βklotho is essential for the anti‐endothelial mesenchymal transition effects of N‐acetyl‐seryl‐aspartyl‐lysyl‐proline

Endothelial–mesenchymal transition (EndMT) has emerged as an essential bioprocess responsible for the development of organ fibrosis. We have previously reported that fibroblast growth factor receptor 1 (FGFR1) is involved in the anti‐EndMT effect of N‐acetyl‐seryl‐aspartyl‐lysyl‐proline (AcSDKP). FGFR1 is expressed on the cell membrane and performs its biological function through interaction with co‐receptors, including βklotho (KLB). However, it remains unknown whether KLB is involved in the anti‐EndMT effects of AcSDKP. Here, we demonstrated that AcSDKP increased KLB expression in an FGFR1‐dependent manner and that KLB deficiency induced AcSDKP‐resistant EndMT via the induction of the mitogen‐activated protein kinase (MAPK) pathway. In cultured endothelial cells, AcSDKP increased KLB protein level in an FGFR1‐dependent manner through induction of the FGFR1–KLB complex. KLB suppression by small interfering RNA transfection did not affect FGFR1 levels and resulted in the induction of EndMT. In contrast to the EndMT observed under FGFR1 deficiency, the EndMT induced by KLB suppression was not accompanied by the induction of Smad3 phosphorylation; instead, KLB‐deficient cells exhibited induced activation of the MAPK/extracellular signal‐regulated kinase (ERK) kinase (MEK) and ERK pathways. Treatment with the specific MEK inhibitor U0126 diminished KLB deficiency‐induced EndMT. Consistent with this finding, AcSDKP did not suppress either EndMT or MEK/ERK activation induced by KLB deficiency. Application of either FGF19 or FGF21 synergistically augmented the anti‐EndMT effects of AcSDKP. Taken together, these results indicate that endogenous peptide AcSDKP exerts its activity through induction of the FGFR1–KLB complex in vascular endothelial cells.

(aSMA), smooth muscle protein 22a (SM22a), fibroblast-specific protein 1 (FSP1), and vimentin. Transforming growth factor bs (TGFbs) are considered to be the pivotal cytokines driving EndMT through the activation of the Smad signaling pathway [2,3]. It has been well documented that fibroblast growth factor receptor 1 (FGFR1) acts as the key inhibitor of EndMT by inhibiting TGFb/Smad signaling [4][5][6]. EndMT has emerged as a crucial source of extracellular matrix-producing mesenchymal cells that are responsible for the development of fibrosis in involved organs, including the kidney and heart [7,8]. Therefore, the identification of approaches targeting EndMT is of great clinical potential for the development of novel therapeutic agents.
N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), an endogenous tetrapeptide, has been shown to be a substance that displays anti-EndMT and antifibrotic effects both in vitro and in vivo by our group and other groups [4,9,10]. AcSDKP is produced from the N-terminal peptide sequence of thymosin b4 by the cleavage activity of prolyl oligopeptidase. AcSDKP is normally present in plasma and is exclusively hydrolyzed by angiotensin-converting enzyme. We reported that AcSDKP inhibited TGFb/Smad signaling [11] and displayed antifibrotic effects in various fibroproliferative rodent models in experimental intervention studies [4,9,10,12]. The restoration of FGFR1 expression induced by AcSDKP was later identified as the essential event responsible for the inhibition of TGFb/Smad signaling as well as for the subsequent EndMT [4,9]. However, the molecular regulation of the anti-EndMT effects of AcSDKP through FGFR1 remains unclear due to the complexity of FGFR1 biology.
The klotho family is a family of newly defined proteins that is composed of aklotho (KLA) [13], bklotho (KLB) [14], and cklotho [15]. Klotho proteins are type I single-pass transmembrane proteins and share homology to family 1 b-glycosidases. The intracellular part of klotho proteins is very short, while the extracellular domain is relatively long and is composed of two internal repeats, termed KL1 and KL2 [16]. The main roles of membrane-bound klotho proteins rely on their functions as co-receptors of FGFRs, facilitating the binding of FGF19 subfamily ligands to FGFRs (e.g., KLA for FGF23 and KLB for FGF19/FGF21) [17,18]. The FGF19 and FGF21 have been emerged as essential metabolism-regulating factors during the past decades. Tomlinson et al. [19] reported that the FGF19 transgenic mice were resistant to adiposity and displayed increased metabolic rate. Furthermore, FGF21 has been implicated in the stimulation of glucose uptake as well as in the resistance to diet-induced obesity [20]. The subsequent in vitro studies demonstrated that the KLB acts as a co-receptor for FGF19 and FGF21, both of which exhibited low affinity to their corresponding FGFRs [21][22][23]. In addition, Adams and colleagues provided clear evidence regarding the involvement of KLB in regulating the metabolic processes of FGF19/21 in experimental animals [24,25].
Among the three of klotho family proteins, KLA has attracted considerable attention in studies due to its anti-aging property [13]. The functions of KLA in endothelial cells as well as cardiovascular disorders have been widely studied [26][27][28][29]. In contrast, the biological role of KLB is not well analyzed. The expression of KLB was detected in human umbilical vein endothelial cells [30] and human brain microvascular endothelial cells [31] and contributes to blood-brain barrier formation. However, the contribution of KLB to EndMT and fibrotic disorders remains poorly understood. Therefore, the roles of KLB in EndMT as well as its responses to AcSDKP treatment were investigated in the current study.

Multiplex staining
Multiplex staining was performed by the opal in situ kit (PerkinElmer, Waltham, MA, USA) according to the manufacturer's instructions. Deparaffinized sections were labeled with the FGFR1/KLB or VE-cadherin/KLB antibodies. The cell nuclei were labeled with DAPI. For the negative controls, blocking solution was utilized to replace the primary antibody.

Western blot analysis
After the indicated treatment, the cells were washed with PBS, and the protein extracts were harvested by adding a RIPA lysis buffer system (containing lysis buffer, PMSF, protease inhibitor cocktail, and sodium orthovanadate; Santa Cruz Biotechnology, SC-24948). The detailed procedure followed a general protocol described elsewhere [4].

Immunofluorescence staining
Immunofluorescence staining of HMVECs cultured on eight-well culture slides (Corning, Discovery Labware, Oak Park, MA, USA) and mouse heart sections followed a method described in previous studies [4].

Duolink in situ assay
A Duolink in situ proximity ligation assay (PLA) was performed following the manufacturer's instructions (Sigma-Aldrich). Briefly, HMVECs were treated with N-FGFR1 (1.5 lgÁmL À1 ) or TGFb2 (5 ngÁmL À1 ) for 48 h with or without a 2-h preincubation with AcSDKP (100 nM). Normal isotype IgG was used as the control. After washing with PBS, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. After blocking, the cells were incubated overnight with primary antibodies against FGFR1 and KLB. Cells without the addition of a primary antibody were used as the negative control. The cells were incubated with the PLA probe solution for 1 h at 37°C, followed by incubation with a ligase solution for 30 min at 37°C and a polymerase amplification solution for 100 min at 37°C. The samples were then mounted with Duolink in situ mounting medium with DAPI for 20 min and analyzed by using an all-in-one BZ-X700 fluorescence microscope (Keyence, Osaka, Japan). For each slide, the images in at least six different fields at 9400 magnification were analyzed [4].

Immunoprecipitation
Human dermal microvascular endothelial cells were subjected to the indicated treatment for 48 h and harvested by the RIPA lysis buffer system and centrifugation; the immunoprecipitation (IP) assay was then performed according to the manufacturer's instructions (Cell Signaling Technology). The detailed procedure was as described in previous studies [4].

Animal experiments
Control, streptozotocin (STZ)-treated CD-1, and AcSDKPtreated STZ mice were prepared according to our previous study [9], and the characteristics of animals were as reported [4]. In brief, 8-week-old male CD-1 mice were subjected to a single intraperitoneal injection of STZ (200 mgÁkg À1 ) to induce diabetes. Twelve weeks later, the diabetic mice were divided into a nontreatment group and an AcSDKP treatment group (500 lgÁkg BW À1 Áday À1 using an osmotic minipump) for another 4 weeks. The experiments described in the methods were carried out in compliance with the animal protocols of Kanazawa Medical University (protocol numbers 2014-89, 2013-114, and 2014-101).

Statistical analysis
The graphs were generated, and statistical analyses were performed using GRAPHPAD PRISM software (Version 7.00, La Jolla, Canada). The data are presented as the means AE SDs. One-way ANOVA followed by Tukey's multiple comparisons test was used for statistical analysis. A P-value of < 0.05 was considered significant. All data were confirmed in three independent experiments.

AcSDKP induced the FGFR1-KLB complex in endothelial cells
We have previously demonstrated that the anti-EndMT effects of AcSDKP mediated by the suppression of the TGFb/Smad pathway were associated with the induction of FGFR1 signaling [4,9]. However, it remains unclear whether KLB, the cofactor of FGFRs, is involved in this process. First, we performed the proximity assay and found that endogenous FGFR1 and KLB are expressed physically near each other in cultured endothelial cells (Fig. 1A,B). In addition, AcSDKP treatment significantly enhanced the proximity of FGFR1 and KLB. The proximity of FGFR1 and KLB was abolished by the co-incubation with neutralizing FGFR1 antibody (N-FGFR1; Fig. 1A) or TGFb2 (Fig. 1B). The decreased proximity of FGFR1 and KLB induced by N-FGFR1 was not altered by AcSDKP; TGFb-induced disruption of the proximity of FGFR1 and KLB was partially rescued by preincubation with AcSDKP (Fig. 1A,B).
A direct physical interaction between KLB and FGFRs was confirmed by Ogawa et al. [33]. Consistent with Ogawa's report, our IP results revealed a physical interaction between KLB and FGFR1 in normal endothelial cells. IP analysis demonstrated that AcSDKP induced the FGFR1-KLB complex; co-incubation with N-FGFR1 abolished AcSDKP-enhanced levels of FGFR1-KLB complex in AcSDKP-treated endothelial cells (Fig. 1C). A comparable result was obtained when the anti-KLB antibody was used as the pull-down antibody (Fig. 1D). The levels of KLB and FGFR1 were elevated upon AcSDKP treatment; AcSDKP did not affect the levels of either FGFR1 or KLB in N-FGFR1-treated cells (Fig. 1E,F), suggesting that AcSDKP increased KLB in an FGFR1-dependent manner and subsequent induction of FGFR1-KLB complex.
We have previously reported that FGFR1 expression was reduced in the heart of STZ-induced diabetes model animals and that the administration of AcSDKP could restore FGFR1 expression [4,9]. As expected, a notable decrease in the FGFR1 and KLB levels was observed in the hearts of STZ-induced diabetic mice compared to the levels of these proteins in the hearts of control mice; administration of AcSDKP significantly restored them (Fig. 1G). In line with this finding, VEcadherin-positive endothelial cells in the hearts of diabetic mice exhibited reduced levels of KLB; however, AcSDKP restored the KLB levels (Fig. 1H).

KLB deficiency led to EndMT in HMVECs
First, we confirmed the levels of KLB and FGFR1 in cells treated either with specific siRNA targeting KLB or with N-FGFR1. N-FGFR1 diminished the levels of both FGFR1 and KLB; KLB siRNA-transfected cells exhibited decreased levels of KLB but no alteration in FGFR1 levels ( Fig. 2A). Immunofluorescence analysis revealed that a deficiency in either KLB or FGFR1 induced an increase in aSMA levels and an alteration of the cell morphology to a spindle shape (Fig. 2B). Consistent with this observation, we found that specific siRNAmediated KLB knockdown resulted in the induction of EndMT, as evidenced by the decreased VE-cadherin levels and enhanced expression of aSMA, SM22a, vimentin, and FSP1; however, AcSDKP had no effect on KLB deficiency-induced EndMT (Fig. 2C,D).

KLB deficiency was not associated with the induction of TGFb/Smad3 signaling
Fibroblast growth factor receptor 1 suppressioninduced EndMT has shown to be associated with the activation of the TGFb/Smad3 pathway [4,5,9]. As reported elsewhere, cells incubated with N-FGFR1 exhibited increased phosphorylation of Smad3 compared to that in cells incubated with control IgG (Fig. 3A,B); as reported, AcSDKP did not affect Smad3 phosphorylation in N-FGFR1-treated cells (Fig. 3A,B). TGFb2-treated cells were used as the positive control for the phosphorylation of Smad3, and AcSDKP suppressed TGFb2-induced Smad3 phosphorylation (Fig. 3C,D) [4]. However, unexpectedly, KLB knockdown did not impact on the Smad3 phosphorylation in HMVECs (Fig. 3C,D).

KLB deficiency-induced EndMT is dependent on the mitogen-activated protein kinase pathway
Liu et al. [34] recently reported that the overexpression of KLB in endometrial adenocarcinoma cells inhibited 17b-estradiol-induced epithelial-mesenchymal transition (EMT) via the suppression of ERK1/2. In basal condition, AcSDKP suppressed both MEK and ERK phosphorylation in culture endothelial cells (Fig. 4A,  B). KLB knockdown by specific siRNA induced a significant increase in the phosphorylation of MEK and ERK; AcSDKP did not inhibit KLB deficiency-induced activation of the MEK/ERK pathway (Fig. 4A,B). Consistent with this finding, the MEK inhibitor U0126 completely inhibited KLB deficiencyinduced EndMT (Fig. 4C,D), suggesting the fundamental role of the MEK/ERK pathway in KLB deficiency-induced EndMT. Supporting with this, the occurrence of EndMT and the activation of MEK/ ERK pathway induced by N-FGFR1 were suppressed by U0126. (Fig. S1A-D).

Discussion
Combating organ fibrosis is an important topic in medical research. The attractive anti-EndMT effects of AcSDKP have been intensively reported by our group and other groups during the last decade [4,[9][10][11][12]. The induction of FGFR1 expression upon AcSDKP treatment has been considered the critical event responsible for the inhibition of the TGFb/Smad signaling pathway and fibrosis [4,9,11]. In current study, we demonstrated that (a) AcSDKP increased the level of KLB via an FGFR1-dependent manner and subsequent induction of FGFR1-KLB complex in endothelial cells; (b) KLB deficiency did not influence FGFR1 levels in endothelial cells; (c) KLB deficiency-induced Smad3 signaling-independent but MAPK-dependent EndMT; (d) AcSDKP had no effect on KLB   Neither AcSDKP-mediated-regulation of KLB norinfluence on the level of FGFR1-KLB complex has been studied. In our study, AcSDKP increased the levels of KLB and subsequent induction of FGFR1-KLB complex; AcSDKP had no effect on the decreased levels of KLB in N-FGFR1-treated endothelial cells, suggesting that AcSDKP-induced KLB expression is FGFR1-dependent. The reduction in the FGFR1 and KLB protein levels was also observed in the hearts of STZ-induced diabetic mice, and AcSDKP restored these levels. A logical question is whether KLB affects the FGFR1 level in endothelial cells; however, we found that the suppression of KLB by specific siRNA did not influence the FGFR1 levels indicating that the FGFR1 level is not regulated by the KLB protein in cultured endothelial cells.
We next explored the signaling pathway through which EndMT is induced by KLB knockdown. Considering the activation of TGFb/Smad3 signaling upon FGFR1 deficiency, the phosphorylation of Smad3 was first analyzed in KLB knockdown endothelial cells. Surprisingly, no obvious alteration in Smad3 phosphorylation was observed. Instead, molecules in the MAPK signaling pathway, MEK1/2 and ERK1/2, were activated, and the MEK inhibitor significantly blocked KLB suppression-induced EndMT. This observation was consistent with Liu's report, which showed that the overexpression of KLB abolished the activation of ERK1/2 and EMT, the epithelial program sharing similar molecular mechanisms to EndMT [34]. Furthermore, our data demonstrated that the activation of the MAPK pathway without the activation of the Smad3 signaling pathway in KLB-deficient cells is sufficient to induce EndMT. Regard with this, ligand of FGFR1-KLB complex, either FGF19 or FGF21, could synergistically suppress EndMT and MEK/ERK pathway in AcSDKP-treated cells, suggesting the biological significance of AcSDKP in endothelial cells through the induction of FGFR1-KLB complex.
In conclusion, the current study highlights the involvement of KLB in EndMT pathobiology and in the anti-EndMT effects of AcSDKP. These data provide new insight into the essential role of KLB, which is important for establishing new therapeutic strategies to combat progressive fibrotic diseases associated with EndMT.
(S2015e3, S2016-3, and S2017-1) from Kanazawa Medical University. Mitsubishi Tanabe Pharma and Ono Pharmaceutical contributed to establishing the Division of Anticipatory Molecular Food Science and Technology. KK is under a consultancy agreement with Boehringer Ingelheim.

Conflict of interest
The authors declare no conflict of interest.

Author contributions
RG performed all the experiments and contributed to manuscript writing. KK proposed the original idea, designed and supervised the experiments, provided intellectual input, and edited the manuscript. JL participated in the establishment of the diabetic mouse model and immunofluorescence staining. MK and TO were involved in the discussions. DK provided intellectual input.