Altered protein expression in gestational diabetes mellitus placentas provides insight into insulin resistance and coagulation/fibrinolysis pathways

Abstract

Objective: To investigate the placental proteome differences between pregnant women complicated with gestational diabetes mellitus (GDM) and those with normal glucose tolerance (NGT).

Methods: We used two-dimensional electrophoresis (2DE) to separate and compare placental protein levels from GDM and NGT groups. Differentially expressed proteins between the two groups were identified by MALDI-TOF/TOF mass spectrometry and further confirmed by Western blotting. The mRNA levels of related proteins were measured by realtime RT-PCR. Immunohistochemistry (IHC) was performed to examine the cellular location of the proteins expressed in placenta villi.

Results: Twenty-one protein spots were differentially expressed between GDM and NGT placenta villi in the tested samples, fifteen of which were successfully identified by mass spectrometry. The molecular functions of these differentially expressed proteins include blood coagulation, signal transduction, anti-apoptosis, ATP binding, phospholipid binding, calcium ion binding, platelet activation, and tryptophan-tRNA ligase activity. Both protein and mRNA levels of Annexin A2, Annexin A5 and 14-3-3 protein ζ/δ were up-regulated, while the expression of the Ras-related protein Rap1A was down-regulated in the GDM placenta group.

Conclusion: Placenta villi derived from GDM pregnant women exhibit significant proteome differences compared to those of NGT mothers. The identified differentially expressed proteins are mainly associated with the development of insulin resistance, transplacental transportation of glucose, hyperglucose-mediated coagulation and fibrinolysis disorders in the GDM placenta villi.

Figure 1. Representative 2DE gel images of placenta villi samples from gestational diabetes mellitus pregnancy and normal glucose tolerance pregnancy.

(A) Gestational diabetes mellitus pregnancy (GDM) Group. (B) Normal glucose tolerance pregnancy (NGT) group. The differentially expressed protein spots are circled on the gels and labeled with unique spot numbers. The spot number on the gels corresponds to the numbers in Table 2. White circles indicate up-regulated proteins in the gels analyzed using ImageMaster Software, while black circles indicate down-regulated proteins.

Figure 2. Identification of Annexin A2 by MALDI TOF/TOF mass spectrometry.

(A) MS/MS spectra of Annexin A2. (B) Amino acid sequence of Annexin A2. Underlined bold text indicates matched peptide sequences.

Figure 3. Protein and mRNA expression of Annexin A2, Annexin A4, Annexin A5, 14-3-3 ζ/δ, and Ras related protein Rap1A in placenta villi from GDM and NGT (Control) groups.

(A) A representative image of Western blots for Annexin A2, Annexin A4, Annexin A5, 14-3-3 ζ/δ, and Ras related protein Rap1A in placenta villi from each group. β-actin was used to verify equivalent loading. (B) Graphic representation of relative abundance of Annexin A2, Annexin A4, Annexin A5, 14-3-3 ζ/δ, and Ras related protein Rap1A normalized to β-actin. Data was given as mean ± S.E.M values of ten samples from each group. * P<0.05 by independent student’s t-test between GDM and NGT (Control) group. (C) Realtime RT-PCR analysis of relative mRNA levels of the genes from the five identified proteins in placenta villi from GDM and NGT (Control) groups. Bar graph shows mean ± S.E.M of 15 samples from each group with three repeats. ANXA2, ANXA4, ANXA5, YWHAZ, and Ras related protein Rap1A indicate the gene names of Annexin A2, Annexin A4, Annexin A5, 14-3-3 ζ/δ, and Ras related protein Rap1A, respectively.

Figure 4. Immunohistochemistry analysis of Annexin A2, Annexin A4, Annexin A5, 14-3-3 ζ/δ, and Ras related protein Rap1A in placenta villi from GDM and NGT (Control) groups.

Annexin A2, Annexin A4, Annexin A5, 14-3-3 ζ/δ and Ras related protein Rap1A are mainly expressed in trophoblast cells of placenta villi. Magnification: 200x Arrows: indicate location of each protein. Bar: 50 µm.

Figure 5. Differentially expressed proteins and insulin resistance.

The insulin signal pathway has been previously described by Pessin (Ref. 15). When stimulated by insulin, the insulin receptor activates insulin receptor substrates (IRS), PI3K, AKT and GLUT4 sequentially, leading to the localization of GLUT4 to the cell membrane for transfer of glucose. 1. Insulin stimulation activates Annexin A2 (Ref. 20) resulting in internalization of the insulin receptor by endosome formation (Ref. 17–20). 2.14-3-3 proteins compete with IR for IRS binding (Ref. 21). 3.14-3-3 proteins decrease PI3K activity (Ref. 22). 4.14-3-3 proteins bind and translocate the IRS/PI3K complex (Ref. 23).

Figure 6. Differentially expressed proteins involved in coagulation and fibrinolysis.

(A) Annexin A5 inhibits coagulation. The coagulation activation process in this panel has been previously described in Harrison’s Principles of Internal Medicine (ref. 27 ). 1. Annexin A5 inhibits thromboplastin (Ref. 29). 2. Annexin A5 competes with factor VIII for phosphatidylserine (PS) binding sites (Ref. 28). 3. Annexin A5 blocks the binding of factor X to thrombin-stimulated platelets (Ref. 30). (B) Ras related protein Rap1A and Annexin A2 on fibrinolysis. The fibrinolysis pathway in this panel has been previously described in References 33∼38. 4. Ras related protein Rap1A inhibits the binding of Ras and Raf in a competitive manner (Ref. 32). Down-regulated Rap1A in GDM placenta villi may decrease the inhibition of the Ras signal pathway, thereby increasing PAI-1 levels. 5. Annexin A2 increases plasmin levels by binding to tissue plasminogen activator (t-PA) and plasminogen(Ref. 39).