PMC

Protein phosphorylation in endothelial cells treated with HKE₂. HUVEC were treated with (A) 500 nM of HKE₂ for 0 to 120 min or (B) 0 to 500 nM of HKE₂ for 30 min, and cellular protein was analyzed by SDS-PAGE and immunoblotting using antibodies against phospho-tyrosine (α-pTyr) and β-actin. Red triangles indicate protein bands that showed increased phosphorylation in response to treatment with HKE₂ compared to vehicle control. C, a human phospho-Receptor Tyrosine Kinase (RTK) array was used to analyze HKE₂-induced phosphorylation of 49 RTKs in HUVEC lysates. X-ray film images of the membranes treated with vehicle and 50, 100, and 500 nM HKE₂ are shown. Protein spots that showed differences in intensity between HKE₂ and vehicle control are boxed. D, quantification of the RTK array results using ImageJ software. The five RTK boxed in panel (C) are marked with a black triangle. The mean of two independent repeats of the RTK array using 0 to 500 nM HKE₂ is shown. HUVEC, human umbilical vein endothelial cell; RTK, receptor tyrosine kinase.

Isolated human leukocytes form HKE₂ and HKD₂. Human leukocytes were stimulated with LPS for 6 h and then with A23187 for 15 min before extraction and LC-ESI-MS-SRM analysis. (A) Standards of HKE₂ (red trace) and HKD₂ (blue) generated by reaction of recombinant human COX-2 with 5S-HETE (black). (B) HKE₂ and HKD₂ formed by the leukocytes following stimulation with LPS and A23187. (C) Addition of exogenous 5S-HETE together with A23187 to the leukocytes increased formation of HKE₂ and HKD₂. (D) Formation of hemiketals was attenuated by preincubation with the COX-2 inhibitor NS-398.

HKD₂ and HKE₂ induce formation of capillary-like structures and migration of endothelial cells. (A) Microvascular endothelial cells were plated onto Matrigel in the presence of 125 or 500 nM of HKD₂ or HKE₂. Representative images of capillary-like structures taken 6 h after plating are shown. (B) Quantification of capillary network formation. Cells incubated with HKD₂ or HKE₂ form tubule-like structures more efficiently than vehicle-treated cells (control). Values are the mean ± SD. calculated for 20 images per treatment. Differences between vehicle versus HKD₂ or HKE₂ treated cells (*), or between 125 versus 500 nM treatment (**) were significant (p < 0.05). (C) Migration of cells toward serum-free medium with or without HKD₂ or HKE₂ or complete medium. Values are fold changes versus serum-free medium from four experiments; (*) p < 0.05 versus control.

Adduction and inhibition of PTP1B by HKE₂.A, recombinant PTP1B (0.5 μM) was treated with HKE₂ or N-ethyl maleimide (NEM) at 5 or 45 μM, respectively, followed by the detection of free remaining cysteine residues with biotin-maleimide and analysis by SDS-PAGE. The image shows Western blot detection using HRP-conjugated avidin (top) or an antibody against PTP1B (bottom). B, the graph shows the ratio of unmodified PTP1B (top panel in A) to total PTP1B (bottom panel in A) from n = 3 independent repeats. Data are presented as means ± S.D. (∗) indicates significant differences (∗∗∗∗p < 0.0001) between control and HKE₂ or NEM-treated samples. C, recombinant PTP1B (0.5 μM) was treated with HKE₂ or N-ethyl maleimide (NEM) at 5 or 45 μM, respectively, followed by the detection of free remaining cysteine residues with PEG-PC-Maleimide and analysis by SDS-PAGE with Coomassie staining. D, the graph shows the relative abundance of shifted bands (band 1–6) and the original PTP1B band for each treatment from n = 3 independent repeats. E, phosphatase activity of PTP1B (1 μM) using pNPP as a substrate from n = 3 independent repeats. Data are presented as means ± S.D. (∗) indicates significant differences (∗∗∗∗p < 0.0001) between control and HKE₂ or NEM-treated samples. F, illustration of the Michael-type addition of HKE₂ to a reactive cysteine residue of PTP1B. NEM, N-ethyl-maleimide; PTP1B, protein tyrosine phosphatase 1B.