Up-regulation of VLDL receptor expression and its signaling pathway induced by VLDL and beta-VLDL.

Very low density lipoprotein receptor (VLDLR) is thought to participate in the pathogenesis of atherosclerosis induced by VLDL and beta-VLDL. The present study was undertaken to elucidate the effects of VLDL and beta-VLDL on VLDLR expression and its signaling pathway. RAW264.7 cells were incubated with VLDL and beta-VLDL. The expression of VLDLR mRNA was detected by RT-PCR. The transcriptional activity of VLDLR gene was detected in recombinant plasmid pGL4.2VR-luciferase transfected RAW264.7. Western blot assay was used to detect the changes of phosphorylated ERK1/2 protein. Inhibitors or activators were used to observe the signal pathway involving VLDLR expression regulation. The results showed that VLDL and beta-VLDL stimulated ERK1/2 activity in a PKC-dependent manner. VLDL or beta-VLDL-induced VLDLR expression on macrophages was extremely abolished by inhibitors ERK1/2 or PKC. Our findings revealed that VLDL or beta-VLDL-induced VLDLR expression via PKC/ERK cascades and the effect was linked to the transcriptional activation of VLDLR gene promoter.

Up-regulation of VLDL Receptor Expression and Its Signaling Pathway Induced by VLDL and β-VLDL / 华中科技大学学报（医学）（英德文版）

Very low density lipoprotein receptor (VLDLR) is thought to participate in the patho-genesis of atherosclerosis induced by VLDL and β-VLDL. The present study was undertaken to elu-cidate the effects of VLDL and β-VLDL on VLDLR expression and its signaling pathway. RAW264.7 cells were incubated with VLDL and β-VLDL. The expression of VLDLR mRNA was detected by RT-PCR. The transcriptional activity of VLDLR gene was detected in recombinant plasmid pGL4.2VR-luciferase transfected RAW264.7. Western blot assay was used to detect the changes of phosphorylated ERK1/2 protein. Inhibitors or activators were used to observe the signal pathway in-volving VLDLR expression regulation. The results showed that VLDL and β-VLDL stimulated ERKI/2 activity in a PKC-dependent manner. VLDL or β-VLDL-induced VLDLR expression on macrophages was extremely abolished by inhibitors ERKI/2 or PKC. Our findings revealed that VLDL or β-VLDL-induced VLDLR expression via PKC/ERK cascades and the effect was linked to the transcriptional activation of VLDLR gene promoter.

The inhibitory effects of an antisense u-PAR vector on invasion of highly invasive human prostate carcinoma PC-3M cell subclones.

To observe the inhibitory effects of an antisense u-PAR vector on invasion of highly invasive PC-3M cell subclones, the effects of the antisense u-PAR on activity of MMP-9 in those highly invasive cell subclones were detected by a quantitative RT-PCR and zymography. The monolayer invasion assay and colony formation assay in soft agar were used. And tumorigenesis rate and invasions by the cell subclones with or without the antisense u-PAR were observed in nude mice. It was found that in vitro growth of highly invasive PC-3M cell subclones transfected with the antisense u-PAR was declined, and the ability of anchorage-independent growth of those cell subclones was found decreased sharply, with the inhibiting rate becoming 79% and 60%, respectively. Although the antisense u-PAR didn't change MMP-9 gene transcription, they could inhibit the activation of MMP-9 of highly invasive PC-3M cell subclones. Moreover, the tumorigenesis rate of the cell subclones with the antisense u-PAR decreased and the growth of a neoplasm also slowed down. The t tests showed the difference between experimental and control groups was statistically significant (P < 0.01). The antisense u-PAR vector could not only inhibit the invasion ability of highly invasive PC-3M cell subclones in vitro but also restrain the growth of those cell subclones in vivo.

Construction of antisense RNA expression plasmid for u-PAR and its transfection to highly invasive PC-3M cell subclones.

To evaluate the specific inhibition of antisense u-PAR on the u-PAR expressions in highly invasive cell subclones and to determine its blocking function in the invasion by those cells, a cDNA fragment of u-PAR obtained by RT-PCR was inserted into a plasmid vector named pcDNA3 in antisense orientation. Then the antisense u-PAR recombinant was transfected into highly invasive cell subclones. The u-PAR expression in neo-resistant cells was examined by RT-PCR and immunohistochemical assay. Compared to the control cells, the content of mRNA and protein of u-PAR in transfected cells decreased sharply, and the rate of inhibition was 53% and 73%, respectively, indicating that an antisense u-PAR might have played a specific inhibitory role in its expression in the cells, which may provide a good cell model for making further investigation of the inhibitory effects of the antisense u-PAR on invasion in highly invasive cell subclones of human prostate carcinoma.

[Effects of induced deletion of repeats in binding domain of the VLDL receptor on its ligand-binding capacity].

The ligand-binding domain of the very low-density lipoprotein receptor (VLDL-R) contains eight cysteine-rich repeat sequences that have been postulated as ligand-binding sites. This is obviously different from that of low-density lipoprotein receptor (LDL-R) that includes seven similar repeats. To make clear the contribution of these repeats to ligand-binding and to explore the reason of both receptors' ligand-binding characteristic, the VLDL-R recombinants lacking different repeat(s) were constructed by oligonucleotide-directed mutagenesis and transfected into ldl-A7 cell. Ligand-binding results showed that repeat 1 and repeat 2 were the most important in binding with apoE-rich lipoprotein(VLDL and beta-VLDL). Repeat 3 and repeat 6 also important for binding VLDL. The results also showed that VLDL-R lacking LBR7 retained partly LDL-R ligand-binding properties. It suggests that LBR7 in VLDL-R may responsible for both receptors' ligand-binding properties differences.