FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2018164908> ?p ?o ?g. }

• W2018164908 endingPage "7118" @default.
• W2018164908 startingPage "7112" @default.
• W2018164908 abstract "Calphostin-C, a protein kinase C inhibitor, induces apoptosis of cultured vascular smooth muscle cells. However, the mechanisms are not completely defined. Because apoptosis of vascular smooth muscle cells is critical in several proliferating vascular diseases such as atherosclerosis and restenosis after angioplasty, we decided to investigate the mechanisms underlying the calphostin-C-induced apoptotic pathway. We show here that apoptosis is inhibited by the addition of exogenous phosphatidic acid, a metabolite of phospholipase D (PLD), and that calphostin-C inhibits completely the activities of both isoforms of PLD, PLD1 and PLD2. Overexpression of either PLD1 or PLD2 prevented the vascular smooth muscle cell apoptosis induced by serum withdrawal but not the calphostin-C-elicited apoptosis. These data suggest that PLDs have anti-apoptotic effects and that complete inhibition of PLD activity by calphostin-C induces smooth muscle cell apoptosis. We also report that calphostin-C induced microtubule disruption and that the addition of exogenous phosphatidic acid inhibits calphostin-C effects on microtubules, suggesting a role for PLD in stabilizing the microtubule network. Overexpressing PLD2 in Chinese hamster ovary cells phenocopies this result, providing strong support for the hypothesis. Finally, taxol, a microtubule stabilizer, not only inhibited the calphostin-C-induced microtubule disruption but also inhibited apoptosis. We therefore conclude that calphostin-C induces apoptosis of cultured vascular smooth muscle cells through inhibiting PLD activity and subsequent microtubule polymerization. Calphostin-C, a protein kinase C inhibitor, induces apoptosis of cultured vascular smooth muscle cells. However, the mechanisms are not completely defined. Because apoptosis of vascular smooth muscle cells is critical in several proliferating vascular diseases such as atherosclerosis and restenosis after angioplasty, we decided to investigate the mechanisms underlying the calphostin-C-induced apoptotic pathway. We show here that apoptosis is inhibited by the addition of exogenous phosphatidic acid, a metabolite of phospholipase D (PLD), and that calphostin-C inhibits completely the activities of both isoforms of PLD, PLD1 and PLD2. Overexpression of either PLD1 or PLD2 prevented the vascular smooth muscle cell apoptosis induced by serum withdrawal but not the calphostin-C-elicited apoptosis. These data suggest that PLDs have anti-apoptotic effects and that complete inhibition of PLD activity by calphostin-C induces smooth muscle cell apoptosis. We also report that calphostin-C induced microtubule disruption and that the addition of exogenous phosphatidic acid inhibits calphostin-C effects on microtubules, suggesting a role for PLD in stabilizing the microtubule network. Overexpressing PLD2 in Chinese hamster ovary cells phenocopies this result, providing strong support for the hypothesis. Finally, taxol, a microtubule stabilizer, not only inhibited the calphostin-C-induced microtubule disruption but also inhibited apoptosis. We therefore conclude that calphostin-C induces apoptosis of cultured vascular smooth muscle cells through inhibiting PLD activity and subsequent microtubule polymerization. Proliferation and apoptosis of vascular smooth muscle cells (VSMCs) 1The abbreviations used are: VSMC, vascular smooth muscle cells; PKC, protein kinase C; JNK, c-Jun N-terminal kinase; PLD, phospholipase D; PA, phosphatidic acid; PI, propidium iodide; CHO, Chinese hamster ovary; Dox, doxycycline; LSC, laser scanning cytometry; PBS, phosphate-buffered saline; GFP, green fluorescent protein; ANOVA, analysis of variance. 1The abbreviations used are: VSMC, vascular smooth muscle cells; PKC, protein kinase C; JNK, c-Jun N-terminal kinase; PLD, phospholipase D; PA, phosphatidic acid; PI, propidium iodide; CHO, Chinese hamster ovary; Dox, doxycycline; LSC, laser scanning cytometry; PBS, phosphate-buffered saline; GFP, green fluorescent protein; ANOVA, analysis of variance. are important in the progression of several vascular diseases including atherosclerosis and restenosis after angioplasty (1Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9929) Google Scholar, 2Rossig L. Dimmeler S. Zeiher A.M. Basic Res. Cardiol. 2001; 96: 11-22Crossref PubMed Scopus (133) Google Scholar). We wished to investigate the apoptosis of VSMCs in vitro and its underlying mechanisms. It is known that calphostin-C, a protein kinase C (PKC) inhibitor, induces apoptosis in VSMCs and in several other cell systems (3Ikemoto H. Tani E. Matsumoto T. Nakano A. Furuyama J. J. Neurosurg. 1995; 83: 1008-1016Crossref PubMed Scopus (77) Google Scholar, 4Spinedi A. Oliverio S. Di Sano F. Piacentini M. Biochem. Pharmacol. 1998; 56: 1489-1492Crossref PubMed Scopus (19) Google Scholar, 5Zhu D.M. Narla R.K. Fang W.H. Chia N.C. Uckun F.M. Clin. Cancer Res. 1998; 4: 2967-2976PubMed Google Scholar, 6Leszczynski D. Zhao Y. Luokkamaki M. Foegh M.L. Am. J. Pathol. 1994; 145: 1265-1270PubMed Google Scholar). However, the underlying mechanisms for calphostin-C-induced apoptosis of VSMCs are not completely defined, although pathways involving the activation of calpain, c-Jun N-terminal kinase (JNK), and p38 may be involved (4Spinedi A. Oliverio S. Di Sano F. Piacentini M. Biochem. Pharmacol. 1998; 56: 1489-1492Crossref PubMed Scopus (19) Google Scholar, 7Ozaki I. Tani E. Ikemoto H. Kitagawa H. Fujikawa H. J. Biol. Chem. 1999; 274: 5310-5317Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Nevertheless, it is clear that apoptosis is induced by calphostin-C not through its inhibition of PKC but through some PKC-independent pathway. A recent study (8Sciorra V.A. Hammond S.M. Morris A.J. Biochemistry. 2001; 40: 2640-2646Crossref PubMed Scopus (46) Google Scholar) reported that calphostin-C is a potent inhibitor of phospholipase D (PLD). Given that PLDs may have anti-apoptotic effects (9Kim J.H. Yoon Y.D. Shin I. Han J.S. IUBMB Life. 1999; 48: 445-452Crossref PubMed Scopus (14) Google Scholar, 10Nozawa Y. Biochim. Biophys. Acta. 2002; 1585: 77-86Crossref PubMed Scopus (65) Google Scholar, 11Park M.A. Lee M.J. Lee S.H. Jung D.K. Kwak J.Y. FEBS Lett. 2002; 519: 45-49Crossref PubMed Scopus (21) Google Scholar, 12Zhong M. Shen Y. Zheng Y. Joseph T. Jackson D. Foster D.A. Biochem. Biophys. Res. Commun. 2003; 302: 615-619Crossref PubMed Scopus (76) Google Scholar), we asked whether calphostin-C induces apoptosis through its inhibition of PLDs in VSMCs.If PLD inhibition is involved in calphostin-C-induced apoptosis, we would ask how PLD inhibition results in apoptosis. It is well known that in the reaction catalyzed by the PLDs, PLD1 and PLD2 produce phosphatidic acid (PA) from phosphatidylcholine and PA is a key signaling molecule that can trigger various cellular functions (13Frohman M.A. Sung T.C. Morris A.J. Biochim. Biophys. Acta. 1999; 1439: 175-186Crossref PubMed Scopus (276) Google Scholar, 14Exton J.H. Rev. Physiol. Biochem. Pharmacol. 2002; 144: 1-94Crossref PubMed Google Scholar). PLD2 has been reported to provoke cytoskeletal reorganization (15Colley W.C. Sung T.C. Roll R. Jenco J. Hammond S.M. Altshuller Y. Bar-Sagi D. Morris A.J. Frohman M.A. Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (632) Google Scholar). Several other studies have described PLD1 interaction with actin and its role in facilitating stress fiber formation (16Kam Y. Exton J.H. Mol. Cell Biol. 2001; 21: 4055-4066Crossref PubMed Scopus (90) Google Scholar, 17Cross M.J. Roberts S. Ridley A.J. Hodgkin M.N. Stewart A. Claesson-Welsh L. Wakelam M.J. Curr. Biol. 1996; 6: 588-597Abstract Full Text Full Text PDF PubMed Google Scholar, 18Lee S. Park J.B. Kim J.H. Kim Y. Kim J.H. Shin K.J. Lee J.S. Ha S.H. Suh P.G. Ryu S.H. J. Biol. Chem. 2001; 276: 28252-28260Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Moreover in plant cells, PLD has been reported to bind microtubules (19Gardiner J.C. Harper J.D. Weerakoon N.D. Collings D.A. Ritchie S. Gilroy S. Cyr R.J. Marc J. Plant Cell. 2001; 13: 2143-2158Crossref PubMed Scopus (223) Google Scholar). Taken together, these studies suggest that PA may regulate the function and/or structure of the cytoskeleton. To our knowledge, however, it is not known whether PA regulates the assembly or disassembly of microtubules in mammalian cells. It is conceivable that a complete removal of PA in cells could interrupt the integrity of the cytoskeleton, e.g. the microtubule network. We have recently found that microtubule disruption using nocodazole induces apoptosis of VSMCs with some similar features to those cells treated with calphostin-C, which promoted us to examine the effects of calphostin-C on the microtubule network. In this report, we show that calphostin-C induces apoptosis of VSMCs through its inhibition of PLD followed by microtubule disruption.EXPERIMENTAL PROCEDURESChemicals and Reagents—Calphostin-C and GF 109203X were obtained from Calbiochem. Monoclonal antibodies for α-tubulin, phosphatidic acid, and propidium iodide (PI) were purchased from Sigma. RPMI 1640 medium and fetal bovine serum were purchased from Invitrogen.Cell Culture and Treatment—Primary culture of VSMCs was carried out using the explant method as described previously (20Chamley-Campbell J. Campbell G.R. Ross R. Physiol. Rev. 1979; 59: 1-61Crossref PubMed Scopus (1257) Google Scholar). The blood vessel tissues were derived from thoracic aorta of male Sprague-Dawley rats (250-300 g). The medial aortic tissue was cut into small pieces (1 mm3), which were then placed on the plastic surface of culture dishes. The tissue explants were maintained in RPMI 1640 medium containing 10% (v/v) fetal calf serum, penicillin (100 units/ml), and streptomycin (100 μg/ml). The culture was carried out at 37 °C in a humidified atmosphere of 5% C02 and 95% air. Cells were usually observed to migrate from tissues between days 4 and 7 after explanting. The islets of outgrowing cells were then treated with 0.25% trypsin and 0.25 mm EDTA solution (Invitrogen) followed by subculture. The cells from passage 6-8 were used for the experiments described. Calphostin-C was dissolved in water and added to culture medium directly to achieve a final concentration of 0.2 μm for 24 h. To test the effects of PA on calphostin-C-induced apoptosis, PA micelles (1 μg/ml) were added to culture medium 30 min before calphostin-C. In serum starvation experiments, cells were cultured in medium containing 0.5% serum for 2 days before any treatment. CHO cells were cultured using Ham's F-12 medium supplemented with 10% (v/v) fetal calf serum essentially like VSMCs. These cells harbored either hemagglutinin-PLD2 or hemagglutinin-PLD2 K758R under control of a tetracycline-inducible T-Rex (Invitrogen). Expression of PLD2 or PLD2K758R was induced by adding 1 μg/ml doxycycline (Dox) for 20 h.Apoptosis and DNA Analysis Using Laser-scanning Cytometry (LSC)—VSMCs were cultured on acid-washed coverslips. After various treatments, cells were fixed with 80% ethanol for 20 min and cold 100% ethanol for another 20 min. After rinsing with PBS (50 mm NaH2PO4, 300 mm NaCl, pH 7.4), cells were incubated with a solution containing 5 mg/ml PI and 200 ng/ml RNase for 20 min. Coverslips were mounted to microscopic slides. DNA analysis and apoptosis of VSMCs in response to calphostin-C were performed with LSC (CompuCyte, Cambridge, MA) as previously described (21Kamentsky L.A. Kamentsky L.D. Cytometry. 1991; 12: 381-387Crossref PubMed Scopus (226) Google Scholar, 22Kamentsky L.A. Burger D.E. Gershman R.J. Kamentsky L.D. Luther E. Acta Cytol. 1997; 41: 123-143Crossref PubMed Scopus (165) Google Scholar). Data were acquired and analyzed with WinCyte acquisition software (version 3.4, CompuCyte). Apoptosis of cultured VSMCs was indicated by the appearance of a sub-G0/G1 apoptotic peak. The apoptotic cells in the sub-G0/G1 peak were confirmed using relocation of LSC to observe cells directly through the CCD camera.DNA Fragmentation Assay—After treatment with or without 0.2 μm or 1 μm calphostin-C for 24 h, cells were collected and solubilized by vigorous vortexing in TE9S buffer (0.5 m EDTA, 10 mm NaCl, 10 mm Tris-HCl, pH 9), 1% (w/v) SDS) containing proteinase K (1 mg/ml). This was followed by a 3-h incubation at 50 °C and DNA extraction using phenol-chloroform. After incubation with RNase A (l mg/ml) for 1 h at 20-22 °C, DNA samples were analyzed using conventional electrophoresis in 1.5% (w/v) agarose gels. The gels were visualized using a gel documentation system (Bio-Rad).GFP-PLD Expression Vector construction and the Establishment of Stably Transfected Cells—The construction of pCGN-PLDlb (GenBank™ accession number U38545), pCGN-PLD1-K898R (inactive allele), pCGN-PLD2 (GenBank™ accession number U87557), and pCGN-PLD2-K758R (inactive allele) was performed as described previously (23Sung T.C. Zhang Y. Morris A.J. Frohman M.A. J. Biol. Chem. 1999; 274: 3659-3666Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 24Sung T.C. Altshuller Y.M. Morris A.J. Frohman M.A. J. Biol. Chem. 1999; 274: 494-502Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). All of the full-length cDNAs were subcloned into pEGFP vectors. To avoid changing the reading frames, PLD1b and PLD2 were cloned into pEGFP-C2 and pEGFP-C3 vectors, respectively (Clontech Laboratories). PLDlb and PLD1-K898R were excised from pCGN-PLDlb and pCGN-PLD1-K898R with HapI and SmaI. PLD2 and PLD2-K758R were excised from pCGN-PLD2 and pCGN-PLD2-K758R with XbaI and Smal. Both pEGFP-C2 and pEGFP-C3 were digested using SmaI. Excised cDNA fragments were purified using a gel extraction kit (Qiagen). The sizes of the excised fragments were confirmed by gel electrophoresis. After construction, restriction mapping confirmed the presence and orientation of the PLD1b and PLD2 cDNAs in the vector.Cells were transiently transfected with each DNA preparation using LipofectAMINE 2000 according to the instructions provided by the manufacturer (Invitrogen). Successful transfection was confirmed by fluorescence microscopy. To make stable cells, transfected cells were selected by adding 400 ng/ml G418 into the culture medium. After forming single colonies in the presence of G418, all of the cells were pooled to make a stable cell mixture and these cells were maintained in 100 ng/ml G418, which was withdrawn 1 week before setting up the experiments. The expression of PLDs in stably transfected cells was confirmed using both immunoprecipitation/Western blot and a PLD activity assay.Immunoprecipitation and Western Blot Analysis of GFP and PLD-GFP Expression—Stably transfected cells after 24-h serum starvation were lyzed for protein extraction. An equal amount of protein (100 μg) from each sample was added to 1 ml of PBS followed by an overnight incubation with antibody for GFP and protein G-Sepharose beads at 4 °C. Immunoprecipitated proteins were analyzed using standard SDS-PAGE. Proteins were then transferred (30 V, overnight, 4 °C) from the gels to a nitrocellulose membrane. Membranes were blocked with 10% (w/v) bovine serum albumin for 1 h at room temperature followed by incubation with anti-GFP mouse monoclonal antibody to detect both GFP and PLD-GFPs. A horseradish peroxidase-coupled goat anti-mouse IgG was used as the secondary antibody. Visualization of signals was achieved by using chemiluminescence (ECL reagent, Amersham Biosciences).PLD Activity Assay—Cultured cells were maintained in cell starvation medium (0.5% serum) for 3 days and then labeled with l μCi/ml [9,10(n)-3H]myristic acid (Amersham Biosciences, catalog number TRK90) for 12 h. Cells were then washed three times with PBS, pH 7.4, and pre-equilibrated at 37 °C in cell starvation medium for 60 min. Butanol (0.5%) was included during the last 10 min of incubation. After termination of the reaction, cells were collected followed by lipid extraction and separation with 0.6 ml of methnol/chloroform/0.1 n HCl (v/v/v, 1:1:1). A fraction of the extracted lipids was measured for total radioactivity using a liquid scintillation counter (Beckman). Samples were subsequently separated on a silica gel TCL sheet. After autoradiography, the spots corresponding to the reference lipid (phosphatidylbutanol) were scraped to scintillation vials for the measurement of radioactivity. The activities of PLDs were presented as a ratio of radioactivity for each spot to the total radioactivity.Immunocytochemical Studies—Cells cultured on coverslips were treated with or without calphostin-C for 48 h. These cells were then rinsed with PBS and fixed with 80% ethanol for 20 min. Cells were subsequently permeabilized with 0.25% Triton X-100 in PBS. After incubation with bovine serum albumin (0.5%) containing PBS for 20 min, cells on coverslips were incubated with a primary antibody to target the protein of interest, washed with PBS, and incubated with Alexa Flour-488 labeled secondary antibodies. Cell nuclei were counterstained with a PI solution (5 μg/ml) that contained RNase (200 ng/ml). Cellular fluorescent signals were detected using either regular fluorescence microscopy or LSC. CHO cells after induction of PLD2 and PLD2 K758R with Dox (1 μg/ml) for 20 h were fixed with cold methanol followed by staining for α-tubulin and overexpressed PLD2.Data Analysis—Data are expressed as the mean ± S.E. The number of replicate (n) represents the number of cell samples used in the studies. Differences between means were evaluated by the Student's t test (paired or independent) when two groups were compared and by analysis of variance (ANOVA) followed by Bonferroni's correction when three or more groups were compared. p < 0.05 or p < 0.01 was considered significant. All statistical analysis was performed using the Statistical Package for the Social Sciences software (SPSS, version 10, Chicago, IL).RESULTSSmooth Muscle Cell Apoptosis Induced by Calphostin-C—We had observed that there was significant cell death with a slight rounding up of cultured rat VSMCs in response to treatment with calphostin-C (0.2 μm) but not GF109203X (3 μm) (data not shown). It has been reported that calphostin-C induces apoptosis in several cell systems including VSMCs in culture. Therefore, we wished to confirm this calphostin-C induced apoptosis in cultured rat aortic VSMCs. To do so, we extracted DNA from cells with or without treatment with calphostin-C (0.2 or 1 μm) for 24 h. The DNA samples were analyzed using agarose gels as described under “Experimental Procedures.” Our results showed that calphostin-C at 0.2 μm induced significant DNA fragmentation, and the fragmentation was even more prominent at a concentration of 1 μm (Fig. 1, panel A). Typical apoptotic nuclei in response to calphostin-C treatment were observed directly with fluorescent microscopy after PI staining (Fig. 1, panels B and C). We further analyzed VSMC apoptosis induced by calphostin-C with LSC, a microscope-based cytometer. As shown in Fig. 1, panels D and E, we observed the typical sub-G0/G1 apoptotic peak in the DNA histograms. Cells in the apoptotic peak were then located using LSC relocation to confirm their apoptotic morphologies as shown in the insert of Fig. 1, panel E. Accumulated data showed that apoptotic cell number (percentage of total cells) significantly increased in response to treatment with calphostin-C (0.2 μm). The apoptosis in VSMCs induced by calphostin-C appeared independent of its PKC inhibition, because inhibition of PKC by GF109203X did not induce an apoptotic response (data not shown). Recently, calphostin-C has been reported to directly inhibit the activities of PLD independent of its inactivation of PKC (8Sciorra V.A. Hammond S.M. Morris A.J. Biochemistry. 2001; 40: 2640-2646Crossref PubMed Scopus (46) Google Scholar). Given that PLD has anti-apoptotic effects in some cell systems, we hypothesized that inhibition of PLD could contribute to the apoptotic action of calphostin-C. Therefore, we explored the following two sets of experiments to test this possibility.Inhibition of PLD Activities by Calphostin-C—To test the involvement of PLDs in calphostin-C induced apoptosis, we first examined whether calphostin-C inhibits PLD activities in cultured rat aortic VSMCs. To do so, we labeled the lipid pool with [9,10(n)-3H]myristic acid and used butanol for transphosphatidylation to measure PLD activities of cells cultured with or without the presence of calphostin-C at 0.2 μm for 24 h. As expected, calphostin-C treatment completely inhibited the activity of PLDs in cells cultured with 10% serum (Fig. 2). Therefore, this observation has confirmed that calphostin-C is a potent PLD inhibitor in VSMCs. Also, it suggests that PLD inhibition may be the mechanism through which calphostin-C induces apoptosis in VSMCs. To further test this hypothesis, we examined the effect of exogenous phosphatidic acid on calphostin-C-induced apoptosis.Fig. 2Inhibition of PLD activity by calphostin-C. The PLD activities in VSMCs cultured in medium containing 10% serum are represented by the formation of phosphatidylbutanol as described under “Experimental Procedures.” The y axis indicates the total PLD activity as a percentage of that in control cells without any treatment. Results are presented as mean ± S.E. (n = 4). The PLD activity of control cells is set as 100. The x axis indicates different groups with various treatments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)PA Inhibits Calphostin-C-induced Apoptosis—Cultured VSMCs were incubated with phosphatidic acid micelles (1 μg/ml) 30 min before treatment with calphostin-C (0.2 μm) for 24 h. As anticipated, the results showed that treatment of cells with PA prevented the appearance of the sub-G0/G1 apoptotic peak induced by calphostin-C as shown in the DNA histograms acquired using LSC (Fig. 3). This inhibition of apoptosis by PA was confirmed using the DNA fragmentation assay (data not shown). These data, along with the results that show PLD inhibition by calphostin-C, strongly suggested that calphostin-C induced apoptosis of cultured VSMCs through its inhibition of PLDs. However, we wished to establish further that PLD activation in cultured VSMCs indeed has an anti-apoptotic effect.Fig. 3Exogenous PA inhibits calphostin-C-induced apoptosis of VSMCs. Cells were cultured on coverslips without or with the presence of calphostin-C (panels A and B) for 24 h followed by analysis using LSC. Cells in panel C were pretreated with PA (1 μg/ml) before the addition of calphostin-C (0.2 μm). The y axis (Count) shows the number of scanned cells, and the x axis (PI Integral) shows the total DNA in each cell. Cells in region 1 are considered to be apoptotic cells. Panel D is a bar figure for accumulated data in which the y axis is the percentage of apoptotic cells in each condition derived from LSC. The x axis indicates the different groups. The asterisk indicates a significant difference (p < 0.01).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Overexpression of Either PLD1b or PLD2 in Cultured VSMCs Inhibits Apoptosis Induced by Serum Starvation—In our preliminary studies, we observed that serum withdrawal induced apoptosis of cultured rat aortic VSMCs as described by others (25Aoki M. Morishita R. Matsushita H. Nakano N. Hayashi S. Tomita N. Yamamoto K. Moriguchi A. Higaki J. Ogihara T. Heart Vessels. 1997; 12 (suppl.): 71-75PubMed Google Scholar). It has been reported that cultured cells have low PLD activities in the absence of serum (26Carnero A. Cuadrado A. Del Peso L. Lacal J.C. Oncogene. 1994; 9: 1387-1395PubMed Google Scholar). The addition of exogenous PA prevented the apoptosis of cultured VSMCs, resulting from serum starvation for 3 days (Fig. 4, panel A). Therefore, we hypothesized that overexpression of either PLD1b or PLD2 would prevent apoptosis resulting from serum withdrawal. To test this possibility, we established stably transfected cells harboring PLD1b, PLD2, or their inactive forms (see “Experimental Procedures”). The expression of PLD-GFP fusion proteins was first confirmed by observing the bright fluorescence in transiently transfected cells (data not shown). Cells were then selected with G418 (400 ng/ml) for 2 weeks. The expression of PLD-GFP fusion proteins in stably transfected cells was further confirmed using immunoprecipitation/Western blot to detect the presence of GFP as shown in Fig. 4, panel B. As anticipated, our data showed that control cells harboring only GFP undergo significant apoptosis after 3 days of culture in serum-free medium as analyzed using LSC (Fig. 4, panels C and H). However, there was no significant apoptosis observed in cells expressing either PLD1b or PLD2 and the rescue is clearly activity-dependent because expression of the inactive forms of PLD1b and PLD2 did not inhibit apoptosis. This activity dependence was further confirmed by measuring PLD activity in the presence and absence of 10% serum. As shown in Fig. 4, panel I, serum withdrawal for 3 days significantly reduced PLD activity in the GFP control cells. In contrast, stably transfected cells harboring PLD1b or PLD2, but not their inactive forms, displayed significantly higher activities than the control cells in the presence or absence of serum. Therefore, these findings demonstrate that both PLD1b and PLD2 have anti-apoptotic effects in cultured VSMCs. Taken together, we conclude that the PLDs have anti-apoptotic effects and that calphostin-C induces apoptosis through its inhibition of PLD. However, we wished to further examine the mechanisms underlying calphostin-C-induced apoptosis. As mentioned earlier, we observed the rounding up of cells in response to calphostin-C treatment. This cell response is similar to what we observed in response to microtubule-disruptive agents such as nocodazole, which also induces VSMC apoptosis. Therefore, we explored whether there was a relationship between PLD and the cytoskeleton.Fig. 4Overexpression of PLD1b and PLD2 inhibits apoptosis induced by serum withdrawal. Serum starvation-induced apoptosis was prevented by the presence of PA (panel A). Cells on coverslips were cultured in serum-free medium without (control) and with (+PA) the presence of PA (1 μg/ml) for 3 days followed by analysis of apoptotic cells using LSC as described under “Experimental Procedures.” The asterisk indicates a significant difference compared with control cells in the absence of serum for 3 days (p < 0.01, n = 4). Panel B, stably transfected cells harboring pEGFP-PLD1b, pEGFP-PLD1b-K898R, pEGFP-PLD2, and pEGFP-PLD2-K758R were characterized using immunoprecipitation/Western blot. Cells after serum starvation for 24 h were lyzed for protein extraction, and 100-μg protein from each population was analyzed with immunoprecipitation/Western blot using an anti-GFP antibody. In panel B, lane 1, cells transfected with the GFP construct; lanes 2 and 3, cells harboring pEGFP-PLD1b and pEGFP-PLD1b-K898R respectively; and lanes 4 and 5, cells harboring pEGFP-PLD2 and pEGFP-PLD2-K758R. To evaluate apoptosis, all of the cells were maintained in serum-free medium for 3 days followed by analysis of apoptosis using LSC. Panels C-G, DNA histograms derived from LSC in which the y axis (Count) shows the number of scanned cells and the x axis (PI Integral) shows the total DNA in each cell. Cells in region 1 are apoptotic cells contributing to the sub-G0/G1 peak. Panel C is from control cells with pEGFP alone. Panels D and E are from cells harboring pEGFP-PLD1b and pEGFP-PLD1b-K898R. Panels F and G are data from cells harboring pEGFP-PLD2 and pEGFP-PLD2-K758R. Panel H is a bar figure for accumulated data to show apoptotic cell number. The y axis is apoptotic cells (percentage of total cells scanned). The x axis indicates the different groups. The asterisk indicates a significant difference (p < 0.01; n = 6). Panel I shows the total PLD activities (percentage of control, the y axis) in various stable cells as indicated in the x axis with and without the presence of 10% serum for 3 days. The asterisk indicates a significant difference compared with the GFP control cells in the presence of serum (p < 0.05, n = 4). The double asterisk shows a significant difference compared with the GFP control cells in the absence of serum for 3 days (p < 0.05; n = 4).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Calphostin-C Induction of Microtubule Disruption and Effects of PA and Taxol—To address whether calphostin-C modulates the microtubule network, we first examined microtubules via immunostaining for α-tubulin. As expected, calphostin-C treatment induced significant disruption of microtubules (Fig. 5, panel B). Importantly, the addition of exogenous PA prevented the disruption of microtubules and the morphologic changes (Fig. 5, panel C). As expected, a microtubule stabilizer, taxol (0.01 μm) (27Axel D.I. Kunert W. Goggelmann C. Oberhoff M. Herdeg C. Kuttner A. Wild D.H. Brehm B.R. Riessen R. Koveker G. Karsch K.R. Circulation. 1997; 96: 636-645Crossref PubMed Scopus (684) Google Scholar, 28Amos L.A. Lowe J. Chem. Biol. 1999; 6: R65-R69Abstract Full Text PDF PubMed Scopus (226) Google Scholar, 29Schiff P.B. Fant J. Horwitz S.B. Nature. 1979; 277: 665-667Crossref PubMed Scopus (3114) Google Scholar), suppressed the effect of calphostin-C on microtubules (Fig. 5, panel D). These d" @default.
• W2018164908 created "2016-06-24" @default.
• W2018164908 creator A5034300226 @default.
• W2018164908 creator A5068972249 @default.
• W2018164908 creator A5075822447 @default.
• W2018164908 creator A5080165366 @default.
• W2018164908 creator A5087789028 @default.
• W2018164908 date "2004-02-01" @default.
• W2018164908 modified "2023-09-27" @default.
• W2018164908 title "Calphostin-C Induction of Vascular Smooth Muscle Cell Apoptosis Proceeds through Phospholipase D and Microtubule Inhibition" @default.
• W2018164908 cites W1537149667 @default.
• W2018164908 cites W1945199177 @default.
• W2018164908 cites W1963490212 @default.
• W2018164908 cites W1971906200 @default.
• W2018164908 cites W1973624256 @default.
• W2018164908 cites W1991293840 @default.
• W2018164908 cites W199474707 @default.
• W2018164908 cites W1998624840 @default.
• W2018164908 cites W1999359428 @default.
• W2018164908 cites W2001656598 @default.
• W2018164908 cites W2004687011 @default.
• W2018164908 cites W2006266549 @default.
• W2018164908 cites W2009856729 @default.
• W2018164908 cites W2015112918 @default.
• W2018164908 cites W2018717545 @default.
• W2018164908 cites W2025691233 @default.
• W2018164908 cites W2029699912 @default.
• W2018164908 cites W2050202210 @default.
• W2018164908 cites W2054067779 @default.
• W2018164908 cites W2066384132 @default.
• W2018164908 cites W2067299834 @default.
• W2018164908 cites W2077538389 @default.
• W2018164908 cites W2077548365 @default.
• W2018164908 cites W2086706837 @default.
• W2018164908 cites W2087081676 @default.
• W2018164908 cites W2106258234 @default.
• W2018164908 cites W2115772141 @default.
• W2018164908 cites W2126567503 @default.
• W2018164908 cites W2132811374 @default.
• W2018164908 cites W2137570167 @default.
• W2018164908 cites W2167031071 @default.
• W2018164908 cites W2168257088 @default.
• W2018164908 doi "https://doi.org/10.1074/jbc.m310721200" @default.
• W2018164908 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/14660552" @default.
• W2018164908 hasPublicationYear "2004" @default.
• W2018164908 type Work @default.
• W2018164908 sameAs 2018164908 @default.
• W2018164908 citedByCount "25" @default.
• W2018164908 countsByYear W20181649082013 @default.
• W2018164908 countsByYear W20181649082014 @default.
• W2018164908 countsByYear W20181649082015 @default.
• W2018164908 countsByYear W20181649082017 @default.
• W2018164908 countsByYear W20181649082021 @default.
• W2018164908 countsByYear W20181649082023 @default.
• W2018164908 crossrefType "journal-article" @default.
• W2018164908 hasAuthorship W2018164908A5034300226 @default.
• W2018164908 hasAuthorship W2018164908A5068972249 @default.
• W2018164908 hasAuthorship W2018164908A5075822447 @default.
• W2018164908 hasAuthorship W2018164908A5080165366 @default.
• W2018164908 hasAuthorship W2018164908A5087789028 @default.
• W2018164908 hasBestOaLocation W20181649081 @default.
• W2018164908 hasConcept C12927208 @default.
• W2018164908 hasConcept C134018914 @default.
• W2018164908 hasConcept C181199279 @default.
• W2018164908 hasConcept C185592680 @default.
• W2018164908 hasConcept C190283241 @default.
• W2018164908 hasConcept C195794163 @default.
• W2018164908 hasConcept C20418707 @default.
• W2018164908 hasConcept C2779395532 @default.
• W2018164908 hasConcept C2780245509 @default.
• W2018164908 hasConcept C2992686903 @default.
• W2018164908 hasConcept C55493867 @default.
• W2018164908 hasConcept C62478195 @default.
• W2018164908 hasConcept C86803240 @default.
• W2018164908 hasConcept C95444343 @default.
• W2018164908 hasConceptScore W2018164908C12927208 @default.
• W2018164908 hasConceptScore W2018164908C134018914 @default.
• W2018164908 hasConceptScore W2018164908C181199279 @default.
• W2018164908 hasConceptScore W2018164908C185592680 @default.
• W2018164908 hasConceptScore W2018164908C190283241 @default.
• W2018164908 hasConceptScore W2018164908C195794163 @default.
• W2018164908 hasConceptScore W2018164908C20418707 @default.
• W2018164908 hasConceptScore W2018164908C2779395532 @default.
• W2018164908 hasConceptScore W2018164908C2780245509 @default.
• W2018164908 hasConceptScore W2018164908C2992686903 @default.
• W2018164908 hasConceptScore W2018164908C55493867 @default.
• W2018164908 hasConceptScore W2018164908C62478195 @default.
• W2018164908 hasConceptScore W2018164908C86803240 @default.
• W2018164908 hasConceptScore W2018164908C95444343 @default.
• W2018164908 hasIssue "8" @default.
• W2018164908 hasLocation W20181649081 @default.
• W2018164908 hasOpenAccess W2018164908 @default.
• W2018164908 hasPrimaryLocation W20181649081 @default.
• W2018164908 hasRelatedWork W2291113408 @default.
• W2018164908 hasRelatedWork W2374602682 @default.
• W2018164908 hasRelatedWork W2376418594 @default.
• W2018164908 hasRelatedWork W2376741113 @default.
• W2018164908 hasRelatedWork W2379086703 @default.

• next