Stable transfectants of smooth muscle cell line lacking the expression of myosin light chain kinase and their characterization with respect to the actomyosin system.

We constructed a plasmid vector having a 1.4-kilobase pair insert of myosin light chain kinase (MLCK) cDNA in an antisense direction to express antisense mRNA. The construct was then transfected to SM3, a cell line from vascular smooth muscle cells, producing a few stable transfectants. The down-regulation of MLCK expression in the transfectants was confirmed by both Northern and Western blots. The control SM3 showed chemotaxic motility to platelet-derived growth factor-BB, which was supported by lamellipodia. However, the transfectants showed neither chemotaxic motility nor developed lamellipodia, indicating the essential role of MLCK in the motility. The specificity for the targeting was assessed by a few tests including the rescue experiment. Despite this importance of MLCK, platelet-derived growth factor-BB failed to induce MLC20 phosphorylation in not only the transfectants but also in SM3. The mode in which MLCK was involved in the development of membrane ruffling is discussed with special reference to the novel property of MLCK that stimulates the ATPase activity of smooth muscle myosin without phosphorylating its light chain (Ye, L.-H., Kishi, H., Nakamura, A., Okagaki, T., Tanaka, T., Oiwa, K., and Kohama, K. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6666-6671).

Commitment of neutrophilic differentiation and proliferation of HL-60 cells coincides with expression of transferrin receptor. Effect of granulocyte colony stimulating factor on differentiation and proliferation.

To examine the regulatory mechanisms of proliferation and maturation in neutrophilic lineage cells, we have tried to sort dimethyl sulfoxide (Me(2)SO)-treated HL-60 cells into transferrin receptor (Trf-R) positive (Trf-R(+)) and negative (Trf-R(-)) cells. Differentiated Trf-R(-) cells expressed more formyl-Met-Leu-Phe receptor (fMLP-receptor) and ability of O-(2) genaration, as markers of differentiation, than Trf-R(+) cells, and Trf-R(-) cell differentiation was markedly accelerated by the incubation with granulocyte colony stimulating factor (G-CSF). On the other hand, Trf-R(+) cells had a tendency to proliferate rather than differentiate, and proliferation was enhanced by G-CSF. These results indicate that Trf-R expression coincides with the commitment to proliferate or differentiate of HL-60 cells, and G-CSF accelerates these commitments. G-CSF-induced tyrosine phosphorylation of STAT 3 in Trf-R(-) cells much more than in Trf-R(+) cells. Protein 70 S6 kinase expression was higher in Trf-R(+) cells than in Trf-R(-) cells. Furthermore, p70 S6 kinase was hyperphosphorylated by G-CSF in Trf-R(+) cells, but not in Trf-R(-) cells. Rapamycin, an inhibitor of p70 S6 kinase activity, inhibited G-CSF-dependent proliferation of Trf-R(+) cells and increased fMLP-R expression on these cells. These results suggest that commitment to proliferation and differentiation in Me(2)SO-treated HL-60 cells is preprogrammed and correlated with Trf-R expression, and G-CSF potentiates the cellular commitment. STAT 3 may promote differentiation of Me(2)SO-treated HL-60 cells into neutrophils, while p70 S6 kinase may promote proliferation and negatively regulate neutrophilic differentiation.

The role of STAT3 in granulocyte colony-stimulating factor-induced enhancement of neutrophilic differentiation of Me2SO-treated HL-60 cells. GM-CSF inhibits the nuclear translocation of tyrosine-phosphorylated STAT3.

The role of granulocyte colony-stimulating factor (G-CSF) on neutrophilic differentiation of Me2SO-treated HL-60 cells was studied. G-CSF augmented the functional maturation of Me2SO-treated HL-60 cells in terms of both O-2-generating ability and expression of the formyl-methionyl-leucyl-phenylalanine receptor. G-CSF induced enhancement of cell growth in Me2SO-treated HL-60 cells. These results indicate that G-CSF is a potent enhancer for the differentiation and proliferation of Me2SO-treated HL-60 cells. G-CSF caused the activation of p70 S6 kinase but not mitogen-activated protein (MAP) kinase. On the other hand, G-CSF rapidly induced tyrosine phosphorylation of signal transducers and activators of transcription-3 (STAT3), but did not induce serine727 phosphorylation. From the analysis of confocal laser scanning fluorescence microscopy and differential centrifugation, it was clearly demonstrated that G-CSF induced nuclear translocation of tyrosine-phosphorylated STAT3. The G-CSF-dependent enhancement of neutrophilic differentiation in Me2SO-HL-60 cells was reversely inhibited by granulocyte-macrophage colony-stimulating factor (GM-CSF). Notably, in the presence of GM-CSF, G-CSF induced the tyrosine phosphorylation of STAT3 but failed to induce the nuclear translocation of tyrosine-phosphorylated STAT3. GM-CSF induced activation of not only p70 S6 kinase, but also of MAP kinase. Furthermore, GM-CSF caused the rapid serine727 phosphorylation of STAT3, both in the presence and absence of G-CSF. PD98059, an MEK1 inhibitor, inhibited the G-CSF-dependent serine727 phosphorylation of STAT3 and blocked the inhibitory effect of GM-CSF on G-CSF-dependent nuclear translocation of STAT3. These results suggest that G-CSF-dependent nuclear translocation of STAT3 coordinates with the promotion of neutrophilic differentiation in Me2SO-treated HL-60 cells.

Arachidonic acid pretreatment enhances smooth muscle cell migration via increased Ca2+ influx.

It is well known that vascular smooth muscle cell (SMC) migration is an initial step in atheromatous plaque formation. In the present study, we investigated the effects of arachidonic acid (AA, 20:4 n-6) on bovine carotid artery SMC migration using the modified Boyden chamber technique. SMCs pretreated with 2.5 microg/ml of AA for 2 days, showed an enhanced migration response to fetal bovine serum. AA pretreatment (0.5-5.0 microg/ml) increased fetal bovine serum-induced SMC migration dose-dependently, and maximum stimulation was observed at a concentration of 2.5 microg/ml. However, AA pretreatment did not enhance fetal bovine serum-induced endothelial cell migration. Using lipid analysis, we found that AA was substantially incorporated into cellular phospholipids. When SMC migration was induced by platelet derived growth factor (PDGF)-BB, instead of serum, the stimulative effect of AA pretreatment was retained. SMCs pretreated with AA showed greater mobilization of intracellular Ca2+ in response to PDGF-BB than SMCs without AA pretreatment (controls). Nifedipine, a Ca2+ channel blocker, and glycoletherdiamine-tetraacetic acid (EGTA) had no effect on PDGF-induced migration of controls but both drugs reduced the enhanced PDGE-induced migration of AA-pretreated SMCs to the control level. Baicalein, an inhibitor of 12-lipoxygenase, reduced PDGF-BB-induced migration of both control and AA pretreated SMCs, however the AA-pretreated cells still showed enhanced migration compared to control cells. These findings suggest that AA accelerates SMC migration in the thickening of the intima during atheroma formation, via stimulation of extracellular Ca2+ influx.

Docosapentaenoic acid (22:5, n-3), an elongation metabolite of eicosapentaenoic acid (20:5, n-3), is a potent stimulator of endothelial cell migration on pretreatment in vitro.

Endothelial cell (EC) migration plays an important role in wound repair of blood vessels. We have previously reported that eicosapentaenoic acid (EPA; 20:5, n-3) pretreatment stimulates migration of ECs but not smooth muscle cells. In the present study, we used the modified Boyden chamber technique to investigate whether the stimulative effect of EPA pretreatment on EC migration is caused by EPA itself or by some metabolites of EPA. When ECs were treated with EPA (5 micrograms/ml) for 2 days, EPA was predominantly elongated to docosapentaenoic acid (DPA; 22:5, n-3), with little docosahexaenoic acid (DHA; 22:6, n-3) being formed. Direct pretreatment of ECs with DPA (0.01-1.0 microgram/ml) resulted in a dose-dependent increase in migration in response to fetal bovine serum. Moreover, maximum stimulation of EC migration by DPA pretreatment (0.5 microgram/ml) was achieved at a concentration one-tenth of that required for maximal stimulation by EPA pretreatment (5.0 micrograms/ml), indicating that DPA is a potent stimulator of EC migration. We have demonstrated by lipid analysis that direct DPA pretreatment (0.5 microgram/ml) sufficiently increased the absolute quantity of phospholipids of ECs. Cyclooxygenase inhibitor and lipoxygenase inhibitor did not abolish the stimulative effect of DPA pretreatment on EC migration. In contrast to EC migration, DPA pretreatment had no effect on smooth muscle cell migration. Together these data suggest that the stimulative effect of EPA on EC migration occurs via DPA, and that DPA may act as a powerful anti-atherogenic factor.

[Endothelial cell function and angiogenesis].

Angiogenesis, a process of new blood vessel formation, is an integral part of development, wound repair and tumor growth. The formation of capillary networks requires a complex series of cellular events, in which endothelial cells locally degrade their basement membrane, migrate into the connective tissue stroma, proliferate at the migrating tip, enlongate and organize into capillary loops. In response to angiogenic stimuli, endothelial cells in culture develop networks of capillary-like tubes. In this paper, we showed the relationship between angiogenesis and diseases, the assay systems of angiogenesis and the reports of angiogenesis published recently.

Eicosapentaenoic acid abolishes the proatherogenic effects of cholesterol: effects on migration of bovine smooth muscle and endothelial cells in vitro.

It is well known that vascular endothelial cell (EC) migration plays a major role in regeneration of the injured endothelium and also that smooth muscle cell (SMC) migration is the important step for atheromatous plaque formation. In the present study, we investigated the effects of cholesterol and eicosapentaenoic acid (EPA) on bovine carotid artery EC and SMC migration using the modified Boyden chamber technique. The migration activity of the cholesterol-enriched ECs loaded with cholesterol-rich liposomes was significantly suppressed, whereas that of the cholesterol-enriched SMCs was enhanced. Next, we examined the effects of EPA pretreatment on the migration of both cell types. When ECs and SMCs were treated with EPA (5 micrograms/ml) for 2 days, the EPA content increased from 0.55 +/- 0.04% to 11.72 +/- 0.19% and 1.22 +/- 0.09% to 9.69 +/- 0.07% in cellular phospholipids, respectively. Although pretreatment of the ECs with EPA caused a significant increase in serum-induced cell migration, pretreatment of SMCs had no effect. If both cell types were concomitantly pretreated with EPA and cholesterol-rich liposomes, EPA abolished the effects of cholesterol on the migration of both cell types, but did not affect the content of cholesterol in both cells. These data indicate the possibility that EPA counteracts the atherogenic effect of cholesterol on EC and SMC migration.