Proteasomal Activity in Placentas from Women with Preeclampsia and Intrauterine Growth Restriction: Implications for Expression of HIF-α Proteins

Abstract

Hypoxia-inducible transcription factor-1α and -2α (HIF-α) proteins and regulated genes are increased in preeclamptic (PE) placentas. Although placental hypoxia likely stabilizes HIF-α proteins, we previously reported that there is also a defect in oxygen-dependent reduction of HIF-α 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-1α 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-1α 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-1α protein in PE placentas. Immunoprecipitation studies revealed comparable association of HIF-1α 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-α proteins. These results suggest that molecular events leading to the formation of the HIF-1α: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-α proteins. In conclusion, accumulation of HIF-α 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-α proteins, which have not been increased by an hypoxic environment.

Introduction

Inadequate trophoblast invasion leading to incomplete spiral artery remodeling and reduced uteroplacental perfusion is widely believed to underlie the human pregnancy disorder, preeclampsia, in many women who develop the disease (reviewed in refs. [1], [2]). Whether cause or consequence of preeclampsia, placental expression of hypoxia-inducible transcription factors-1α and -2α proteins, and target genes, fms-like tyrosine kinase 1 (FLT-1), soluble FLT-1 and tyrosine hydroxylase are significantly increased in preeclamptic placentas relative to normal-term and gestationally aged-matched control placentas [3], [4], [5]. Other downstream genes that are regulated by HIF-α are also increased in preeclamptic placentas [6], [7], [8], [9], [10], [11], [12], [13]. DNA microarray and suppressive-subtractive hybridization analyses reinforce the concept of a global increase in hypoxia-activated genes in preeclamptic placentas [14], [15]. Thus, upregulation of HIF-α in the preeclamptic placenta is likely to be critical to the over-expression of various factors that are secreted into the maternal (and possibly fetal) circulation, thereby disrupting the vascular endothelium leading to disease manifestations, e.g., sFLT-1 [9] and soluble endoglin [13].

A similar placental etiology has been proposed for many women with normotensive pregnancies complicated by idiopathic intrauterine growth restriction, i.e., Inadequate trophoblast invasion leading to incomplete spiral artery remodeling and reduced uteroplacental perfusion (IUGR; [1], [2]). However, we recently discovered that HIF-α proteins and target genes, FLT-1 and sFLT-1, are not significantly increased in placentas from pregnancies complicated by IUGR at >37 weeks of gestation (accounting for ∼90% of all IUGR cases [16]) despite the delivery of asymmetrically grown babies [4]. (To our knowledge, the status of HIF-α proteins and regulated genes in early onset IUGR is presently unknown.)

Although placental ischemia-hypoxia is likely to be a major cause of HIF-α protein stabilization in preeclampsia [17], impaired oxygen-dependent reduction of HIF-α proteins in preeclamptic placentas may also contribute as we previously reported [18]. Thus, the overarching goal of the present work was to elucidate potential mechanisms for reduced oxygen-dependent reduction of HIF-α proteins in preeclamptic placentas as normally HIF-α proteins are rapidly and virtually completely degraded in an oxygen environment. We first tested whether the impaired oxygen-dependent reduction of HIF-α proteins in placental villous explants from preeclamptic placentas is secondary to enhanced HIF-α protein synthesis or to impaired HIF-α protein degradation. To accomplish this objective, we took a pharmacological approach using cyclohexamide and lacto-clastocystin to inhibit protein synthesis and proteasomal degradation, respectively. Degradation of HIF-α protein is triggered by oxygen, which activates the prolyl hydroxylase enzymes (PHDs) that, in turn, hydroxylate proline residues 402 and 564 of HIF-α. These hydroxylated proline residues are requisite for recognition and binding of the chaperone, von Hippel Lindau protein, which is part of a larger complex of proteins that ubiquitinates HIF-α, thereby targeting it for proteasomal destruction (reviewed in refs. [19], [20]). Accordingly, we next investigated a pivotal molecular event leading to the ubiquitinylation of HIF-α, and consequently, its proteasomal destruction, i.e., the interaction between HIF-α and von Hippel Lindau protein. Finally, we measured placental proteasomal activities, in order to ascertain whether the proteasome itself is impaired in preeclamptic placentas, thereby contributing to reduced HIF-α degradation and consequent over-expression. Because HIF-α proteins are not significantly increased in placentas from pregnancies complicated by IUGR at >37 weeks of gestation (4), we also measured proteasomal activities in these placentas as another “control”, in addition to normal term placentas.