Isoprenylcysteine carboxyl methyltransferase activity modulates endothelial cell apoptosis

Abstract

Extracellular ATP, adenosine (Ado), and adenosine plus homocysteine (Ado/HC) cause apoptosis of cultured pulmonary artery endothelial cells through the enhanced formation of intracellular S-adenosylhomocysteine and disruption of focal adhesion complexes. Because an increased intracellular ratio of S-adenosylhomocysteine/S-adenosylmethionine favors inhibition of methylation, we hypothesized that Ado/HC might act by inhibition of isoprenylcysteine-O-carboxyl methyltransferase (ICMT). We found that N-acetyl-S-geranylgeranyl-L-cysteine (AGGC) and N-acetyl-S-farnesyl-L-cysteine (AFC), which inhibit ICMT by competing with endogenous substrates for methylation, caused apoptosis. Transient overexpression of ICMT inhibited apoptosis caused by Ado/HC, UV light exposure, or tumor necrosis factor-alpha. Because the small GTPase, Ras, is a substrate for ICMT and may modulate apoptosis, we also hypothesized that inhibition of ICMT with Ado/HC or AGGC might cause endothelial apoptosis by altering Ras activation. We found that ICMT inhibition decreased Ras methylation and activity and the activation of the downstream signaling molecules Akt, ERK-1, and ERK-2. Furthermore, overexpression of wild-type or dominant active H-Ras blocked Ado/HC-induced apoptosis. These findings suggest that inhibition of ICMT causes endothelial cell apoptosis by attenuation of Ras GTPase methylation and activation and its downstream antiapoptotic signaling pathway.

Figure 1

Schematic representation of the intracellular pathways involved in adenosine metabolism. Dashed line represents product inhibition of SAM-dependent methyltransferases.

Figure 2

Endothelial cell apoptosis is induced by ICMT inhibition. PAECs were incubated with buffer in the absence (control) or presence of 20 μM AGC, 20 μM AGGC, or 20 μM AFC (a) or in the absence (control) or presence of 50 μM FTA or 10 μM FTS (b) for 4 h. Cells were visualized by fluorescence microscopy and scored for change in nuclear morphology consistent with apoptosis. c, Asp-Glu-Val-Asp-ase (DEVDase) activity was determined in equivalent quantities of PAEC lysate treated with buffer (control), 100 μM Ado/HC, 20 μM AGC, or 20 μM AGGC for varying times. All data are expressed as mean ± SEM. n = 4. *p < 0.05 compared with control.

Figure 3

Overexpression of ICMT-GFP in endothelial cells. PAECs transiently overexpressing pGFP-C1 (a) or pICMT-GFP (b) cDNAs were visualized by fluorescence microscopy. In parallel, protein overexpression was confirmed by immunoblot analysis for GFP (c), and overexpression of a functional enzyme was confirmed by assaying for methyltransferase (ICMT) activity (d). Data in d are expressed as mean ± SEM. n = 3. *p < 0.05 compared with untransfected cultures or cultures transfected with pGFP-C1.

Figure 4

ICMT overexpression protects against endothelial cell apoptosis. PAECs were transfected with pGFP-C1 or pICMT-GFP and incubated with: a, buffer (control) or 100 μM Ado/HC; b, exposed to UV light for indicated time; or c, serum-free MEM (control) or 20 ng/ml TNF-α and incubated for 20 h at 37°C. The cells were then fixed and stained with Hoechst. Cells were visualized by fluorescence microscopy and scored for change in nuclear morphology consistent with apoptosis. Data are expressed as mean ± SEM. n = 3. *p < 0.05 compared with control or 0 min UV light treatment (in a, c, or b, respectively). **p < 0.05 compared with pGFP-C1 transfected endothelial cells with respective treatment.

Figure 5

ICMT inhibition attenuates Ras GTPase activity. PAECs were incubated in buffer in the absence (control) or presence of 20 μM AGC or 20 μM AGGC for 4 h or 100 μM Ado/HC overnight at 37°C. a, Cell lysates were harvested and active Ras GTPase was purified with GST-fused Raf-1 protein bound to glutathione agarose beads. Parallel gels were run with corresponding crude lysates. All gels were immunoblotted for Ras GTPase. Immunoblot signals were quantified by densitometry and are presented as the mean ± SEM of the ratio of GST-Raf-1–bound Ras GTPase to total Ras GTPase present in crude lysate. b, Membrane and cytosolic fractions were harvested, resolved by SDS-PAGE, and immunoblotted for Ras. Immunoblot signals were quantified and are presented as the mean ± SEM of the ratio of membrane/cytosol Ras normalized to buffer control. c, To determine the level of carboxyl methylation, Ras GTPase was immunoprecipitated, resolved on SDS-PAGE, and immunoblotted for respective GTPase. Illuminated bands were excised and hydrolyzed, and the level of [³H]methyl incorporation was determined. n = 3. *p < 0.05.

Figure 6

Ras GTPase activity and carboxyl methylation are blunted by UV light. PAECs were exposed to UV light for indicated times and incubated in serum-free MEM overnight at 37°C. The levels of active Ras GTPase (a) and Ras GTPase carboxyl methylation (b) were measured and quantified as stated in Figure 5 legend. n = 3–4. *p < 0.05.

Figure 7

Akt and ERK-1/ERK-2 activities are blunted by ICMT inhibition. PAECs were incubated in buffer in the absence (control) or presence of 20 μM AGC or 20 μM AGGC at 37°C for indicated times. Equivalent volumes of protein were resolved on SDS-PAGE and immunoblotted for phosphorylated Akt (a) or ERK-1/2 (b). Respective immunoblots were subsequently stripped and reprobed for total Akt or ERK-1/2 protein. Immunoblot signals were quantified by densitometry and are presented as the mean ± SEM of the ratio of phosphorylated protein to total protein. Representative immunoblots are shown. n = 5 for control and AGC; n = 3 for AGGC. *p < 0.05 compared with time 0 of respective treatment (a) or compared with control or AGC (b).

Figure 8

H-Ras GTPase overexpression protects against Ado/HC-induced endothelial cell apoptosis. PAECs transiently cotransfected with pGFP-C1 and vector (pUSEamp), wild-type [pUSE H-Ras(wt)], dominant active [pUSE H-Ras(L61)], or dominant negative [pUSE H-Ras(dn)] H-Ras GTPase cDNAs were incubated in buffer in the absence or presence of 100 μM Ado/HC. The cells were then fixed and stained with Hoechst. Endothelial cells were visualized with a fluorescence microscope, and GFP expressing cells were scored for change in nuclear morphology consistent with apoptosis. Data are expressed as mean ± SEM. n = 3. *p < 0.05 compared with cotransfected endothelial cells with respective buffer treatment. **p < 0.05 compared with vector and dominant negative H-Ras GTPase–overexpressing endothelial cells.