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1.  Growth differentiation factor 15 in erythroid health and disease 
Current opinion in hematology  2010;17(3):184-190.
Purpose of review
Growth differentiation factor 15 (GDF15) was identified as a hepcidin-suppression factor that is expressed at high levels in patients with ineffective erythropoiesis. This review addresses the regulation, expression and potential functions of GDF15 in the context of erythroid biology.
Recent findings
GDF15 expression during late erythroid differentiation was discovered as part of an erythroblast transcriptome project. Since GDF15 expression is associated with cellular stress or apoptosis, further investigation of the cytokine was focused upon its involvement in ineffective erythropoiesis. Remarkably high serum levels were detected in patients with thalassemia syndromes, congenital dyserythropoiesis and some acquired sideroblastic anemias. Similarly high-level GDF15 expression is not a feature of normal erythropoiesis, or erythroid recovery after bone marrow transplantation. Since GDF15 is a TGF-β superfamily member, it was investigated as an effector of ineffective erythropoiesis that suppresses hepcidin expression despite iron overloading.
Summary
In contrast to the low-levels of GDF15 expressed during normal erythropoiesis, ineffective erythropoiesis causes high-level expression of GDF15. In patients with thalassemia and related anemias, GDF15 expression may contribute to iron overloading or other features of the disease phenotype.
doi:10.1097/MOH.0b013e328337b52f
PMCID: PMC2884377  PMID: 20182355
GDF15; ineffective erythropoiesis; iron regulation
2.  Suppressed hepcidin expression correlates with hypotransferrinemia in copper-deficient rat pups but not dams 
Genes & Nutrition  2012;7(3):405-414.
Copper deficiency leads to anemia but the mechanism is unknown. Copper deficiency also leads to hypoferremia, which may limit erythropoiesis. The hypoferremia may be due to limited function of multicopper oxidases (MCO) hephaestin in enterocytes or GPI-ceruloplasmin in macrophages of liver and spleen whose function as a ferroxidase is thought essential for iron transfer out of cells. Iron release may also be limited by ferroportin (Fpn), the iron efflux transporter. Fpn may be lower following copper deficiency because of impaired ferroxidase activity of MCO. Fpn is also dependent on the liver hormone hepcidin as Fpn is degraded when hepcidin binds to Fpn. Anemia and hypoferremia both down regulate hepcidin by separate mechanisms. Current studies confirmed and extended earlier studies with copper-deficient (CuD) rats that suggested low hepicidin resulted in augmented Fpn. However, current studies in CuD dams failed to confirm a correlation that hepcidin expression was associated with low transferrin receptor 2 (TfR2) levels and also challenged the dogma that holotransferrin can explain the correlation with hepcidin. CuD dams exhibited hypoferremia, low liver TfR2, anemia in some rats, yet no depression in Hamp expression, the hepcidin gene. Normal levels of GDF-15, the putative erythroid cytokine that suppresses hepcidin, were detected in plasma of CuD and iron-deficient (FeD) dams. Importantly, FeD dams did display greatly lower Hamp expression. Normal hepcidin in these CuD dams is puzzling since these rats may need extra iron to meet needs of lactation and the impaired iron transfer noted previously.
doi:10.1007/s12263-012-0293-7
PMCID: PMC3380187  PMID: 22457245
Copper deficiency; Rats; Transferrin iron; Hepcidin; Anemia
3.  Mechanism underlying the transient increase of serum iron during FOLFOX/FOLFIRI therapy 
Molecular and Clinical Oncology  2014;2(6):968-972.
In patients with advanced colorectal cancer (CRC), a transient significant increase of serum iron is observed during chemotherapy with leucovorin and fluorouracil plus oxaliplatin (FOLFOX) or leucovorin and fluorouracil plus irinotecan (FOLFIRI). Serum iron may be a useful and convenient predictor of the response to chemotherapy; however, the mechanism underlying its increase has not been fully elucidated. Accordingly, the mechanism underlying the elevation of serum iron during chemotherapy was investigated in 20 patients with advanced CRC who were treated between September, 2012 and July, 2013. The levels of iron, ferritin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), hemoglobin (Hb), hepcidin-25, interleukin (IL)-6 and soluble transferrin receptor (sTfR) were measured before and 48 h after chemotherapy. The serum levels of iron and hepcidin-25 were found to be significantly increased after chemotherapy (P<0.0001), whereas those of IL-6 were significantly decreased (P=0.0057). There were no significant changes in any of the other parameters. The lack of significant changes in AST, ALT and Hb suggested that the elevation of serum iron was not due to the destruction of hepatocytes, whereas the stable sTfR level suggested no destruction of erythroblasts. Hepcidin-25 regulates iron metabolism and decreases serum iron levels; it is increased by an iron load and IL-6, but is decreased under anemic or hypoxic conditions. The suppression of erythropoiesis increases serum iron levels and chemotherapy suppresses erythropoiesis. As serum iron and hepcidin-25 were both significantly increased and IL-6 was significantly decreased, with no significant changes in sTfR, it appears that the elevation of serum iron during chemotherapy may be secondary to reduced iron consumption by erythropoiesis, leading to increased expression of hepcidin-25 and suppression of Il-6 via negative feedback.
doi:10.3892/mco.2014.385
PMCID: PMC4179814  PMID: 25279183
serum iron; hepcidin; interleukin-6; soluble transferrin receptor; chemotherapy
4.  GDF15 Is a Novel Biomarker for Impaired Fasting Glucose 
Diabetes & Metabolism Journal  2014;38(6):472-479.
Background
Growth differentiation factor-15 (GDF15) is a protein that belongs to the transforming growth factor β superfamily. An elevated serum level of GDF15 was found to be associated with type 2 diabetes mellitus (T2DM). T2DM is an inflammatory disease that progresses from normal glucose tolerance (NGT) to impaired fasting glucose (IFG). Hence, we aimed to validate the relationship between GDF15 and IFG.
Methods
The participants were divided into the following three groups: NGT (n=137), IFG (n=29), and T2DM (n=75). The controls and T2DM outpatients visited the hospital for routine health check-ups. We used fasting blood glucose to detect IFG in nondiabetic patients. We checked the body mass index (BMI), C-reactive protein level, metabolic parameters, and fasting serum GDF15 level.
Results
Age, BMI, triglyceride, insulin, glucose, homeostatic model assessment-insulin resistance (HOMA-IR), and GDF15 levels were elevated in the IFG and T2DM groups compared to the NGT group. In the correlation analysis between metabolic parameters and GDF15, age and HOMA-IR had a significant positive correlation with GDF15 levels. GDF15 significantly discriminated between IFG and NGT, independent of age, BMI, and HOMA-IR. The serum levels of GDF15 were more elevated in men than in women. As a biomarker for IFG based on the receiver operating characteristic curve analysis, the cutoff value of GDF15 was 510 pg/mL in males and 400 pg/mL in females.
Conclusion
GDF15 had a positive correlation with IR independent of age and BMI, and the serum level of GDF15 was increased in the IFG and T2DM groups. GDF15 may be a novel biomarker for detecting IFG in nondiabetic patients.
doi:10.4093/dmj.2014.38.6.472
PMCID: PMC4273034  PMID: 25541611
Biological markers; Diabetes mellitus, type 2; Growth differentiation factor 15; Prediabetic state
5.  Growth Differentiation Factor 15 Is Induced by Hepatitis C Virus Infection and Regulates Hepatocellular Carcinoma-Related Genes 
PLoS ONE  2011;6(5):e19967.
Liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) are commonly induced by chronic hepatitis C virus (HCV) infection. We aimed to identify and characterize the involvement of previously screened cytokine GDF15 in HCV pathogenesis. We examined the GDF15 expression after HCV infection both in vitro and in vivo. Cultured JFH-1 HCV was used to determine the GDF15 function on virus propagation. GDF15 overexpression and RNA interference were employed to profile the GDF15-regulated genes, signaling pathways and cell biology phenotypes. The mRNA expression and protein secretion of GDF15 was dramatically increased in HCV-infected hepatoma cells, which maybe a host response to viral proteins or infection-induced cell stress. Patients infected with HCV had an average 15-fold higher blood GDF15 level than that of healthy volunteers. Three HCC individuals in the HCV cohort showed extremely high GDF15 concentrations. Transfection or exogenously supplied GDF15 enhanced HCV propagation, whereas knockdown of endogenous GDF15 resulted in inhibition of virus replication. Overexpressed GDF15 led to Akt activation and the phosphorylation of Akt downstream targeted GSK-3β and Raf. Several HCC-related molecules, such as E-cadherin, β-catenin, Cyclin A2/B1/D1, were up-regulated by GDF15 stimulation in vitro. Overexpression of GDF15 in hepatoma cells resulted in increased DNA synthesis, promoted cell proliferation, and importantly enhanced invasiveness of the cells. In conclusion, these results suggest that an elevated serum GDF15 level is a potential diagnostic marker for viral hepatitis, and GDF15 may contribute to HCV pathogenesis by altering the signaling and growth of host cells.
doi:10.1371/journal.pone.0019967
PMCID: PMC3100307  PMID: 21625435
6.  A Multi-Scale Model of Hepcidin Promoter Regulation Reveals Factors Controlling Systemic Iron Homeostasis 
PLoS Computational Biology  2014;10(1):e1003421.
Systemic iron homeostasis involves a negative feedback circuit in which the expression level of the peptide hormone hepcidin depends on and controls the iron blood levels. Hepcidin expression is regulated by the BMP6/SMAD and IL6/STAT signaling cascades. Deregulation of either pathway causes iron-related diseases such as hemochromatosis or anemia of inflammation. We quantitatively analyzed how BMP6 and IL6 control hepcidin expression. Transcription factor (TF) phosphorylation and reporter gene expression were measured under co-stimulation conditions, and the promoter was perturbed by mutagenesis. Using mathematical modeling, we systematically analyzed potential mechanisms of cooperative and competitive promoter regulation by the transcription factors, and experimentally validated the model predictions. Our results reveal that hepcidin cross-regulation primarily occurs by combinatorial transcription factor binding to the promoter, whereas signaling crosstalk is insignificant. We find that the presence of two BMP-responsive elements enhances the steepness of the promoter response towards the iron-sensing BMP signaling axis, which promotes iron homeostasis in vivo. IL6 co-stimulation reduces the promoter sensitivity towards the BMP signal, because the SMAD and STAT transcription factors compete for recruiting RNA polymerase to the transcription start site. This may explain why inflammatory signals disturb iron homeostasis in anemia of inflammation. Taken together, our results reveal why the iron homeostasis circuit is sensitive to perturbations implicated in disease.
Author Summary
The nutritional iron uptake is tightly regulated because the body has limited capacity of iron excretion. Mammals maintain iron homeostasis by a negative feedback loop, in which the peptide hepcidin senses the iron blood level and controls iron resorption. Molecular perturbations in the homeostasis loop lead to iron-related diseases such as hemochromatosis or anemia of inflammation. Quantitative studies are required to understand the dynamics of the iron homeostasis circuitry in health and disease. We investigated how the biological activity of hepcidin is regulated by combining experiments with mathematical modeling. We present a multi-scale model that describes the signaling network and the gene promoter controlling hepcidin expression. Possible scenarios of hepcidin regulation were systematically tested against experimental data, and interpreted using a network model of iron metabolism in vivo. The analysis showed that the presence of multiple redundant regulatory elements in the hepcidin gene promoter facilitates homeostasis, because changes in iron blood levels are sensed with high sensitivity. We further suggest that inflammatory signals establish molecular competition at the hepcidin promoter, thereby reducing its iron sensitivity and leading to a loss of homeostasis in anemia of inflammation. We conclude that quantitative insights into hepcidin expression regulation explain features of systemic iron homeostasis.
doi:10.1371/journal.pcbi.1003421
PMCID: PMC3879105  PMID: 24391488
7.  Growth differentiation factor 15 deficiency protects against atherosclerosis by attenuating CCR2-mediated macrophage chemotaxis 
The TGF-β family member GDF-15 promotes lesion formation and plaque instability in atherosclerosis-prone LDLr-deficient mice.
Growth differentiation factor (GDF) 15 is a member of the transforming growth factor β (TGF-β) superfamily, which operates in acute phase responses through a currently unknown receptor. Elevated GDF-15 serum levels were recently identified as a risk factor for acute coronary syndromes. We show that GDF-15 expression is up-regulated as disease progresses in murine atherosclerosis and primarily colocalizes with plaque macrophages. Hematopoietic GDF-15 deficiency in low density lipoprotein receptor−/− mice led to impaired initial lesion formation and increased collagen in later lesions. Although lesion burden in GDF-15−/− chimeras was unaltered, plaques had reduced macrophage infiltrates and decreased necrotic core formation, all features of improved plaque stability. In vitro studies pointed to a TGFβRII-dependent regulatory role of GDF-15 in cell death regulation. Importantly, GDF-15−/− macrophages displayed reduced CCR2 expression, whereas GDF-15 promoted macrophage chemotaxis in a strictly CCR2- and TGFβRII-dependent manner, a phenomenon which was not observed in G protein–coupled receptor kinase 2+/− macrophages. In conclusion, GDF-15 deletion has a beneficial effect both in early and later atherosclerosis by inhibition of CCR2-mediated chemotaxis and by modulating cell death. Our study is the first to identify GDF-15 as an acute phase modifier of CCR2/TGFβRII-dependent inflammatory responses to vascular injury.
doi:10.1084/jem.20100370
PMCID: PMC3039852  PMID: 21242297
8.  A seven day running training period increases basal urinary hepcidin levels as compared to cycling 
Background
This investigation compared the effects of an extended period of weight-bearing (running) vs. non-weight-bearing (cycling) exercise on hepcidin production and its implications for iron status.
Methods
Ten active males performed two separate exercise training blocks with either running (RTB) or cycling (CTB) as the exercise mode. Each block consisted of five training sessions (Day 1, 2, 4, 5, 6) performed over a seven day period that were matched for exercise intensity. Basal venous blood samples were obtained on Day 1 (D1), and on Recovery Days 3 (R3) and 7 (R7) to assess iron status, while basal and 3 h post-exercise urinary hepcidin levels were measured on D1, D2, D6, as well as R3 and R7 (basal levels only) for each condition.
Results
Basal urinary hepcidin levels were significantly elevated (p ≤ 0.05) at D2, R3 and R7 as compared to D1 in RTB. Furthermore, 3 h post-exercise urinary hepcidin levels on D1 were also significantly higher in RTB compared to CTB (p ≤ 0.05). In CTB, urinary hepcidin levels were not statistically different on D1 as compared to R7. Iron parameters were not significantly different at D1 compared to R3 and R7 during both conditions.
Conclusions
These results suggest that basal hepcidin levels may increase over the course of an extended training program, especially if a weight-bearing exercise modality is undertaken. However, despite any variations in hepcidin production, serum iron parameters in both RTB and CTB were unaffected, possibly due to the short duration of each training block. In comparing running to cycling, non-weight-bearing activity may require more training sessions, or sessions of extended duration, before any significant changes in basal hepcidin levels appear. Chronic elevations in hepcidin levels may help to explain the high incidence of iron deficiency in athletes.
doi:10.1186/1550-2783-11-14
PMCID: PMC3991905  PMID: 24716892
Iron deficiency; Weight-bearing exercise; Non-weight-bearing exercise; Cytokines
9.  Decreased Serum Hepcidin Concentration Correlates with Brain Iron Deposition in Patients with HBV-Related Cirrhosis 
PLoS ONE  2013;8(6):e65551.
Purpose
Excessive brain iron accumulation contributes to cognitive impairments in hepatitis B virus (HBV)-related cirrhotic patients. The underlying mechanism remains unclear. Hepcidin, a liver-produced, 25-aminoacid peptide, is the major regulator of systemic iron metabolism. Abnormal hepcidin level is a key factor in some body iron accumulation or deficiency disorders, especially in those associated with liver diseases. Our study was aimed to explore the relationship between brain iron content in patients with HBV-related cirrhosis and serum hepcidin level.
Methods
Seventy HBV-related cirrhotic patients and forty age- sex-matched healthy controls were enrolled. Brain iron content was quantified by susceptibility weighted phase imaging technique. Serum hepcidin as well as serum iron, serum transferrin, ferritin, soluble transferrin receptor, total iron binding capacity, and transferrin saturation were tested in thirty cirrhotic patients and nineteen healthy controls. Pearson correlation analysis was performed to investigate correlation between brain iron concentrations and serum hepcidin, or other iron parameters.
Results
Cirrhotic patients had increased brain iron accumulation compared to controls in the left red nuclear, the bilateral substantia nigra, the bilateral thalamus, the right caudate, and the right putamen. Cirrhotic patients had significantly decreased serum hepcidin concentration, as well as lower serum transferring level, lower total iron binding capacity and higher transferrin saturation, compared to controls. Serum hepcidin level negatively correlated with the iron content in the right caudate, while serum ferritin level positively correlated with the iron content in the bilateral putamen in cirrhotic patients.
Conclusions
Decreased serum hepcidin level correlated with excessive iron accumulation in the basal ganglia in HBV-related cirrhotic patients. Our results indicated that systemic iron overload underlined regional brain iron repletion. Serum hepcidin may be a clinical biomarker for brain iron deposition in cirrhotic patients, which may have therapeutic potential.
doi:10.1371/journal.pone.0065551
PMCID: PMC3679136  PMID: 23776499
10.  Therapeutic effects of adenovirus-mediated growth and differentiation factor-5 in a mice disc degeneration model induced by annulus needle puncture 
BACKGROUND CONTEXT
The therapeutic strategies that have thus far been employed for the treatment of intervertebral disc degeneration (IDD) have focused on relieving the symptoms, while reversal of the degeneration remains an important challenge for the effective treatment of IDD. Growth and differentiation factor-5 (GDF5), of which deficiency leads to early disc degeneration changes, has the potential to increase proliferation of disc cells and expression of extracellular matrix proteins.
PURPOSE
To develop a lumbar disc degeneration model in mice and determine the effect of adenoviral GDF5 gene therapy.
STUDY DESIGNE
Compare the degeneration changes of discs punctured by different size needles to develop a mice lumbar disc degeneration model. Evaluate the effects of in vivo gene therapy for the mice disc degeneration model by an adenoviral vector carrying GDF5 gene.
METHODS
A lumbar disc degeneration model was developed by needle punctures to the discs in Balb/c mice. Afterwards, a gene therapy treatment to disc degeneration was evaluated. Two of the mice lumbar discs were randomly chosen to be punctured by a 30- gauge needle and then injected with adenovirus that had been engineered to express either the luciferase gene (Ad-Luc) or the GDF5 gene (Ad-GDF5). Animals were analyzed by bioluminescent imaging, radiographic and MRI scanning, then sacrificed at 1-, 2-, 4-, or 8- week post operation and subjected to histological and biochemical assays.
RESULTS
By the detection of T2-weighted MRI scanning and histological study, the degeneration was found in all of the discs punctured by different size needles. But the development of the degeneration in the discs injured by 30-gauge needle was more reliable and moderate compared with other groups. The detection of luciferase activity by bioluminescent imaging revealed that adenovirus survived and the introduced genes were expressed over 6 weeks after injection. There were no T2-weighted MRI signals in either the Ad-Luc or Ad-GDF5 injected mice up to 4 weeks post operation. At 6 and 8 weeks, T2-weighted signals were detected in the Ad-GDF5 group, but none in the Ad-Luc control group. The percent disc height index (%DHI) was significantly decreased (~ 20%) by 1 week following injury in both groups, indicating the development of disc degeneration. At 2 weeks, the %DHI in the mice injected with Ad-GDF5 increased significantly compared with that of the mice injected with Ad-Luc group; the increase was sustained for the rest of experiment period. The disc histology treated with Ad-GDF5 was improved compared with that in control group. Glycosaminoglycan (GAG) levels were significantly decreased in the Ad-Luc injection group since 2 weeks after injury, and the DNA content had diminished by 4 weeks after the operation. In contrast, in the discs injected with Ad-GDF5, there was no decrease in the GAG and DNA levels following injury throughout the 8 weeks treatment period.
CONCLUSIONS
Disc degeneration animal model can be developed by using needle puncture to the discs in mice. The adenovirus is an effective vehicle for gene delivery with rapid and prolonged expression of target protein, and resulting improvement in markers of disc degeneration. Ad-GDF5 gene therapy could restore the functions of injured discs and has the potential to be an effective treatment.
doi:10.1016/j.spinee.2009.10.006
PMCID: PMC2818300  PMID: 19926342
Intervertebral disc; degeneration; gene therapy; growth factor
11.  A Low-Molecular-Weight Compound K7174 Represses Hepcidin: Possible Therapeutic Strategy against Anemia of Chronic Disease 
PLoS ONE  2013;8(9):e75568.
Hepcidin is the principal iron regulatory hormone, controlling the systemic absorption and remobilization of iron from intracellular stores. The expression of the hepcidin gene, HAMP, is increased in patients with anemia of chronic disease. Previously, the synthetic compound K7174 was identified through chemical screening as a novel inhibitor of the adhesion of monocytes to cytokine-stimulated endothelial cells. K7174 also ameliorated anemia induced by inflammatory cytokines in mice, which suggests a possible involvement of hepcidin regulation. The present study was performed to assess the impact of K7174 on hepcidin expression in a human hematoma cell line and in mice in vivo. We first demonstrated that K7174 treatment in HepG2 cells significantly decreased HAMP expression. Then, we conducted microarray analysis to determine the molecular mechanism by which K7174 inhibits HAMP expression. Transcriptional profiling confirmed the downregulation of HAMP. Surprisingly, we found that K7174 strongly induced GDF15, known as a negative regulator of HAMP expression. Western blotting analysis as well as ELISA confirmed the induction of GDF15 by K7174 treatment. Furthermore, K7174-mediated HAMP suppression was rescued by the silencing of GDF15 expression. Interestingly, we found that K7174 also upregulates CEBPB. Promoter analysis and chromatin immunoprecipitation analysis revealed that CEBPB could contribute to K7174-mediated transcriptional activation of GDF15. Subsequently, we also examined whether K7174 inhibits hepcidin expression in mice. Quantitative RT-PCR analysis with liver samples from K7174-treated mice demonstrated significant upregulation of Gdf15 and downregulation of Hamp expression, as compared to control mice. Furthermore, serum hepcidin concentration was also significantly decreased in K7174-treated mice. In conclusion, K7174 inhibits hepcidin expression partly by inducing GDF15. K-7174 may be a potential therapeutic option to treat anemia of chronic disease.
doi:10.1371/journal.pone.0075568
PMCID: PMC3785497  PMID: 24086573
12.  A Novel Immunological Assay for Hepcidin Quantification in Human Serum 
PLoS ONE  2009;4(2):e4581.
Background
Hepcidin is a 25-aminoacid cysteine-rich iron regulating peptide. Increased hepcidin concentrations lead to iron sequestration in macrophages, contributing to the pathogenesis of anaemia of chronic disease whereas decreased hepcidin is observed in iron deficiency and primary iron overload diseases such as hereditary hemochromatosis. Hepcidin quantification in human blood or urine may provide further insights for the pathogenesis of disorders of iron homeostasis and might prove a valuable tool for clinicians for the differential diagnosis of anaemia. This study describes a specific and non-operator demanding immunoassay for hepcidin quantification in human sera.
Methods and Findings
An ELISA assay was developed for measuring hepcidin serum concentration using a recombinant hepcidin25-His peptide and a polyclonal antibody against this peptide, which was able to identify native hepcidin. The ELISA assay had a detection range of 10–1500 µg/L and a detection limit of 5.4 µg/L. The intra- and interassay coefficients of variance ranged from 8–15% and 5–16%, respectively. Mean linearity and recovery were 101% and 107%, respectively. Mean hepcidin levels were significantly lower in 7 patients with juvenile hemochromatosis (12.8 µg/L) and 10 patients with iron deficiency anemia (15.7 µg/L) and higher in 7 patients with Hodgkin lymphoma (116.7 µg/L) compared to 32 age-matched healthy controls (42.7 µg/L).
Conclusions
We describe a new simple ELISA assay for measuring hepcidin in human serum with sufficient accuracy and reproducibility.
doi:10.1371/journal.pone.0004581
PMCID: PMC2640459  PMID: 19238200
13.  Growth Differentiation Factor‐15 Deficiency Inhibits Atherosclerosis Progression by Regulating Interleukin‐6–Dependent Inflammatory Response to Vascular Injury 
Background
Growth differentiation factor (GDF)‐15 is a distant and divergent member of the transforming growth factor‐β superfamily (TGF‐β) . There is growing evidence indicating the involvement of GDF‐15 in various pathologies. Expression of GDF‐15 is induced under conditions of inflammation and increased GDF‐15 serum levels are suggested as a risk factor for cardiovascular diseases.
Methods and Results
We show here that GDF‐15 and proinflammatory cytokine interleukin (IL)‐6 levels are highly increased (5‐fold) in cultured oxidized low‐density lipoproteins–stimulated peritoneal macrophages derived from GDF‐15+/+/apolipoprotein (apo) E−/−, mice. Notably, IL‐6 induction on oxidized low‐density lipoproteins stimulation is completely abolished in the absence of GDF‐15. Consistent with our in vitro data GDF‐15 mRNA expression and protein levels are upregulated (2.5‐ to 6‐fold) in the atherosclerotic vessel wall of GDF‐15+/+/apoE−/− mice after a cholesterol‐enriched diet. GDF‐15 deficiency inhibits lumen stenosis (52%) and 18FDG uptake (34%) in the aortic arch despite increased serum triglyceride/cholesterol levels and elevated body weight. Immunohistomorphometric investigations of atherosclerotic lesions reveal a decreased percentage of inflammatory CD11b+ (57%) or IL‐6+, leukocytes, and apoptotic cells (74%) after 20 weeks. However, the total number of macrophages and cell density in atherosclerotic lesions of the innominate artery are increased in GDF‐15−/−/apoE−/− mice.
Conclusions
Our data suggest that GDF‐15 is involved in orchestrating atherosclerotic lesion progression by regulating apoptotic cell death and IL‐6–dependent inflammatory responses to vascular injury.
doi:10.1161/JAHA.112.002550
PMCID: PMC3540664  PMID: 23316317
atherosclerosis; GDF‐15; inflammation; interleukins
14.  Hepcidin levels in diabetes mellitus and polycystic ovary syndrome 
Diabetic Medicine  2013;30(12):1495-1499.
Aim
Increased body iron is associated with insulin resistance. Hepcidin is the key hormone that negatively regulates iron homeostasis. We hypothesized that individuals with insulin resistance have inadequate hepcidin levels for their iron load.
Methods
Serum concentrations of the active form of hepcidin (hepcidin-25) and hepcidin:ferritin ratio were evaluated in participants with Type 2 diabetes (n = 33, control subjects matched for age, gender and BMI,n = 33) and participants with polycystic ovary syndrome (n = 27, control subjects matched for age and BMI,n = 16). To investigate whether any changes observed were associated with insulin resistance rather than insulin deficiency or hyperglycaemia per se, the same measurements were made in participants with Type 1 diabetes (n = 28, control subjects matched for age, gender and BMI,n = 30). Finally, the relationship between homeostasis model assessment of insulin resistance and serum hepcidin:ferritin ratio was explored in overweight or obese participants without diabetes (n = 16).
Results
Participants with Type 2 diabetes had significantly lower hepcidin and hepcidin:ferritin ratio than control subjects (P < 0.05 and P < 0.01, respectively). Participants with polycystic ovary syndrome had a significantly lower hepcidin:ferritin ratio than control subjects (P < 0.05). There was no significant difference in hepcidin or hepcidin:ferritin ratio between participants with Type 1 diabetes and control subjects (P = 0.88 and P = 0.94). Serum hepcidin:ferritin ratio inversely correlated with homeostasis model assessment of insulin resistance (r = –0.59, P < 0.05).
Conclusion
Insulin resistance, but not insulin deficiency or hyperglycaemia per se, is associated with inadequate hepcidin levels. Reduced hepcidin concentrations may cause increased body iron stores in insulin-resistant states.
doi:10.1111/dme.12262
PMCID: PMC4232927  PMID: 23796160
15.  Circulating and Placental Growth-Differentiation Factor 15 in Preeclampsia and in Pregnancy Complicated by Diabetes Mellitus 
Hypertension  2009;54(1):106-112.
Abstract
Growth-differentiation factor 15 (GDF-15), a stress-responsive transforming growth factor-β–related cytokine, is emerging as a new risk marker in patients with cardiovascular disease. We explored GDF-15 in preeclampsia and in diabetic pregnancies, because these conditions are associated with augmented risk for cardiovascular disease, both in mother and in offspring. Plasma from pregnant women (n=267; controls: n=59, preeclampsia: n=85, diabetes mellitus: n=112, and superimposed preeclampsia in diabetes mellitus: n=11), fetal plasma (n=72), and amniotic fluid (n=99) were analyzed by immunoassay for GDF-15. Placental GDF-15 mRNA and protein expression levels were analyzed by quantitative real-time PCR and immunoblots in 78 and 18 pregnancies, respectively. Conditioned media from preeclamptic (n=6) and control (n=6) villous placenta explants were analyzed by immunoassay for GDF-15. Median maternal GDF-15 concentration was elevated in those with diabetes mellitus, as compared with controls (91 549 versus 79 875 ng/L; P=0.02). Median GDF-15 concentration was higher in patients with preeclampsia than in controls in term maternal blood samples (127 061 versus 80 319 ng/L; P<0.001). In the fetal circulation and amniotic fluid, GDF-15 was elevated in preeclampsia and superimposed preeclampsia in diabetes mellitus, as compared with controls. GDF-15 placental mRNA expression was elevated in preeclampsia, as compared with controls (P=0.002). Placenta immunoblots confirmed a single GDF-15 protein band, and a time-dependent increase in GDF-15 protein was detected in the conditioned media. Our study is the first to show that GDF-15 is dysregulated, both in preeclampsia and in diabetic pregnancies. The mechanisms and diagnostic implications of these findings remain to be explored.
doi:10.1161/HYPERTENSIONAHA.109.130583
PMCID: PMC4167791  PMID: 19470878
cardiovascular disease; growth differentiation factor 15; preeclampsia; diabetes mellitus; pregnancy
16.  Evidence for a Lack of a Direct Transcriptional Suppression of the Iron Regulatory Peptide Hepcidin by Hypoxia-Inducible Factors 
PLoS ONE  2009;4(11):e7875.
Background
Hepcidin is a major regulator of iron metabolism and plays a key role in anemia of chronic disease, reducing intestinal iron uptake and release from body iron stores. Hypoxia and chemical stabilizers of the hypoxia-inducible transcription factor (HIF) have been shown to suppress hepcidin expression. We therefore investigated the role of HIF in hepcidin regulation.
Methodology/Principal Findings
Hepcidin mRNA was down-regulated in hepatoma cells by chemical HIF stabilizers and iron chelators, respectively. In contrast, the response to hypoxia was variable. The decrease in hepcidin mRNA was not reversed by HIF-1α or HIF-2α knock-down or by depletion of the HIF and iron regulatory protein (IRP) target transferrin receptor 1 (TfR1). However, the response of hepcidin to hypoxia and chemical HIF inducers paralleled the regulation of transferrin receptor 2 (TfR2), one of the genes critical to hepcidin expression. Hepcidin expression was also markedly and rapidly decreased by serum deprivation, independent of transferrin-bound iron, and by the phosphatidylinositol 3 (PI3) kinase inhibitor LY294002, indicating that growth factors are required for hepcidin expression in vitro. Hepcidin promoter constructs mirrored the response of mRNA levels to interleukin-6 and bone morphogenetic proteins, but not consistently to hypoxia or HIF stabilizers, and deletion of the putative HIF binding motifs did not alter the response to different hypoxic stimuli. In mice exposed to carbon monoxide, hypoxia or the chemical HIF inducer N-oxalylglycine, liver hepcidin 1 mRNA was elevated rather than decreased.
Conclusions/Significance
Taken together, these data indicate that hepcidin is neither a direct target of HIF, nor indirectly regulated by HIF through induction of TfR1 expression. Hepcidin mRNA expression in vitro is highly sensitive to the presence of serum factors and PI3 kinase inhibition and parallels TfR2 expression.
doi:10.1371/journal.pone.0007875
PMCID: PMC2773926  PMID: 19924283
17.  Is hemojuvelin a possible new player in iron metabolism in hemodialysis patients? 
International Urology and Nephrology  2011;44(6):1805-1811.
Introduction
Hemojuvelin (HJV) is highly expressed in the liver, skeletal muscles, and heart, seems to play a role in iron absorption and release from cells, and has anti-inflammatory properties. Moreover, HJV plays an essential role in the regulation of hepcidin expression, specifically in the iron-sensing pathway. Hepcidin has emerged as a key regulator of iron homeostasis. In this study we tested for the first time the hypothesis that HJV is related to iron metabolism in hemodialysis (HD) patients.
Methods
Iron status, complete blood count, and serum creatinine, albumin, and lipids were assessed, using standard laboratory methods. Serum levels of soluble transferrin receptor (sTFR), high-sensitivity CRP, IL-6, hepcidin, and HJV were measured using commercially available kits.
Results
Serum HJV, hepcidin, ferritin, IL-6, hsCRP, and serum creatinine were significantly higher (all P < 0.001), whereas serum iron, sTFR, transferrin, hemoglobin, and erythrocyte count were significantly lower in HD patients, compared to healthy volunteers (all P < 0.001). In univariate analysis, HJV was strongly correlated (P < 0.001) with ferritin, transferrin saturation, and TIBC, as well as with hsCRP, hepcidin, Kt/V (P < 0.01) and residual renal function, the presence of diabetes, APKD, and coronary heart disease. Predictors of HJV level in multiple regression analysis were ferritin (beta value was 0.50, P = 0.00004) and transferrin saturation (beta value was 0.47, P = 0.0002), explaining 81% of the HJV variations.
Conclusions
Serum HJV is elevated in HD patients and related predominantly to kidney function and iron metabolism. However, HJV is probably not correlated to inflammation. HJV appears to be a new player in iron metabolism in these patients.
doi:10.1007/s11255-011-0084-x
PMCID: PMC3510395  PMID: 22130959
Iron metabolism; Hemodialysis; Inflammation; Hepcidin; Hemojuvelin
18.  Body iron delocalization: the serious drawback in iron disorders in both developing and developed countries 
Pathogens and Global Health  2012;106(4):200-216.
Over 2 billion people in both developing as well as developed countries – over 30% of the world’s population – are anaemic. With the classical preconception that oral iron administration or the intake of foods rich in iron increase haemoglobin concentration and reduce the prevalence of anaemia, specific programs have been designed, but iron supplementations have been less effective than expected. Of note, this hazardous simplification on iron status neglects its distribution in the body. The correct balance of iron, defined iron homeostasis, involves a physiological ratio of iron between tissues/secretions and blood, thus avoiding its delocalization as iron accumulation in tissues/secretions and iron deficiency in blood. Changes in iron status can affect the inflammatory response in multiple ways, particularly in the context of infection, an idea that is worth remembering when considering the value of iron supplementation in areas of the world where infections are highly prevalent. The enhanced availability of free iron can increase susceptibility and severity of microbial and parasitic infections.
The discovery of the hepcidin–ferroportin (Fpn) complex, which greatly clarified the enigmatic mechanism that supervises the iron homeostasis, should prompt to a critical review on iron supplementation, ineffective on the expression of the most important proteins of iron metabolism. Therefore, it is imperative to consider new safe and efficient therapeutic interventions to cure iron deficiency (ID) and ID anaemia (IDA) associated or not to the inflammation.
In this respect, lactoferrin (Lf) is emerging as an important regulator of both iron and inflammatory homeostasis. Oral administration of Lf in subjects suffering of ID and IDA is safe and effective in significantly increasing haematological parameters and contemporary decreasing serum IL-6 levels, thus restoring iron localization through the direct or indirect modulation of hepcidin and ferroportin synthesis. Of note, the nuclear localization of Lf suggests that this molecule may be involved in the transcriptional regulation of some genes of host inflammatory response.
We recently also reported that combined administration of oral and intravaginal Lf on ID and IDA pregnant women with preterm delivery threat, significantly increased haematological parameters, reduced IL-6 levels in both serum and cervicovaginal fluid, cervicovaginal prostaglandin PGF2α, and suppressed uterine contractility. Moreover, Lf combined administration blocked further the shortening of cervical length and the increase of foetal fibronectin, thus prolonging the length of pregnancy until the 37th–38th week of gestation.
These new Lf functions effective in curing ID and IDA through the restoring of iron and inflammatory homeostasis and in preventing preterm delivery, could have a great relevance in developing countries, where ID and IDA and inflammation-associated anaemia represent the major risk factors of preterm delivery and maternal and neonatal death.
doi:10.1179/2047773212Y.0000000043
PMCID: PMC4001586  PMID: 23265420
anaemia; iron deficiency; lactoferrin; pregnancy; malaria; iron homeostasis; inflammatory homeostasis
19.  Rethinking Iron Regulation and Assessment in Iron Deficiency, Anemia of Chronic Disease, and Obesity: Introducing Hepcidin 
Adequate iron availability is essential to human development and overall health. Iron is a key component of oxygen-carrying proteins, has a pivotal role in cellular metabolism, and is essential to cell growth and differentiation. Inadequate dietary iron intake, chronic and acute inflammatory conditions, and obesity are each associated with alterations in iron homeostasis. Tight regulation of iron is necessary because iron is highly toxic and human beings can only excrete small amounts through sweat, skin and enterocyte sloughing, and fecal and menstrual blood loss. Hepcidin, a small peptide hormone produced mainly by the liver, acts as the key regulator of systemic iron homeostasis. Hepcidin controls movement of iron into plasma by regulating the activity of the sole known iron exporter ferroportin-1. Downregulation of the ferroportin-1 exporter results in sequestration of iron within intestinal enterocytes, hepatocytes, and iron-storing macrophages reducing iron bioavailability. Hepcidin expression is increased by higher body iron levels and inflammation and decreased by anemia and hypoxia. Importantly, existing data illustrate that hepcidin may play a significant role in the development of several iron-related disorders, including the anemia of chronic disease and the iron dysregulation observed in obesity. Therefore, the purpose of this article is to discuss iron regulation, with specific emphasis on systemic regulation by hepcidin, and examine the role of hepcidin within several disease states, including iron deficiency, anemia of chronic disease, and obesity. The relationship between obesity and iron depletion and the clinical assessment of iron status will also be reviewed.
doi:10.1016/j.jada.2011.08.038
PMCID: PMC3381289  PMID: 22717199
20.  Iron status in patients with chronic heart failure 
European Heart Journal  2012;34(11):827-834.
Aims
The changes in iron status occurring during the course of heart failure (HF) and the underlying pathomechanisms are largely unknown. Hepcidin, the major regulatory protein for iron metabolism, may play a causative role. We investigated iron status in a broad spectrum of patients with systolic HF in order to determine the changes in iron status in parallel with disease progression, and to associate iron status with long-term prognosis.
Methods and results
Serum concentrations of ferritin, transferrin saturation (Tsat), soluble transferrin receptor (sTfR), and hepcidin were assessed as the biomarkers of iron status in 321 patients with chronic systolic HF [age: 61 ± 11 years, men: 84%, left ventricular ejection fraction: 31 ± 9%, New York Heart Association (NYHA) class: 72/144/87/18] at a tertiary cardiology centre and 66 age- and gender-matched healthy subjects. Compared with healthy subjects, asymptomatic HF patients had similar haematological status, but increased iron stores (evidenced by higher serum ferritin without distinct inflammation, P < 0.01) with markedly elevated serum hepcidin (P < 0.001). With increasing HF severity, patients in advanced NYHA classes had iron deficiency (ID) (reduced serum ferritin, low Tsat, high sTfR), iron-restricted erythropoiesis (reduced haemoglobin, high red cell distribution width), and inflammation (high serum high-sensitivity-C-reactive protein and interleukin 6), which was accompanied by decreased circulating hepcidin (all P < 0.001). In multivariable Cox models, low hepcidin was independently associated with increased 3-year mortality among HF patients (P < 0.001).
Conclusions
Increased level of circulating hepcidin characterizes an early stage of HF, and is not accompanied by either anaemia or inflammation. The progression of HF is associated with the decline in circulating hepcidin and the development of ID. Low hepcidin independently relates to unfavourable outcome.
doi:10.1093/eurheartj/ehs377
PMCID: PMC3697803  PMID: 23178646
Heart failure; Iron deficiency; Ferritin; Hepcidin; Prognosis
21.  Hepcidin Levels and Their Determinants in Different Types of Myelodysplastic Syndromes 
PLoS ONE  2011;6(8):e23109.
Iron overload may represent an additional clinical problem in patients with Myelodysplastic Syndromes (MDS), with recent data suggesting prognostic implications. Beyond red blood cells transfusions, dysregulation of hepcidin, the key iron hormone, may play a role, but studies until now have been hampered by technical problems. Using a recently validated assay, we measured serum hepcidin in 113 patients with different MDS subtypes. Mean hepcidin levels were consistently heterogeneous across different MDS subtypes, with the lowest levels in refractory anemia with ringed sideroblasts (RARS, 1.43 nM) and the highest in refractory anemia with excess blasts (RAEB, 11.3 nM) or in chronic myelomonocytic leukemia (CMML, 10.04 nM) (P = 0.003 by ANOVA). MDS subtypes remained significant predictors of hepcidin in multivariate analyses adjusted for ferritin and transfusion history. Consistently with current knowledge on hepcidin action/regulation, RARS patients had the highest levels of toxic non-transferrin-bound-iron, while RAEB and CMML patients had substantial elevation of C-Reactive Protein as compared to other MDS subtypes, and showed lost of homeostatic regulation by iron. Growth differentiation factor 15 did not appear as a primary hepcidin regulator in this series. If confirmed, these results may help to calibrate future treatments with chelating agents and/or hepcidin modulators in MDS patients.
doi:10.1371/journal.pone.0023109
PMCID: PMC3158762  PMID: 21886780
22.  The A736V TMPRSS6 polymorphism influences hepcidin and iron metabolism in chronic hemodialysis patients: TMPRSS6 and hepcidin in hemodialysis 
BMC Nephrology  2013;14:48.
Background
Aim of this study was to evaluate whether the A736V TMPRSS6 polymorphism, a major genetic determinant of iron metabolism in healthy subjects, influences serum levels of hepcidin, the hormone regulating iron metabolism, and erythropoiesis in chronic hemodialysis (CHD).
Methods
To this end, we considered 199 CHD patients from Northern Italy (157 with hepcidin evaluation), and 188 healthy controls without iron deficiency, matched for age and gender. Genetic polymorphisms were evaluated by allele specific polymerase chain reaction assays, and hepcidin quantified by mass spectrometry.
Results
Serum hepcidin levels were not different between the whole CHD population and controls (median 7.1, interquartile range (IQR) 0.55-17.1 vs. 7.4, 4.5-17.9 nM, respectively), but were higher in the CHD subgroup after exclusion of subjects with relative iron deficiency (p = 0.04). In CHD patients, the A736V TMPRSS6 polymorphism influenced serum hepcidin levels in individuals positive for mutations in the HFE gene of hereditary hemochromatosis (p < 0.0001). In particular, the TMPRSS6 736 V variant was associated with higher hepcidin levels (p = 0.017). At multivariate analysis, HFE and A736V TMPRSS6 genotypes predicted serum hepcidin independently of ferritin and C reactive protein (p = 0.048). In patients without acute inflammation and overt iron deficiency (C reactive protein <1 mg/dl and ferritin >30 ng/ml; n = 86), hepcidin was associated with lower mean corpuscular volume (p = 0.002), suggesting that it contributed to iron-restricted erythropoiesis. In line with previous results, in patients without acute inflammation and severe iron deficiency the “high hepcidin” 736 V TMPRSS6 variant was associated with higher erythropoietin maintenance dose (p = 0.016), independently of subclinical inflammation (p = 0.02).
Conclusions
The A736V TMPRSS6 genotype influences hepcidin levels, erythropoiesis, and anemia management in CHD patients. Evaluation of the effect of TMPRSS6 genotype on clinical outcomes in prospective studies in CHD may be useful to predict the outcomes of hepcidin manipulation, and to guide treatment personalization by optimizing anemia management.
doi:10.1186/1471-2369-14-48
PMCID: PMC3585892  PMID: 23433094
Anemia; Chronic kidney disease; Erythropoietin; Genetics; Inflammation; Iron; Hemodialysis; Hepcidin; Hfe gene; Matriptase-2; Tmprss6
23.  Iron Status and Systemic Inflammation, but Not Gut Inflammation, Strongly Predict Gender-Specific Concentrations of Serum Hepcidin in Infants in Rural Kenya 
PLoS ONE  2013;8(2):e57513.
Hepcidin regulation by competing stimuli such as infection and iron deficiency has not been studied in infants and it’s yet unknown whether hepcidin regulatory pathways are fully functional in infants. In this cross-sectional study including 339 Kenyan infants aged 6.0±1.1 months (mean±SD), we assessed serum hepcidin-25, biomarkers of iron status and inflammation, and fecal calprotectin. Prevalence of inflammation, anemia, and iron deficiency was 31%, 71%, 26%, respectively. Geometric mean (±SD) serum hepcidin was 6.0 (±3.4) ng/mL, and was significantly lower in males than females. Inflammation (C-reactive protein and interleukin-6) and iron status (serum ferritin, zinc protoporphyrin and soluble transferrin receptor) were significant predictors of serum hepcidin, explaining nearly 60% of its variance. There were small, but significant differences in serum hepcidin comparing iron deficient anemic (IDA) infants without inflammation to iron-deficient anemic infants with inflammation (1.2 (±4.9) vs. 3.4 (±4.9) ng/mL; P<0.001). Fecal calprotectin correlated with blood/mucus in the stool but not with hepcidin. Similarly, the gut-linked cytokines IL-12 and IL-17 did not correlate with hepcidin. We conclude that hepcidin regulatory pathways are already functional in infancy, but serum hepcidin alone may not clearly discriminate between iron-deficient anemic infants with and without infection. We propose gender-specific reference values for serum hepcidin in iron-replete infants without inflammation.
doi:10.1371/journal.pone.0057513
PMCID: PMC3583867  PMID: 23460869
24.  Effects of a 7-day military training exercise on inflammatory biomarkers, serum hepcidin, and iron status 
Nutrition Journal  2013;12:141.
Background
Hepcidin, a peptide that is released into the blood in response to inflammation, prevents cellular iron export and results in declines in iron status. Elevated serum and urinary levels of hepcidin have been observed in athletes following exercise, and declines in iron status have been reported following prolonged periods of training. The objective of this observational study was to characterize the effects of an occupational task, military training, on iron status, inflammation, and serum hepcidin.
Findings
Volunteers (n = 21 males) included Norwegian Soldiers participating in a 7-day winter training exercise that culminated in a 3-day, 54 km ski march. Fasted blood samples were collected at baseline, on day 4 (PRE, prior to the ski march), and again on day 7 (POST, following the ski march). Samples were analyzed for hemoglobin, serum ferritin, soluble transferrin receptor (sTfR), interleukin-6 (IL-6), and serum hepcidin. Military training affected inflammation and serum hepcidin levels, as IL-6 and hepcidin concentrations increased (P < 0.05) from the baseline to POST (mean ± SD, 9.1 ± 4.9 vs. 14.5 ± 8.4 pg/mL and 6.5 ± 3.5 vs. 10.2 ± 6.9 ng/mL, respectively). Iron status was not affected by the training exercise, as sTfR levels did not change over the course of the 7-day study.
Conclusions
Military training resulted in significant elevations in IL-6 and serum hepcidin. Future studies should strive to identify the role of hepcidin in the adaptive response to exercise, as well as countermeasures for the prevention of chronic or repeated elevations in serum hepcidin due to exercise or sustained occupational tasks which may result in longer term decrements in iron status.
doi:10.1186/1475-2891-12-141
PMCID: PMC3830559  PMID: 24188143
Physical activity; Operational stress; Military; Ferritin; Inflammation; Iron absorption; Soluble transferrin receptor
25.  The Identification of Trans-acting Factors That Regulate the Expression of GDF5 via the Osteoarthritis Susceptibility SNP rs143383 
PLoS Genetics  2013;9(6):e1003557.
rs143383 is a C to T transition SNP located in the 5′untranslated region (5′UTR) of the growth differentiation factor 5 gene GDF5. The T allele of the SNP is associated with increased risk of osteoarthritis (OA) in Europeans and in Asians. This susceptibility is mediated by the T allele producing less GDF5 transcript relative to the C allele, a phenomenon known as differential allelic expression (DAE). The aim of this study was to identify trans-acting factors that bind to rs143383 and which regulate this GDF5 DAE. Protein binding to the gene was investigated by two experimental approaches: 1) competition and supershift electrophoretic mobility shift assays (EMSAs) and 2) an oligonucleotide pull down assay followed by quantitative mass spectrometry. Binding was then confirmed in vivo by chromatin immunoprecipitation (ChIP), and the functional effects of candidate proteins investigated by RNA interference (RNAi) and over expression. Using these approaches the trans-acting factors Sp1, Sp3, P15, and DEAF-1 were identified as interacting with the GDF5 5′UTR. Knockdown and over expression of the factors demonstrated that Sp1, Sp3, and DEAF-1 are repressors of GDF5 expression. Depletion of DEAF-1 modulated the DAE of GDF5 and this differential allelic effect was confirmed following over expression, with the rs143383 T allele being repressed to a significantly greater extent than the rs143383 C allele. In combination, Sp1 and DEAF-1 had the greatest repressive activity. In conclusion, we have identified four trans-acting factors that are binding to GDF5, three of which are modulating GDF5 expression via the OA susceptibility locus rs143383.
Author Summary
GDF5 is an important growth factor that plays a vital role in the development and repair of articulating joints. rs143383 is a polymorphism within the regulatory region of the GDF5 gene and has two allelic forms, C and T. Genetic studies have demonstrated that the T allele is associated with an increased risk of osteoarthritis in a range of ethnic populations whilst previous functional studies revealed that this allele mediates its effect by producing less GDF5 transcript than the C allele. In this study, we sought to identify transcription factors that are binding to rs143383 and that are responsible for mediating this differential level of expression. Using two different approaches we have identified four factors and our functional studies have revealed that three of these factors repress GDF5 expression and that DEAF-1 modulates the differential expression of the two rs143383 alleles. The factors that we have identified could serve as novel therapeutic targets, with their depletion restoring the expression levels of GDF5 in patients with the osteoarthritis susceptibility T allele. The relevance of our results extends beyond osteoarthritis, since the T allele of rs143383 is also a risk factor for a number of other musculoskeletal diseases.
doi:10.1371/journal.pgen.1003557
PMCID: PMC3694828  PMID: 23825960

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