Serum hepcidin and growth differentiation factor‐15 ( <scp>GDF</scp> ‐15) levels in polycythemia vera and essential thrombocythemia

Objectives: Hepcidin plays a regulatory role in systemic iron homeostasis. GDF‐15 has been found to be expressed from matured erythroblasts and very high levels of GDF‐15 suppresses hepcidin secretion. In this study, we evaluated hepcidin and GDF‐15 levels in polycythemia vera (PV) and essential thrombocythemia (ET). Methods: The study included 29 patients and 21 healthy controls. The patient group included 13 patients with ET and 16 patients with PV. Serum hepcidin and GDF‐15 levels were measured at the time of diagnosis, before the initiation of any therapy. Results: Hepcidin levels did not differ significantly in patients with chronic myeloproliferative disease (CMPD) and healthy controls. However, GDF‐15 levels were significantly increased in patients with CMPD (P = 0.038). No difference could be found between patients with PV and ET in terms of hepcidin and GDF‐15 levels. Patients with JAK2‐V617F mutation had increased GDF‐15 levels when compared with patients without this mutation (P: 0.006). Conclusions: The levels of GDF‐15 were higher in CMPD, which are characterized by increased erythropoiesis, and this effect was more pronounced particularly in individuals with JAK2‐V617F mutation. Hepcidin levels were not suppressed despite the increased erythroid activity and GDF‐15 levels may be protective against the clinical complications of the disease such as thrombosis. This study revealed that, hepcidin levels were not suppressed despite increased erythroid activity and high GDF‐15 levels in CMPD. We hypothesized that, this may be an attempt to prevent further amplification of erythropoietic activity by reducing iron utilization.

polycythemia vera; essential thrombocythemia; hepcidin; growth differentiation factor‐15

Chronic myeloproliferative diseases (CMPD) are clonal hematopoietic disorders associated with hyperproliferation in one or multiple blood cell lineages. Polycythemia vera (PV) and essential thrombocythemia (ET) are chronic myeloproliferative diseases (CMPD), which are characterized by bone marrow hyperplasia with increased erythrocyte and platelet counts, respectively. This overproduction of cellular elements of the blood is thought to be a result of defects in pluripotent hematopoietic stem cells [1] , [2] .

The discovery of the somatic mutation in hematopoietic progenitors in 2005 leads to a better understanding of the etiopathogenesis of BCR‐ABL1‐negative myeloproliferative diseases. JAK2‐V617F mutation involves conversion of guanine to thymine through substitution of valine with phenylalanine at the 617th codon in the pseudokinase domain of JAK2. A structural kinase activity occurs with this mutation and, in the absence of erythropoietin, EPO receptor signal pathway gains the capability of being activated. JAK2‐V617F mutation is acquired and clonal [3] . The incidence of JAK2‐V617F mutation has been reported as 90–95% in PV and 50–70% in ET [4] .

Hepcidin plays a regulatory role in systemic iron homeostasis. It inhibits iron absorption by the intestines and prevents release of iron from macrophages [5] . Hepcidin production is regulated by iron and erythropoietic activity. Elevations in iron levels enhance hepcidin production, while iron deficiency and increased erythropoietic activity suppress hepcidin production. In addition, inflammation and infections as a defense strategy of the host also result in increased levels of hepcidin [6] . High serum hepcidin levels have also been identified in many disease states including acute injury, inflammation, and cancer [7] .

GDF‐15 (growth differentiation factor‐15) is a protein with a molecular weight of 25 kDa and is a member of the transforming growth factor‐β superfamily [8] . Transforming growth factor‐β superfamily is involved in several processes including cell differentiation, development, and apoptosis [9] . GDF‐15 has been found to be expressed from matured erythroblasts, which occur in diseases of increased ineffective erythropoiesis such as congenital dyserythropoietic anemia, pyruvate kinase deficiency, and particularly in thalassemias. It has been demonstrated that GDF‐15 protein suppresses hepcidin secretion and that very high levels of GDF‐15 are required for this suppression [10] , [11] .

There is no study in the literature investigating the relationship between CMPD and GDF‐15, which is thought to have a suppressor effect on hepcidin that has significant involvement in the regulation of iron metabolism. The present study investigated the relationships between JAK2‐V617F mutation, hepcidin, and GDF‐15 levels in PV and ET, which are diseases characterized by increased hematopoiesis.

Twenty‐nine patients who presented to the hematology outpatients clinic of our university hospital and diagnosed as PV (16 patients) and ET (13 patients) between 2010 and 2012 were enrolled to this study. Patients' diagnoses were based on the diagnostic criteria of the World Health Organization [12] . All of the patients were newly diagnosed and were free of any kind of medication for the last 6 months, and none of them had treated with phlebotomy previously. The healthy control group consists of 21 individuals without any diagnosed diseases who admitted to our internal medicine department for check‐up. Individuals with any hematological and malignant diseases other than PV and ET, those with hepatic and/or renal diseases, decompensated heart disease and active infection were excluded. The study was approved by the local ethics committee, and written informed consents were obtained from all of the participants.

Blood samples following a 12‐h fasting were collected from the patients and controls for hematological and biochemical analyses. Complete blood count analysis, fasting blood glucose, urea, creatinine, AST, ALT, total bilirubin, indirect bilirubin, uric acid, iron, iron‐binding capacity, transferrin saturation, ferritin, and lactate dehydrogenase (LDH) serum level measurements were taken from the blood samples. JAK2‐V617F mutation were analyzed by real‐time PCR method. However, we were unable to evaluate other potential JAK2 mutations (e.g. exon 2, MPL mutations) or soluble transferrin receptor (sTfR) as these assays were not available.

Characteristics of the patients and healthy controls are demonstrated in Table [NaN] . There was a statistically significant difference in female/male ratio, creatinine levels, LDH levels, hemoglobin levels, hemotocrit ratios, MCV levels, leukocyte counts, platelet counts, serum iron, and ferritin levels between patients and healthy controls (Table [NaN] ).

Characteristics of patients with CMPD and healthy controls

1 P < 0.05 statistically significant.

• 2 Independent sample t‐test.
• 3 The Mann–Whitney U‐test.

Characteristics of patients with PV and ETare demonstrated in Table [NaN] . There was a statistically significant difference between hemoglobin levels, hemotocrit ratios, MCV levels, platelet counts of patients with PV and ET, as expected (Table [NaN] ).

Characteristics of patients with PV and ET

• 4 P < 0.05 statistically significant. (The Mann–Whitney U‐test).
• 5 Chi‐squared test.

For hepcidin and GDF‐15 analyses, 5 cc blood was collected from each patient and healthy control, and these samples were centrifuged for 10 min at 1370 g, and the separated sera were stored at −80°C. DRG® Hepcidin hormone ELISA (EIA‐4705)” (enzyme‐linked immunosorbent assay) DRG International Inc., (Mountainside, NJ, USA) kit was used for hepcidin measurements. Serum GDF‐15 levels were measured using ‘Quantikine Human GDF‐15 ELISA (Cat. Num. DGD150)” (enzyme‐linked immunosorbent assay) R&D Systems Inc. (Minneapolis, MN, USA) kit. The results were read on the DYNEX‐DSX™ Four‐Plate Automated ELISA Processing System (Research Triangle Park, NC, USA).

SPSS 16.0 software Inc., Chicago, IL, USA was used for statistical analyses. The variables were investigated using visual (histogram and probability plots) and analytical methods (Shapiro–Wilk tests) to determine whether or not they are normally distributed. Logarithmic transformation was applied to nonparametric data. The independent sample t‐test was used to compare of parametric variables between the two groups. The Mann–Whitney U‐test was used to compare of nonparametric variables between the two groups. Correlation analyses were performed using Pearson's and Spearman's correlation. A P‐value of < 0.05 was considered as a statistically significant result.

Hepcidin levels in CMPD group (72.48 ± 15.43 ng/mL) were not significantly different from healthy controls (81.99 ± 19.95 ng/mL) (P: 0.063) (Fig. [NaN] ). However, GDF‐15 levels were found to be significantly increased in CMPD group (1629.87 ± 1382.01 pg/mL) in comparison with healthy controls (855.98 ± 431.45 pg/mL) (P: 0.038) (Fig. [NaN] , Table [NaN] ). Hepcidin levels were higher in the PV group (77.09 ± 18.04 ng/mL) compared with ET group (66.78 ± 9.19 ng/mL), although the difference was not statistically significant (P = 0.075). Likewise, no statistically significant difference could be found in GDF‐15 levels between patients with PV and ET (P = 0.983) (Table [NaN] ).

Hepcidin and GDF ‐15 levels in CMPD , healthy control, PV and ET groups

• 6 The Mann–Whitney U‐test and independent sample t‐test were used.
• 7 P < 0.05 statistically significant.

When hepcidin and GDF‐15 levels were analyzed in the CMPD group according to JAK2‐V617F mutation status, GDF‐15 levels were found to be significantly higher in patients with JAK2‐V617F mutation compared with those without JAK2‐V617F mutation (P: 0.006) (Table [NaN] ). Although hepcidin levels were higher in patients without JAK2‐V617F mutation, the difference was not statistically significant (P: 0.835) (Table [NaN] ).

Relationships between presence of JAK 2‐ V 617 F mutation and erythropoietin levels with hepcidin and GDF ‐15 levels

• 8 The Mann–Whitney U‐test was used to compare of between the two groups.
• 9 P < 0.05 statistically significant.

Sixteen patients with PV were subdivided into two groups in terms of their erythropoietin (EPO) levels, as those with low EPO levels and those within normal ranges. Eleven patients had low and five patients had normal EPO levels. Hepcidin and GDF‐15 levels did not differ significantly in PV patients with low and normal EPO levels (P: 0.441 and P: 0.661, respectively). (Table 5).

Pearson and Spearman correlation analyses of the group of patients with CMPD did not yield any significant correlations between hepcidin levels and platelet counts, GDF‐15 levels, lactate dehydrogenase levels, leukocyte counts, hemoglobin levels, ferritin levels (P > 0.05). However, when GDF‐15 levels are concerned, there was a near‐significant positive correlation between GDF‐15 levels and leukocyte counts (P = 0.002, r = 0.425). There was a moderate positive correlation between age and GDF‐15 levels in all study population including both healthy controls and patients with CMPD (P < 0.001 r = 0.52). Hepcidin or GDF‐15 levels did not differ significantly with gender, existence of splenomegaly, or iron deficiency (P > 0.05).

In this study, we measured serum levels of hepcidin and GDF‐15 in patients with CMPD, namely PV and ET and healthy controls. Hemoglobin levels, platelet counts were increased, and ferritin levels were decreased in patients with CMPD when compared with healthy controls, as expected. Hepcidin levels did not differ significantly in patients with CMPD and healthy controls. However, GDF‐15 levels were significantly increased in patients with CMPD. No difference could be found between patients with PV and ET in terms of hepcidin and GDF‐15 levels. Patients with JAK2‐V617F mutation had increased GDF‐15 levels when compared with patients without this mutation; while a positive correlation could be found between age and GDF‐15 levels both in patients with CMPD and healthy controls, a positive correlation between leukocyte count and GDF‐15 levels could be detected only in patients with CMPD.

The hormone hepcidin, a 25‐amino‐acid (aa) peptide, is the principal regulator of iron absorption and its distribution to tissues. Hepcidin is synthesized predominantly in hepatocytes, but it is expressed at low levels in other cells and tissues, such as macrophages, adipocytes, and brain. Hepcidin is the main regulator of plasma iron concentrations and, on the other hand, hepcidin production is regulated by iron and erythropoietic activity. Iron excess stimulates hepcidin production, and increased concentrations of the hormone in turn decrease dietary iron absorption and consequently prevents further iron loading. Conversely, hepcidin production is decreased in iron deficiency, thus allowing for increased absorption of dietary iron. Increased erythropoietic activity also suppresses hepcidin production. Apart from enhancing iron absorption, this enables the rapid release of stored iron from macrophages and hepatocytes and augments the supply of iron for erythropoiesis. Hepcidin is also increased in inflammation and infection, and it is presumed that this regulation evolved as a host defense strategy to limit iron availability to microorganisms [5] .

There are two studies in literature investigating hepcidin levels in CMPD. In one of these studies, urinary hepcidin levels were measured in four patients with myelofibrosis, and the results revealed suppression of urinary hepcidin in those patients [13] . In the other study, prohepcidin levels were evaluated in patients with PV and it demonstrated that prohepcidin levels were significantly lower in these patients and that low prohepcidin levels were more pronounced in patients with iron deficiency. Authors have suggested that these results may be an adaptive response for compensation of the organism [14] . Our study is the first study in literature, investigating serum hepcidin levels in patients with PV and ET. This study revealed no difference in terms of hepsidin levels between patients with CMPD and healthy controls. Additionally, hepcidin levels did not differ significantly between patients with PV and ET. In contrast to the study conducted by Kwapisz et al., we could not demonstrate a decrease in hepcidin levels even in patients with iron deficiency in PV group. This inconsistency could be due to the fact that Kwapisz et al. measured serum prohepcidin levels; while in our study, we measured serum hepcidin levels. Previous studies demonstrated that prohepcidin levels do not correlate with urinary and serum hepcidin and they do not respond to relevant physiological stimuli [14] , [15] . Besides, even though there are no studies in literature which can support our hypothesis, the attempt to prevent further amplification of erythropoietic activity by reducing iron utilization could be the explanation for the lack of detection of decreased levels of hepcidin levels even in patients with iron deficiency anemia in PV patient group.

Two proteins produced by erythroid precursors, GDF 15 and twisted gastrulation protein (TWSG1), have been proposed to mediate hepcidin suppression in anemias with ineffective erythropoiesis [10] . GDF‐15 is a member of transforming growth factor‐β (TGF‐β) superfamily that comprises more than 40 members [9] . Mature erythroblasts were suggested to be the source of GDF‐15 as high levels of GDF‐15 gene expression were detected in mature erythroblasts cultures [10] . In literature, serum GDF‐15 levels in patients with thalassemia intermedia and congenital dyserythropoietic anemia type I (CDAI) were demonstrated to be dramatically elevated compared with healthy volunteers or patients with the thalassemia trait [10] , [16] , [17] . On the other hand, studies in patients with effective erythropoiesis after bone marrow transplantation or after injection of erythropoietin failed to demonstrate major increases in serum GDF‐15 [18] , [19] . In our study, we detected significantly high GDF‐15 levels in CMPD group when compared with healthy controls. However, GDF‐15 levels did not differ significantly between patients with PV and ET. We suggested that high GDF‐15 levels in CMPD group could be related to the increased erythropoietic activity. There are no other studies in literature investigating serum levels of GDF‐15 in CMPD.

In literature, there are several studies demonstrating the potential role of GDF‐15 in the regulation of hepcidin [10] , [17] , [20] . Hepcidin is suggested to be regulated by members of the TGF‐β superfamily, specifically BMPs 2, 4, and 9 that act through phosphorylation of receptor SMADs 1, 5, and 8, which, on association with SMAD 4, translocate to the nucleus and stimulate expression of hepcidin. In a study conducted by Kautz et al. revealed that increases in body iron levels enhance the expression of BMP6 and eventually lead to increase in hepcidin expression [21] . Although no study about BMP expression in patients with PV and ET in literature exists, Bock et al. [22] demonstrated that BMP6 expression was increased in advanced stages of myelofibrosis compared with controls. In addition to this, GDF‐15, which is a member of transforming growth factor‐β (TGF‐β) superfamily has been shown to induce SMAD 2/3 transcriptional activity [23] . Tanno et al. demonstrated that patients with β‐thalassemia who have ineffective erythropoiesis have elevated serum GDF‐15 levels and on the contrary, they have inappropriately low hepcidin levels. Besides, in this study, they showed that in cultured human hepatocytes, both serum GDF‐15 from patients with thalassemia and recombinant GDF‐15 suppressed hepcidin expression. However, high levels (>5000 pg/mL) of GDF‐15 are required for this suppression, and the suppression was incomplete even after the addition of the highest dose of 100 000 pg/mL [10] . In the study conducted by Mast et al., [20] reduced hepcidin levels but no significant increase in GDF‐15 levels had been demonstrated in high‐intensity blood donors in whom effective erythropoiesis is expected. To date, there are no studies in literature demonstrating both GDF‐15 and hepcidin levels in CMPD. In our study, we found GDF‐15 levels were significantly increased in patients with CMPD when compared with healthy controls, but hepcidin levels did not differ between the patients and healthy controls. Besides, we failed to demonstrate a correlation between GDF‐15 and hepcidin levels in contrast to data in literature. These results may be related to the fact that the levels of GDF‐15 levels in the patient group included in our study seem to be not high enough to suppress hepcidin levels. The other explanation could be, it is the consequence of increase in BMP6 expression in myeloproliferative diseases or it may just be due to limited number of patients enrolled to this study.

JAK2 gene, firstly detected in 1992, is located on the short arm of chromosome nine (9p24). The gene codes for a tyrosine kinase protein named as JAK [24] . JAK is composed of four intracellular proteins binding to receptors of specific hematopoietic growth factors (e.g. erythropoietin, thrombopoietin, prolactin, interleukin‐3, interferon‐γ, GM‐CSF, and G‐CSF) [25] . After JAK2 protein binds to its receptor, it will be activated by phosphorylation leading to subsequent activation of signaling molecules, such as STATs. Consequently, when these signaling molecules are transported into the nucleus, they stimulate transcriptional factors. JAK2‐V617F is a point mutation, affecting the JAK/STAT pathway and in turn influences cell proliferation, migration, activation, and apoptosis [26] . Ferroportin levels are strictly regulated by its ligand hepcidin. The binding of hepcidin to ferroportin triggers the internalization and degradation of the receptor‐ligand complex and is responsible for phosphorylation of ferroportin [27] . Domenico et al. demonstrated that JAK2 is required for this phosphorylation and that binding of JAK2 to ferroportin was hepcidin‐dependent. The study also revealed that for hepcidin‐mediated internalization of ferroportin, JAK2 was essential, but not sufficient [28] . However, in the following years, Ross et al. [29] demonstrated that JAK2 is not required for hepcidin‐mediated ferroportin internalization and JAK/STAT pathway is not involved in ferroportin phosphorylation. Because of conflicting data, the role of JAK2, and consequently JAK2 mutation on iron metabolism is still not clear. In this study, hepcidin levels did not differ significantly in patients with JAK2‐V617F mutation and patients without JAK2‐V617F mutation. However, we demonstrated that GDF‐15 was significantly higher in patients with JAK2‐V617F mutation. This is the first study demonstrating the relation between GDF‐15 and JAK2‐V617F mutation. Although this study failed to reveal a correlation between iron hemostasis and hepcidin and GDF‐15 levels, further studies are required to bring to light the underlying mechanism of the relation between GDF‐15 and JAK2‐V617F mutation.

In conclusion, the present study demonstrated that the levels of GDF‐15 was higher in CMPD, which involve increased erythropoietic compartment and that this effect was more pronounced particularly in individuals with JAK2‐V617F mutation. This study revealed that hepcidin levels were not suppressed despite increased erythroid activity and high GDF‐15 levels. We hypothesized that this may be an attempt to prevent further amplification of erythropoietic activity by reducing iron utilization. Clinical studies enrolling larger numbers of patients are obviously required to support the current data. We hope the current study will lead to further studies about relationship between JAK2‐V617F mutation and GDF‐15 levels and iron homeostasis.

We would like to thank Nicholas Facey for English editing of our manuscript.

The authors declare no competing financial interests.

Graph: Hepcidin levels of CMPD and healthy control groups ( P : 0.063).

Graph: GDF ‐15 levels of CMPD and healthy control groups. * P : 0.038 ( P  <   0.05 statistically significant).