Growth Differentiation Factor 15 Production Research Articles

Nausea, vomiting and conflict in pregnancy: The adaptive significance of Growth-Differentiation Factor 15.

Nausea and vomiting in pregnancy (NVP) is heritable, common and aversive, and its extreme, hyperemesis gravidarum (HG), can be highly deleterious to the mother and fetus. Recent influential studies have demonstrated that HG is caused predominantly by high levels of Growth-Differentiation Factor 15 (GDF15), a hormone produced by the placenta in substantial amounts. This work has led to calls for therapeutic modulation of this hormone to reduce GDF15 levels and ameliorate HG risk. I describe three main lines of evidence relevant to the hypothesis that GDF15 production is typically adaptive for the fetus, in the context of enhanced placental invasion, reduced rates of miscarriage and preterm birth and higher birth weight. These considerations highlight the medical implications of maternal-fetal conflict, in the context of tradeoffs between aversive symptoms during gestation, rare disorders of pregnancy with major adverse effects and moderate fitness-enhancing benefits to fetuses.

GDF15 is a major determinant of ketogenic diet-induced weight loss

A ketogenic diet (KD) has been promoted as an obesity management diet, yet its underlying mechanism remains elusive. Here we show that KD reduces energy intake and body weight in humans, pigs, and mice, accompanied by elevated circulating growth differentiation factor 15 (GDF15). In GDF15- or its receptor GFRAL-deficient mice, these effects of KD disappeared, demonstrating an essential role of GDF15-GFRAL signaling in KD-mediated weight loss. Gdf15 mRNA level increases in hepatocytes upon KD feeding, and knockdown of Gdf15 by AAV8 abrogated the obesity management effect of KD in mice, corroborating a hepatic origin of GDF15 production. We show that KD activates hepatic PPARγ, which directly binds to the regulatory region of Gdf15, increasing its transcription and production. Hepatic Pparγ-knockout mice show low levels of plasma GDF15 and significantly diminished obesity management effects of KD, which could be restored by either hepatic Gdf15 overexpression or recombinant GDF15 administration. Collectively, our study reveals a previously unexplored GDF15-dependent mechanism underlying KD-mediated obesity management.

261-LB: Hepatocyte Gdf15 Protects against CCL4-Induced Liver Injury Independent of Changes in Feeding and Inflammation

Growth differentiation factor 15 (GDF15) is a stress-induced secreted protein whose circulating levels increase in obesity and fibrotic liver disease, like NAFLD. GDF15 KO mice develop more severe fibrosis after carbon tetrachloride (CCL4) treatment, suggesting a protective effect of GDF15. Despite this, the tissue specific sites of GDF15 production during liver injury are unknown and the potential for local action is understudied. To determine the hepatocyte specific contribution of Gdf15 to changes in circulating GDF15 and effects on fibrosis, we utilized a Gdf15 KO-first mouse strain where endogenous expression of Gdf15 can be restored in a tissue specific fashion via Flp recombinase (FLP). Gdf15 KO-first mice received AAV8-TBG-Gfp (KO+GFP) or AAV8-TBG-Flp (liver specific Gdf15 expression; KO+FLP) and were treated with CCL4 3X per week for 3 weeks alongside wild type (WT) controls. CCL4 increased circulating GDF15 7X in WT and 3.5X in KO+FLP compared with WT vehicle (VEH), and levels were undetected in KO+GFP mice. CCL4 caused significant BW loss in WT and KO+FLP mice and had no effect on KO+GFP mice. CCL4 reduced feeding similarly in all groups despite marked differences in circulating GDF15. Liver gene expression markers of fibrosis were upregulated in KO+GFP mice compared with WT and KO+FLP mice. Interestingly, despite differences in fibrosis, circulating cytokines and liver expression of inflammatory markers were reduced in KO+GFP and KO+FLP compared with WT mice. These data demonstrate that hepatocyte Gdf15 protects against CCL4-induced fibrotic liver injury independent of feeding and inflammation. They also suggest that GDF15 may have local, GFRAL-independent effects within liver to remodel the extracellular matrix that require further study. Disclosure J. Kastroll: None. M. J. Jurczak: None. F. Bello: None. A. Vandevender: None. I. J. Sipula: None. J. Alder: None.

Hepatocyte-derived GDF15 suppresses feeding and improves insulin sensitivity in obese mice.

Growth differentiation factor 15 (GDF15) is a stress-induced secreted protein whose circulating levels are increased in the context of obesity. Recombinant GDF15 reduces body weight and improves glycemia in obese models, which is largely attributed to the central action of GDF15 to suppress feeding and reduce body weight. Despite these advances in knowledge, the tissue-specific sites of GDF15 production during obesity are unknown, and the effects of modulating circulating GDF15 levels on insulin sensitivity have not been evaluated directly. Here, we demonstrate that hepatocyte Gdf15 expression is sufficient for changes in circulating levels of GDF15 during obesity and that restoring Gdf15 expression specifically in hepatocytes of Gdf15 knockout mice results in marked improvements in hyperinsulinemia, hepatic insulin sensitivity, and to a lesser extent peripheral insulin sensitivity. These data support that liver hepatocytes are the primary source of circulating GDF15 in obesity.

Growth differentiation factor-15 modulates adrenocorticotropic hormone synthesis in murine AtT-20 corticotroph cells

Growth differentiation factor-15 (GDF15) is a stress-responsive cytokine that plays important roles in regulation of inflammatory responses, cell growth, and cell differentiation. However, the nature of these roles remains unclear. Here, we aimed to examine the regulatory effects of dexamethasone on Gdf15 expression in murine AtT-20 corticotroph cells. Human Gdf15 promoter-driven luciferase reporter constructs were transfected into corticotroph cells to analyze their promoter activity. The effects of time and concentration of dexamethasone on Gdf15 and proopiomelanocortin (Pomc) mRNA levels were assessed using quantitative real-time polymerase chain reaction. Dexamethasone induced Gdf15 transcription and mRNA levels as well as GDF15 production in transfected cells, whereas reduced the Pomc mRNA levels. GDF15 modulated adrenocorticotropic hormone (ACTH) synthesis, and the dexamethasone-mediated reduction in Pomc mRNA levels were partially relieved upon Gdf15 knockdown. We concluded that GDF15 modulated ACTH production in pituitary corticotrophs in an autocrine manner by suppressing Pomc expression and subsequently mediating the negative feedback effect of glucocorticoids, thereby contributing to pituitary stress response and homeostasis.

374-P: Identifying the Role and Mechanism by Which GDF15 Enhances the Counterregulatory Response to Hypoglycemia

Severe hypoglycemia is the leading acute mortality risk in patients with diabetes. The counterregulatory gluconeogenic and glycogenolytic response to hypoglycemia is the primary adaptive mechanism to enable survival. To better understand the hormonal response that orchestrates hypoglycemia counterregulation, we employed the insulin tolerance test (ITT) and hyperinsulinemic-hypoglycemic clamp to mimic the two common settings in which hypoglycemia can occur in patients: postprandial insulin overdose and elevated basal insulin infusion, respectively. We found that Growth Differentiation Factor 15 (GDF15) can be induced (33.5±2.1 vs. 172.4±29.2 pg/ml, P<0.in ITT and 46.2±11.6 vs. 2865±547.3 pg/ml, P<0.0in the clamp) . GDF15 mRNA and protein are upregulated in kidney, and the GDF15 tdTomato reporter mouse demonstrates the expression of GDF15 in the S3 segment of the proximal tubule. GDF15 increases hepatic gluconeogenesis (26.1±2.2 vs. 33.5±1.6 mg/kg/min, P<0.05) by increasing intrahepatic lipolysis in a beta-adrenergic receptor-2 (Adrb2) -dependent manner. To investigate the role of GDF15 counterregulation in diabetes, we utilized a streptozotocin (STZ) -induced type 1 diabetes (T1D) model. Mice with T1D exhibit impaired GDF15 production in an ITT. Additionally, we found that recurrent hypoglycemia impairs endogenous glucose production during hypoglycemia in mice (27.5±4 vs. 10.5±1.3 mg/kg/min, P<0.01) , which may result from lower GDF15 production (1372±248.2 vs. 648.5±71.3 pg/ml, P<0.05) . In humans, hypoglycemic clamps in healthy volunteers can induce GDF15 production (p=0.01) , although this induction was absent in patients with T1D. Conclusion: These data show the kidney can produce GDF15 in response to hypoglycemia, thereby increasing hepatic gluconeogenesis to enable survival. However, T1D patients are unable to initiate a functional GDF15 signaling pathway resulting in an impaired glucose counterregulatory response to hypoglycemia. Disclosure Z.Li: None. C.Zhang: None. R.Chang: None. J.J.Hwang: None. A.Wang: Research Support; NGM Biopharmaceuticals. R.J.Perry: None. X.Zhang: None. W.Zhu: None. Q.Xu: None. C.Yu: None. B.G.Costa lima: None. X.Li: None. K.Israni-winger: None. A.Nasiri: None. Funding National Institutes of Health (P30 DK045735) National Institutes of Health (R37 CA258271-01A1)

The Role of GDF15 as a Myomitokine.

Growth differentiation factor 15 (GDF15) is a cytokine best known for affecting systemic energy metabolism through its anorectic action. GDF15 expression and secretion from various organs and tissues is induced in different physiological and pathophysiological states, often linked to mitochondrial stress, leading to highly variable circulating GDF15 levels. In skeletal muscle and the heart, the basal expression of GDF15 is very low compared to other organs, but GDF15 expression and secretion can be induced in various stress conditions, such as intense exercise and acute myocardial infarction, respectively. GDF15 is thus considered as a myokine and cardiokine. GFRAL, the exclusive receptor for GDF15, is expressed in hindbrain neurons and activation of the GDF15–GFRAL pathway is linked to an increased sympathetic outflow and possibly an activation of the hypothalamic-pituitary-adrenal (HPA) stress axis. There is also evidence for peripheral, direct effects of GDF15 on adipose tissue lipolysis and possible autocrine cardiac effects. Metabolic and behavioral outcomes of GDF15 signaling can be beneficial or detrimental, likely depending on the magnitude and duration of the GDF15 signal. This is especially apparent for GDF15 production in muscle, which can be induced both by exercise and by muscle disease states such as sarcopenia and mitochondrial myopathy.

Immunization With RANKL Inhibits Osteolytic Bone Metastasis in Breast Cancer.

Breast cancer cells often metastasize to bone. Accumulating evidence suggests that inhibiting the receptor activator of nuclear factor-κB ligand (RANKL) not only leads to reduced bone metastasis of breast cancer but also has antitumoral effects. Here, we used mutant receptor activator of nuclear factor-κB ligand (RANKLM) as a vaccine for active immunization to induce antibodies for immunotherapy of bone metastatic cancer. We investigated whether anti-RANKL antibodies inhibit osteolytic bone metastasis in vitro and in a murine model. MC3T3 cells stimulated by MDA-MB-231 culture medium secreted growth differentiation factor-15 (GDF-15), which induced the nuclear factor-κB signaling cascade. In addition, RANKLM treatment-induced reduction of intraosseous growth of MDA-MB-231 cells correlated with decreased GDF-15 expression, a reduced number of osteolytic lesions, and slower tumor progression. In addition, vaccination with RANKLM led to significant improvement in overall survival and skeletal metastasis in tumor-bearing mice. Induction of anti-RANKL antibodies by RANKLM decreased GDF-15 production by deactivating nuclear factor-κB signaling, which in turn inhibited metastasis of MDA-MB-231 cells to bone. Taken together, the results demonstrate a role for RANKLM immunization in preventing bone metastasis of breast cancer.

GDF15: emerging biology and therapeutic applications for obesity and cardiometabolic disease.

Growth differentiation factor 15 (GDF15) is a member of the TGFβ superfamily whose expression is increased in response to cellular stress and disease as well as by metformin. Elevations in GDF15 reduce food intake and body mass in animal models through binding to glial cell-derived neurotrophic factor family receptor alpha-like (GFRAL) and the recruitment of the receptor tyrosine kinase RET in the hindbrain. This effect is largely independent of other appetite-regulating hormones (for example, leptin, ghrelin or glucagon-like peptide 1). Consistent with an important role for the GDF15-GFRAL signalling axis, some human genetic studies support an interrelationship with human obesity. Furthermore, findings in both mice and humans have shown that metformin and exercise increase circulating levels of GDF15. GDF15 might also exert anti-inflammatory effects through mechanisms that are not fully understood. These unique and distinct mechanisms for suppressing food intake and inflammation makes GDF15 an appealing candidate to treat many metabolic diseases, including obesity, type 2 diabetes mellitus, non-alcoholic fatty liver disease, cardiovascular disease and cancer cachexia. Here, we review the mechanisms regulating GDF15 production and secretion, GDF15 signalling in different cell types, and how GDF15-targeted pharmaceutical approaches might be effective in the treatment of metabolic diseases.

Growth differentiation factor 15 protects against the aging-mediated systemic inflammatory response in humans and mice.

Mitochondrial dysfunction is associated with aging‐mediated inflammatory responses, leading to metabolic deterioration, development of insulin resistance, and type 2 diabetes. Growth differentiation factor 15 (GDF15) is an important mitokine generated in response to mitochondrial stress and dysfunction; however, the implications of GDF15 to the aging process are poorly understood in mammals. In this study, we identified a link between mitochondrial stress‐induced GDF15 production and protection from tissue inflammation on aging in humans and mice. We observed an increase in serum levels and hepatic expression of GDF15 as well as pro‐inflammatory cytokines in elderly subjects. Circulating levels of cell‐free mitochondrial DNA were significantly higher in elderly subjects with elevated serum levels of GDF15. In the BXD mouse reference population, mice with metabolic impairments and shorter survival were found to exhibit higher hepatic Gdf15 expression. Mendelian randomization links reduced GDF15 expression in human blood to increased body weight and inflammation. GDF15 deficiency promotes tissue inflammation by increasing the activation of resident immune cells in metabolic organs, such as in the liver and adipose tissues of 20‐month‐old mice. Aging also results in more severe liver injury and hepatic fat deposition in Gdf15‐deficient mice. Although GDF15 is not required for Th17 cell differentiation and IL‐17 production in Th17 cells, GDF15 contributes to regulatory T‐cell‐mediated suppression of conventional T‐cell activation and inflammatory cytokines. Taken together, these data reveal that GDF15 is indispensable for attenuating aging‐mediated local and systemic inflammation, thereby maintaining glucose homeostasis and insulin sensitivity in humans and mice.

GDF15 secreted by senescent endothelial cells improves vascular progenitor cell functions.

Endothelial dysfunction (ED) is part of the first steps in the development of cardiovascular diseases (CVD). Growth Differentiation Factor 15 (GDF15) is a cytokine belonging to the Transforming Growth Factor β superfamily and its expression is increased both during ED and in CVD. Because high blood levels of GDF15 have been reported during ED, we hypothesized that GDF15 could be produced by endothelial cells in response to a vascular stress, possibly to attenuate endothelial function loss. Since senescence is mainly involved in both vascular stress and endothelial function loss, we used Endothelial Colony Forming Cells generated from adult blood (AB-ECFCs) as a model of endothelial cells to investigate GDF15 expression during cellular senescence. Then, we analyzed the potential role of GDF15 in AB-ECFC functions and senescence. When AB-ECFCs become senescent, they secrete increased levels of GDF15. We investigated GDF15 paracrine effects on non-senescent AB-ECFCs and showed that GDF15 enhanced proliferation, migration, NO production and activated several signaling pathways including AKT, ERK1/2 and SMAD2 without triggering any oxidative stress. Taken together, our results suggest that GDF15 production by senescent AB-ECFCs could act in a paracrine manner on non-senescent AB-ECFCs, and that this interaction could be beneficial to its model cells. Therefore, GDF15 could play a beneficial role in a dysfunctional vascular system as previously reported in patients with CVD, by limiting ED related to vascular stress occurring in these diseases.

Treatment-damaged hepatocellular carcinoma promotes activities of hepatic stellate cells and fibrosis through GDF15

The aim of this study was to investigate whether treatment-damaged hepatocellular carcinoma (HCC) would accelerate liver cirrhosis through promoting the activities of hepatic stellate cells (HSCs). HCC cells were exposed to chemotherapeutic agent or hypoxia to mimic the transarterial chemoembolization (TACE)-like treatment. Growth differentiation factor 15 (GDF15) expression was increased in cisplatin- or hypoxia-treated HCC cells. Treatment-induced GDF15 increase in HCC cells was mediated by p38MAPK, JNK, ERK1/2 activation. GDF15 from treatment-damaged HCC cells enhanced the proliferation and collagen synthesis of HSCs through ERK1/2- and Smad3-dependent pathways. Metformin significantly reduced the GDF15 production from treatment-damaged HCC cells by targeting JNK. The use of metformin could attenuate the in vivo fibrotic activities of HSCs promoted by treatment-damaged HCC cells and inhibit GDF15 expression. In conclusion, treatment-damaged HCC accelerates fibrosis by promoting the activities of HSCs via GDF15 secretion, which could be reversed by metformin. This provides a potential therapeutic target for alleviating TACE-aggravated liver cirrhosis.

GDF15 deficiency exacerbates chronic alcohol- and carbon tetrachloride-induced liver injury

Growth differentiation factor 15 (GDF15) has recently been shown to have an important role in the regulation of mitochondrial function and in the pathogenesis of complex human diseases. Nevertheless, the role of GDF15 in alcohol-induced or fibrotic liver diseases has yet to be determined. In this study, we demonstrate that alcohol- or carbon tetrachloride (CCl4)-mediated hepatic GDF15 production ameliorates liver inflammation and fibrosis. Alcohol directly enhanced GDF15 expression in primary hepatocytes, which led to increased oxygen consumption. Moreover, GDF15 reduced the expression of pro-inflammatory cytokines in liver-resident macrophages, leading to an improvement in inflammation and fibrosis in the liver. GDF15 knockout (KO) mice had more TNF-α-producing T cells and more activated CD4+ and CD8+ T cells in the liver than wild-type mice. Liver-infiltrating monocytes and neutrophils were also increased in the GDF15 KO mice during liver fibrogenesis. These changes in hepatic immune cells were associated with increased tissue inflammation and fibrosis. Finally, recombinant GDF15 decreased the expression of pro-inflammatory cytokines and fibrotic mediators and prevented the activation of T cells in the livers of mice with CCl4-induced liver fibrosis. These results suggest that GDF15 could be a potential therapeutic target for the treatment of alcohol-induced and fibrotic liver diseases.

Erythropoiesis-driven regulation of hepcidin in human red cell disorders is better reflected through concentrations of soluble transferrin receptor rather than growth differentiation factor 15.

Growth differentiation factor 15 (GDF-15) is a bone marrow-derived cytokine whose ability to suppress iron regulator hepcidin in vitro and increased concentrations found in patients with ineffective erythropoiesis (IE)suggest that hepcidin deficiency mediated by GDF-15 may be the pathophysiological explanation for nontransfusional iron overload. We aimed to compare GDF-15 production in anemic states with different types of erythropoietic dysfunction. Complete blood counts, biochemical markers of iron status, plasma hepcidin, GDF-15, and known hepcidin regulators [interleukin-6 and erythropoietin (EPO)] were measured in 87 patients with red cell disorders comprising IE and hemolytic states: thalassemia, sickle cell anemia, and cobalamin deficiency. Healthy volunteers were also evaluated for comparison. Neither overall increased EPO,nor variable GDF-15 concentrations correlated with circulating hepcidin concentrations (P = 0.265 and P = 0.872). Relative hepcidin deficiency was found in disorders presenting with concurrent elevation of GDF-15 and soluble transferrin receptor (sTfR), a biomarker of erythropoiesis, and sTfR had the strongest correlation with hepcidin (r(s) = 0.584, P < 0.0001). Our data show that high concentrations of GDF-15 in vivo are not necessarily associated with pathological hepcidin reduction, and hepcidin deficiency was only found when associated with sTfR overproduction. sTfR elevation may be a necessary common denominator of erythropoiesis-driven mechanisms to favor iron absorption in anemic states and appears a suitable target for investigative approaches to iron disorders.

Circulating growth differentiation factor‐15 correlates with myocardial fibrosis in patients with non‐ischaemic dilated cardiomyopathy and decreases rapidly after left ventricular assist device support

Growth differentiation factor-15 (GDF-15) is a stress-responsive cytokine and is emerging as a biomarker of cardiac remodelling. Left ventricular assist devices (LVADs) provide unloading of the left ventricle, resulting in partial reverse remodelling. Our aim was to study GDF-15 in patients with a non-ischaemic dilated cardiomyopathy (DCM) during LVAD support. We analysed circulating GDF-15 in 30 patients before and 1, 3, and 6 months after LVAD implantation and before heart transplantation or explantation. In addition, mRNA and protein expression of GDF-15 were evaluated in myocardial tissue obtained prior to and after LVAD support. Circulating GDF-15 was significantly higher before LVAD implantation as compared with healthy controls (P < 0.001). After 1 month of mechanical support, GDF-15 levels were significantly decreased compared with pre-implantation levels (P < 0.001) and remained stable thereafter. Circulating GDF-15 was significantly correlated with kidney function and the severity of myocardial fibrosis. Interestingly, GDF-15 mRNA and protein expression in the myocardium were hardly detectable. High circulating levels of GDF-15 in patients with end-stage non-ischaemic DCM correlate with myocardial fibrosis and kidney function and decline strongly after 1 month of mechanical unloading, remaining stable thereafter. However, cardiac mRNA and protein expression of GDF-15 are very low, suggesting that the heart is not an important source of GDF-15 production in these patients.

Identification of Follistatin-Like 1 by Expression Cloning as an Activator of the Growth Differentiation Factor 15 Gene and a Prognostic Biomarker in Acute Coronary Syndrome

Growth differentiation factor 15 (GDF15) is a stress-responsive cytokine and biomarker that is produced after myocardial infarction and that is related to prognosis in acute coronary syndrome (ACS). We hypothesized that secreted proteins that activate GDF15 production may represent new ACS biomarkers. We expressed clones from an infarcted mouse heart cDNA library in COS1 cells and assayed for activation of a luciferase reporter gene controlled by a 642-bp fragment of the mouse growth differentiation factor 15 (GDF15) gene promoter. We measured the circulating concentrations of follistatin-like 1 (FSTL1) and GDF15 in 1369 patients with ACS. One cDNA clone that activated the GDF15 promoter-luciferase reporter encoded the secreted protein FSTL1. Treatment with FSTL1 activated GDF15 production in cultured cardiomyocytes. Transgenic production of FSTL1 stimulated GDF15 production in the murine heart, whereas cardiomyocyte-selective deletion of FSTL1 decreased production of GDF15 in cardiomyocytes, indicating that FSTL1 is sufficient and required for GDF15 production. In ACS, FSTL1 emerged as the strongest independent correlate of GDF15 (partial R(2) = 0.26). A total of 106 patients died of a cardiovascular cause during a median follow-up of 252 days. Patients with an FSTL1 concentration in the top quartile had a 3.7-fold higher risk of cardiovascular death compared with patients in the first 3 quartiles (P < 0.001). FSTL1 remained associated with cardiovascular death after adjustment for clinical, angiographic, and biochemical variables. Our study is the first to use expression cloning for biomarker discovery upstream of a gene of interest and to identify FSTL1 as an independent prognostic biomarker in ACS.

Cigarette smoke induces growth differentiation factor 15 production in human lung epithelial cells: Implication in mucin over-expression

Excessive mucus is a hallmark of chronic obstructive pulmonary disease (COPD). There is an emerging interest in the role of TGF-β signaling in the initiation and progression of COPD. Growth differentiation factor 15 (GDF15) is a divergent member of TGF-β superfamily. However, whether cigarette smoke induces airway epithelial GDF15 production and its functions in the airways have not been revealed. Therefore, we first analyzed GDF15 protein expression in airway epithelium of human COPD smokers versus normal non-smokers. We then examined the regulation and function of GDF15 in human airway epithelial cells in response to cigarette smoke exposure. We found increased GDF15 protein expression in airway epithelium (mainly in ciliated cells) of human COPD smokers compared with normal non-smokers. Furthermore, cigarette smoke exposure consistently up-regulated GDF15 expression in human airway epithelial cells. Moreover, GDF15 was shown to play a critical role in cigarette smoke-induced airway epithelial MUC5AC expression. Lastly, activation of phosphoinositide 3-kinase (PI3K) pathway was largely responsible for GDF15-induced airway epithelial MUC5AC expression. Our findings indicate that human airway epithelial cells can produce GDF15 during cigarette smoke exposure, which subsequently activates PI3K pathway to promote mucin (e.g. MUC5AC) expression. This highlights a novel role of GDF15 in regulating airway mucosal immunity (e.g. mucin) in cigarette smoke-exposed lungs.

Effect of High Levels of Growth Differentiation Factor 15 (GDF15) On Hepcidin Expression in Monocytes of β-Thalassemia Intermedia Patients.

Abstract 4061Poster Board III-996Ineffective erythropoiesis in thalassemia has been associated with inappropriate suppression of hepatic synthesis of the key iron regulatory peptide hepcidin, leading to spontaneous iron overload. Hepcidin mRNA was found to be suppressed in hepatocyte cultures by high levels of growth differentiation factor 15 (GDF15) detected in sera from patients with thalassemic syndromes. GDF15 may inhibit hepcidin production by antagonizing positive regulatory cytokines such as bone morphogenic protein 6 (BMP6), shown to stimulate hepatic hepcidin expression in mouse models. Although mainly produced in the liver, human hepcidin production occurs to a lesser extent in circulating monocytes. Studies with monocytes in patients with anemia of chronic disease showed increased hepcidin expression and iron retention, but to the best of our knowledge, monocyte-derived hepcidin has not yet been characterized in iron-loading anemias such as beta-thalassemia intermedia (TI). We evaluated GDF15 plasmatic levels and correlated these to hemoglobin (Hb) levels, reticulocyte counts and gene expressions in monocytes from transfusion-independent, non-chelated TI patients, homozygous for the IVS-I-6 T→C mutation (n=18), healthy, age-matched controls with no iron deficiency or overload (n=10) and transfusion-independent sickle cell anemia (SCA) patients in steady state (n=5), as a positive control group in which hyperexpression of hepcidin in mononuclear cells has been previously demonstrated. Total RNA was extracted from monocytes isolated from peripheral blood mononuclear cells and determination of BMP6 and hepcidin gene expression was performed by Real Time Polymerase Chain Reaction. Plasma GDF15 levels were determined by ELISA. Mean TI patient Hb and serum ferritin levels were 7.05±0.21g/dL and 1846±346.4μg/L, respectively. Mean absolute reticulocyte count in TI was 163.9±21.5×103/mm3. Mean GDF15 plasma levels differences were statistically significant among TI, SCA and healthy control groups (8390±827, 1780±460 and 196±21pg/mL, p<0.0001, respectively). Hepcidin gene expression did not differ significantly between TI and healthy control groups (0.007±0.006 vs. 0.05±0.03, p>0.05, respectively) but was elevated in our positive control SCA patient group (0.56±0.20; p=0.04). BMP6 gene expression was significantly decreased in TI patients compared to healthy controls (1.17±0.15 vs. 0.51±0.11, p=0.01, respectively). GDF15 concentrations correlated positively with reticulocyte counts (r=0.47; p=0.007) and negatively with hemoglobin levels (r=-0.74; p<0.0001) and BMP6 gene expression (r=-0.62; p=0.006). Our data show very high GDF15 plasma levels in a relatively homogenous population of patients with iron overload secondary to beta-thalassemia intermedia. Correlation of GDF15 with hematimetric parameters reinforces its relation to the degree of erythropoietic activity in beta thalassemia due to ineffective erythropoiesis. In addition, our study demonstrates that monocyte-derived hepcidin, like its hepatic counterpart, is inappropriately suppressed in iron-overloaded beta thalassemia intermedia patients in the presence of increased GDF15 production, correlated to decreased levels of BMP6 expression. This supports the possibility that GDF15/BMP interaction regulates hepcidin production in monocytes and hepatocytes in a similar manner, and further studies of monocyte-derived hepcidin regulation may prove to be a suitable, non-invasive tool for the investigation of human liver-derived hepcidin pathways in thalassemia and other iron-loading anemias. Disclosures:No relevant conflicts of interest to declare.

Growth differentiation factor 15 production is necessary for normal erythroid differentiation and is increased in refractory anaemia with ring‐sideroblasts

The disturbed erythropoiesis in patients with refractory anaemia with ring-sideroblasts (RARS) is characterized by intramedullary apoptosis of erythroid precursors and increased iron accumulation in mitochondria. To gain insight into these pathophysiological mechanisms we compared the gene expression profile (GEP) of erythroid precursors from RARS patients to the GEP of normal erythroid precursors. Three hundred sixty four probe sets were up-, and 253 probe sets downregulated in RARS cells. Interestingly, Growth Differentiation factor 15 (GDF15), a cytokine from the TGFbeta family, was dramatically upregulated in all RARS patients. Measurement of GDF15 in the sera from twenty RARS patients confirmed this finding by showing significantly, 7.2-fold, increased protein levels (3254 +/- 1400 ng/ml vs. 451 +/- 87 ng/ml in normals). In vitro studies demonstrated erythroid-specific production of GDF15 and dependence on erythropoietin. Induction of apoptosis by arsenic trioxide, a drug which acts via reduction of the mitochondrial membrane potential, also stimulated GDF15 production. Downregulation of endogenous GDF15 production in erythoblasts by specific siRNA led to diminished erythroid differentiation. Taken together, our findings demonstrate a new role for GDF15 in normal erythropoiesis as well as in the ineffective erythropoiesis of RARS patients.

Cleavage of growth differentiation factor 15 (GDF15) by membrane type 1‐matrix metalloproteinase abrogates GDF15‐mediated suppression of tumor cell growth

Growth differentiation factor 15 (GDF15), a transforming growth factor (TGF)-beta superfamily member, has been cloned from a placenta cDNA library as a gene product that has promoted activation of pro-matrix metalloproteinase (MMP)2 mediated by membrane type (MT)1-MMP. Expression of MT1-MMP in HEK293T cells caused cleavage of the GDF15 mature form at N(252)-M(253) to produce a 6-kDa C-terminal fragment. Treatment of MCF7 cells with GDF15 induced activation of p53 and enhanced expression of p21, which was abrogated by MT1-MMP expression. GDF15 mRNA synthesis was also shown to be induced by treatment of cells with GDF15. Treatment of MCF7 cells with GDF15 caused suppression of cell proliferation. However, proliferation of MCF7 cells transfected with the MT1-MMP gene was not affected by GDF15 treatment, but was suppressed in the presence of the MMP inhibitor BB94. HT1080 cells transfected with the GDF15 gene, which endogenously express MT1-MMP, synthesize a high-level GDF15 precursor form and a low-level mature form, and treatment of cells with BB94 enhanced production of the GDF15 mature form. Consistent with GDF15 production, HT1080 cells transfected with the GDF15 gene proliferated almost equally with control cells, and addition of BB94 effectively suppressed growth of HT1080 cells transfected with the GDF15 gene concomitant with the accumulation of the GDF15 mature form, but not control cells. These results suggest that MT1-MMP contributes to tumor cell proliferation through the cleavage of GDF15, which down-regulates cell proliferation by inducing activation of p53 and p21 synthesis.