Elevated FGF21 leads to attenuated postnatal linear growth in preterm infants through GH resistance in chondrocytes.

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Elevated FGF21 Leads to Attenuated Postnatal Linear Growth in Preterm Infants Through GH Resistance in Chondrocytes Leonardo Guasti,* Sanna Silvennoinen,* Neil W. Bulstrode, Patrizia Ferretti, Ulla Sankilampi,* and Leo Dunkel* Centre for Endocrinology (L.G., L.D.), William Harvey Research Institute, Barts and the London, Queen Mary University of London, London EC1M 6BQ, United Kingdom; Department of Pediatrics (S.S., U.S.), Kuopio University Hospital, 70210 Kuopio, Finland; Department of Plastic Surgery (N.W.B.), Great Ormond Street Hospital for Children National Health Service Trust, London WC1N 3JH, United Kingdom; and Developmental Biology Unit (P.F.), University College London Institute of Child Health, London WC1N 1EH, United Kingdom

Context: The hormone fibroblast growth factor 21 (FGF21) is a key metabolic regulator in the adaptation to fasting. In food-restricted mice, inhibition of skeletal growth is mediated by the antagonistic effect of FGF21 on GH action in the liver and growth plate. Objective: The objective of the study was to assess the role of FGF21 in growth regulation in humans using postnatal growth failure of very preterm infants as a model. Design: FGF21 levels were measured serially in very preterm infants, and their linear growth evaluated from birth to term-equivalent age. Primary chondrocytes obtained from pediatric donors were used to test whether FGF21 can directly interfere with GH signaling. Results: A negative association (␤ ⫺.415, P ⬍ .005, linear regression model) of FGF21 levels with the change in SD score for length was found. In primary chondrocytes, FGF21 upregulated basal and GH-induced SOCS2 expression and inhibited GH-induced signal transducer and activator of transcription 5 (STAT5) phosphorylation as well as GH-induced COLII and ALP expression. Finally, FGF21 inhibited GH-induced IGF-1 expression and cell proliferation, indicating GH resistance. However, FGF21 did not affect IGF-1–induced cell proliferation. Conclusions: Elevated FGF21 serum levels during the first weeks of life are independently associated with postnatal growth failure in preterm infants. Furthermore, our data provide mechanistic insights into GH resistance secondary to prematurity and may offer an explanation for the growth failure commonly seen in chronic conditions of childhood. (J Clin Endocrinol Metab 99: E2198–E2206, 2014)

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pproximately 5% to 10% of children are born prematurely, and 1% of children are born ⬍32 weeks gestation, as very preterm (VPT) infants. Despite improvements in neonatal intensive care including standardized nutrition, growth retardation is common in these infants, and final height is often compromised (1, 2). Postnatally reduced GH/IGF-1 action impairs linear growth both in term (3) and preterm (4) infants, but at the same time, even healthy infants display biochemical features of GH resis-

tance (high GH and low IGF-1 levels) through yet unknown mechanisms (5, 6). Recently, a new mechanism of GH resistance and growth failure was proposed in mice. In a seminal study by Kubicky et al (7), induction of fibroblast growth factor 21 (FGF21) by undernutrition was shown to cause GH resistance. It was also shown that FGF21 antagonized GH actions on chondrogenesis directly at the growth plate and that high concentrations of FGF21 directly suppress

ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received February 26, 2014. Accepted July 18, 2014. First Published Online August 19, 2014

* L.G., S.S., U.S., and L.D. contributed equally to this work. Abbreviations: CV, coefficient of variation; FBS, fetal bovine serum; FGF21, fibroblast growth factor 21; FGFR, FGF receptor; GHR, GH receptor; RT-qPCR, quantitative RT-PCR; SDS, SD score; STAT5, signal transducer and activator of transcription 5; SOCS2, suppressor of cytokine signaling 2; VPT, very preterm.

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J Clin Endocrinol Metab, November 2014, 99(11):E2198 –E2206

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growth plate chondrocyte proliferation and differentiation (8). Importantly, growth failure due to undernutrition was attenuated in FGF21-knockout compared with wildtype mice (7). FGF21, along with FGF15/19, lacks the FGF heparinbinding domain; therefore, it can be released from the site of synthesis and function as an endocrine factor (9). During fasting, increased hepatic secretion of FGF21 induces gluconeogenesis, fatty acid oxidation, and ketogenesis. Thus, FGF21 is considered a key regulator of the metabolic adaptation to fasting (10). In target tissues, FGF21 binds to FGF receptors (FGFRs). A preferential binding to the complex FGFR1/␤-klotho has been described (11, 12). In this study, the role of FGF21 in mediating GH resistance was examined in a cohort of VPT infants. The molecular mechanisms underlying FGF21 actions were investigated using an in vitro model of primary human chondrocytes. We demonstrate for the first time that elevated FGF21 levels impair linear growth in VPT infants via a mechanism likely involving direct inhibition of GH action on chondrocytes. This suggests an important role for FGF21 in the regulation of postnatal linear growth in humans.


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Table 1. Clinical Characteristics of the 32 VPT Infants (19 Males) Median/n Range At birth Gestational age, wk Weight, kg Weight, SDa Length, cm Length, SDa At term-equivalent age Postmenstrual age, wk Weight, kg Weight, SDb Length, cm Length, SDb Nutrition at sampling Week 1 (n ⫽ 29) Enteral Combined parenteral and enteral Total parenteral Week 3 (n ⫽ 32) Enteral Combined parenteral and enteral Total parenteral Week 5 (n ⫽ 28) Enteral Combined parenteral and enteral Total parenteral

28.2 0.99 ⫺0.2 36.8 ⫺0.1

23.4 –31.9 0.48 –1.92 ⫺2.2 to 1.7 28.0 – 44.0 ⫺2.6 to 1.6

40.0 2.99 ⫺1.2 47.5 ⫺1.1

37.0 – 41.6 1.55–3.79 ⫺4.3 to 0.4 40.0 –51.5 ⫺4.5 to 0.7

5 17 7 19 11 2 22 5 1

a Birth weight and length were converted to SDS using the populationbased birth size reference for singletons or twins (16).

Patients and Methods

b Weight and length at term-equivalent age were converted to SDS using the population-based birth size reference for singletons (16).

Patients As part of the Finnish PreBaby study on metabolism and growth in VPT infants, 32 infants (19 boys, 57%) were recruited during the first week of life at the Kuopio University Hospital neonatal intensive care unit (Table 1). The median gestational age at birth was 28.2 (range 23.4 – 31.9, SD 2.9) weeks, and the median length of hospitalization was 67 (range 28 –182, SD 45.4) days. At discharge, the median postmenstrual age was 38.6 (range 35.4 –51.0, SD 4.1) weeks. Two sets of unidentical twins and one set of unidentical triplets were included in the study cohort. All the VPT infants survived, but presented with typical morbidity associated with prematurity: 5 infants (16%) had early or late-onset septic infection, three (9%) had necrotizing enterocolitis, 8 (25%) developed bronchopulmonary dysplasia (diagnosed at 36 postmenstrual weeks by the oxygen reduction test) (13), 2 (6%) had intraventricular hemorrhage grade III or IV, and 2 infants (6%) developed retinopathy of prematurity. Their nutrition adhered to current recommendations (14, 15). The VPT infants were fed immediately with a high-dose iv protein and carbohydrate infusion, an early lipid infusion, and minimal enteral feeding to avoid undernutrition and starving and to achieve full enteral feeding as early as possible. Data on nutrition (enteral nutrition, combined enteral and parenteral nutrition, or total parenteral nutrition) were registered. The median age when the full enteral nutrition was achieved was 10 (range 3–101, SD 22.9) days. Weight and recumbent length were measured at birth, at weeks 1, 3, and 5, and at term-equivalent age using the Giraffe OmniBed inbed scale (Ohmeda Medical) in intensive care or a baby scale (Seca model 376) and neonatometer (Pedihealth). These measures were trans-

formed into SD scores (SDSs) using the contemporary population-based reference (16). Small for gestational age was defined as birth weight or length at least 2 SD below the sex- and gestational age-specific reference mean (17). Extrauterine growth retardation was defined as weight or length at least 2 SD below the sex-specific reference mean at the term-equivalent age. Only 2 of the 32 VPT infants had birth weight or length below ⫺2.0 SD (ie, were small for gestational age at birth). Mixed umbilical blood samples were obtained immediately after birth and peripheral venous or arterial samples in weeks 1, 3, and 5. The plasma and serum samples were prepared by centrifugation after blood collection, separated into aliquots, and stored at ⫺70°C until analyzed. Because of the extremely small size of our patients, blood samples were not obtained after overnight fasting. Plasma FGF21 concentrations were measured by an FGF21 human ELISA kit (BioVendor) according to the manufacturer’s instructions. Plasma was diluted 1:1.5 or 1:2. The measuring range with 1:1.5 dilution was 11.3 to 2880 pg/mL and with 1:2 dilution 15 to 3840 pg/mL. Within-run coefficient of variation (CV) was 4.6% (mean FGF21, 223 pg/mL), and between-run CV was 13.9% (mean 611 pg/mL). Serum IGF-1 concentrations were measured by an ELISA kit (Mediagnost GmbH). The within-run CV was 4.7% (mean IGF-1 4.8 nmol/L), and between-run CV for control serum was 6% (mean IGF-1, 13.6 nmol/L) in the range of 1.84 to 65 nmol/L.


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FGF21 in Preterm Infants

Cells Chondrocyte cultures were established from rib cartilage of consented pediatric patients undergoing facial reconstruction (because of hemifacial microsomia with microtia) at Great Ormond Street Hospital, London, UK. In particular, patients (n ⫽ 3 males, aged 9, 10, and 11 years old) underwent autologous costal cartilage to ear graft, and they had normal linear growth. The protocol to establish primary cultures, their markers’ expression, and chondrogenic potential has been described previously (18, 19). Cells were grown in high-glucose (4500 mg/L) DMEM (Life Technologies) supplemented with 10% embryonic stem cells qualified fetal bovine serum (FBS) (Life Technologies) and 1% penicillin/ streptomycin (Life Technologies) at 37°C in a humidified incubator with 5% CO2. Cells were used at passage 4 or lower.

RT-PCR and quantitative RT-PCR Human brain RNA was obtained from Agilent Technologies. Human liver RNA was from Clontech. Human rib cartilage RNA from pediatric donors was extracted using TRIzol (Life Technologies) followed by RNeasy Mini kit (QIAGEN). RNA from primary chondrocytes was extracted using RNeasy Mini kit. cDNAs were synthesized using Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Promega) according to the manufacturer’s instructions. PCR to detect FGFR1 (isoforms IIIb and IIIc), ␤KLOTHO, GHR, and GAPDH was performed in a GS1 thermocycler (GStorm) using 1.25 U Taq polymerase (New England Biolabs), 200␮M of each dNTP, 1.5mM MgCl2, and 0.5␮M specific primers. For quantitative RT-PCR (RT-qPCR), amplification was in a 10-␮L reaction containing 2 ␮L cDNA template, 5 ␮L 2⫻ SYBR Green I Master Mix (KAPA Biosystems), 0.2 ␮L low ROX (KAPA Biosystems), 0.5␮M of each primer and 2.3 ␮L nucleasefree H2O and performed using an Mx3000 thermocycler (Stratagene). Data were analyzed with MxPro software (Stratagene). For RT-PCR and RT-qPCR, GAPDH was used as housekeeping gene. Primers and amplification conditions are reported in Supplemental Table 1. Each reaction was carried out in triplicate, and each experiment was repeated 3 times. Normalization and quantification of RT-qPCR data were performed using the relative cycle threshold method. Data are expressed as fold change relative to GAPDH.

Cell treatments GH and IGF-1 responsiveness Cells (1 ⫻ 105 cells per well in 24-well plates) were serumstarved and when 80% confluent (24 hours later) treated with recombinant GH (500 ng/mL; Life Technologies) or IGF-1 (100 ng/mL; Life Technologies). Cells were lysed in 2⫻ Laemmli buffer (Sigma) after 0, 5, 10, 20, 30, and 60 minutes and then processed for Western blotting. In another set of experiments, cells were serum-starved for 24 hours in the absence or presence of recombinant FGF21 (5 ␮g/mL; Abnova) before GH or IGF-1 challenge for 20 minutes.


free medium) for 24 hours, cells were challenged with recombinant human GH (500 ng/mL) for 30 minutes or 8, 16, or 24 hours before RNA extraction. FGF21 was kept in the medium during GH treatment. FGF21 concentrations lower than 0.5 ␮g/mL did not elicit any SOCS2 or IGF-1 transcriptional response (not shown). To assess the effect of FGF21 in GH-induced chondrogenic differentiation, cells that had been confluent for 72 hours were treated with recombinant GH (500 ng/mL) in DMEM/1% FBS in the absence or presence of recombinant FGF21 (5 ␮g/mL) for 7 days before RNA extraction. Medium was changed every 2 days.

Proliferation Cells (1 ⫻ 104 cells per well in 96-well plates) were serumstarved for 24 hours and then incubated with or without 5 ␮g/mL recombinant FGF21 for 24 hours. Cells were then incubated with or without 500 ng/mL GH or 100 ng/mL recombinant IGF-1, all diluted in DMEM/1% FBS, for 96 hours. FGF21 was kept in the medium during GH and IGF-1 treatment. Cells were fixed in 4% paraformaldehyde in ice-cold PBS for 30 minutes and then stained with methylene blue (Sigma) in 0.01M boric acid (pH 8.5) (1% wt/vol). After washes with boric acid, the dye was extracted by incubating plates for 16 hours with a solution consisting of 50% ethanol and 50% 0.1M HCl. Absorbance was measured at 650 nm with a Microplate Reader (Bio-Rad).

Protein analysis Anti-FGF21 (Abnova) and control rabbit IgG (Insight Biotechnology Limited) were cross-linked to the Thermo Scientific Ammonolink Plus coupling resin (part of the Microlink protein coupling kit; Pierce Thermo Scientific) following the instructions provided. Pooled serum (100 ␮L) from VPT infants was diluted 10 times with PBS and incubated with the columns overnight at 4°C before washes and elution as reported in the kit protocol. Aliquots of eluates were mixed with reducing or nonreducing Laemmli buffer. Samples were size-separated through a NuPAGE Novex BisTris 4% to 12% gradient gel (Life Technologies), along with the Novex Sharp prestained protein ladder (Life Technologies) and blotted onto nitrocellulose membranes (Fisher). Membranes were incubated with blocking buffer, consisting in 5% nonfat dry milk (Asda) in PBS containing 0.1% Tween 20 for 2 hours at room temperature. They were then incubated with primary antibodies (information in Supplemental Table 2) diluted in blocking buffer. After washes, membranes were incubated with goat antimouse IRDye800 and goat antirabbit IRDye680 (Life Technologies; 1:10000 dilution). Immunoblots were scanned using the Odyssey Infrared Imaging System (LI-COR). In other sets of experiments, 1 ␮g recombinant FGF21 was size-separated as described above and gels silver stained (silver stain for mass spectrometry kit; Pierce), following the instructions provided.

Study approval Quantitative RT-PCR Cells (1 ⫻ 105 cells per well in 24-well plates) were serumstarved for 24 hours before treatment with recombinant proteins or VPT patient serum with a low or high endogenous FGF21. After recombinant FGF21 treatment (0.5 or 5 ␮g/mL in serum-

This study was carried out with ethical approval (Ethics Committees of the Pohjois-Savo Health Care District, Finland, and Camden and Islington Community Local Research Ethics Committee). Informed consent was obtained from both parents of all study participants.


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SPSS software version 19.0 (SPSS Inc) was used for statistical analysis. P values ⬍.05 were considered significant. For in vitro experiments, statistical significance was evaluated by ANOVA and Student’s t test (Vassar Stats Online Calculator) and P values ⬍.05 were considered significant. Data are presented as means ⫾ SEM. Each experiment was performed in triplicate on chondrocytes from at least 3 donors.

Results FGF21 levels and growth failure in VPT infants FGF21 levels were below the sensitivity of the assay in 85% of infants in the umbilical blood, and in 31%, 25%, and 18% of the infants at weeks 1, 3, and 5, respectively. A significant increase in FGF21 level was Figure 1. FGF21 levels increased by age and were significantly associated with the change in observed from week 1 (median 33.2, ⌬SDS for length but not for weight. A, A significant increase in FGF21 level from week 1 (median 33.2, range ⬍11.3–1921.0 pg/mL) to week 3 (median 44.0, range ⬍11.3–928.2) and to week 5 range ⬍ 11.3–1921.0 pg/mL) to (median 92.6, range ⬍11.3–2384.0 pg/mL) was observed. B and C, Association of the postnatal week 3 (median 44.0, range ⬍ 11.3– FGF21 level (mean of log-transformed FGF21 concentrations at weeks 1, 3, and 5) and ⌬SDS for 928.2 pg/mL; P ⫽ .001), and from length from birth to term-equivalent age (B) and ⌬SDS for weight from birth to term-equivalent age (C) in 32 VPT infants. Standardized regression coefficients (␤) and P values were obtained weeks 3 to 5 (median 92.6, range from the linear regression model adjusted for gestational age at birth, birth size, sex, nutrition, ⬍11.3–2384.0 pg/mL; P ⫽ .008) and average postnatal IGF-1 level at weeks 1, 3, and 5. (Figure 1A). The clinical characteristics of our cohort are reported in Table 1. At Statistics FGF21 and IGF-1 concentrations that were below the limit of birth, the median birth weight SDS was ⫺0.2 (range ⫺2.2 detection of the assays, were substituted with a constant value to 1.7; 2 infants had a birth weight SDS ⬍ ⫺2). At termthat was half of the limit of detection (for FGF21, 5.6 pg/mL, and equivalent age (range 37.0 – 41.6 weeks), the median for IGF-1, 0.92 nmol/L, respectively) to avoid their dropping out weight SDS was ⫺1.2 (range ⫺4.3 to 0.4; 11 infants had of the statistical analyses. To identify factors associated with a weight SDS ⬍ ⫺2). Mean ⌬SDS (individual changes) for FGF21 levels in VPT infants, between- and within-group comweight was ⫺1.3 (range ⫺3.8 to 0.0). Median birth length parisons were done using a mixed-models analysis, suitable because of the potential correlation structure of the data caused by SD was ⫺0.1 (range ⫺2.6 to 1.6; only 1 VPT infant had the twins/triplets and repeated measurements. Gestational age, sex, birth length SDS ⬍ ⫺2). At term-equivalent age, median time point at sampling, birth weight SDS, birth length SDS, parlength SD was ⫺1.1 (range ⫺4.5 to 0.7; 11 infants had enteral nutrition (yes/no), enteral nutrition (yes/no), and IGF-1 length SDS below ⫺2). Mean ⌬SDS for length (individual concentration were included in the model as fixed effects, and the changes) was ⫺1.5 (range ⫺4.2 to 0.2). A negative ⌬SDS subject and pair (twins or triplets) as random effects. The FGF21 data were right-skewed and therefore transformed logarithmifor length and weight was observed in every VPT infant cally to achieve normality of residuals in the mixed-models after birth. analysis. Factors potentially contributing to growth failure after To assess factors associated with postnatal growth failure birth were analyzed and are shown in Table 2. Higher from birth to term-equivalent age, linear regression analysis was gestational age at birth was associated with a less severe carried out. The outcome measures were ⌬SDS for weight and length from birth to term. As explanatory factors, birth weight growth failure postnatally (standardized regression coefSDS, birth length SDS, parenteral nutrition (yes/no), enteral nuficient for ⌬SDS for length was 0.475, P ⬍ .005, and for trition (yes/no), gestational age, sex, IGF-1, and FGF21 were ⌬SDS for weight was 0.441, P ⬍ .005), whereas a higher included. The average FGF21 and IGF-1 before term-equivalent birth weight SDS was negatively associated with ⌬SDS for age (mean of log-transformed FGF21 at weeks 1, 3, and 5 and weight (⫺.518, P ⬍ .001) but not with ⌬SDS for length. mean of IGF-1 at weeks 1, 3, and 5) were used in the analysis as Need for total parenteral nutrition at week 1, 3, or 5 was a summary measure.


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Table 2. Associations of Gestational Age at Birth, Birth Weight and Length SDSs, Sex, Postnatal FGF21 and IGF-1 Levels, and Nutrition With the Postnatal Growth From Birth Until Term-Equivalent Age in 32 VPT Infants ⌬SDS for Length From Birth to Term-Equivalent Age

⌬SDS for Weight From Birth to Term-Equivalent Age

Linear Regression Model

Standardized Regression Coefficients

P Value

Standardized Regression Coefficients

P Value

Gestational age at birth Birth length, SD Birth weight, SD Female sex Any enteral nutrition Total parenteral nutrition FGF21a IGF-1a

.475 ⫺.249 .192 ⫺.035 .026 ⫺.180 ⫺.415 .057

.000 .055 .179 .555 .683 .006 .000 .440

.441 .273 ⫺.518 ⫺.208 .028 ⫺.182 ⫺.045 .144

.000 .059 .001 .002 .686 .012 .571 .081

a Mean of weeks 1, 3, and 5 was used to reflect the average postnatal FGF21 (log-transformed values to normalize the distribution for the linear regression model) and IGF-1 levels.

negatively associated with ⌬SDS for both weight and length (⫺.182, P ⫽ .012, and ⫺.180, P ⫽ .006, respectively). A significant negative association between the average FGF21 level during the first 5 weeks of life and ⌬SDS for length (Figure 1B) was found (standardized regression coefficient -.415 and P ⬍ .005 after adjustment for gestational age at birth, birth length SDS, birth weight SDS, sex, nutrition, and IGF-1). The association of FGF21 levels with ⌬SDS for weight was not statistically significant (standardized regression coefficient ⫺.045, P ⫽ .571, Figure 1C). Average IGF-1 level during the first 5 weeks of life was not associated with ⌬SDS for length or weight (standardized regression coefficient .057, P ⫽ .440, and .144, P ⫽ .081, respectively). Effect of FGF21 on GH signaling in human primary chondrocytes We hypothesized that FGF21 acts on chondrocytes, as shown in mouse studies (7, 8). We tested the effect of recombinant FGF21 and patients’ serum on primary chondrocyte cultures established from pediatric patients with normal linear growth. These cells express lower levels of cartilage markers compared with cartilage, have high chondrogenic potential, and are also highly proliferative (18, 19); therefore, they appeared to be suitable in vitro correlates of the growth plate because in the latter, cells with high GH receptor (GHR) and signal transducer and activator of transcription 5 (STAT5)b levels are proliferative/prehypertrophic (20, 21). Moreover, chondrocyte primary cultures responded canonically to recombinant GH and IGF-1 (Supplemental Figure 1). These cells also expressed the FGF21 receptor complex FGFR1/␤-KLOTHO (11, 12) (Figure 2A), indicating that they can respond to FGF21. However, their FGF21 expression levels were significantly lower compared with that of liver (Supplemental Figure 2). Suppressor of cyto-

kine signaling 2 (SOCS2) is an important negative regulator of the GH signaling cascade, and its expression is stimulated by GH and regulated by GH-dependent STAT5 activity (22). Interestingly, a significant increase of Socs2 mRNA and Socs2 protein is found in the liver of Fgf21transgenic mice (10). In primary chondrocytes, FGF21 upregulated basal and GH-induced SOCS2 expression (Figure 2B, middle panel). SOCS1 and SOCS3 expression (Figure 2B, left and right panels, respectively) showed a similar trend, albeit not significant. Remarkably, patient serum of VPT infants, containing high endogenous levels of FGF21, increased SOCS2 expression in chondrocytes (Figure 2C). FGF21 also inhibited GH-induced STAT5 phosphorylation but had no apparent affect on GHinduced AKT phosphorylation, whereas GH-induced ERK1/2 phosphorylation increased upon FGF21 treatment (Figure 2D). FGF21 had no effect on IGF-1–induced AKT and ERK1/2 phosphorylation (not shown). The increased levels of ALP and COLII expression during longterm GH treatment (23) were also attenuated by FGF21 (Figure 2E), whereas neither GH nor FGF21 had any effect on COLX expression (Figure 2E, right panel). FGF21 also reduced both basal and GH-induced IGF-1 expression (Figure 2F) and inhibited GH- but not IGF-1–induced chondrocyte proliferation (Figure 3G). To further elucidate the fact that the concentrations of FGF21 needed to elicit a biological response in vitro were ⬎3000 times higher than those normally seen in the circulation, we immunoisolated FGF21 from a pooled serum sample. When eluates were subjected to Western blot with anti-FGF21 antibody, a band with an apparent molecular mass of ⬃50 kDa could be detected both in reducing and nonreducing conditions (Figure 3, left panel). No bands were present in eluates from columns conjugated with a control antibody. An aliquot of recombinant FGF21 run


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Figure 2. FGF21 affects GH signaling in primary chondrocytes. A, RT-PCR analysis of FGFR1 IIIb and IIIc isoforms, ␤-KLOTHO, and GAPDH expression in human chondrocytes and liver; negative controls (⫺con) were PCR samples where cDNA was omitted. Human brain cDNA was used as positive control for both IIIb and IIIc isoforms. B, Cells were incubated with FGF21 (0.5 and 5 ␮g/mL) for 24 hours and then challenged with GH (500 ng/mL) for 1 hour before analysis of SOCS1, SOCS2, and SOCS3 expression by RT-qPCR. C, Cells were incubated for 1 hour with VPT infants’ pooled sera with low (66.12 pg/mL) and high (746.67 pg/mL) FGF21 levels before SOCS2 RT-qPCR. The levels of GH were similar in the 2 samples (37.5 and 38.6 ng/mL). D, Cells were incubated in the absence or presence of FGF21 (5 ␮g/mL) for 24 hours and then challenged with GH (500 ng/mL) for 20 minutes before analysis of STAT5, AKT, and ERK1/2 phosphorylation by Western blot. Data indicate the ratio of phosphorylated vs total protein and are normalized to GH treatment alone. E, Cells were incubated with GH (500 ng/mL) with or without FGF21 (5 ␮g/mL) for 7 days before analysis of COLII, ALP, and COLX expression by RT-qPCR. F, Cells were incubated with FGF21 (5 ␮g/mL) for 24 hours and then challenged with GH (500 ng/mL) for 30 minutes or 8, 16, or 24 hours before analysis of IGF-1 expression by RT-qPCR. G, Cells were incubated with FGF21 (5 ␮g/mL) for 24 hours and then challenged with GH (500 ng/mL) or IGF-1 (100 ng/mL) for 96 hours before methylene blue assay. The experiments were performed on cells obtained from 3 patients, in triplicate. Error bars represent the mean ⫾ SEM. *, P ⬍ .05; **, P ⬍ .01; ***, P ⬍ .001.


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to GH resistance have yet to be elucidated. Extrauterine growth failure in VPT infants is a well-characterized model of GH resistance of unknown origin. Lack of association between IGF-1 levels and postnatal growth failure in VPT infants can be best explained by the fact that circulating IGF-1 levels reflect only hepatic production, whereas the GH Figure 3. Left panel, FGF21 was immunoisolated from pooled sera of VPT infants with antiresistance is generalized and is also FGF21 antibodies. Rabbit IgG were used as control. Eluates were size-separated under reducing (R) or nonreducing (NR) conditions and Western blotted with anti-FGF21 antibodies; 0.1 ␮g localized in the growth plates. recombinant FGF21 was run alongside these samples. Middle panel, Recombinant FGF21 (1 ␮g) We have shown that FGF21 inrun under R and NR conditions was Western blotted with a different anti-FGF21 antibody. Right hibits GH-induced proliferation in panel, Silver staining of 1 ␮g recombinant FGF21 run under NR conditions. primary chondrocytes by acting downstream of GHR signaling, specifically alongside resulted in 2 bands, a major one of ⬃25 kDa and at the level of SOCS2 expression. Because both SOCS2 a very faint one at ⬃50 kDa. This last finding was cor- activity (22) and extracellular GH levels (34) have been roborated with a different antibody to FGF21 (Figure 3, shown to induce loss of GHR in the plasma membrane, it center panel), with the 50-kDa band being sensitive to is intriguing that FGF21 can potentiate GH-dependent reducing conditions. These results indicate that FGF21 SOCS2 expression in primary chondrocytes; one likely forms dimers in vivo and that ⬎ 95% of recombinant scenario is that the concomitant high levels of GH and FGF21 is monomeric. The dimer could not be detected in FGF21 might induce a state of GH resistance through a silver-stained gel after size separation of recombinant short-term inhibition of GH signaling and/or increased FGF21 (Figure 3, right panel). We speculate that monoGHR turnover, eventually resulting in decreased chonmeric FGF21 has a lower bioactivity as shown for FGF2 drocyte proliferation. FGF21 also reduced GH-induced (24 –26). chondrocyte maturation, as seen by the attenuation of COLII and ALP expression, suggesting that chronic high levels of FGF21 could affect various stages of chondrocyte Discussion maturation/differentiation. In this prospective clinical cohort study, we measured Even though we detected extremely low FGF21 expresplasma FGF21 levels in VPT infants below 32 gestation sion in primary chondrocytes compared with the liver weeks at birth and at 3 serial time points during the first (Supplemental Figure 2), the ability to express FGF21 weeks of life. Elevated FGF21 during the first 5 weeks of from the human growth plate in vivo must not be excluded life was specifically associated with failure in linear because it has been reported in mice (8). Importantly, our growth but not with weight gain. data indicate that patients’ serum, containing high endogPremature infants provide an applicable model in clin- enous levels of FGF21, increased the levels of SOCS2 in ical studies on mechanisms of growth failure in children. chondrocytes in a dose-dependent manner, further conDespite vast improvements in neonatal intensive care, firming mechanistic data obtained with recombinant postnatal growth failure in VPT infants is still a frequent FGF21. phenomenon that is usually followed by a period of We also report here for the first time that FGF21 likely catch-up growth (27, 28). However, despite this, adult forms dimers in vivo. It is well established that FGF2 height is often compromised. A limitation of this study was that data on daily caloric dimerization is crucial to enhance its receptor binding and and protein intake was unavailable, as well as other fac- biological activity such as the activation of downstream tors that might contribute to the growth failure in VPT signaling (24 –26). We speculate that the almost exclusive presence of monomeric FGF21 in the recombinant prepinfants (29 –31). The GH–IGF-1 axis has been shown to be important for aration explains the inability of FGF21 to elicit significant linear growth in infancy (3, 32, 33), and there is evidence cellular responses when used at physiological concentrathat impaired linear growth is due to GH resistance (nor- tions in this study and in others (8). Biochemical and crysmal or elevated GH and low IGF-1). However, the precise tallography studies, along with computerized molecular molecular mechanisms by which chronic conditions lead docking prediction of FGF21 with FGFR1/␤-klotho will


doi: 10.1210/jc.2014-1566

help understand the relative bioactivity of monomeric vs dimeric FGF21. However, the need to use higher concentrations of FGF21 in our in vitro system to trigger a biological response could have other explanations such as 1) higher levels of FGF21 cleavage and/or 2) the inability of recombinant FGF21 to be concentrated near/around its receptor complex in a 2-dimensional environment with low expression of extracellular matrix components (18, 19), which are known to potentiate signaling of FGF cytokines. GH and IGF-1 are the 2 key hormones regulating linear growth and its associated metabolic processes. Both hormones have independent, overlapping, and complementary effects on bone, cartilage, muscle, and fat to promote linear growth, increase lean body mass, and decrease fat mass. Additionally, GH and IGF-1 have distinct cellular targets at the level of the growth plate chondrocyte. For example, in mice lacking both GHR and IGF-1, the growth phenotype is more severe than in mice lacking either gene (35). Because IGF-1–induced cell proliferation is unaffected by FGF21 (unlike GH-induced cell proliferation), we propose that IGF-1 treatment could provide a novel therapeutic strategy to increase growth in patients with growth failure due to elevated FGF21 levels. Although further studies both in experimental animals and humans are needed to fully elucidate the effects of FGF21 on growth at the systemic level and in the growth plate, our findings have identified novel mechanisms of GH-resistant growth failure in humans that could lead to development of new growth-promoting treatment modalities.

Acknowledgments Address all correspondence and requests for reprints to: Leo Dunkel, Centre for Endocrinology, William Harvey Research Institute, Barts and the London, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK. E-mail: [email protected]. Disclosure Summary: The authors have declared that no conflict of interests exists.

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