling may contribute to the downregulation of Orai1 by soluble αKlotho.
Accumulating evidence demonstrates that the upregulation of Orai1/STIM1-mediated SOCE is associated with tumor progression and poor prognosis in multiple cancers including breast, lung, and renal cancer [14,35,44]. Hyperactivation of the PI3K/Akt signaling pathway promotes tumor cell migration. Currently, targeting growth factor receptor-driven PI3K signaling pathway with pharmaceutical agents have been suggested as a therapeutic solution for treating cancers and applied in clinical trials [8,37,38]. αKlotho suppresses growth factor-stimulated cell migration by inhibiting PI3K/Akt pathway in multiple tumors such as breast and renal cancers [15,39]. This study provides compelling evidence supporting αKlotho targeting PI3K-stimulated SOCE function as a tumor suppressor. SOCE is critical for finetuning ER Ca 2+ stores for cellular signaling and function and its altered activity leads to pathologies [31,32]. Hence, αKlotho-based approaches may be attractive targets for treating SOCE-related pathologies including tumors.

Compliance with ethical standards
This article does not contain any studies with animal and human participants performed by any of the authors.

Conflict of interest
The authors declare that they have no conflict of interest. Abbreviations ANOVA, analysis of variance; BFA, Brefeldin A; cDNA, complementary DNA; CPA, cyclopiazonic acid; CRAC, calcium releaseactivated calcium; ER, endoplasmic reticulum; FBS, fetal bovine serum; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; hERG, Ether-a-go-go-related gene; Kv1.3, voltage-gated potassium channel, shaker-related subfamily, member 3; KCNQ1, voltage-gated potassium channel subfamily Q member 1; KCNE1, voltage-gated potassium channel subfamily E regulatory subunit 1; KLFL, transmembrane fulllength mouse αKlotho; KL△TM, Extracellular domain of mouse αKlotho; GFP, Green fluorescent protein; Lctl, lactase-like; P i , inorganic phosphate; PI3K, phosphoinositide-3-kinase; PLCγ, phospholipase C gamma; IP 3 , inositol trisphosphate; RIPA, radioimmunoprecipitation assay; ROMK, renal outer medullary potassium channel; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEM, standard error of the mean; SNARE, SNAP receptor; STIM1, stromal interaction molecule 1; SOCE, store-operated Ca 2+ entry; TeNT, tetanus toxin A; TRP, transient receptor potential; TRPC, transient receptor potential canonical type; TRPV, transient receptor potential vanilloid type; VEGFR, vascular endothelial growth factor receptor; WMN, Wortmannin; YFP, Yellow fluorescent protein Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.