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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 18, 2003 as doi:10.1096/fj.02-1134fje. |
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, or L-NMMA1
* Division of Hematology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan;
Center for Tsukuba Advanced Research Alliance and Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan;
Kowa Research Institute, Kowa Co., Ltd., Tokyo 103-8433, Japan; and
Laboratory for Systems Biology and Medicine Research Center for Advanced Science and Technology, University of Tokyo, Tokyo 153-8904, Japan
2Correspondence: Division of Hematology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan. E-mail: simigawa{at}md.tsukuba.ac.jp
SPECIFIC AIM
One common pathogenesis of anemia of chronic diseases (ACD) and with renal disease appears to be stimulation of GATA binding activity by interleukin-1ß (IL-1ß), tumor necrosis factor-
(TNF-), or NG-monomethyl L-arginine (L-NMMA), which inhibits erythropoietin (Epo) promoter activity. We investigated the ability of K-7174 (a GATA-specific inhibitor) to improve Epo production after inhibition by IL-1ß, TNF-, or L-NMMA treatment in Hep3B cells in vitro and in an in vivo mouse assay.
PRINCIPAL FINDINGS
1. Epo protein production, which is inhibited by IL-1ß or TNF-, is rescued by K-7174
Hypoxia (1% O2) induced Epo protein production from aliquots of 3 x 106 Hep3B cells over 24 h. However, 15 U/mL IL-1ß and 220 U/mL TNF- each inhibited Epo protein production, and addition of 10–20 µM K-7174 rescued these inhibitions in a dose-dependent manner (Fig. 1
).
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2. Epo promoter activity, which is inhibited by IL-1ß, TNF-, or L-NMMA, is rescued with K-7174
Hep3B cells were transiently transfected with a construct spanning regions of the human Epo enhancer containing hypoxia-inducible factor 1 (HIF-1) and a promoter containing a GATA element (Pwt: wild-type). Hypoxic induction of Epo promoter activity from Pwt was 33.7-fold higher than that from normoxic Pwt. Addition of 15 U/mL IL-1ß, 220 U/mL TNF-, or 10-3 M L-NMMA inhibited induction from Pwt with lowered hypoxic/normoxic ratios of 19.9-, 20.7-, or 19.8-fold, respectively. However, the addition of 10 µM K-7174 increased induction from hypoxic Pwt to 40.3-fold higher than that from normoxic Pwt. Furthermore, the combination of 10 µM K-7174 with + IL-1ß, + TNF-, or + L-NMMA rescued hypoxic inductions from Pwt, with increased hypoxia/normoxia ratios of 43.0-, 49.6-, or 35.6-fold, respectively. These results indicate that K-7174 rescues the suppression of Epo gene expression by IL-1ß, TNF-, or L-NMMA through the Epo gene regulatory regions.
3. The binding activity of GATA, which is enhanced by IL-1ß, TNF-, or L-NMMA, is inhibited with K-7174
Hep3B cells showed strong GATA DNA binding by an electrophoretic mobility shift assay (EMSA). The addition of K-7174 decreased the intensity of this band dose dependently. Stimulation with 15 U/mL IL-1ß, 220 U/mL TNF-, or 10-3 M L-NMMA for 24 h led to an increase in GATA binding. These increments of GATA binding activity induced by IL-1ß, TNF-, or L-NMMA were inhibited by the addition of 10 µM K-7174. Thus, IL-1ß, TNF-, and L-NMMA all enhance GATA binding activity, which in turn inhibits Epo promoter activity and protein production.
4. K-7174 increases Epo production by mice in vivo and rescues the inhibition of hemoglobin (Hb) concentrations and reticulocyte counts by IL-1ß or TNF-
To elucidate the effects of K-7174, IL-1ß, and TNF- on Epo production, Hb concentrations, and reticulocyte counts in an in vivo mouse assay, blood samples (0.3 mL) were obtained from the orbital vein of ICR mice at 0, 12, 24, and 36 h. Compared with PBS control, intraperitoneal injection of IL-1ß decreased Epo production, Hb concentrations, and reticulocyte counts on days 3, 6, and 10 (Fig. 2
). Injection of TNF- also inhibited Epo production, Hb concentrations, and reticulocyte counts on the same days (Fig. 2)
. Compared with these results, injection of K-7174 significantly increased Epo production, Hb concentrations, and reticulocyte counts on the same days, while injection of K-7174 significantly rescued the inhibitions of Epo production, Hb concentrations, and reticulocyte counts by IL-1ß or TNF- (Fig. 2)
. Thus, K-7174 treatment increased Epo production, Hb concentrations, and reticulocyte counts in vivo.
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CONCLUSIONS AND SIGNIFICANCE
By in vitro cell assay, both 15 U/mL IL-1ß and 220 U/mL TNF-, inhibited Epo protein production, and this was rescued by 10 µM K-7174. The three treatments (15 U/mL IL-1ß, 220 U/mL TNF-, and 10-3 M L-NMMA) each inhibited Epo promoter activity and the addition of K-7174 reversed this. The EMSA showed that addition of K-7174 decreased GATA binding activity, which in turn was increased with the addition of IL-1ß, TNF-, or L-NMMA. Intraperitoneal injections of IL-1ß or TNF- into mice decreased Hb concentrations and reticulocyte counts. However, the addition of K-7174 reversed this anemia produced by IL-1ß or TNF-. K-7174 was developed as a low molecular weight anti-inflammatory drug. It is known to be a specific inhibitor of GATA. Intraperitoneal injection of 30 mg/kg K-7174 into ICR mice produced no significant change in the weight of the spleen or the numbers of white blood cells and platelets (data not shown). A dose of 300 mg/kg for rats (10-fold higher than the dose used in this study) revealed no adverse effects on the liver, kidney, or on hematopoiesis (data not shown). Furthermore, K-7174 decreases the production of circulating glutamic-oxaloacetic transaminase (GOT) and lactate dehydrogenase (LDH), which are increased by the injection of IL-1ß and TNF- into ICR mice (data not shown). Thus, the anemia induced by IL-1ß or TNF- can be prevented by simultaneous administration of K-7174 (Fig. 3
). This study raises the possibility of using K-7174 as a novel drug for treating ACD and for anemia associated with renal disease. We are presently investigating the effects of K-7174 using DBA/2FG-pcy mice as a model of anemia associated with renal disease.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1134fje; doi: 10.1096/fj.02-1134fje 
This article has been cited by other articles:
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  Y. Nakano, S. Imagawa, K. Matsumoto, C. Stockmann, N. Obara, N. Suzuki, T. Doi, T. Kodama, S. Takahashi, T. Nagasawa, et al. Oral administration of K-11706 inhibits GATA binding activity, enhances hypoxia-inducible factor 1 binding activity, and restores indicators in an in vivo mouse model of anemia of chronic disease Blood, December 15, 2004; 104(13): 4300 - 4307. [Abstract] [Full Text] [PDF]
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