Mechanistic Target of Rapamycin Complex 1 Signaling Links Hypoxia to Increased IGFBP-1 Phosphorylation in Primary Human Decidualized Endometrial Stromal Cells

Abstract

Insulin-like growth factor-1 (IGF-1) bioavailability in pregnancy is governed by IGF binding protein (IGFBP-1) and its phosphorylation, which enhances the affinity of IGFBP-1 for the growth factor. The decidua is the predominant source of maternal IGFBP-1; however, the mechanisms regulating decidual IGFBP-1 secretion/phosphorylation are poorly understood. Using decidualized primary human endometrial stromal cells (HESCs) from first-trimester placenta, we tested the hypothesis that mTORC1 signaling mechanistically links hypoxia to decidual IGFBP-1 secretion/phosphorylation. Hypoxia inhibited mechanistic target of rapamycin (mTORC1) (p-P70-S6K/Thr389, −47%, p = 0.038; p-4E-BP1/Thr70, −55%, p = 0.012) and increased IGFBP-1 (total, +35%, p = 0.005; phosphorylated, Ser101/+82%, p = 0.018; Ser119/+88%, p = 0.039; Ser 169/+157%, p = 0.019). Targeted parallel reaction monitoring-mass spectrometry (PRM-MS) additionally demonstrated markedly increased dual IGFBP-1 phosphorylation (pSer98+Ser101; pSer169+Ser174) in hypoxia. IGFBP-1 hyperphosphorylation inhibited IGF-1 receptor autophosphorylation/ Tyr1135 (−29%, p = 0.002). Furthermore, silencing of tuberous sclerosis complex 2 (TSC2) activated mTORC1 (p-P70-S6K/Thr389, +68%, p = 0.038; p-4E-BP1/Thr70, +30%, p = 0.002) and reduced total/site-specific IGFBP-1 phosphorylation. Importantly, TSC2 siRNA prevented inhibition of mTORC1 and the increase in secretion/site-specific IGFBP-1 phosphorylation in hypoxia. PRM-MS indicated concomitant changes in protein kinase autophosphorylation (CK2/Tyr182; PKC/Thr497; PKC/Ser657). Overall, mTORC1 signaling mechanistically links hypoxia to IGFBP-1 secretion/phosphorylation in primary HESC, implicating decidual mTORC1 inhibition as a novel mechanism linking uteroplacental hypoxia to fetal growth restriction.

Increased Colocalization and Interaction Between Decidual Protein Kinase A and Insulin-like Growth Factor–Binding Protein-1 in Intrauterine Growth Restriction

“…To provide support for a functional interaction between decidual PKA and IGFBP-1, we performed IP of IGFBP-1 as in our recent study using primary decidualized HESC cultured in hypoxia or normoxia. 29 Using IP samples of decidualized primary HESC, we performed PRM-MS analysis to detect the presence and autophosphorylation status of PKA. PKA-specific peptides from the IGFBP-1 immunoprecipitates collected from cultured decidual HESC samples were successfully detected (Fig.…”

“…11 In addition, involvement of PKC together with CK2 was reported in decidualized primary HESC cultured under hypoxic conditions. 29 Decidual IGFBP-1 regulates the bioavailability of IGF-1 within the local environment of the placenta. 1 In the current study, we have not directly shown that PKA-mediated phosphorylation affects the affinity of IGFBP-1 to bind IGF-1 and hence IGF-1 bioavailability.…”

“…Isolation and culture of primary HESC from termination placenta and details of in vitro decidualization have been reported previously. 29 In brief, cells were decidualized and serum starved overnight and subjected to either hypoxia (1% O 2 ) or normoxia (incubator air) for 72 hr. To achieve hypoxia, cells were placed in a hypoxic chamber (Billups-Rothenburg Inc.; San Diego, CA) which was flushed with 1% O 2 , 5% CO 2 , and balanced N 2 gas mixture (Linde Canada Inc.; Mississauga, ON, Canada) for 15 min to ensure saturation.…”

“…Independent PRM-MS experiments (n=4) were performed using freshly extracted HESC from a pooled sample of n=3 placentas as described in detail previously. 29 In brief, decidualized primary HESC incubated under normoxia or hypoxia were immunoprecipitated (IP) with IGFBP-1 mouse monoclonal anti-human (mAb) 6303 antibody (Medix Biochemica; Kauniainen, Finland) using Protein A Sepharose beads (50 µl, 50% slurry; GE Healthcare Bio Sciences AB, Uppsala, Sweden) as described previously. 29 PRM-MS Analyses of IGFBP-1 Peptides and PKA Autophosphorylation.…”

“…Here, we applied targeted parallel reaction monitoring-mass spectrometry (PRM-MS), dual immunofluorescence (IF), and proximity ligation assay (PLA) approaches to determine PKA and IGFBP-1 interactions. We used fully decidualized primary human endometrial stromal cells (HESC) 29 and immortalized decidualized HESC [transformed HESC (T-HESC)] 22 as in vitro models. In addition, we used human decidua from IUGR and healthy pregnancies with normal fetal growth and decidual samples from our non-human baboon model of IUGR due to MNR at GD120 and GD165 (full term GD185).…”