Fibroblast Growth Factor 23 and Klotho Contribute to Airway Inflammation

Abstract

Circulating levels of fibroblast growth factor (FGF) 23 are associated with systemic inflammation and increased mortality in chronic kidney disease. α-klotho, a co-receptor for FGF23, is downregulated in chronic obstructive pulmonary disease (COPD). However, whether FGF23 and klotho-mediated FGFR activation delineates a pathophysiologic mechanism in COPD remains unclear. We hypothesized that FGF23 can potentiate airway inflammation via klotho independent FGFR4 activation. FGF23 and its effect were studied using plasma and transbronchial biopsies from COPD and control patients and primary human bronchial epithelial cells isolated from COPD patients as well as a murine COPD model.

Plasma FGF23 levels were significantly elevated in COPD patients. Exposure of airway epithelial cells to cigarette smoke and FGF23 led to a significant increase in IL-1β release via klotho-independent FGFR4-mediated activation of phospholipase Cγ (PLCγ)/nuclear factor of activated T-cells (NFAT) signaling. In addition, klotho knockout mice developed COPD and showed airway inflammation and elevated FGFR4 expression in their lungs, whereas overexpression of klotho led to an attenuation of airway inflammation.

In conclusion, cigarette smoke induces airway inflammation by downregulation of klotho and activation of FGFR4 in the airway epithelium in COPD. Inhibition of FGF23 or FGFR4 might serve as a novel anti-inflammatory strategy in COPD.

INTRODUCTION

Chronic obstructive pulmonary disease (COPD) represents the third leading cause of global mortality according to the World Health Organization (http://www.who.int/gho/en/). Exposure to cigarette smoke is responsible for the majority of COPD cases and is the world’s leading cause of avoidable premature mortality (1). Various studies have shown inflammatory changes in the airway epithelium in COPD and due to cigarette smoke, including goblet hyperplasia and increased expression of pro-inflammatory cytokines (2-4).

Fibroblast Growth Factor (FGF) 23, a 27 kDa protein secreted by osteocytes, plays an essential role in maintaining serum phosphate homeostasis (5-7). In chronic kidney disease (CKD), plasma concentrations of FGF23 are elevated and strongly associated with the risk of CKD progression, cardiovascular events and mortality (8,9). To date, direct effects of FGF23 on the lung have not been reported. However, it has been shown that klotho expression is reduced in airways from COPD patients (10). Recombinant klotho, a single-transmembrane protein that exists in a membrane bound and soluble form and acts as a FGF23 co-receptor, protects the alveolar epithelium against oxidative damage (11-13). Furthermore, mice lacking klotho develop an aging phenotype with widened alveolar spaces, consistent with pulmonary emphysema (14).

FGF receptors (FGFR) are a sub-family of receptor tyrosine kinases and consist of four isoforms (FGFR1-4) (15). In its classic target organs, such as kidney and the parathyroid gland, FGF23 signals via FGFR1 and requires klotho. By binding to FGFR1/klotho, FGF23 activates the Ras/mitogen-activated protein kinase (MAPK) signaling pathway (16). In the absence of klotho, FGF23 can activate FGFR4 and induce subsequent PLCγ/NFAT signaling. This mechanism mediates pathophysiologic actions of FGF23 that include the induction of hypertrophic growth in cardiac myocytes and the increased production of inflammatory cytokines in hepatocytes (17,18).

We hypothesized that cigarette smoke contribute to increased production and activation of FGF23 signaling in COPD. Here, we examine whether cigarette smoke reduces klotho and increases FGFR4 expression resulting in pro-inflammatory PLCγ/NFAT signaling in human primary bronchial epithelial cells. We postulate that FGF23 can potentiate inflammation in the lung via activation of FGFR4 and downregulation of klotho.

RESULTS

Plasma FGF23 levels are increased in individuals with COPD

We measured FGF23 in plasma samples from 28 individuals, including 18 with COPD and 10 age-matched non-COPD patients. COPD was defined as a forced expiratory volume in 1-second to forced vital capacity (FEV1/FVC) < 0.70. The post-bronchodilator FEV1 percent predicted (FEV1%) was used to measure the severity of airflow obstruction, with a FEV1 ≥ 50% and ≤ 1 COPD exacerbation within the last year, indicating mild-to-moderate COPD (9 individuals) and a FEV1 < 50% and ≥ 2 COPD exacerbations within the last year, indicating severe COPD (9 individuals). The clinical characteristics, including age, sex, smoking history and percent predicted FEV1 (FEV1%) are shown in supplemental Table 1. As expected, the post bronchodilator FEV1% was significantly lower in both COPD groups when compared to controls.

Individuals with mild-to-moderate COPD had higher FGF23 plasma levels when compared to patients without COPD (59±12 versus 42±10 pg/ml, P=0.004; Fig. 1a). However, FGF23 was not significantly increased in severe COPD compared to controls in this study population (51 ± 18 versus 42±10 pg/ml, P=0.26). We therefore focused on mild-to-moderate COPD for further experimentations. We detected no difference between active smokers and non-smokers (Suppl. Fig. 1a).

Figure 1.

FGF23 levels are increased in patients with mild-to-moderate COPD who have goblet cell hyperplasia and increased levels of pro-inflammatory cytokines. (a) FGF23 plasma levels in non-COPD and COPD patients divided in mild-to-moderate and severe COPD and comparison of their FGF23 plasma levels. (b) Hematoxylin (upper row) and anti-FGF23 (lower row) staining of representative endobronchial biopsies from 1 control subject (left row) and 2 (middle and right row) subjects with mild-to-moderate COPD (20X magnification) with scale bar = 40 μm. (c) Quantification of goblet cells per field of view shows a significant increase in mild-to-moderate COPD when compared to subjects without COPD (n=6 of each group). (d) IL-6 plasma levels (left panel) and IL-8 levels in BALF (right panel) and dot blots showing their correlation with FGF23 levels in control subjects and patients with mild COPD (n = 6 of each group). Statistical analysis was done using Student’s t test showing mean ± S.E.M. or ANOVA with *P<0.05 and **P<0.01 and for correlation analysis the Pearson correlation coefficient.

Endobronchial biopsies from the mild-to-moderate subgroup with significantly elevated FGF23 levels showed bronchial epithelial cell metaplasia (Fig. 1b, upper panel). Tracheal sections from control and these COPD donors showed distinct FGF23 staining of the epithelial layer (Fig. 1b, lower panel). As expected, the COPD group had significantly increased goblet cell numbers when compared to the control group (Fig. 1c).

We observed significant increases in plasma IL-6 levels (Fig. 1d, left panel), and bronchoalveolar lavage fluid (BALF) IL-8 levels (Fig. 1d, right panel) were elevated in COPD patients. Determination of the Pearson correlation coefficient showed a significant positive correlation between plasma FGF23 levels and plasma IL-6 levels (R²= 0.2224 with p=0.0415, Fig. 1d, lower left panel)) but no significant correlation between plasma FGF23 levels and BALF IL-8 levels (R²= 0.01542 with p=0.7006, Fig. 1d, lower right panel). Combined, these data suggest that COPD patients develop increased plasma levels of FGF23, which coincide with elevated levels of inflammatory cytokines in the lung and circulation.

FGF23 and cigarette smoke induce IL-1β secretion in bronchial epithelial cells from individuals with COPD

To determine direct effects of FGF23 in combination with cigarette smoke, we exposed primary bronchial epithelial cells isolated from four different individuals with COPD (COPD-BEC) to cigarette smoke and FGF23 and assessed expression of IL-6, IL-8 and IL-1β by quantitative real time PCR. Overall, we observed a considerable heterogeneity in cytokine expression. IL-6 mRNA increased to FGF23 ± cigarette smoke with high variability (Fig. 2a); IL-8 was significantly increased with FGF23 ± cigarette smoke, but the increase was less variable and more modest (Fig. 2b). IL-1β mRNA levels were not significantly increased by FGF23 alone, but the combination of both stimuli led to an over 5-fold elevation (Fig. 2c). Exposure of COPD-BEC to either stimulus, however, caused a significant increase in basolateral secretion of IL-1β, which was further elevated, when both stimuli were combined (Fig. 2d). These findings indicate that, in combination with smoke, FGF23 can directly target COPD-BEC and induce the expression and release of IL-1β.

Figure 2.

Effect of cigarette smoke and FGF23 on expression of pro-inflammatory cytokines in COPD-BEC. (a) mRNA levels of IL-6 and (b) IL-8 in COPD-BEC 24h after stimulation with cigarette smoke (4 cigarettes) ± FGF23 (25 ng/ml). (c) IL-1β mRNA and (d) secreted protein levels in the basolateral media from COPD-BEC after stimulation with FGF23, CS or both. Data are represented as fold change in mRNA or protein expression with n = 3 independent experiments from 6 different lungs. All bar graphs are mean ± S.E.M. *P<0.05, **P<0.01 and ***P<0.005, compared to control (ctrl) group.

FGF23 activates FGFR4 and PLCγ/calcineurin/NFAT signaling in COPD-BEC resulting in increased IL-1β secretion

As shown in other tissues, FGF23 can induce both phosphorylation of PLCγ (19) and ERK (20) depending on the absence or presence of klotho. To determine whether FGF23 can activate FGFR4 and subsequent PLCγ/calcineurin/NFAT signaling and induce an inflammatory response in the lung, as we have recently shown in the liver (18), we analyzed FGFR4 binding to PLCγ after treatment with FGF23.

In COPD-BEC, stimulation with FGF23 + cigarette smoke caused phosphorylation of PLCγ, consistent with the premise that these effects might be mediated by klotho-independent FGFR4 signaling. On the other hand, FGF23 alone did not result in phosphorylation of ERK, an effect clearly associated with smoke exposure (Fig. 3a, left panel).

Figure 3.

FGF23 induces activation of FGFR4/PLCγ/NFAT signaling in COPD-BEC. (a) Left panel: Representative immunoblot and densitometric analysis (p-PLCγ/total PLCγ/actin ratio with n = 3) shows FGF23 + CS-induced phosphorylation of PLCγ (FGF23- and smoke dependent) and of ERK (purely smoke dependent). Right panel: Immunoblot of co-immunoprecipitation with FGFR4 using an anti-PLCγ antibody shows activation of PLCγ after FGF23 stimulation in COPD-BEC, which could be inhibited by pre-incubation with anti-FGFR4. (b) CS + FGF23 induce PLCγ and ERK phosphorylation as seen above but anti-FGFR4 only inhibits PLCγ phosphorylation. Both anti-FGFR4 and cyclosporine A (CsA), a calcineurin inhibitor, inhibit CS + FGF23-mediated induction of IL-1β mRNA and secreted protein levels. (c) CsA also attenuates induction of COX2 mRNA. FGF23, CSE alone and both activate NFAT, as assessed by a luciferase-based reporter gene assay in 16HBE cells, which can be blocked by CsA. Anti-FGFR4 inhibits FGF23-induced NFAT activation. The right two bar diagrams indicate mRNA levels of NFAT isoforms in both HBEC from nonsmokers and COPD patients as well as 16HBE cells. (d) siRNA targeting NFAT isoforms NFAT2c and NFAT3c were transfected into 16HBE cells. siNFAT2c reduces the expression of NFAT2c and NFAT3c, whereas siNFAT3c is specific for NFAT3c (left panel). When 16HBE cells were stimulated with FGF23 (20 ng/ml), CSE (5%) or both for 24 hours, IL-1β increased (second to right panel). Finally, the effects of siNFAT2c (knockdown of both the 2c and 3c isoforms) and siNFAT3c transfection were assessed after stimulation with CSE+FGF23 (middle panel). CSE±FGF23 also led to significant upregulation of FGFR4 protein expression in 16HBE cells. All n = 3 independent experiments (from 3 different lungs in the case of COPD-BEC) showing mean ± S.E. with *P<0.05, **P<0.01 and ^***P<0.005.

In order to verify this finding, endogenous PLCγ was immunoprecipitated and eluates analyzed by immunoblotting with an anti-FGFR4 antibody. Compared with eluates from control cells, levels of co-purified FGFR4 were elevated in FGF23-treated COPD-BEC while total PLCγ expression levels were unchanged. Pre-treatment with a FGFR4-specific blocking antibody (anti-FGFR4) (17) inhibited the co-immunoprecipitation of FGFR4 and PLCγ (Fig. 3a, right panel). Combined exposure of COPD-BEC to FGF23 and cigarette smoke also led to the phosphorylation of PLCγ which was blocked by anti-FGFR4 (Fig. 3b, left panel). Cigarette smoke, but not FGF23 stimulation by itself, caused a marked increase in ERK phosphorylation which has been shown before in bronchial epithelial cells (21). However, ERK phosphorylation was unchanged by pre-incubation with anti-FGFR4 (Fig. 3b left panel). These findings indicate that FGF23 signaling in the bronchial epithelium occurs mainly via activation of PLCγ rather than ERK and that this effect is mediated by FGFR4.

To determine if the FGFR4/PLCγ/calcineurin/NFAT signaling cascade mediates FGF23 effects on IL-1β expression, we pre-incubated COPD-BEC with anti-FGFR4 followed by stimulation with FGF23 and cigarette smoke. Anti-FGFR4 significantly reduced elevations in mRNA as well as secreted levels of IL-1β (Fig. 3b, middle panel). To establish the link between FGF23 and NFAT activation, we employed the widely used calcineurin inhibitor, cyclosporine A (CsA) (17,18) and showed that CsA also inhibited the FGF23 and CS induced IL-1β responses (Fig. 3b, right panel). Furthermore, mRNA levels of cyclooxygenase (COX) 2, a described NFAT target gene in the bronchial epithelium (22), were increased following co-stimulation with FGF23 and smoke. This effect was inhibited when cells were pre-treated with CsA (Fig. 3c). Additionally, we used the bronchial epithelial cell line 16HBE and transfected these cells with a NFAT reporter gene luciferase construct. An increase in NFAT activity was seen after stimulation with FGF23, which was significantly inhibited when cells were pre-incubated with anti-FGFR4 and CsA. Furthermore, cigarette smoke extract (CSE) and CSE+FGF23 also induced NFAT activation, which could be blocked by CsA (Fig. 3c, middle panel).

We identified NFAT2c and 3c as the most abundant isoforms in COPD-BEC, HBEC and 16HBE cells (Fig. 3c, middle and right panel). To further validate the specificity of FGF23 induced NFAT activation, both isoforms were knocked down by using siRNA in 16HBE cells (Fig. 3d). Since these 16HBE cells showed a similar IL-1β response as the primary cells, we stimulated control, siNFAT2c and 3c transfected 16 HBE cells with CSE and FGF23. SiNFAT2c decreased both NFAT2c and 3c, while siNFAT3c was isoform-specific. Compared to si control transfected cells, the CSE+FGF23 induced IL-1β response was blunted in siNFAT2c and siNFAT3c transfected cells (Fig. 3d, middle panel). This indicates that both NFAT2c and NFAT3c are mediators of FGF23-induced signaling. Furthermore, CSE ± FGF23 led to a significant increase in FGFR4 protein expression levels in 16HBE cells (Fig. 3d, right panel). Combined, our results show that FGF23 directly activates FGFR4/PLCγ/calcineurin/NFAT signaling in bronchial epithelial cells thereby inducing IL-1β.

Exposure to cigarette smoke downregulates klotho expression and upregulates FGFR4 in bronchial epithelial cells from individuals with COPD

Since klotho expression is decreased in COPD airways (10), we analyzed klotho expression in our primary air liquid interface bronchial epithelial cell culture system. Exposure of COPD-BEC to cigarette smoke caused a significant decrease in klotho protein and mRNA levels, whereas FGF23 alone had no effect (Fig. 4a). Compared to HBEC derived from non-smoker and non-COPD lungs, klotho mRNA and protein levels were significantly reduced in COPD-BEC (Fig. 4b). These data show that in COPD-BEC, klotho expression is decreased and further reduced by cigarette smoke, indicating that both COPD and smoke might serve as factors that promote klotho-independent FGF23 signaling in the lung.

Figure 4.

Cigarette smoke exposure regulates expression of klotho and FGFR4 in COPD airway epithelia. (a) Representative immunoblot showing downregulation of klotho protein and bar graphs indicating mRNA levels from COPD-BEC after treatment with CS, FGF23 or both stimuli for 24h. (b) Representative immunoblot and klotho mRNA levels of HBEC from nonsmokers compared to COPD-BEC. (n = 3 independent experiments from 6 different lungs. (c) Immunohistochemistry for FGFR4 and counterstaining with hematoxylin of endobronchial biopsies from 2 representative control patients and 2 COPD patients, compared to a negative control (upper left corner, 20X magnification). Bar graphs showing increased FGFR4 mRNA expression in COPD-BECs compared to control lungs. (d) Immunoblot analysis of FGFR4 protein levels (representative blot from 1 COPD patient, tested in 3 different COPD lungs) and quantitative real time PCR of FGFR4 mRNA levels in COPD-BECs 24h after exposure to 2, 4 and 6 cigarettes (1 cigarette = 6 puffs). (All n = 3 independent experiments from 3 different lungs showing mean ± S.E.M. with **P<0.01 and ***P<0.001).

We measured FGFR4 expression in the lung, as FGFR4 has been previously shown to mediate the klotho-independent FGF23 signaling in the heart and liver (17,18). Paraffin embedded endobronchial biopsies were used for hematoxylin staining and FGFR4 immunohistochemistry. FGFR4 staining was localized to the bronchial epithelium and appeared to be increased in endobronchial tissue biopsies from COPD patients (Fig. 4c, left panel). To better quantify FGFR4 expression, FGFR4 mRNA levels were determined and showed a significant increase in COPD-BEC compared to control HBEC (Fig. 4c, right panel).

Next, COPD-BEC were exposed to an increasing number of cigarette puffs. A dose dependent upregulation of FGFR4 protein and mRNA levels was detected after 24 hours (Fig. 4d). Taken together, FGFR4 is expressed in the bronchial epithelium and upregulated in COPD patients and upon exposure to cigarette smoke.

Klotho deficiency leads to increased FGFR4 activation and inflammation

As genetic mouse models lacking klotho develop elevated serum FGF23 levels, we wanted to determine if klotho-independent FGF23 signaling and associated inflammation occur in the lungs of these animals. As shown by others before (12), we observed widened airway spaces on H&E staining (Fig. 5a) by immunohistochemical characterization of the klotho hypomorphic mice (kl^−/−) (Fig. 5a).

Figure 5.

Loss of klotho expression causes increased airway inflammation in mice. (a) Hematoxylin and anti-FGF23 staining of lung tissue from wild type (kl^+/+) and hypomorphic klotho (kl^−/−) mice (4X magnification). (b) Mean linear intercept analysis demonstrates widened airspaces in the kl^−/− lung samples and FGF23 mRNA levels are upregulated in whole lung tissue from kl^−/− when compared to kl^+/+ mice (6 mice for each group) (left panel). Immunohistochemical staining using anti-FGF23 in paraffin embedded mouse lung tissue sections indicates a signal in the bronchial epithelium (right panel). (c) Assessment of total cells in BALF from kl^+/+ versus kl^−/− mice (4 mice for each group). Also shown are BALF macrophage/monocyte count, BALF lymphocyte count × 10⁴ per ml BAL fluid and IL-6 fold changes in BALF from kl^+/+ versus kl^−/− mice. (d) Co-immunoprecipitation of endogenous FGFR4 by using anti-PLC-γ or anti-GFP (control) in lung tissue lysates from either kl^−/− or kl^+/+ mice indicates increased fgfr4 in kl^−/− lungs. qRT-PCR for FGFR4 mRNA in kl^−/− MTEC showed a significant upregulation when compared to wild type MTEC and IL-6 protein levels in basolateral media from kl^+/+ and kl^−/− MTEC. (Shown are means ± S.E.M. with *P<0.05, **P<0.01 and ***P<0.001; for mouse experiments, three different independent experiments were done; for each experiment, cells were pooled from 3-4 animals and morphometry and BALF analysis was done from 4 mice in each group).

Mean linear intercept (MLI × 1000 in μm) analysis was used to quantify airspace enlargement, which was significantly higher in klotho deficient mouse lungs compared to wild type (kl^+/+) littermates (Fig. 5a, upper panel). Furthermore, FGF23 mRNA levels were upregulated in in kl^−/− lungs when compared to kl^+/+ (Fig. 5a, lower panel). Immunhistochemical analysis of lung sections using anti-FGF23 showed an intense signal in the bronchial epithelium (Fig. 5b, right panel). When compared to their wild type litter mates, bronchoalveolar lavage fluid (BALF) from kl^−/− mice contained more cells, specifically neutrophils (23) and macrophages/monocytes (Fig. 5c). There was no difference in the number of lymphocytes between both groups (Fig. 5c), but IL-6 mRNA levels were also increased (Fig. 5c). To determine the activity of FGFR4/PLCγ, we immunoprecipitated PLCγ from mouse lung tissue extracts followed by immunoblotting of eluates for FGFR4. We found an increase in co-purified FGFR4 in lungs from kl^−/− mice when compared to wild type littermates (Fig. 5d, left panel). Furthermore, FGFR4 mRNA levels were increased in cultured, fully differentiated murine tracheal epithelial cells (MTEC) from kl^−/− mice (Fig. 5d, middle panel). They also showed a significantly increased basolateral secretion of IL-6 (Fig. 5d, right panel). These findings indicate that in the absence of klotho and in the presence of elevated FGF23, FGFR4/PLCγ signaling and production of inflammatory cytokines are elevated in the mouse lung.

Soluble klotho protects COPD-BECs from pro-inflammatory effects of FGF23 and smoke

The extracellular domain of klotho can exist in a soluble form (24). It has been shown that soluble klotho has protective, anti-inflammatory effects on different cell types (25,26). To test if soluble klotho interferes with the effects of FGF23 in the lung, we co-incubated COPD-BEC with FGF23, cigarette smoke and with the ectodomain of recombinant human α-klotho at 0.1 μg/ml for 30 minutes. As shown before, FGF23+CS caused an increase in PLCγ phosphorylation without affecting total PLCγ levels. In the presence of FGF23 and cigarette smoke, treatment of COPD-BEC with soluble klotho also led to a decrease in IL-1β secretion (Fig. 6b).

Figure 6.

Supplementation of human recombinant klotho inhibits CS and FGF23-induced IL-ϊβ secretion. (a) Representative immunoblots show FGF23 (25 ng/ml for 30 min) induced phosphorylation of PLCγ. Inhibition of PLCγ phosphorylation occurs by pre-incubation with klotho (1 µg /ml, lane 3 and 0.1 µg /m, lane 4). (b) Soluble klotho inhibits CS+FGF23-mediated increases of IL-1β protein levels, assessed by ELISA from basolateral media. (All n = 3 independent experiments from 3 different lungs showing mean ± S.E. with **P<0.01).

Overexpression or knockdown of klotho in mice alters FGF23/FGFR4 signaling.

Since mice that were exposed to chronic cigarette smoke (27) did not show an upregulation of FGF23 serum levels (Suppl. Fig. 1b), we used an acute mouse model for cigarette smoke exposure. Mice were exposed to one week of cigarette smoke to induce acute airway inflammation. Western blot analyses of total lung tissue showed in some mice an increase in FGF23 levels (Fig. 7a: 6/11 mice in smoke exposure, 3/11 mice in controls) (Fig. 7a). However, there was no significant increase in serum FGF23 levels between the control and smoked-exposed groups (Fig 7a), and there was no significant difference in mRNA levels of klotho or FGFR4 in lung tissue (Fig. 7a). When comparing wild type mice and klotho overexpressing mice (WT, OE) (28), the later group showed increased klotho mRNA levels in the lung, which was independent of smoke exposure (Fig. 7b). We detected no difference in serum FGF23 levels and in BALF total cell count (Fig. 7b). Smoke-induced increase of IL-6 in the lung was decreased in the klotho overexpressing mice when compared to wild type littermates (Fig. 7b). To test the effect of klotho deficiency and since kl^−/− mice were too sick to be exposed to cigarette smoke, MTECs were isolated from both kl^+/+ and kl^−/− mice. At baseline, kl^−/− MTECs had higher mRNA levels of IL-6 and fgfr4 when compared to wild type cells (Fig. 7c).

Figure 7.

Effect of klotho overexpression and klotho deficiency on FGF23 signaling in mice. (a) Representative immunoblots show FGF23 protein levels in total lung lysates from control mice and mice acutely exposed to cigarette smoke (CS). Bar graphs demonstrating serum FGF23 levels, total lung klotho and FGFR4 mRNA levels in both mouse groups (b) Klotho mRNA levels, serum FGF23 levels, BAL fluid total cell count and CS-induced fold increase in total mouse lung IL-6 expression after exposure to acute CS and compared to non-exposed control mice (both wild type and klotho overexpressing mice). c) Bar graphs indicate IL-6 and FGFR4 transcript levels of MTECs isolated from kl^+/+ and kl^−/− mice (Means are shown ± SEM with **p<0.01 and ***p<0.005).

To further characterize the link between klotho deficiency, FGF23 increase and elevated FGFR4 expression as direct or indirect, MTECs from fgfr4 constitutively active transgenic (TG) mice were analyzed and did not show any difference in IL-6 levels compared to control MTEcs. Interestingly, there was also a trend toward an increase in klotho mRNA levels in these fgfr4 TG MTECs (Suppl Fig. 2a and b). MTEcs from fgfr4^−/− mice though showed protection from FGF23 and CS induced IL-6 increase accompanied by a downregulation of klotho expression (Suppl. Fig. 2c and d).

In summary, our findings indicate that in COPD, there is smoke induced upregulation of FGFR4 expression and a decrease in soluble klotho, enabling elevated FGF23 to signal via FGFR4/PLCγ/NFAT to induce airway inflammation (Fig. 8). Furthermore, co-administration with soluble klotho can block FGF23-mediated signaling in COPD-BEC thereby inhibiting the pro-inflammatory actions of FGF23.

Figure 8.

Possible signaling mechanism for cigarette smoke and FGF23-mediated regulation of FGFR4/PLCγ/calcineurin/NFAT pathway. Diagram illustrating the direct effects of FGF23 and cigarette smoke on the COPD airway epithelium: cigarette smoke induces upregulation of FGFR4 and decreases klotho expression in the bronchial epithelium. In combination with elevated FGF23 plasma levels in COPD activates FGFR4/PLCγ/calcineurin/NFAT signaling to release IL-1β thereby inducing inflammation. Definition of Abbreviations: IL=interleukin, FGF=fibroblast growth factor, FGFR= FGF receptor, NFAT= Nuclear factor of activated T-cells, PLC=phospholipase C.

DISCUSSION

Here, we show for the first time FGF23-induced signaling in the bronchial epithelium and provide new insights into the opposing roles of FGF23 and its co-receptor klotho in cigarette smoke-induced chronic bronchitis. We demonstrate that 1) plasma FGF23 levels are elevated in patients with mild-to-moderate COPD and an associated inflammatory phenotype with goblet cell hyperplasia, 2) cigarette smoke exposure in combination with FGF23 induces IL-1β secretion from primary human bronchial epithelial cell cultures from COPD patients, 3) cigarette smoke induces an increase of FGFR4 and a decrease of klotho expression thereby causing activation of PLCγ/NFAT signaling and inflammation, and 4) the presence of soluble klotho attenuates smoke-induced IL-1β secretion.

A positive correlation between plasma FGF23 levels and smoking has been described previously using a multivariable regression analysis of 604 patients with CKD (29). However, in this study the smokers were not further characterized regarding COPD. In a different report, increased FGF23 levels in COPD patients were linked to hypophosphatemia, but a mechanism for elevation and the source of circulating FGF23 was not identified (30). Here, we show that FGF23 levels appear not to correlate with disease severity per se, since there is no systemic FGF23 elevation in patients with severe COPD. One explanation could be that FGF23/klotho perturbations may decrease in advanced disease due to “burn-out” as the residual mass of viable lung epithelium decreases. If this is the case, FGF23/klotho dysregulation may effectively represent a marker not only of disease but also for “at risk” lung tissue mass. Therefore, we focused on the group of these patients with mild-to-moderate COPD by assessing inflammation in their plasma and transbronchial biopsy specimens. Klotho, a co-receptor for FGF23, is downregulated in airway epithelial cells from COPD patients (10). In addition, previous reports showed that gene silencing of klotho in a bronchial epithelial cell line induces IL-8 secretion which is further enhanced by cigarette smoke extract (10).

Our data also show that in a COPD mouse model (due to deficiency of klotho), there is increased bronchial inflammation, which is at least partially generated by the bronchial epithelial cells and an influx of macrophages and neutrophils. We further characterize the underlying novel signaling pathway for smoke-induced inflammatory airway disease that has the potential for future therapeutic intervention, namely FGFR4 activation and consecutive PLCγ/calcineurin/NFAT signaling which occurs in a state of klotho deficiency and upregulation of FGF23. It is challenging to experimentally characterize the link between klotho deficiency, and our data indicate that regulation of both is tightly linked. It has been shown previously that klotho knockout mice have elevated FGF23 levels (19). Currently, it is not clear how soluble klotho blocks the FGF23 effects in bronchial epithelial cells, since ERK phosphorylation is not altered. However, we showed that soluble klotho is not a circulating co-receptor for FGF23 in the lung that mediates ERK signaling. Therefore, further studies are needed.

Interestingly, we also observed ERK activation in cigarette smoke-induced IL-1β expression, which appears to be FGF23/FGFR4 independent, as there was no change after pre-incubation with a FGFR4 inhibitor (Fig. 3b). Since cigarette smoke downregulates klotho expression, FGF23 signaling occurs via FGFR4 as we have previously demonstrated in the liver and in the myocardium (17,18). This ultimately leads to an increase in IL-1β secretion, a key cytokine in virus-induced lung disease (31). Administration of an IL-1β antibody reduces lung inflammation in a mouse model of influenza (32). Furthermore, IL-1β is elevated in total lung tissue and sputum from COPD patients (33) and associated with frequent COPD exacerbations as well as an IL-1β-associated sputum proteomic signature (34,35).

The importance of identifying new COPD-related signaling pathways is required for future therapeutic strategies. FGFRs are ubiquitously expressed, whereas FGFR4 is mainly found in lung, heart, kidney and liver. Signaling via FGFR4 is associated with inflammatory changes and we demonstrate several links of FGFR4 signaling to airspace remodeling and inflammation. In addition, a FGFR4 blocking antibody and small molecule inhibitors have been developed for the treatment of hepatocellular carcinomas (36,37). Therefore, therapeutically targeting FGFR4 or soluble klotho levels in the inflammatory subtypes of COPD may represent novel therapeutic options.

METHODS

Please refer to Supplementary Material for further methodological details.

Study Approval

The clinical study was conducted according to the principles of the Declaration of Helsinki and was approved by the University of Miami Institutional Review Board. Written informed consent was received from each participant prior to inclusion in the study. Human airways were obtained from organ donors whose lungs were rejected for transplant. Institutional Review Board-approved consent for research was obtained by the Life Alliance Organ Recovery Agency of the University of Miami or the LifeCenter Northwest and conformed to the Declaration of Helsinki. All animal protocols were approved by the Institutional Animal Care and Use Committees at the University of Miami and the University of Alabama at Birmingham.

IL-1β, IL-6 and IL-8 ELISA and mRNA assessment

An ultrasensitive IL-1β and IL-6 enzyme-linked immunosorbent assay (ELISA) from Invitrogen (Thermo Fisher Scientific) was used. HBEC were stimulated with FGF23 ± cigarette smoke (25 ng/ml and 4 cigarettes for 24 h) and 100 μl of the basolateral medium (undiluted) was used for measurements. Gene expression was performed by qPCR using Taqman probes (Life technologies/Applied Biosystems, Carlsbad, CA, USA).

Statistics

Data were analyzed with Prism5 (GraphPad Software, Inc., La Jolla, CA) and shown as mean ± SEM using Student's t test and analysis of variance or Kruskal Wallis H test with one-way ANOVA with appropriate post tests for at least three independent experiments. Significance was accepted at p < 0.05.