Thyroid hormone induced angiogenesis through the integrin αvβ3/protein kinase D/histone deacetylase 5 signaling pathway
- Key Laboratory of Hormones and Development (Ministry of Health), Metabolic Diseases Hospital and Tianjin Institute of Endocrinology, 2011 Collaborative Innovation Center of Tianjin for Medical Epigenetics, Tianjin Medical University, 300070 Tianjin, China
- Correspondence should be addressed to L Li; Email: lily{at}tijmu.edu.cn
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Figure 1Effects of T4 and Tetrac on migration of HUVECs in wound-healing assay. HUVECs were grown to 70% confluence in six-well plates. The monolayer was wounded and imaged immediately (0 h). Growth media containing a vehicle (PBS) or T4, Tetrac, or and Tetrac+T4 was added to every well. In the Tetrac+T4 group, HUVECs were pretreated with Tetrac for 30 min, then treated with T4. The concentration of T4 and Tetrac were both 100 nmol/l. Wound closure was recorded at 8, 12, and 24 h. The width of the wound was the average of nine determinations per time point. Distance of migration was calculated at each time point as described in Materials and methods. Phase-contrast images of the wounds were taken at ×100 magnification. The experiment was repeated three times and results of a representative experiment are shown. *P<0.05 when compared with the control group, #P<0.05 when compared with the T4 group at the same time.
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Figure 2
Effects of T4 on tubulogenesis of HUVECs. HUVECs were mixed with Matrigel (total 50 μl) for 30 min at 37 °C in an atmosphere of 5% CO2 to allow formation of the gel. This was followed by the addition of 250 μl HUVEC cell medium (5×103 cells per well) and incubation at 37 °C for 48 h. After washing with PBS, the cell medium was replaced with HUVECs medium mixed with thyroid hormone (T4: 100 nmol/l), which was replaced every 24 h for 48 h. The tube-like structures inside the wells were photographed. The total length and number of the capillary-like structures (indicated by arrows) that were formed within the Matrigel were measured. Compared with the control group, the number in the T4 group was greater (*,P<0.05 compared with the control group), and the length of tube-like structures (P>0.05) was longer, but the difference was not statistically significant.
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Figure 3
PKD and HDAC5 phosphorylation in HUVECs. (A) T4-stimulated PKD phosphorylation. HUVECs were starved without serum for 24 h and incubated with T4 (100 nmol/l) for 15, 30, and 45 min. Cell lysates were prepared and subjected to western blotting using a specific antibody. GAPDH was used as a loading control. Quantification of bands was done by densitometric analysis. The results are representative of three independent experiments. *P<0.05 when compared with the control group. (B) Tetrac inhibited PKD phosphorylation induced by T4. HUVECs were starved without serum for 24 h, then Tetrac (10−7, 10−6, and 10−5 mol/l) was added 30 min before T4 (100 nmol/l, 30 min) addition. The results are representative of three independent experiments.*P<0.05 when compared with the control group. #P<0.05 when compared with the T4 group. (C) PKC inhibitor (bisindolymaleimide I, Bis) inhibited PKD phosphorylation induced by T4. HUVECs were starved without serum for 24 h, and then they were pretreated with Bis (2.5 μmol/l) for 30 min and finally with T4 (100 nmol/l) for 15, 30, and 45 min. The results were representative of three independent experiments. *P<0.05 when compared with the control group. #P<0.05 when compared with the T4 group treated at the same time. (D) Effects of T4 and Tetrac on phosphorylation of HDAC5. HUVECs were starved without serum for 24 h, and then they were pretreated with Tetrac (10−7, 10−6, and 10−5 mol/l) for 30 min and finally with T4 (100 nmol/l) for 30 min. The results were representative of three independent experiments.*P<0.05 when compared with the control group, #P<0.05 when compared with the T4 group.
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Figure 4
Expression of phosphorylated HDAC5 in the cytoplasm. HUVECs were starved without serum for 24 h, and then they were pretreated with Tetrac (100 nmol/l) or Bis (2.5 μmol/l) for 30 min and finally with T4 (100 nmol/l) for 30 min. Cytoplasmic protein was extracted, and phosphorylation of HDAC5 was observed in the cytoplasm. GAPDH was used as a loading control. The results are representative of three independent experiments. *P<0.05 when compared with the control group, #P<0.05 when compared with the T4 group.
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Figure 5
Effects of PKD siRNA on phosphorylation of HDAC5 induced by T4. (A) Expression of PKD mRNA in HUVECs after transfection with PKD siRNA. HUVECs were starved without serum for 24 h. Transfection reagent with negative control siRNA (25 nmol/l) or one of the three PKD siRNAs (25 nmol/l) was added to cell culture medium for 6 h, then the transfection reagent was replaced with culture medium. HUVECs were cultured for another 24 h, and PKD and UBC cDNAs were isolated. The levels of PKD cDNA were corrected for variations in UBC cDNA. (B) Expression of PKD protein in HUVECs after transfection with PKD siRNA. HUVECs were transfected with a vehicle, negative siRNA (25 nmol/l) or PKD siRNA (25 nmol/l) for 6 h, and then the transfection reagent was removed. After culturing for 48 h, HUVECs were treated with T4 (100 nmol/l) for 15 min. β-actin was used as a loading control. *P<0.05 when compared with the control group. #P<0.05 when compared with the T4 group. (C) Phosphorylation of HDAC5 induced by T4 was inhibited by PKD siRNA. After HUVECs were transfected with a negative siRNA (25 nmol/l, control) or PKD siRNA (25 nmol/l), culture was continued for 48 h, and then cells were exposed to T4 (100 nmol/l) for 15 and 30 min. Representative immunoblot data are shown (n=3). *P<0.05 when compared with the control group, #P<0.05 when compared with the T4 group.
- © 2014 Society for Endocrinology
