Oxygen-dependent expression of the erythropoietin gene in rat hepatocytes in vitro

Eckardt, Kai‐Uwe; Pugh, Christopher W.; Ratcliffe, Peter J.; Kurtz, Armin

doi:10.1007/bf00374928

Cited by 30 publications

(18 citation statements)

References 43 publications

(52 reference statements)

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“…In agreement with previous results [16] total R N A synthesis in hepatocytes, as determined by [3H]uridine Fig.3. Effect of vasopressin on EPO mRNA levels in isolated hepatocytes incubated at 3% 02 for 2.5 h. Values are related to controls incubated in parallel at 3% O2 in the absence of vasopressin (mean +_ SEM, n = 3).…”

Section: Effect Of Phorbol Esters On Epo Mrna Accumulation and Total supporting

confidence: 93%

“…Although cells were isolated from normoxic animals EPO mRNA was readily detectable after cell isolation, which is in accordance with previous data from in vivo studies [15]. When cells were preincubated at 20% oxygen for 4 h, as in previous experiments [16] average concentrations of EPO mRNA did not change significantly (not shown), but when preincubation of cells was performed at 40% oxygen, EPO mRNA levels after 4 h were significantly lower than immediately after isolation, and in many experiments became undetectable. Reducing the oxygen tension in the incubator to 3% after the end of the preincubation period resulted in a marked increase of EPO mRNA levels within 2.5 h. Despite continued hypoxic exposure, a reduction of EPO mRNA levels occurred between 5 and 9 h, before the concentration of EPO mRNA remained fairly constant.…”

Section: Measurement Of Total Rna Synthesis [3h]uridine Incorporationsupporting

confidence: 89%

“…Hepatocytes have recently been identified as the predominant site of oxygen-dependent EPO production in rats during the early postnatal period [15,32,45], and we have previously observed that the ability of hepatocytes to modulate EPO production in an oxygen-dependent fashion is maintained after isolation of the cells [16]. Extending these observations, studies of the time course of EPO mRNA levels in isolated hepatocytes (Fig.…”

Section: Discussionmentioning

confidence: 55%

“…It appears likely therefore that the effect of these kinase inhibitors is not due to inhibition of PKC, but that another, as yet undefined, kinase activity is an important element in the signal transduction that determines oxygen-dependent control of hepatic EPO mRNA levels. We have previously shown that addition of cAMP and cGMP to hepatocytes incubated at ambient oxygen pressure does not increase EPO mRNA levels, indicating that cAMP-and cGMP-dependent kinases do not directly mediate hypoxic signalling in these cells [16], but this does not exclude a possible permissive role. Moreover, it is important in this respect, that a haem protein was suggested to operate as oxygen sensor in the control of EPO formation [23] and that in Rhizobium meliloti an oxygen-sensing haem protein with kinase activity was identified [21].…”

Section: Discussionmentioning

confidence: 94%

“…Since in juvenile rats the liver is the predominant site of EPO formation [15], and since hepatocytes have recently been identified as the main cellular site of EPO production in the liver [32,45] we have studied EPO production in primary cultures of juvenile rat hepatocytes. Recently we have shown that steady-state levels of EPO mRNA in isolated hepatocytes increase approximately 15-fold after incubation at reduced oxygen tensions for 18 h [16]. This indicates that primary cultures of hepatocytes can be used to investigate oxygen-dependent control of EPO formation in vitro.…”

Section: Introductionmentioning

confidence: 96%

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Hypoxia-induced accumulation of erythropoietin mRNA in isolated hepatocytes is inhibited by protein kinase C

et al. 1994

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Abstract.To define the role of protein kinase C (PKC) in oxygen-dependent production of erythropoietin (EPO) in the liver, we have determined EPO messenger ribonucleic acid (mRNA) expression in primary cultures of juvenile rat hepatocytes incubated at different oxygen tensions in the absence and presence of phorbol esters, vasopressin, and structurally different kinase inhibitors. Upon reduction of oxygen concentrations from 40% to 3% EPO mRNA in cultured hepatocytes increased markedly within 1.25 h, reached maximal values after 2.5 h and remained elevated for up to 72 h. Treatment of hepatocytes during 1.25-5 h of hypoxic exposure with phorbol 12-myristate-13 acetate (PMA) attenuated hypoxiainduced EPO mRNA levels dose-dependently by a maximum of approximately 50%. This inhibitory effect of PMA disappeared upon treatment for more than 5 h and was completely lost after incubation for 9 and 18 h in the presence of 10 -6 M and 10 -7 M PMA, respectively. Phorbol 12,13-dibutyrate and vasopressin also inhibited EPO mRNA accumulation, whereas 4 alpha-phorbol 12,13-didecanoate was ineffective. Western blot analysis of PKC isozymes revealed the presence of PKC alpha, beta II, delta, epsilon and zeta and provided no evidence that the PMA-induced inhibition of EPO expression was associated with depletion of any of these isozymes. Conversely, PMA-induced inhibition of EPO mRNA accumulation was paralleled by translocation of PKC alpha from cytosol to membranes and the time-and dose-dependent attenuation of the inhibitory effect of PMA on EPO mRNA levels was paralleled by down-regulation of PKC alpha. A dose-dependent inhibition of EPO mRNA formation, independent of effects on total RNA synthesis, as determined by [3H]uridine incorporation, was also found in the presence of the kinase inhibitor staurosporine (EDso --2 • 10 s M) and three structurally related derivatives with increased selectivity for PKC (RO 317549, EDso --1 • -6 M; RO 318220, EDso --lX

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Section: Effect Of Phorbol Esters On Epo Mrna Accumulation and Total supporting

confidence: 93%

Section: Measurement Of Total Rna Synthesis [3h]uridine Incorporationsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 55%

Section: Discussionmentioning

confidence: 94%

Section: Introductionmentioning

confidence: 96%

See 3 more Smart Citations

Hypoxia-induced accumulation of erythropoietin mRNA in isolated hepatocytes is inhibited by protein kinase C

et al. 1994

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Hypoxia signalling in the control of erythropoietin gene expression in rat hepatocytes

et al. 1996

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This study aimed to investigate the sequence of events involved in the stimulation of erythropoietin (EPO) gene expression by hypoxia in hepatocytes. To this end, primary cultures of rat hepatocytes were kept at either high (40% O2) or low (3% O2) oxygen tensions for 2.5 h. Hypoxia increased EPO mRNA about fifteen-fold, whilst the divalent cation cobalt (50-100 microM) or the iron chelator desferrioxamine (10-200 microM) did not increase EPO mRNA levels. Addition of hydrogen peroxide (100-500 microM) to the culture medium did also not change EPO mRNA levels at high or low oxygen tension. Addition of catalase (50-200 micrograms/ml) to the culture medium resulted in a lower level of hypoxia-induced EPO mRNA. Inhibition of protein synthesis by cycloheximide (100 microM) completely abolished the increase of EPO mRNA in response to hypoxia. Hypoxia but not cobalt increased the appearance of the hypoxia-inducible factor 1 (HIF-1), and this increase was blunted by cycloheximide. Taken together, these findings suggest that a classic heme protein and a related oxygen-dependent production of oxygen radicals is less likely to be involved in the regulation of the EPO gene by oxygen in hepatocytes. On the other hand, intact protein synthesis is an absolute requirement for the hypoxia-induced appearance of HIF-1 and for hypoxia-stimulated expression of the EPO gene in hepatocytes.

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Erythropoietin

Wenger

Kurtz

2011

Comprehensive Physiology

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The hormone erythropoietin (Epo) is the main humoral regulator of erythropoiesis. It binds to specific receptors belonging to the cytokine receptor superfamily. Epo stimulates proliferation and differentiation of erythroid precursor cells, but may also bind to and exert some additional effects in nonhemopoietic tissues. It is mainly produced in the kidneys and to minor extents also in the liver and in the brain. The plasma concentration of erthyropoietin is inversely related to the oxygen content of the blood. The secretion of Epo into the circulation and hence its plasma concentrations are mainly determined by the transcription rate of the Epo gene, which itself is essentially under control of the cellular oxygen concentration. Sinks of the oxygen concentrations increase the activity of the hypoxia-inducible transcription factor (HIF), which in turn triggers Epo gene transcription. Disorders of kidney function lead to inappropriate Epo production, what may result in anemia or polycythemia.

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Oxygen-dependent expression of the erythropoietin gene in rat hepatocytes in vitro

Cited by 30 publications

References 43 publications

Hypoxia-induced accumulation of erythropoietin mRNA in isolated hepatocytes is inhibited by protein kinase C

Hypoxia-induced accumulation of erythropoietin mRNA in isolated hepatocytes is inhibited by protein kinase C

Hypoxia signalling in the control of erythropoietin gene expression in rat hepatocytes

Erythropoietin

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