Sarah A. Hale

Methylation-Controlled J Protein Promotes c-Jun Degradation To Prevent ABCB1 Transporter Expression

ABSTRACT Methylation-controlled J protein (MCJ) is a newly identified member of the DnaJ family of cochaperones. Hypermethylation-mediated transcriptional silencing of the MCJ gene has been associated with increased chemotherapeutic resistance in ovarian cancer. However, the biology and function of MCJ remain unknown. Here we show that MCJ is a type II transmembrane cochaperone localized in the Golgi network and present only in vertebrates. MCJ is expressed in drug-sensitive breast cancer cells but not in multidrug-resistant cells. The inhibition of MCJ expression increases resistance to specific drugs by inducing expression of the ABCB1 drug transporter that prevents intracellular drug accumulation. The induction of ABCB1 gene expression is mediated by increased levels of c-Jun due to an impaired degradation of this transcription factor in the absence of MCJ. Thus, MCJ is required in these cells to prevent c-Jun-mediated expression of ABCB1 and maintain drug response.

NEGATIVE REGULATION OF HIF-1α BY AN FBW7-MEDIATED DEGRADATION PATHWAY DURING HYPOXIA

Hypoxia inducible factor‐1α (HIF‐1α) stimulates expression of genes associated with angiogenesis and is associated with poor outcomes in ovarian and other cancers. In normoxia, HIF‐1α is ubiquitinated and degraded through the E3 ubiquitin ligase, von Hippel–Lindau; however, little is known about the regulation of HIF‐1α in hypoxic conditions. FBW7 is an E3 ubiquitin ligase that recognizes proteins phosphorylated by glycogen synthase kinase 3β (GSK3β) and targets them for destruction. This study used an ovarian cancer cell model to test the hypothesis that HIF‐1α phosphorylation by GSK3β in hypoxia leads to interaction with FBW7 and ubiquitin‐dependent degradation. Expression of constitutively active GSK3β reduced HIF‐1α protein and transcriptional activity and increased ubiquitination of HIF‐1α in hypoxia, whereas pharmacologic inhibition of GSK3 or expression of siGSK3β promoted HIF‐1α stabilization and activity. A mechanism through FBW7 was supported by the observed decrease in HIF‐1α stabilization when FBW7 was overexpressed and both the elevation of HIF‐1α levels and decrease in ubiquitinated HIF‐1α when FBW7 was suppressed. Furthermore, HIF‐1α associated with FBW7γ by co‐immunoprecipitation, and the interaction was weakened by inhibition of GSK3 or mutation of GSK3β phosphorylation sites. The relevance of this pathway to angiogenic signaling was supported by the finding that endothelial cell tube maturation was increased by conditioned media from hypoxic SK‐OV‐3 cell lines expressing suppressed GSK3β or FBW7. These data introduce a new mechanism for regulation of HIF‐1α during hypoxia that utilizes phosphorylation to target HIF‐1α for ubiquitin‐dependent degradation through FBW7 and may identify new targets in the regulation of angiogenesis. J. Cell. Biochem. 112: 3882–3890, 2011.

Pregnancy Induces Persistent Changes in Vascular Compliance in Primiparous Women

OBJECTIVE Pregnancy induces rapid, progressive, and substantial changes to the cardiovascular system. The low recurrence risk of preeclampsia, despite familial predisposition, suggests an adaptation associated with pregnancy that attenuates the risk for subsequent preeclampsia. We aimed to evaluate the persistent effect of pregnancy on maternal cardiovascular physiology. STUDY DESIGN Forty-five healthy nulliparous women underwent baseline cardiovascular assessment before conception and repeated an average of 30 months later. After baseline evaluation, 17 women conceived singleton pregnancies and all delivered at term. The remaining 28 women comprised the nonpregnant control group. We measured mean arterial blood pressure, cardiac output, plasma volume, pulse wave velocity, uterine blood flow, and flow-mediated vasodilation at each visit. RESULTS There was a significant decrease in mean arterial pressure from the prepregnancy visit to postpartum in women with an interval pregnancy (prepregnancy, 85.3±1.8; postpartum, 80.5±1.8 mm Hg), with no change in nonpregnant control subjects (visit 1, 80.3±1.4; visit 2, 82.8±1.4 mm Hg) (P=.002). Pulse wave velocity was significantly decreased in women with an interval pregnancy (prepregnancy, 2.73±0.05; postpartum, 2.49±0.05 m/s), as compared with those without an interval pregnancy (visit 1, 2.56±0.04; visit 2, 2.50±0.04 m/s) (P=.005). We did not observe a residual effect of pregnancy on cardiac output, plasma volume, uterine blood flow, or flow-mediated vasodilation. CONCLUSION Our observations of decreased mean arterial pressure and reduced arterial stiffness following pregnancy suggest a significant favorable effect of pregnancy on maternal cardiovascular remodeling. These findings may represent a mechanism by which preeclampsia risk is reduced in subsequent pregnancies.

Reduced NO signaling during pregnancy attenuates outward uterine artery remodeling by altering MMP expression and collagen and elastin deposition

Recent findings indicate that endothelial nitric oxide (NO) plays a key role in uterine artery outward circumferential remodeling during pregnancy. Although the underlying mechanisms are not known, they likely involve matrix metalloproteinases (MMPs). The goal of this study was to examine the linkage among NO inhibition, expansive remodeling, and MMP expression within the uterine vascular wall. Adult female rats were treated with N(G)-nitro-L-arginine methyl ester [L-NAME (LPLN)] beginning on day 10 of pregnancy and until death at day 20 and compared with age-matched controls [late pregnant (LP)]. Mean arterial pressure of LPLN rats was significantly higher than controls. LPLN fetal and placental weights were significantly reduced compared with controls. Main uterine arteries (mUA) were collected to determine dimensional properties (lumen area and wall thickness), collagen and elastin content, and levels of endothelial nitric oxide synthase (eNOS) and MMP expression. Circumferential remodeling was attenuated, as evidenced by significantly smaller lumen diameters. eNOS RNA and protein were significantly (>90%) decreased in the LPLN mUA compared with LP. Collagen and elastin contents were significantly increased in LPLN rats by ∼10 and 25%, respectively, compared with LP (P < 0.05). Both MMP-2 and tissue inhibitors of metalloproteinase-2 as assessed by immunofluorescence were lower in the endothelium (reduction of 60%) and adventitia (reduction of 50%) of LPLN compared with LP mUA. Membrane bound MMP-1 (MT1-MMP) as assessed by immunoblot was significantly decreased in LPLN. These data suggest a novel contribution of MMPs to gestational uterine vascular remodeling and substantiate the linkage between NO signaling and gestational remodeling of the uterine circulation via altered MMP, TIMP-2, and MT1-MMP expression and activity.

Sildenafil Increases Uterine Blood Flow in Nonpregnant Nulliparous Women

This study investigated the effect of sildenafil on uterine volumetric blood flow (UVF) and vascular impedance in nonpregnant, nulliparous women. Fifteen women were randomized in a double-blind fashion to receive either placebo or sildenafil (25 or 100 mg) during the luteal phase of the menstrual cycle. Color Doppler ultrasound of both uterine arteries was performed at baseline and at 1 and 3 hours postdosing to calculate resistance index (RI) and UVF. Those who received sildenafil significantly increased UVF and decreased RI over the 3-hour monitoring period. When UVF responses to sildenafil were examined as a function of baseline UVF, a significant increase in UVF was observed in only those participants with higher baseline UVF. Overall, women in the luteal phase demonstrated a significant increase in UVF in response to sildenafil. However, this increase appears to be directly associated with basal UVF.

Relationship between Prepregnancy and Early Pregnancy Uterine Blood Flow and Resistance Index

We evaluated the relationship between prepregnancy and early pregnancy uterine blood flow (UBF) and resistance index (RI). Nineteen nulliparous participants were studied during cycle day 8 + 4, and early pregnancy (13.4 + 1.6 weeks). Color Doppler ultrasound of both uterine arteries and maternal heart was performed to calculate uterine RI, volumetric UBF, and cardiac output (CO), respectively. We observed a strong negative association of uterine RI with prepregnancy UBF (r = —.82, P < .001) that weakened, but remained significant in early pregnancy (r =—.48, P = .04). Prepregnancy uterine index (UBF/CO) was significantly associated with early pregnancy uterine index; r = .48, P = .04). There was also a trend associating prepregnancy and early pregnancy volumetric UBF (r = .44, P = .068). Prepregnancy UBF may be a determinant of early pregnancy UBF and UBF may have independent value as a predictor of adverse pregnancy outcome.

Pulse Pressure and Arterial Compliance Prior to Pregnancy and the Development of Complicated Hypertension during Pregnancy

We examined the relationship between prepregnant pulse pressure (PP), mean arterial pressure (MAP), cardiac output (CO)/PP, a measure of arterial compliance, and development of complicated hypertension (CH) during pregnancy with the goal of identifying a potential predictor of CH. Twenty nulliparous participants were studied before pregnancy; 17 had normal pregnancies (control; CTL) and 3 CH. Blood pressure monitoring was performed using tonometry. Cardiac output was determined by Doppler echocardiograph. Data are expressed as mean ± SD. Prepregnant PP was significantly higher in CH participants (CH: 58.3 ± 6.3, CTL: 46.2 ± 1.7 mm Hg; P = .02). Cardiac output /pulse pressure was significantly lower in CH participants (CH: 6.9 ± 1.8, CTL: 10.6 ± 2.8; P = .04). Mean arterial pressure was not significantly different. Increased PP before pregnancy may suggest increased risk for CH. With accurate prediction of CH before pregnancy, initiation of preventative measures could begin earlier, either prior to or in early pregnancy, potentially increasing preventative efficacy and decreasing CH.

The relationship of a family history for hypertension, myocardial infarction, or stroke with cardiovascular physiology in young women.

Cardiovascular disease (CVD) and preeclampsia share several pathophysiologic risk factors. We examined family history (FH) and physiologic status in 60 healthy, nulliparous women to determine the relationship between FH and known risk factors for CVD. Data are presented as mean ± standard error (SE). Decreased uterine blood flow was observed in women with FH of hypertension (+FH: 21.5 ± 1.7, no FH: 33.3 ± 9.0 mL/min; P = .04). Women reporting an FH of stroke showed increased alpha- and beta-adrenergic response, as measured by Valsalva maneuver (α: FH: 24.7 ± 1.9, −FH: 18.9 ± 1.1 mm Hg, P = .02; β: FH: 22.0 ± 2.1, −FH: 16.9 ± 1.4 mm Hg; P = .04), and increased cardiac output (4.83 ± 0.22 vs 4.31 ± 0.12 L/min; P = .01). We identified no significant physiologic associations linked to an FH of myocardial infarction. Our observations show significant differences in physiologic characteristics in women with specific CVD family histories. These data, coupled with known heritable contributions to CVD and preeclampsia, suggest a distinct physiologic phenotype that may link preeclampsia risk with FH of CVD, independent of pregnancy.

Differences in cardiovascular function comparing prior preeclamptics with nulliparous controls

OBJECTIVE The objective of the current study was to evaluate cardiovascular function; including blood pressure, cardiac output, pulse wave velocity and vascular compliance in nonpregnant nulliparous women compared to women with a history of preterm preeclampsia. STUDY DESIGN This was a case control study. Blood pressure was measured using the Finapres Pro. Baseline cardiac output was determined by echocardiography. Pulse wave velocity was estimated using simultaneous electrocardiographic tracings and ultrasound determined arterial flow waveforms and calculated as estimated distance divided by the time interval between EKG r-wave peak and ultrasound derived peak popliteal artery flow. During volume challenge, 500mL of lactated Ringers solution was infused through an indwelling antecubital catheter over 10min. Cardiac output and blood pressure during and 15min after the infusion were estimated using the Finapres Pro. MAIN OUTCOME MEASURES Indices of arterial stiffness and vascular compliance. RESULTS Previous preeclamptics exhibited a significant increase in pulse pressure and cardiac output in response to volume challenge when compared with nulliparous controls. Prior preeclamptics had a strong positive correlation between blood pressure indices (r=0.50-0.68, p⩽0.01) and pulse pressure (r=0.58, P=0.008) with pulse wave velocity that was not evident in control women. CONCLUSIONS In women with prior preterm preeclampsia a relationship between blood pressure, intravascular volume and arterial stiffness, is evident in the nonpregnant state and in the absence of hypertension or overt cardiovascular disease. This supports an overarching hypothesis that nonpregnant physiology is an important contributor to pregnancy adaptations.

Abstract 3092: FBW7 targets hypoxia inducible factor-1α (HIF-1α) for proteasomal degradation during hypoxia

Hypoxia Inducible Factor-1α (HIF-1α) mediates expression of genes associated with endothelial cell-mediated angiogenesis and is associated with poor outcomes in a variety of cancers. In normoxia, HIF-1α is ubiquitinated and degraded through interactions with the E3 ubiqutin ligase, von Hippel-Lindau (vHL); however, little is known about the negative regulation of HIF-1α in hypoxia. FBW7, an E3 ubiquitin ligase, has been shown to interact with several transcription factors including those phosphorylated by glycogen synthase kinase 3β (GSK3β). The current study tested the hypothesis that phosphorylation of HIF-1α by GSK3β increases the FBW7-mediated ubiquitination and degradation of HIF-1α, thereby resulting in suppression of the hypoxia-mediated angiogenic response in SKOV-3 ovarian cancer cells. HIF-1α protein and VEGF transcript levels in hypoxia were increased when GSK3β activity was inhibited and were reduced by expression of constitutively active GSK3β (GSK3S9A). Additionally, expression of GSK3S9A increased HIF-1α ubiquitination. Conditioned media from SKOV-3 cells expressing shRNA against GSK3β (shGSK3) had an enhanced effect on endothelial tube formation in a Matrigel matrix, suggesting that GSK3β exerts an inhibitory effect on hypoxia-mediated angiogenesis. Overexpression of the β and γ isoforms of FBW7 decreased HIF-1α stabilization, and HIF-1α interacts with FBW7 by co-immunoprecipitation. Furthermore, knockdown of FBW7 using siRNA resulted in increased HIF-1α levels in hypoxia. These data suggest a new mechanism for negative regulation of HIF-1α during hypoxia that utilizes phosphorylation by GSK3β and interaction with FBW7 leading to ubiquitination and proteasomal degradation. Results of this study better define the signaling pathways necessary for HIF-1α-mediated signaling and may identify new targets that mediate angiogenesis in disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3092. doi:10.1158/1538-7445.AM2011-3092