Maternal hyperglycemia inhibits pulmonary vasculogenesis during mouse fetal lung development by promoting GβL Ubiquitination-dependent mammalian target of Rapamycin assembly

Abstract

Background: Gestational diabetes mellitus (GDM) is associated with retarded lung development and poor lung health in offspring. Mammalian target of rapamycin (mTOR) is a key regulator of vasculogenesis and angiogenesis. The aim of this study was to investigate the role mTOR plays in pulmonary vasculogenesis during fetal lung development under maternal hyperglycemia.

Methods: First, GDM was induced via streptozotocin injection in pregnant C57BL/6 mice before the radial alveolar count (RAC) in the fetal lungs was assessed using hematoxylin and eosin staining. The angiogenic ability of the cultured primary mouse fetal lung endothelial cells (MFLECs) was then assessed using the tube formation assay technique, while western blot and real-time polymerase chain reaction were performed to determine the expression of mTOR, regulatory-associated protein of mTOR (Raptor), rapamycin-insensitive companion of mTOR (Rictor), stress-activated protein kinase interacting protein 1 (Sin1), G protein beta subunit-like protein (GβL), Akt, tumor necrosis receptor associated factor-2 (TRAF2), and OTU deubiquitinase 7B (OTUD7B) in both the fetal lung tissues and the cultured MFLECs. Immunoprecipitation assays were conducted to evaluate the status of GβL-ubiquitination and the association between GβL and mTOR, Raptor, Rictor, and Sin1 in the cultured MFLECs.

Results: The GDM fetal lungs exhibited a decreased RAC and reduced expression of von Willebrand factor, CD31, and microvessel density. The high glucose level reduced the tube formation ability in the MFLECs, with the mTOR, p-mTOR, p-Raptor, and TRAF2 expression upregulated and the p-Rictor, p-Sin1, p-Akt, and OTUD7B expression downregulated in both the GDM fetal lungs and the high-glucose-treated MFLECs. Meanwhile, GβL-ubiquitination was upregulated in the high-glucose-treated MFLECs along with an increased GβL/Raptor association and decreased GβL/Rictor and GβL/Sin1 association. Furthermore, TRAF2 knockdown inhibited the high-glucose-induced GβL-ubiquitination and GβL/Raptor association and restored the tube formation ability of the MFLECs.

Conclusion: Maternal hyperglycemia inhibits pulmonary vasculogenesis during fetal lung development by promoting GβL-ubiquitination-dependent mTORC1 assembly.

Keywords: Gestational diabetes mellitus; Pulmonary development; Ubiquitination; mTORC1; mTORC2.

Fig. 1

The GDM fetal lungs exhibiting deficient alveolar and vascular development. A The RAC determined via H&E staining of the control and GDM fetal lung tissues. B The IHC staining of the control and GDM fetal lung tissues for vWF expression. C The IF staining of the control and GDM fetal lung tissues for CD31 expression. D The MVD evaluation in terms of the GDM and control groups (n = 6 in each group, *p < 0.05, **p < 0.001)

Fig. 2

Upregulation of mTORC1 and downregulation of mTORC2 in the GDM fetal lungs. A The protein expression of TRAF2, OTUD7B, p-mTOR, mTOR, and GβL in the control and GDM fetal lung tissues. B The determined protein levels of p-Sin1, Sin1, p-Rictor, Rictor, p-Raptor, Raptor, p-Akt, and Akt in the control and GDM fetal lung tissues. C The relative protein levels of TRAF2, OTUD7B, p-mTOR, mTOR, and GL in the control and GDM fetal lung tissues. D The relative protein levels of p-Sin1, Sin1, p-Rictor, Rictor, p-Raptor, Raptor, p-Akt, and Akt in the control and GDM fetal lung tissues (n = 3 in triplicate, **p < 0.01, ***p < 0.001)

Fig. 3

High-glucose-inhibited MFLEC angiogenesis through mTOR pathway in vitro. The MFLECs were pre-incubated in a culture medium containing 5 mM glucose (control) or 30 mM glucose (high glucose) or 30 mM glucose with 100ng/ml rapamycin (HG + RAPA) for 72 h and loaded into Matrigel. The cells treated with high glucose levels exhibited a dramatic decrease in the total number of branch points while the number was almost restored to control level in HG + RAPA group. (HG: high glucose, RAPA: rapamycin, n = 3 in triplicate, ***p < 0.001, * p < 0.05)

Fig. 4

High-glucose-stimulated GβL-ubiquitination and mTORC1 assembly in the cultured MFLECs. A The protein levels of TRAF2, OTUD7B, p-mTOR, mTOR, and GβL. B The protein levels of p-Sin1, Sin1, p-Rictor, Rictor, p-Raptor, Raptor, p-Akt and Akt. C The cell lysates were incubated with anti-GβL antibody-coated beads. The mTOR, Raptor, Rictor, Sin1, and ubiquitin levels in the precipitates were determined via western blot analysis (n = 3 in triplicate, **p < 0.01, ***p < 0.001)

Fig. 5

The TRAF2 knockdown restored the MFLEC angiogenesis impaired by the high glucose. A Expression level of TRAF2 protein. B Expression level of TRAF2 mRNA. C The association between GβL and mTOR, Raptor, Rictor, Sin1, and ubiquitin was evaluated using the immunoprecipitation assay. D The cell angiogenesis ability under high-glucose conditions (HG: high glucose, n = 3 in triplicate, ***p < 0.001)

Fig. 6

The proposed mechanism of hyperglycemia inhibits pulmonary vasculogenesis. Maternal hyperglycemia can up-regulate the expression level of TRAF2 in mouse fetal lung, which acts as an E3 ligase in the regulation of mTORC1 and mTORC2 formation. By promoting the polyubiquitination of GβL, up-regulated TRAF2interferes with the interaction of GβL with SIN1 (a unique component of mTORC2), which promotes the formation of mTORC1, thus inhibiting mouse fetal lung angiogenesis.↑represents the up-regulated expression levels of TRAF2, GβL-ubiquitination, and mTORC1 assembly;↓represents down-regulation of mTORC2 assembly; indicates promotion of the events; ⊥ indicates inhibition of the events.