Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Biochemistry

Supervisor

Gupta, Madhulika B

Affiliation

Children's Health Research Institute

Abstract

Fetal growth restriction (FGR) is associated with decreased nutrient availability and reduced insulin-like growth factor (IGF)-I bioavailability via increased IGF binding protein (IGFBP)-1 phosphorylation. While protein kinase C (PKC) is implicated in IGFBP-1 hyperphosphorylation in nutrient deprivation, the mechanisms remain unclear. I hypothesized that the interaction of PKCα with protein kinase CK2β and activation of PKCα under leucine deprivation (L0) mediate fetal hepatic IGFBP-1 hyperphosphorylation. Parallel Reaction Monitoring Mass Spectrometry (PRM-MS) followed by PKCα knockdown demonstrated the PKCα isoform interacts with IGFBP-1 and CK2β under L0. Pharmacological PKCα activation with phorbol 12-myristate 13-acetate (PMA) increased whereas inhibition with bisindolylmaleimide II (Bis II) decreased IGFBP-1 phosphorylation (Ser101/119/169, Ser98+101 and Ser169+174), respectively. Furthermore, PMA mimicked L0-induced PKCα translocation and IGFBP-1 expression. PKCα expression was increased in baboon fetal liver in FGR, providing biological relevance in vivo. In summary, I report a novel nutrient-sensitive mechanism for PKCα in mediating IGFBP-1 hyperphosphorylation in FGR.

Summary for Lay Audience

Fetal growth restriction (FGR) affects approximately 5-10% of all pregnancies and occurs when the fetus fails to reach its growth potential in the uterus. The most common cause of FGR is placental insufficiency, a pathological condition where the placenta fails to adequately supply oxygen and nutrients to the developing fetus. FGR is the second most common cause of perinatal mortality and is responsible for 30% of stillbirths. Additionally, FGR may negatively affect physical and neurological development of the infant resulting in increased risk of metabolic, cardiac and neurological disorders which may not resolve in adulthood.

Fetal growth is primarily mediated by fetal liver secreted insulin-like growth factor I (IGF-I), a peptide hormone which is bound by insulin-like growth factor binding proteins (IGFBPs) which extends the half-life of IGF-I in circulation and mediates its functions. IGFBP-1 is additionally secreted by the fetal liver and is predominantly expressed during pregnancy where it serves its primary role of binding to and inhibiting IGF-I regulated fetal growth. Phosphorylation of IGFBP-1 increases its binding affinity for IGF-I resulting in greater inhibition of IGF-I mediated actions. In FGR, IGFBP-1 phosphorylation is increased and IGF-I bioavailability is reduced which are associated with decreased fetal nutrient provision. We have previously utilized the hepatoblastoma derived HepG2 cell line deprived of leucine as a model for the nutritionally deprived fetal liver to induce IGFBP-1 phosphorylation. However, the roles of the kinases which mediate the phosphorylation of IGFBP-1 in this model have not yet been elucidated.

In this study, we hypothesized that leucine deprivation (L0) activates protein kinase C (PKC) in a nutrient sensitive manner and induces its interaction with the putative IGFBP-1 kinase, protein kinase CK2 which results in increased IGFBP-1 phosphorylation. I found that PKC interacts with IGFBP-1 and CK2 in L0 and that PKC is activated in L0. Additionally, I demonstrate that PKC mediates IGFBP-1 phosphorylation in L0. To establish translational relevance, I further demonstrated global maternal nutrient restriction induces PKC expression within the fetal liver which is associated with increased IGFBP-1 phosphorylation.

Download

Included in

Biochemistry Commons

Share

COinS