Wen Chang

Comparative effect of lipoxygenase products of arachidonic acid on rat aortic smooth muscle cell migration.

We investigated the effects of mono-hydroxyeicosatetraenoic acids (HETEs) and N-formyl-methionyl-leucyl-phenylalanine (F-Met-Leu-Phe) on rat aortic smooth muscle cell migration in modified Boyden chambers. 12-HETE showed the most potent stimulatory effect on smooth muscle cell migration among the mono-HETEs tested. The optimal concentrations for cell migration were 3 X 10(-15) and 3 X 10(-13) g/ml for 12-HETE and 10(-8) g/ml for 15-HETE, 5-HETE and F-Met-Leu-Phe were inactive with these cells. As 12-HETE is biosynthesized from arachidonic acid by the 12-lipoxygenase pathway in platelets and macrophages, and 15-HETE by the 15-lipoxygenase pathway in granulocytes, the present results indicate an important role for such cells in the early phase of atherosclerosis.

Nuclear translocation of epidermal growth factor receptor by Akt-dependent phosphorylation enhances breast cancer-resistant protein expression in gefitinib-resistant cells

Epidermal growth factor receptor (EGFR), an aberrantly overexpressed or activated receptor-tyrosine kinase in many cancers, plays a pivotal role in cancer progression and has been an attractive target for cancer therapy. Gefitinib and erlotinib, two EGFR-tyrosine kinase inhibitors, have been approved for non-small cell lung cancer. However, durable clinical efficacy of these EGFR inhibitors is severely limited by the emergence of acquired resistance. For example, the expression of breast cancer-resistant protein (BCRP/ABCG2) has been shown to confer acquired resistance of wild-type EGFR (wtEGFR)-expressing cancer cells to gefitinib. However, the underlying molecular mechanisms still remain unclear. Here, we show that wtEGFR expression is elevated in the nucleus of acquired gefitinib-resistant cancer cells. Moreover, nuclear translocation of EGFR requires phosphorylation at Ser-229 by Akt. In the nucleus, EGFR then targets the proximal promoter of BCRP/ABCG2 and thereby enhances its gene transcription. The nuclear EGFR-mediated BCRP/ABCG2 expression may contribute at least in part to the acquired resistance of wtEGFR-expressing cancer cells to gefitinib. Our findings shed light on the role of nuclear EGFR in the sensitivity of wtEGFR-expressing cancer cells to EGFR tyrosine kinase inhibitors and also deciphered a putative molecular mechanism contributing to gefitinib resistance through BCRP/ABCG2 expression.

Functional cooperation of simian virus 40 promoter factor 1 and CCAAT/enhancer-binding protein β and δ in lipopolysaccharide-induced gene activation of IL-10 in mouse macrophages

Previous studies have revealed that LPS can activate transcription of the IL-10 gene promoter through an SV40 promoter factor 1 (Sp1) binding site in mouse macrophage RAW264.7. In this study, we determined that, in addition to Sp1, C/EBPβ and δ were also involved in LPS-induced gene expression of IL-10. By transient transfection with 5′-deletion mutants of the IL-10 promoter, we found that there were two LPS-responsive elements in the promoter of the mouse IL-10 gene. Analysis of these two regions by gel shift assay suggested that Sp1 and C/EBPβ and δ were bound to these two regions, respectively. By site-directed mutagenesis, we found that disruption at both the Sp1 and C/EBP binding sites almost completely blocked the LPS response. By gel shift assay and Western blotting, we found that the DNA binding complex and protein expression of C/EBPβ and δ were increased by LPS treatment, but these results were not found for Sp1. Overexpression of C/EBPβ or C/EBPδ, respectively, activated the promoter of the IL-10 gene, and they were enhanced by LPS. Coimmunoprecipitation experiments in intact cells indicated that LPS stimulated interaction between Sp1 and C/EBPβ and δ. These results suggested that the interaction between Sp1 and C/EBPβ and δ induced by LPS cooperatively activated expression of the IL-10 gene. The increase of C/EBPβ and δ proteins and the enhancement of transactivation activity of C/EBPβ and δ by LPS treatment, at least in part, explain the activation of IL-10 gene expression.

Testosterone inhibits prostacyclin production by rat aortic smooth muscle cells in culture

The effects of testosterone on cell proliferation and prostacyclin production were investigated using rat aortic smooth muscle cells in culture. Testosterone at 10(-10)-10(-6) M did not have any significant effect on cell proliferation, but it significantly inhibited prostacyclin production by the cells. Maximal inhibition of prostacyclin production (70%) was observed when cells were treated with a physiological concentration of 19(-8) M testosterone for 5 consecutive days. These results suggest that testosterone may stimulate thrombus formation and accelerate atherosclerosis by suppressing prostacyclin production in arterial smooth muscle cells.

Phosphorylation by c-Jun NH2-terminal Kinase 1 Regulates the Stability of Transcription Factor Sp1 during Mitosis

The transcription factor Sp1 is ubiquitously expressed in different cells and thereby regulates the expression of genes involved in many cellular processes. This study reveals that Sp1 was phosphorylated during the mitotic stage in three epithelial tumor cell lines and one glioma cell line. By using different kinase inhibitors, we found that during mitosis in HeLa cells, the c-Jun NH(2)-terminal kinase (JNK) 1 was activated that was then required for the phosphorylation of Sp1. In addition, blockade of the Sp1 phosphorylation via inhibition JNK1 activity in mitosis resulted in the ubiquitination and degradation of Sp1. JNK1 phosphorylated Sp1 at Thr278/739. The Sp1 mutated at Thr278/739 was unstable during mitosis, possessing less transcriptional activity for the 12(S)-lipoxygenase expression and exhibiting a decreased cell growth rate compared with wild-type Sp1 in HeLa cells. In N-methyl-N-nitrosourea-induced mammary tumors, JNK1 activation provided a potential relevance with the accumulation of Sp1. Together, our results indicate that JNK1 activation is necessary to phosphorylate Sp1 and to shield Sp1 from the ubiquitin-dependent degradation pathway during mitosis in tumor cell lines.

Calcium dependency of aortic smooth muscle cell migration induced by 12-l-hydroxy-5,8,10,14-eicosatetraenoic acid: Effects of A23187, Nicardipine and Trifluoperazine

We have previously reported that 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), a 12-lipoxygenase product of arachidonic acid in platelets, is a potent chemoattractant for rat aortic smooth muscle cells. In the present study, the mechanism involved in 12-HETE-associated smooth muscle cell migration was investigated in relation to calcium mobilization in the cells. Migration of smooth muscle cells was measured by a filter membrane technique in modified Boyden chambers. Smooth muscle cell migration induced by 12-HETE increased with the increase of extracellular Ca2+ concentration and became maximal at the physiological Ca2+ concentration of 1.25 mM. The calcium ionophore A23187, at concentrations of 0.2 and 2.0 microM, significantly stimulated cell migration. Nicardipine, a potent calcium-entry blocker, significantly inhibited 12-HETE-associated smooth muscle cell migration at concentrations from 10(-9) to 10(-5) M. Concentrations of trifluoperazine from 10(-9) to 10(-5) M and W-7 at 10(-5) M, which are specific inhibitors of calmodulin, also significantly inhibited cell migration induced by 12-HETE. Cytochalasin B at 1.0 and 10 microM, and colchicine at 0.1 and 1.0 microM concentrations drastically inhibited cell migration, indicating that actin-containing microfilaments and microtubules are involved in smooth muscle cell migration. These findings indicated that the stimulation of smooth muscle cell migration by 12-HETE is a highly calcium-dependent process and suggest that 12-HETE might act at the initial stage of smooth muscle cell migration through enhancing calcium influx through plasma membrane and thus stimulating cell migration.

Platelets stimulate aortic smooth muscle cell migration in vitro: Involvement of 12-l-hydroxy-5,8,10,14-eicosatetraenoic acid

The migration of rat aortic smooth muscle cells was measured in modified Boyden chambers. Smooth muscle cells were motile in vitro and their migration was stimulated (time- and dose-dependently) by a platelet-derived factor. Treatment of platelets with indomethacin resulted in a significant increase in smooth muscle cell migration, whereas treatment with 5,8,10,14-eicosatetraenoic acid inhibited it. Purified 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid at a very low concentration (6 x 10(-15)-6 x 10(-13) g/ml) significantly stimulated smooth muscle cell migration. The locomotion induced by 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid was chemokinetic. These findings point to the physiological importance of a platelet 12-lipoxygenase product of arachidonic acid in the early phase of atherosclerosis.

A new prostaglandin transformed from arachidonic acid in carrageenin-induced granuloma.

Summary A main product (PII) transformed from arachidonic acid in carrageenin-induced granuloma in rats was structurally analysed. PII was converted to a more polar substance by the reduction of sodium borohydride. According to the analysis of gas chromatography-mass spectrometry, its structure was speculated to be 9α,11α,15α-trihydroxy-6-oxo-prostenoic acid.

Sumoylation of Specificity Protein 1 Augments Its Degradation by Changing the Localization and Increasing the Specificity Protein 1 Proteolytic Process

Although specificity protein 1 (Sp1) accumulation has been found in various tumor strains, its mechanism is still not very clear. Herein, we found that modification of Sp1 by SUMO-1 facilitates Sp1 degradation. Our findings revealed that, although the amounts of Sp1 and Sp1 mutant (K16R) [Sp1(K16R)] mRNA in cells were equal, the protein level of Sp1(K16R) was higher than that of wild-type Sp1. We also proved that this sumoylation site was not the residue at which ubiquitination occurred. Invitro and in vivo pull-down assays revealed that more sumoylated Sp1 was localized in the cytoplasm, and the interaction between SUMO-1-Sp1 and the proteasome subunit rpt6 in HeLa cells was enhanced. In addition, although Sp1 accumulated in the tumorous cervical tissue, it was not prone to sumoylation. Finally, by overexpression of HA (hemagglutinin)-SUMO-1-Sp1-myc, HA-Sp1-myc, and HA-Sp1(K16R), we found that modification of Sp1 by SUMO-1 was important for Sp1 proteolysis. In conclusion, modification of Sp1 by SUMO-1 altered its localization and then increased its interaction with rpt6. This interaction increased the efficiency of Sp1 proteolytic processing and ubiquitination and then resulted in Sp1 degradation. Therefore, sumoylation of Sp1 is attenuated during tumorigenesis in order to increase Sp1 stability.

Estradiol-binding sites in rat aortic smooth muscle cells in culture

To determine whether aortic smooth muscle cells possess estradiol receptors or not, an estradiol binding study has been performed in cultured rat aortic smooth muscle cells by using an intact-cell assay. Specific binding for estradiol is shown by the cells, and the binding reaches a plateau after 15 min of incubation at 37 degrees C. The binding curve shows a saturable binding process. Scatchard analysis reveals a single population for estradiol binding with a dissociation constant of 5.0 X 10(-8) M and specific binding sites of 64 fmole per 10(6) cells (38,500 sites per cell). Molecular specificity shows highly specific binding with estradiol. Among various steroids tested, only testosterone at 10(-5) M competes slightly (9.3%) with the estradiol-specific binding. These data indicate the existence of specific, high-affinity and limited-capacity binding sites for estradiol in rat aortic smooth muscle cells. This accords with the concept that estrogen responsiveness in aortic smooth muscle cells is mediated through the estrogen-receptor binding in the cells.