Erythroid Differentiation Factor Research Articles

Hsa‑miR‑455‑3P as a predictive biomarker of anemia in patients with non‑small cell lung cancer treated with carboplatin plus paclitaxel.

Lung cancer is the leading cause of cancer-related morbidity and mortality worldwide. The initial treatment of lung cancer depends on the definition of the tumor type and its staging. The most common treatment is chemotherapy, and the first-line treatment is a combination of carboplatin and paclitaxel. Although this treatment has good efficacy, there is a high prevalence of adverse events, particularly hematological reactions. Studies on new biomarkers related to these adverse events, such as circulating microRNAs (miRNAs/miRs), are important for optimizing the quality of life of patients. miRNAs have high stability in several biological fluids and they have specific expressions in different tissues or pathologies. Thus, the present study aimed to assess the relationship between circulating miRNAs and adverse hematologic reactions caused by treatment with carboplatin + paclitaxel in patients with lung cancer. Blood was collected from patients before and 15 days after chemotherapy for hematological adverse reaction analysis, microarray and quantitative (q)PCR validation. Adverse reactions were classified according to the Common Terminology Criteria for Adverse Events v4.0. Microarray analysis was performed using plasma from six patients without anemia and six patients with anemia, and nine miRNAs were differentially expressed. miR-1273g-3p, miR-3613-5p and miR-455-3p, identified using microarray, were assessed using qPCR in 20 patients without anemia and 26 patients with anemia. Bioinformatic analyses of miR-455-3p were performed using miRWalk, the Database for Annotation, Visualization and Integrated Discovery and GeneMania software. Microarray analysis of patients with and without anemia revealed nine significant differentially-expressed plasma miRNAs among these patients. Of these, miR-1273g-3p, miR-3613-5p and miR-455-3p were chosen for further assessment. Only miR-455-3p demonstrated a significant reduction in expression (P=0.04) between the groups before chemotherapy with carboplatin + paclitaxel. Bioinformatics analysis of miR-455-3p revealed a relationship between this miRNA and the hematopoietic pathway, particularly with respect to the RUNX family transcription factor 1 (RUNX1) and TAL bHLH transcription factor 1, erythroid differentiation factor (TAL1) genes. The most prevalent adverse reactions in patients with lung cancer treated with carboplatin + paclitaxel were hematological, particularly anemia. This adverse reaction, caused by dysfunction of the hematopoietic system, may be explained by a possible association between the important genes in this system, RUNX1 and TAL1, and hsa-miR-455-3p.

KPT-8602 Induced Hematopoietic Differentiation in U2AF1 S34F Mutant Cells and Myelodysplastic Syndromes Bone Marrow Stem and Progenitor Cells

Myelodysplastic syndromes (MDS) are a group of clonal bone marrow (BM) neoplasms characterized by ineffective hematopoiesis, cytopenia's, and a high risk of progression to acute myeloid leukemia. Current standard therapies include erythropoiesis stimulating agents for lower risk disease and DNMTi for higher risk MDS; however, less than half of patients respond and even the best responses are transient and non-curative. The majority of MDS patients harbor at least one of ~40 commonly found somatic gene mutations, most of which occur in splicing factor genes such as U2AF1, which also have non-canonical roles in nuclear export. KPT-8602 (Eltanexor) is a novel selective inhibitor of nuclear export (SINE) that acts by disrupting nuclear export of cargo relevant in MDS. We hypothesized that U2AF1 S34 F mutant cells would be selectively sensitive to treatment with KPT-8602. We created immortalized murine hematopoietic progenitor cells by retroviral transduction of stimulated bone marrow (BM) cells isolated from U2AF1 WT or U2AF1 S34F mice with a HOXB8_ER vector. Estrogen drives the hematopoietic transcription factor immortalizing the cells in a progenitor state, however, upon estrogen withdrawal the cells will differentiate and eventually die. This differentiation can be driven by treatment with specific growth factors such as erythropoietin (EPO) for erythroid differentiation or granulocyte macrophage colony stimulating factor (GM-CSF) for myeloid differentiation. KPT-8602 exposure induced differentiation and increased hematopoiesis in murine cell models and primary MDS samples. U2AF1 WT and U2AF1 S34F cells were plated in medium lacking estrogen and treated with GM-CSF (10nM) or EPO (1nM) with or without KPT-8602 (10nM) for 72h, then stained with Ly6C, CD11b, Ter119, and CD71 for flow cytometry analysis. In addition, we performed colony forming capacity assays in both the murine cells and primary MDS BM stem and progenitor cells and age-comparable healthy donors, with 14 day KPT-8602 exposure. Treatment with GM-CSF significantly increased the expression of Ly6C in both U2AF1 WT and U2AF1 S34F cells (WT fold change untreated vs GMCSF =1.9, p=0.03; S34F fold change untreated vs GMCSF =2.3, p<0.01). However, addition of KPT-8602 caused significantly more Ly6C expression than GM-CSF alone in the mutant cells (WT fold change GMCSF vs combination =3.1, p=0.06; S34F fold change GMCSF vs combination =4.0, p<0.01). There were no significant changes in CD11b expression with KPT-8602 treatment. We did not find a statistically significant increase in erythroid markers assessed by Ter-119 and CD71 after EPO treatment, however, KPT-8602 treatment alone showed a trend of increased expression of Ter-119 and CD71 in the mutant cells that fell just below statistical significance (Ter119 p=0.08 for WT, p=0.09 for S34F; CD71 p=0.21 for WT, p=0.06 for S34F). Additionally, we looked at selective toxicity of U2AF1 S34Fby WST-1 reagent and found no differences in toxicity between the WT or mutant cells with KPT-8602. However, U2AF1 WT and U2AF1 S34F progenitor cells showed an increase in colony forming capacity (n=1 with technical duplicates) when plated in the presence of KPT-8602 showing an average fold change in colony growth of 1.4 for U2AF1 WT and 1.3 for U2AF1 S34F underscoring the ability of KPT-8602 to induce differentiation. To confirm these results were not cell line artifacts, we next treated primary BM stem and progenitor cells from MDS patients (n=2) and healthy donors (n=4). MDS patient 1 (age 73y) was diagnosed with MDS-MLD-EB1 and harbored mutations in TP53, ASXL1, BCOR, TET2 and PPM1D. MDS patient 2 (age 79y) was diagnosed with MDS/MPN and harbored an SF3B1 mutation. Healthy donors (n=4) were all male and had a median age of 65y. Treatment with KPT-8602 (10nM) significantly increased burst and colony forming unit-erythroid (BFU-E/CFU-E) in MDS primary cells (p=0.02) with no significant increase in the healthy donors (p=0.57) (BFU-E/CFU-E fold increase was 1.1 for healthy donors and 3.5 for MDS). Collectively, these findings suggest that KPT-8602 promotes myeloid and erythroid differentiation and further validates the use of KPT-8602 as a therapeutic strategy for MDS. Future studies will fully elucidate the specific mechanisms by which KPT-8602 acts in the context of splicing factor mutations, and other commonly found MDS mutation types.

Hemoglobin Response to Zinc Supplementation in Patients with Zinc Deficiency and Chronic Anemia

Background: Zinc is a necessary micronutrient used as a catalyst, structural element, and regulatory ion in many metabolic processes including erythropoiesis. Chronic anemia has an estimated prevalence of 5.6% within the US according to NHANES. Iron deficiency is considered the most common nutritional deficiency leading to anemia, but other nutritional deficiencies have been implicated in anemia including deficiencies of Vitamin A, B12, B6, C, D, and E, folate, Riboflavin, Copper, and Zinc. While zinc fortification has been studied in developing countries and has been associated with a decrease in the overall prevalence of anemia within the pediatric and maternal populations, there have been no studies in adults with chronic anemic in the developed world. The mechanism of Zinc in erythropoiesis is likely multifactorial with zinc acting as a catalyst in alpha-aminolevulinic acid dehydratase enzyme in iron metabolism and heme synthesis. Zinc's incorporation in the structure of the growth factor independent 1B (Gfi-1B) Zinc finger protein and GATA-1 Zinc finger transcription factor, which functions as a regulator in erythroid cell growth by modulating gene expression specific to erythroid series including the SMAD pathways and TGF-β signaling. Zinc's interaction in signaling cascades such as the growth hormone and insulin-like growth factor-1 pathways also result in increased erythropoietin transcription and therefore increased erythropoiesis. In addition, zinc is regarded as an important erythroid differentiation factor. This descriptive, retrospective study describes zinc deficient patients with chronic anemia, who underwent zinc supplementation, and their changes in hemoglobin (HGB) over a 12-month period in an academic outpatient hematology center in California, United States of America. Objectives: Primary endpoints: determine the rate of anemia resolution over 12 months following supplementation of zinc in patients with zinc deficiency and chronic anemia. Secondary endpoints: determine if zinc supplementation in zinc deficient patients with chronic anemia will improve HGB by at least 1.0 g.dL by 1, 3, 6, and 12 month time points. Methods: Ninety Three patients with chronic anemia (HGB <12 for women, HGB <13 for men for >3 months) over the age of 18 were identified to have zinc deficiency (plasma zinc concentration <65 ug/dL) in an academic outpatient hematology center in California. Patients did not start new treatment for their anemia, including initiating new treatments for malignancy 3 months preceding, and within the data collection period. Patients were supplemented with Zinc Sulfate 50 mg daily and their HGB was followed over 12 months. A retrospective analysis was completed for prior medical history, lab data, and HGB was tabulated at the time markers of 1, 3, 6, and 12 months following initiation of zinc supplementation. ANOVA, Wilcoxon rank-sum, and unpaired Students and t tests were performed to determine significant differences where p <0.05 is considered statistically significant. Results Prior to supplementation, average HGB among patients was 9.83 g/dL, WBC was 6.8 g/dL, MCV was 89 fl, PLTS were 211 g/dL. Following 1 month of supplementation average HGB was 10.46 g/dL (p = 0.015, n = 82), at 3 months average HGB was 10.75 g/dL (p = 0.0002, n = 83), at 6 months average HGB was 11.34 g/dL (p= 0.000000004, n = 74), and at 12 months average HGB was 11.48 g/dL (p = 0.00000002, n= 57). Primary endpoint of anemia resolution was found in 39 of 93 patients (42%). Secondary endpoint of HGB improvement by at least 1.0 g/dL was reached in 31% of patients at 1 month, 36% of patients at 3 months, 58% of patients at 6 months and 68% of patients at 12 months. Conclusions: Zinc supplementation significantly improved HGB levels in patients with zinc deficiency and chronic anemia, with resolution of anemia in 42% of patients. There was statistically significant improvement from baseline HGB at the1, 3, 6, and 12 month time markers. Further study is warranted to determine the specific mechanism of zinc deficiency in anemia, as well as clinical studies determining zinc supplementation doses, monitoring, and toxicity.

Hemoglobin Response to Zinc Supplementation in Chronic Kidney Disease in Patients with Zinc Deficiency and Anemia

Background: Chronic anemia has an estimated prevalence of 5.6% within the US, where 25.3% of patients with chronic kidney disease (CKD) 3-5 have anemia of chronic kidney disease according to NHANES. Anemia in CKD has long been attributed to a decrease in EPO production due to their kidney disease. While iron deficiency is treated in anemia of chronic kidney disease, other micronutrients are less commonly tested or supplemented. Zinc may be an additional micronutrient of interest in anemia of chronic kidney disease as zinc has numerous proposed mechanisms in erythropoeisis including acting as a catalyst in the iron metabolism and heme synthesis through the activity of alpha-aminolevulinic acid dehydratase enzyme. Zinc is found in the structure of the growth factor independent 1B (Gfi-1B) Zinc finger protein and GATA-1 Zinz finger transcription factor, which functions as a regulator in erythroid cell growth by modulating gene expression specific to erythroid series including the SMAD pathways and TGF-β signaling. The interaction with zinc in signaling cascades such as the growth hormone and insulin-like growth factor-1 pathways also result in increased erythropoietin transcription and therefore increased erythropoiesis. In addition, zinc is regarded as an important erythroid differentiation factor which progress maturation of erythrocytes. Zinc has also shown to stimulate EPO production in nephrectomized rats and Zinc supplementation increased reticulocyte production in human subjects. This descriptive, retrospective study describes zinc deficient patients with CKD 3-5 and chronic anemia; who underwent oral zinc supplementation, and their changes in hemoglobin (HGB) over a 12 month period in an academic outpatient hematology clinic in California, United States of America. Objectives: Primary endpoints: determine the rate of anemia resolution over 12 months following supplementation of zinc in patients with zinc deficiency, CKD, and anemia of chronic kidney disease. Secondary endpoints: determine if zinc supplementation in zinc deficient patients with chronic anemia and CKD 3-5 will improve HGB by at least 1.0 g.dL by 1, 3, 6, and 12 month time points. Methods: Hematology patients from Loma Linda University Cancer Center over the age of 18 who had CKD 3 or above (GFR <59 ml/min) with chronic anemia (HGB <12 for women, HGB <13 for men for >3 months) were identified to have zinc deficiency (plasma zinc concentration <65 ug/dL). Patients did not start new treatment for their anemia, including initiating new treatments for malignancy 3 months preceding, and within the data collection period. Patients were supplemented with Zinc Sulfate 50 mg daily and their HGB was followed over 12 months. A retrospective analysis was completed for prior medical history, lab data, and hemoglobin was tabulated at the time markers of 1, 3, 6, and 12 months following initiation of zinc supplementation. ANOVA, Wilcoxon rank-sum, and unpaired Students and t tests were performed to determine significant differences. p <0.05 is considered statistically significant. Results Thirty-Four patients were identified to have CKD 3-5 with chronic anemia and zinc deficiency. They were found to have no statistically significant baseline characteristics, hematologic values at time of diagnosis, or micronutrient and hormonal etiologies for anemia. HGB prior to supplementation averaged 9.96 g/dL, WBC averaged 5.8, and PLTS averaged 149. Following supplementation HGB at 1 month average was 10.47 g/dL (p=0.19 n=30), at 3 months average of 10.49 (p=0.12 n=31), at 6 months average of 11.03 (p=0.011 n=27), at 1 year 10.93 (p=0.03, n=17). Primary endpoint of HGB resolution was found in 12 of the 34 patients (35%). Secondary endpoint of HGB improvement by at least 1.0 g/dL was reached in 27% of patients at 1 month, 32% of patients at 3 months, 59% of patients at 6 months and 64% of patients at 12 months. Conclusions: Zinc supplementation improved patients' anemia with resolution of anemia in 35% of patients with incremental improvement in HGB over a 1 year time frame. There was statistically significant improvement from baseline HGB at the 6 and 12 month time markers. Further study is warranted to determine the specific mechanism of zinc deficiency in anemia with chronic kidney disease, as well as clinical studies determining zinc supplementation doses, monitoring, and toxicity.

Zinc As an Erythrocyte Stimulating Agent in Myelodysplastic Syndrome

Background: The anemia highly associated with myelodysplastic syndrome (MDS) has been linked to numerous transcriptional factor regulation abnormalities, specifically SMAD 2/3 upregulation that forms an inhibitory feedback loop to downregulate erythrocyte stimulation (ESA), which can have therapeutic implications. There is evidence that zinc contributes to erythropoiesis through multifactorial mechanisms involving regulation and transportation of necessary micronutrients including copper and iron. Zinc is involved in cell signaling pathways for erythropoiesis including acting as a cofactor in the SMAD 2/3 complex, TGF-β signaling, and incorporation of zinc into GATA-1 (Zinc finger transcription factor). The interaction with zinc in signaling cascades such as the growth hormone and insulin-like growth factor-1 pathways also result in increased erythropoietin transcription and therefore increased erythropoiesis. In addition, zinc is regarded as an important erythroid differentiation factor which progress maturation of erythrocytes. Due to the multifactorial role of zinc in erythropoiesis, including acting as a cofactor in the SMAD 2/3 complex, it may represent an inexpensive, and well tolerated ESA or additional micronutrient to test and supplement in MDS. This descriptive, retrospective study describes zinc deficient patients with MDS, who underwent zinc supplementation, and their changes in hemoglobin (HGB) over a 12-month period in an academic outpatient hematology center in California, United States of America. Objectives: Primary endpoints: determine if zinc supplementation in zinc deficient MDS patients will improve HGB by at least 1.0 g.dL by 1, 3, 6, and 12 month time points. Methods: Eight adult patients with MDS were identified to have zinc deficiency (plasma zinc concentration <65 ug/dL). Patients were not started on ESAs or their treatments were not changed for the management of their MDS. Patients were supplemented with Zinc Sulfate 50 mg daily and their HGB was followed over 12 months. A retrospective analysis for medical history and lab data at time of zinc deficiency diagnosis and at 1, 3, 6 and 12 month following initiation of zinc supplementation was completed. ANOVA, Wilcoxon rank-sum, and unpaired Students and t tests were performed to determine significant differences where p <0.05 is considered statistically significant. Results: Prior to supplementation, average HGB among patients was 8.01 g/dL, WBC was 3.65 g/dL, MCV was 97.6 fl, PLTS were 140g/dL. Following 1 month of supplementation average HGB was 8.5 g/dL (p = 0.5, n = 7), at 3 months average HGB was 9.27 g/dL (p = 0.087, n = 7), at 6 months average HGB was 10.3 g/dL (p= 0.006, n = 7), and at 12 months average HGB was 10.28 g/dL (p = 0.04, n= 7). Primary endpoint of HGB improvement by at least 1.0 g/dL was reached in 29% of patients at 1 month, 43% of patients at 3 months, 86% of patients at 6 months and 71% of patients at 12 months. Resolution of anemia was reached in 1 patient (12.5%) of patients within 12 months. Conclusions: Zinc supplementation improved anemia in MDS patients with incremental improvement in HGB over a 1 year time frame. There was statistically significant improvement from baseline HGB at the 3, 6, and 12 month time markers. Further study is warranted to determine the specific mechanism of zinc deficiency in MDS and its mechanism as an erythrocyte stimulating agent as well as further study in non-zinc deficient patients, various zinc supplementation doses, monitoring, and toxicity.

HPV E7 affects the function of cervical cancer cells via the TAL1/lnc‑EBIC/KLHDC7B axis.

High‑risk human papillomavirus (HPV)16 and 18 are the primary cause of cervical cancer (CC) and long non‑coding RNAs (lncRNAs/lncs) are often abnormally expressed in patients with CC. The authors' previous study indicated that oncogenic enhancer of zeste homolog2 (EZH2)‑binding lncRNA in cervical cancer (lnc‑EBIC) serves a role in the tumorigenic activity of the HPV E6 protein in patients with CC. However, whether HPV E7 affects the development of CC through lnc‑EBIC, and the potential mechanisms underlying this remains unclear. Therefore, the present study investigated the effects of lnc‑EBIC and HPV E7 in cervical cancer cell lines HeLa, CaSki and C33A invitro. CCK‑8, EdU and DAPI staining assays, flow cytometry, RT‑qPCR, western blotting and Transwell assay were performed on these cell lines. The results revealed that exogenous expression of HPV16/18 E7 significantly promoted lnc‑EBIC expression, and conversely, lnc‑EBIC was downregulated by silencing endogenous HPV16/18 E7 expression in corresponding CaSki and HeLa cells. Overexpression of lnc‑EBIC significantly increased cellular proliferation, migration and invasion, and inhibited apoptosis in HPV‑ C33A cells. The tumorigenic effects of HPV16/18 E7 in corresponding CaSki and HeLa cells were significantly blocked by the silencing of lnc‑EBIC expression. Molecular analysis revealed that HPV16/18 E7 depended on TAL BHLH transcription factor 1, erythroid differentiation factor inhibition to promote lnc‑EBIC expression, which also resulted in the upregulation of oncogenic Kelch domain‑containing 7B (KLHDC7B) in corresponding CaSki and HeLa cells. Additionally, KLHDC7B knockdown blocked the tumor‑promotive effects of lnc‑EBIC in HPV‑ C33A cells. Collectively, the results of the present study indicated that lnc‑EBIC acts as an oncogenic lncRNA by enhancing KLHDC7B expression in HPV+ and HPV‑ CC cells, and can be exploited by HPV16/18 E7 to accelerate tumorigenic activity in CC. These results further revealed that the lnc‑EBIC/KLHDC7B axis represents a novel molecular mechanism and potential therapeutic target for CC.

Mesenchymal stem cells protect against malaria pathogenesis by reprogramming erythropoiesis in the bone marrow

Malaria remains a major public health problem worldwide. The immune mechanisms that mediate protection against malaria are still unclear. Previously, we reported that mesenchymal stem cells (MSCs) play a critical role in host protection against malaria by altering the dynamic balance of T regulatory cells and effector T cells producing inflammatory cytokines. Here, we report that MSCs reprogram haematopoiesis in primary (bone marrow) and secondary (spleen) lymphoid organs to provide host protection against malaria. Adoptive transfer of MSCs from malaria-infected mice to naïve recipient mice that were subsequently infected with malaria parasites dramatically accelerated the formation of colony-forming units-erythroid cells in the bone marrow. Adoptively transferred MSCs also induced expression of the key erythroid cell differentiation factor GATA-1 in the spleen of recipient animals. Interestingly, we further observed a subtle increase in the CD34+ hematopoietic stem and progenitor cells in lymphoid organs, including spleen and lymph nodes. Infusion of MSCs also enhanced T cell proliferation, resulting in increased numbers of both CD4+ and CD8+ T cells in the spleen. MSCs also inhibited the induction of the negative co-stimulatory receptor programmed death-1 by T cells in recipient animals upon infection with malaria parasites. Taken together, our findings suggest that MSCs play a critical role in host protection against malaria infection by modulating erythropoiesis and lymphopoiesis.

Ingenuity pathway analysis of human facet joint tissues: Insight into facet joint osteoarthritis.

Facet joint osteoarthritis (FJOA) is a common degenerative joint disorder with high prevalence in the elderly. FJOA causes lower back pain and lower extremity pain, and thus severely impacts the quality of life of affected patients. Emerging studies have focused on the histomorphological and histomorphometric changes in FJOA. However, the dynamic genetic changes in FJOA have remained to be clearly determined. In the present study, previously obtained RNA deep sequencing data were subjected to an ingenuity pathway analysis (IPA) and canonical signaling pathways of differentially expressed genes (DEGs) in FJOA were studied. The top 25 enriched canonical signaling pathways were identified and canonical signaling pathways with high absolute values of z-scores, specifically leukocyte extravasation signaling, Tec kinase signaling and osteoarthritis pathway, were investigated in detail. DEGs were further categorized by disease, biological function and toxicity (tox) function. The genetic networks between DEGs as well as hub genes in these functional networks were also investigated. It was demonstrated that C-X-C motif chemokine ligand 8, elastase, neutrophil expressed, growth factor independent 1 transcriptional repressor, Spi-1 proto-oncogene, CCAAT enhancer binding protein epsilon, GATA binding protein 1, TAL bHLH transcription factor 1, erythroid differentiation factor, minichromosome maintenance complex component 4, BTG anti-proliferation factor 2, BRCA1 DNA repair-associated, cyclin D1, chromatin assembly factor 1 subunit A, triggering receptor expressed on myeloid cells 1 and tumor protein p63 were hub genes in the top 5 IPA networks (with a score >30). The present study provides insight into the pathological processes of FJOA from a genetic perspective and may thus benefit the clinical treatment of FJOA.

Erythroid Growth Differentiation Factor 15 and Ferroportin Expression During Gestational Iron Deficiency Anemia

Abstract 5286Iron homeostasis during pregnancy is modulated to meet the increased iron needs but how this is achieved is not very clear. Growth Differentiation Factor (GDF15) produced by the expanded erythroid compartment in β thalassemia has been shown to increase iron absorption by suppressing hepcidin. GDF15 is also highly expressed in the placenta and increasing levels of GDF15 are seen with advancing gestational age of pregnancy. But the role of GDF15 in iron homeostasis in pregnancy has not been elucidated till date. Ferroportin (FPN) is the only known protein involved in iron export and it is the target of hepcidin, the central regulator of iron homeostasis. In this study we analyzed the expression of GDF15 and FPN in pregnant women with iron deficiency anemia.Fourteen pregnant women with proven iron deficiency anemia (IDAP) [Hb<11g/dL and Ferritin <12ng/ul] and thirteen healthy subjects as controls (NC) were enrolled as part of an ongoing study. Serum GDF15 and hepcidin levels were measured by ELISA kits from R&D systems and Bachem, UK respectively. Reticulocytes were isolated and total RNA was purified using Trizol. GDF15 and FPN transcripts were quantified using Taqman Gene expression assays using GAPDH as an internal control. Gene expression values were calculated on the basis of the 2-ΔΔCt method.The mean age of the pregnant women was 22.5±2.5 years. The median ferritin in IDAP was 1.4 and ranged from 0.2 to 8.3 ng/ml. The hepcidin levels were very low [<2ng/ml] in IDAP. Serum GDF15 levels in IDAP was significantly higher as compared to controls [IDAP-3333.71±409 pg/ml vs. NC-309.7±127.0 pg/ml; p=0.000]. Reticulocyte GDF15 mRNA expression was significantly lower [IDAP-25.09 (1.28–239.8) vs. NC-910.4 (0.28–1962); p=0.004] and FPN expression was significantly higher in pregnancy [IDAP-209.8 (48.33–1201) vs. NC-77.96(17.21–281.3); p=0.001] than in the controls. GDF15 mRNA as well as serum GDF15 levels significantly correlated with FPN expression in IDAP [RNA r=0.895; p=0.000; Protein r=0.555, p=0.049] Eight patients were followed up after 8 weeks of supplementation and there was no significant change in the serum GDF15 concentration (3235±468.26pg/ml; p=1.000). However their serum ferritin and hepcidin levels were significantly higher [Ferritin-11.60 (9.80–21.30), p=0.0021; Hepcidin-17.86(0.29–38.50), p=0.015]. There was no significant correlation between GDF15 protein levels and hepcidin (r=0.429, p=0.354).Molecular mechanisms of iron homeostasis in pregnancy are poorly understood. IDAP had very low hepcidin levels which normalized after iron stores were replenished. Elevated GDF15 protein levels in IDAP inspite of low reticulocyte expression indicate that erythroid contribution is minimal and placenta is the main source of GDF15. The significant correlation between GDF15 (mRNA and protein) with FPN expression and absence of correlation with hepcidin levels indicate a possible role for GDF15 in iron homeostasis in pregnancy. These findings has to be validated and the role of GDF15 in modulating FPN and there by iron absorption has to be further elucidated. Disclosures:No relevant conflicts of interest to declare.

The endogenous peptides of normal human serum extracted from the acetonitrile-insoluble precipitate using modified aqueous buffer with analysis by LC–ESI–Paul ion trap and Qq-TOF

Many peptides of biological or medicinal importance may be derived from proteolytic actions and are found at low concentrations in human blood fluids. Endogenous polypeptides from human serum were precipitated in acetonitrile and the precipitate was then selectively extracted with water modified by organic solvents and collected over C18 resin. Extraction of serum with C18 alone, and the acetonitrile supernatant or ultrafiltration collected over C18, served as controls. The samples were analyzed by SDS-PAGE, or C18 high pressure liquid chromatography with electrospray ionization using a Paul ion trap and Qq-TOF. Spectra were correlated without specifying an enzyme using the X!TANDEM or the Paragon algorithms. Multiple endogenous peptides from plasminogen, coagulation factors, collagens, serum amyloid, receptors, zinc finger/bromo peptide proteins, ryanodine receptor, calmodulin binding activator, erythroid differentiation factor, testes cancer antigen, extracellular matrix protein, myeloid/lymphoid leukemia 2 and many low abundance proteins were correlated by X!TANDEM with protein expect values of approximately E-16 or less. Proteins with binding sites for nucleic acids, phosphoinositides, and other cellular locations were also observed using the Qq-TOF and Paragon algorithm. Proteins with low expectation scores and overlapping peptides sequences were observed. The existence of these proteins in serum has been confirmed by tryptic digestion and LC-ESI-MS/MS. The presence of plasminogen, serum amyloid and zinc finger RNA binding proteins were confirmed by Western blot. There was agreement on the detection of endogenous peptides from low abundance proteins associated with the biology of cancer from the examination of the blood peptides by ion trap and Qq-TOF, tryptic digests of blood proteins, and Western blot.

Regulator of differentiation 1 (ROD1) binds to the amphipathic C‐terminal peptide of thrombospondin‐4 and is involved in its mitogenic activity

The matrix protein thrombospondin-4 has an acidic amphipathic C-terminal peptide (C21) which stimulates erythroid cell proliferation. Here we show that C21 stimulates red cell formation in anemic mice in vivo. In vitro experiments indicated that the peptide-mediated increase of erythroid colony formation in cultures of human CD34+ hematopoietic progenitor cells was possible only under continuous presence of erythropoietin. In the absence of this cytokine, C21 stimulated exclusively myeloid colony formation. Therefore, the peptide is not a specific erythroid differentiation factor. In fact, it is mitogenic in non-erythroid cells, such as skin fibroblasts and kidney epithelial cells. In erythroleukemic TF-1 cells, it actually decreased the production of the erythroid differentiation marker glycophorin A. C21-affinity chromatography revealed regulator of differentiation 1 (ROD1) as a major C21-binding protein. ROD1 is the hematopoietic cell paralog of polypyrimidine tract binding proteins (PTBs), RNA splice regulators which regulate differentiation by repressing tissue-specific exons. ROD1 binding to C21 was strongly inhibited by synthetic RNAs in the order poly A > poly U > poly G = poly C and was weakly inhibited by a synthetic phosphorylated peptide mimicking the C-terminal domain of RNA polymerase II. Cellular overexpression or knockdown experiments of ROD1 suggest a role for this protein in the mitogenic activity of C21. Since the nuclear proteins ROD1 and PTBs regulate differentiation at a posttranscriptional level and there is a fast nuclear uptake of C21, we put forward the idea that the peptide is internalized, goes to the nucleus and maintains cells in a proliferative state by supporting ROD1-mediated inhibition of differentiation.

Iron absorption and metabolism

Intestinal iron absorption is an essential physiological process that is regulated by the liver-derived peptide hepcidin. This review will describe recent advances in hepcidin biology and enterocyte iron transport. Hepcidin acts as a repressor of iron absorption and its expression in turn reflects a range of systemic cues, including iron status, hypoxia, erythropoiesis and inflammation. These act through proteins on the hepatocyte plasma membrane such as HFE, hemojuvelin and transferrin receptor 2 to alter transcription of the hepcidin gene. Bone morphogenetic protein-SMAD signaling provides a key pathway of hepcidin activation, whereas the membrane-bound serine protease matriptase-2 and the erythroid factor growth differentiation factor 15 have emerged as important negative regulators of hepcidin expression. At the enterocyte itself, the recent demonstration of a chaperone for delivering iron to ferritin and new data on iron release from the hepcidin target ferroportin are helping to define the pathway of iron movement across the intestinal epithelium. Disturbances in the hepcidin regulatory pathway underlie a range of iron metabolism disorders, from iron deficiency to iron loading, and there is considerable promise that the exciting recent advances in understanding hepcidin action will be translated into improved diagnostic and therapeutic modalities in the near future.

Chaperonin 10 as an endothelial‐derived differentiation factor: Role of glycogen synthase kinase‐3

The anti-inflammatory peptide early pregnancy factor/chaperonin 10 (cpn10) was identified by 2D-electrophoresis/mass spectrometry as one of the proteins increased in human umbilical cord endothelial cells (HUVEC) after treatment with erythropoietin (EPO). EPO increased the amount of cpn10 released into the medium of HUVEC cultures, despite the absence of a secretory signal peptide. Although immunosupressive agents would represent an indirect advantage for red cell formation under conditions of infection and inflammation, it is possible that cpn10 may have direct effects on erythroid cells. We show that the chaperonin decreased cell proliferation in cultures of the erythroleukemia cell line K562 and increased the amounts of the erythroid differentiation markers glycophorin A and hemoglobin in TF-1 cells. Nevertheless, cpn10 is not a specific erythroid cell differentiation factor, because monolayers of skin fibroblasts overexpressing cpn10 had significantly higher levels of the differentiation marker collagen I than normal fibroblasts. Nothing is known about the mechanism of action of cpn10 in its capacity as a general differentiation factor. An analysis of early changes taking place in K562 cells after incubation with cpn10 using antibody microarrays identified several phosphorylation events, including a decrease of GSK-3alpha phosphorylation. Further studies in TF-1 cells indicated that cpn10 decreased the phosphorylation of cofilin-1 while stimulating that of GSK-3beta. Furthermore, glycophorin A production decreased in the presence of a GSK-3 inhibitor in the same cells. These experiments support the idea that GSK-3-regulated signal transduction pathways are not only important for stem cell maintenance but may be involved in events controlling cell differentiation.

Microarray Analysis of Differentially Expressed Genes in the Brains of Tubby Mice

The tubby mouse is characterized by progressive retinal and cochlear degeneration and late-onset obesity. These phenotypes are caused by a loss-of-function mutation in the tub gene and are shared with several human syndromes, suggesting the importance of tubby protein in central nervous system (CNS) functioning. Although evidence suggests that tubby may act as a transcription factor mediating G-protein coupled receptor (GPCR) signaling, any downstream gene regulated by tubby has yet to be identified. To explore potential target genes of tubby with region-specific transcription patterns in the brain, we performed a microarray analysis using the cerebral cortex and hypothalamus of tubby mice. We also validated the changes of gene expression level observed with the microarray analysis using real-time RT-PCR. We found that expression of erythroid differentiation factor 1 (Erdr1) and caspase 1 (Casp1) increased, while p21-activated kinase 1 (Pak1) and cholecystokinin 2 receptor (Cck2r) expression decreased in the cerebral cortex of tubby mice. In the hypothalamic region, Casp 1 was up-regulated and micro-crystallin (CRYM) was down-regulated. Based on the reported functions of the differentially expressed genes, these individual or grouped genes may account for the phenotype of tubby mice. We discussed how altered expression of genes in tubby mice might be understood as the underlying mechanism behind tubby phenotypes.

ACE-011, a Soluble Activin Receptor Type IIa IgG-Fc Fusion Protein, Increases Hemoglobin and Hematocrit Levels in Postmenopausal Healthy Women

ACE-011 is a soluble fusion protein consisting of the extracellular domain of activin receptor IIA linked to the Fc portion of human IgG1 and is a potent activin antagonist. ACE-011 is currently in clinical development for the treatment of bone loss in a variety of disease indications. In addition to its effect on bone forming cells, activin is also known as erythroid differentiation factor (EDF) and has been reported to have proerythrocytic effects and induced terminal differentiation of red blood cells (RBC). In preclinical studies, ACE-011 administration to mice and cynomolgus monkeys is associated with increases in erythropoiesis.A randomized, double-blind, placebo-controlled, multiple-dose, dose-escalation study was conducted in healthy postmenopausal women to evaluate the safety, tolerability and pharmacodynamics of ACE-011 in 4 cohorts of 10 subjects (8 active: 2 placebo). Subjects were to receive 4 monthly doses of ACE-011 at 0.1, 0.3, 1.0 and 2.0 mg/kg or placebo by subcutaneous (SC) route of administration and followed up for 3 months. Safety evaluations were conducted on each cohort prior to dose escalation.A total of 31 subjects received at least one dose of ACE-011 or placebo; 9 subjects in cohort 1 (0.1 mg/kg) received all 4 doses, 10 subjects in cohort 2 (0.3 mg/kg) received 3 doses and 9 subjects in cohort 3 (1 mg/kg) received 2 doses of either ACE-011 or placebo. A dose and time dependent increase in hemoglobin values was observed in all treatment groups; these elevations were statistically significant in the 0.3 and 1.0 mg/kg cohorts. A maximum tolerated dose level was determined to be 1 mg/kg after one subject experienced progressive and persistent hypertension that was attributed to a rapid and significant rise in hemoglobin levels approximately 1 week following her second dose of ACE-011. Increases in hemoglobin and hematocrit represent the dose limiting pharmacodynamic effects of ACE-011, and further dose escalation to the 2 mg/kg dose was suspended. These effects were seen in the red cell lineage; no significant effects on white blood cells or platelets were observed. JAK2 kinase activity was measured in 3 subjects with elevated hemoglobin levels after ACE-011 treatment and was negative.Preliminary analysis of the data, 29 days after the administration of the first dose, is shown below.Δ Hemoglobin (g/dL) from baselinePlacebo (n=7)0.1 mg/kg (n=8)0.3 mg/kg (n=8)1 mg/kg (n=8)Day 8Mean (SD)0.171 (0.399)0.675 (0.403)0.85 (0.563)*1.213 (0.506)**Median0.30.650.751.25Min, Max−0.3, 0.70.20, 1.20.2, 1.60.4, 2.0Day 15Mean (SD)−0.35 (0.695)0.425 (0.413)0.438 (0.912)*1.75 (0.685)**Median−0.550.60.31.5Min, Max−1.1, 0.8−0.3, 0.9−0.6, 2.40.9, 3.1Day 29Mean (SD)0.34 (0.207)0.613 (0.323)1.212 (0.909) *2.675 (0.997)**Median0.40.71.152.45Min, Max0, 0.5−0.1, 1.00.1, 2.81.7, 4.4* p<0.05, ** p<0.01 compared to placeboIn one of the subjects with a history of chronic anemia, as a result of iron deficiency, the hemoglobin level increased by almost 2 g/dL from a baseline value of 8.4 g/dL following the first SC dose of 1 mg/kg ACE-011, and reached a level of 11 g/dL within 3 weeks after the second dose of ACE-011.ACE-011 was generally well tolerated, except for the case described above with uncontrolled hypertension at the 1 mg/kg dose level. The majority of the non-hematological treatment-emergent adverse events were mild in severity and not related to study drug. A dose-dependent decrease in serum FSH levels, a biological marker of activin inhibition, was also observed in postmenopausal subjects following treatment with ACE-011.These data indicate that ACE-011 is associated with increases in hemoglobin and hematocrit levels in both healthy volunteers as well as in a subject with iron deficiency anemia and may be a potential novel agent for the treatment of patients with impaired erythropoiesis.

Focus on TGF-β signalling

Transforming growth factor-b (TGF-b) is the index member of a large family of multifunctional growth factors. They were named originally for their ability to induce normal rat kidney fibroblasts to grow in soft agar in the presence of Epidermal Growth Factor (EGF) (De Larco & Todaro 1978, Roberts et al. 1981). There are 5 subtypes, all products of separate genes, three of which, TGF-b1, b2 and b3, have been shown to be expressed in mammalian ovarian cells (Derynck et al. 1988, Hernandez et al. 1990, Mulheron & Schomberg 1990). These factors are synthesized in inactive precursor forms that undergo cleavage to produce monomers, which can dimerize to 25 kDa forms through the conserved cysteine regions. The active regions of the TGF-b monomers share 98–100% identity. The isolation and cloning of inhibin and erythroid differentiation factor (subsequently called activin), and Mullerian inhibiting substance (MIS) led to the observation that these peptides are also members of the TGF-b superfamily. The family has grown to consist of more than 40 members (for a review see Chang et al. 2002), including the growth differentiation factors (GDFs) and bonemorphogenetic proteins (BMPs) (Fig. 1).Members of the family are related by sequence homology (30–40%) and conservation of the cysteine residues. Many also share a similar signal transduction mechanism involving serine–threonine kinase receptors with downstream Smad signalling pathways (for reviews see Miyazawa et al. 2002, Shi & Massague 2003, Lin et al. 2006). The concept that TGF-b superfamily members function as local regulators, either paracrine or autocrine, gained focus after a review on the topic was published by Sporn and Todaro (1980). Further to this, it had been realized that ‘there are events of gametogenesis and endocrine function .which do not necessarily have a direct relationship to the known hormonal milieu being determined by pituitary secretion rates and the afferent blood supply’ (Findlay & Risbridger 1987), supporting the concept of local regulation. It very quickly became apparent that many family members were acting as local regulators in reproductive tissues. In this issue of Reproduction, TGF-b signalling in reproductive tissues is reviewed. This focus issue begins with a chapter on the signal transduction systems that

Down-Regulation of Activin A by Lymphoma: A Possible Mechanism of Bone Marrow Involvement.

Scientific background: Increasing evidence points to the crucial role of malignant lymphoma interaction with the tumor microenvironment. Our previous investigations indicated that the stromal cytokine activin A induces apoptotic death of myeloma cells due to its antagonism with the growth promoting effect of interleukin-6. Activin A is a homodimer of the ßA inhibin polypeptide chain. Like other transforming growth factor (TGF)ß superfamily members, it is a pleiotropic molecule, which is widely expressed. It has initially been studied in the reproductive system, but has also been implicated in the regulation of hemopoiesis; it is an erythroid differentiation factor and is expressed within the bone marrow microenvironment. Activin A functions are tightly regulated by the competitive inhibitor inhibin A (a heterodimer, /ßA) and the binding inhibitors, follistatins. We previously showed that abundance of activin A was restrictive for B cell production in vitro and that within human nasal polyps activin A expression was widespread but absent from foci of B lineage cells. We were therefore interested to find out whether activin A plays a role in the occurrence of BM (bone marrow) involvement in malignant lymphomas.Materials and methods: The patient population consisted of 17 patients with lymphoma and 3 patients without lymphoma served as controls. Paraffin embedded sections were prepared and immunohistochemical staining was performed using an antibody to the bA chain. The slides were reviewed by team of 5 investigators and graded separately.Results: Out of 17 lymphoma cases, 10 patients showed BM involvement. In patients with BM involvement the level of activin A was significantly decreased in the area surrounding the lymphoid infiltrate (Fig 1a). This was seen uniformly in all the patients except for one. The level of activin A in the rest of the BM was similar to the level seen in specimens of reactive BM. In all 7 patients that had no BM involvement we found a diffuse staining for activin A (similar to what we saw in patients with reactive BM) (Fig.1b).Discussion: It is interesting that only some of the patients with malignant lymphomas have BM involvement. This could stem from a difference in the migratory abilities of the lymphoid cells, which is unlikely, or from a difference in their ability to home and flourish in the BM microenvironment. We have demonstrated that activin A, is significantly down regulated in the vicinity of the lymphoid infiltrate in the BM, as opposed to what occurs in normal inflammatory BM. This suggests that an interaction between the lymphoma cell and the BM microenvironment leads to down-regulation of activin A expression and possibly promotes the survival of the lymphoid cells. [Display omitted]

Targeted histone deacetylase inhibition for cancer therapy.

The histone deacetylase inhibitors are a new class of cytostatic agents that inhibit the proliferation of tumor cells in culture and in vivo by inducing cell cycle arrest, differentiation and/or apoptosis. Histone acetylation and deacetylation play important roles in the modulation of chromatin topology and the regulation of gene transcription. Histone deacetylase inhibition induces the accumulation of hyperacetyl-ated nucleosome core histones in most regions of chromatin but affects the expression of only a small subset of genes, leading to transcriptional activation of some genes, but repression of an equal or larger number of other genes. Non-histone proteins such as transcription factors are also targets for acetylation with varying functional effects. Ace-tylation enhances the activity of some transcription factors such as the tumor suppressor p53 and the erythroid differentiation factor GATA-1 but may repress transcriptional activity of others including T cell factor and the co-activator ACTR. Recent studies in our laboratory and others have shown that the estrogen receptor alpha (ERalpha) can be hyperacetylated in response to histone deacetylase inhibition, suppressing ligand sensitivity and regulating transcriptional activation by histone deacetylase inhibitors. Conservation of the acetylated ERalpha motif in other nuclear receptors suggests that acetylation may play an important regulatory role in diverse nuclear receptor signaling functions. A number of structurally diverse histone deacetylase inhibitors have shown potent antitumor efficacy with little toxicity in vivo in animal models. Several compounds are currently in early phase clinical development as potential treatments for solid and hematological cancers both as monotherapy and in combination with cytotoxics and differentiation agents. This report reviews the biology and clinical development of histone deacetylase inhibitors for cancer therapy.

Expression and Possible Roles of Activin A in Proliferative Vitreoretinal Diseases

Purpose: To examine the expression of activin A in eyes and to determine the possible functions of activin A in proliferative vitreoretinal diseases. Methods: The activin A concentration in vitreous specimens obtained from eyes with or without retinal ischemia was measured by a bioassay using erythroid differentiation factor effects of activin A. The expression of activin A and activin receptors in the preretinal membranes was observed by immunohistochemical analysis. Results: The mean concentration of activin A in the eyes with proliferative diabetic retinopathy was 1.50 ± 1.27 ng/mL (mean ± SD; n = 10), and that in the nondiabetic eyes without retinal ischemia (macular hole and epiretinal membrane) was 0.90 ± 0.55 ng/mL (n = 5). Neither difference was significant. Activin A and its receptors were detected in the vascular endothelial cells, fibroblast-like cells and round-shaped macrophage-like cells in preretinal proliferative membranes by immunohistochemical analysis. Conclusions: Activin A is involved in the proliferative membrane formation in both ischemic and nonischemic vitreoretinal proliferative diseases. Activin A, a member of TGF-β superfamily, regulates angiogenesis and tissue fibrosis in the wound healing process.

Cloning and characterization of goldfish activin β A subunit

We have cloned a full-length cDNA coding for activin β A subunit from a goldfish brain and pituitary cDNA library, which represents the first for activin β A in fish. Sequence analysis of goldfish activin β A shows that this peptide is highly conserved across vertebrates. The mature region of goldfish activin β A shares 81% amino acid identity with that of humans. Messenger RNA of goldfish activin β A is expressed in a variety of tissues including ovary, testis, brain and liver, suggesting a wide range of physiological roles for activin A in the goldfish. The identity of the cloned goldfish activin β A was confirmed by expressing the protein in the Chinese hamster ovary (CHO) cells followed by detection of the specific activin activity in the culture medium using erythroid differentiation factor (EDF) assay with F5-5 cells. Stable CHO cell lines producing high level of recombinant goldfish activin A were established and characterized.