Proteasomal activity in placentas from women with preeclampsia and intrauterine growth restriction: implications for expression of HIF-alpha proteins.
Hypoxia-inducible transcription factor-1alpha and -2alpha (HIF-alpha) proteins and regulated genes are increased in preeclamptic (PE) placentas. Although placental hypoxia likely stabilizes HIF-alpha proteins, we previously reported that there is also a defect in oxygen-dependent reduction of HIF-alpha proteins in PE relative to normal pregnant (NP) placentas that could contribute to their over-expression. After a 4-h exposure to 2% oxygen, placental villous explants were exposed to 21% oxygen over 90 min. As assessed by Western analysis, the defective oxygen-dependent reduction of HIF-1alpha protein in villous explants from PE placenta was unaffected by the protein synthesis inhibitor, cycloheximide. However, after incubation with the proteasomal inhibitor, clasto-lactacystin, oxygen-dependent reduction of HIF-1alpha protein was markedly and similarly impaired in the villous explants from both normal and PE placentas. Thus, impairment of protein degradation rather than increased synthesis causes inadequate oxygen-dependent reduction of HIF-1alpha protein in PE placentas. Immunoprecipitation studies revealed comparable association of HIF-1alpha with von Hippel Lindau (VHL) protein in placentas from NP and PE women. Furthermore, prolyl hydroxylase-3 protein was appropriately upregulated in the PE placentas as determined by Western analysis paralleling the increases of HIF-alpha proteins. These results suggest that molecular events leading to the formation of the HIF-1alpha:VHL:ubiquitin ligase complex are most likely not impaired in PE placentas. Finally, proteasomal trypsin, chymotrypsin, and peptidyl glutamyl-like activities were significantly reduced by approximately 1/3 in PE placentas by using specific peptide substrates coupled to a fluorescent tag. Unexpectedly, however, they were even further decreased in placentas from normotensive women delivering growth restricted babies >37 weeks gestation-placentas which do not have elevated HIF-alpha proteins. In conclusion, accumulation of HIF-alpha proteins in PE placentas may occur as a consequence of both increased formation secondary to relative ischemia/hypoxia and reduced degradation after reperfusion/oxygenation consequent to proteasomal dysfunction. In contrast, in placentas from normotensive women delivering growth restricted babies >37 weeks gestation, proteasomal activity, albeit markedly reduced, is adequate to cope with degradation of HIF-alpha proteins, which have not been increased by an hypoxic environment.
Rajakumar
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Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine and Magee Women's Research Institute and Foundation, Pittsburgh, PA 15213, USA.
Michael
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HM
Daftary
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Jeyabalan
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A
Gilmour
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Conrad
K P
KP
D000328:Adult; D001724:Birth Weight; D015687:Cell Hypoxia / Q000502:physiology; D002478:Cells, Cultured; D002824:Chorionic Villi / Q000378:metabolism / Q000473:pathology; D005260:Female; D005317:Fetal Growth Retardation / Q000378:metabolism* / Q000473:pathology; D005865:Gestational Age; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D007231:Infant, Newborn; D009924:Organ Culture Techniques; D010101:Oxygen Consumption / Q000502:physiology; D010920:Placenta / Q000378:metabolism*; D011225:Pre-Eclampsia / Q000378:metabolism* / Q000473:pathology; D011247:Pregnancy; D011393:Procollagen-Proline Dioxygenase / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011499:Protein Processing, Post-Translational
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D011393:Procollagen-Proline Dioxygenase; C030606:proline, 2-oxoglutarate 3-dioxygenase; D046988:Proteasome Endopeptidase Complex