Study On The Variation And Function Of Very Low Density Lipoprotein Receptor Subtypes In Cancer Cells

Very low density lipoprotein receptors (VLDLR), a member of low density lipoprotein receptor (LDLR) superfamily, consist of two subtypes, type I VLDLR and type II VLDLR. It is generally accepted that VLDLR plays a major role in the metabolism of triglyceride through binding lipoproteins enriched in apoE and has an intimate relation with atherosclerosis. There have been many attempts to study the biological phenotype in homozygous VLDLR knockout mice. However, the physiological and pathological importance of these receptors has not been clearly identified. Recently, VLDLR and many other members of the low density lipoprotein receptor family are found to bind different ligands besides lipoproteins, causing endocytosis and affecting many cellular functions. For example, VLDLR impacts the immigration and location of nerve cells during the early stages of embryonic development by binding the signaling molecule Reln. It can also inhibit cell proliferation by interacting with tissue factor pathway inhibitor (TFPI). In addition, VLDLR plays a certain role in the invasion and metaptosis of tumor cells. And now, VLDLR and the other members of LDLR are known as the multifunctional receptor like"Swiss army knife". Nevertheless, the functional differences between the two VLDLR subtypes need to be further clarified, especially the distinctive biological function of typeⅡVLDLR lacking the o-linked sugar domain has not been illuminated.Studies about the distribution of these two VLDLR subtypes suggested that their distribution presents obvious tissue- and cell-specificity. Type I VLDLR is most highly expressed in heart, skeletal muscle and adipose tissue with active fatty acid metabolism, while type II VLDLR is predominant in non-muscle tissue, including kidney, spleen, adrenal gland, et al. Recent studies have shown that the two VLDLR subtypes are associated with the differentiation and development of tissues and cells. The expression pattern of the two VLDLR subtypes can be changed during embryonic development of chicken and human brain. The type II VLDLR has been found to be the major receptor expressed in early phase of embryonic or fetal brain development, whereas type I VLDLR is mainly present in adult tissues. Other reports indicate that some tumor tissues and cells also express two VLDLR subtypes with inhomogeneity. The expression of the type II VLDLR increases obviously in poorly differentiated adenocarcinomas. Our previous study has also found that the type II VLDLR is mainly expressed in poorly- or moderately- differentiated human gastric adenocarcinoma cell lines, but its expression is relatively low or even can not be detected in well differentiated human gastric adenocarcinoma cell lines. Our recent studies also found that the expression of type II VLDLR is higher in uterine cervix cancer tissues than that in adjacent tissues. Type I VLDLR is the major receptor in senile plaques of Alzheimer diseased brain and type II VLDLR in congestive fibrotic spleen disappeared from the patients with liver cirrhosis. Genomic loss and epigenetic silencing of very-low-density lipoprotein receptor involved in gastric carcinogenesis and the O-linked sugar domain of VLDLR was demonstrated to relate with cell growth inhibition. These studies suggest that the type II VLDLR activities may be related to certain cellular functions other than its involvement in lipoprotein metabolism.Tissue factor pathway inhibitor (TFPI) and urokinase-type plasminogen activator and plasminogen activator inhibitor 1 (uPA-PAI-1) complex are the ligands of VLDLR, which affect different cell function through VLDLR. But whether VLDLR affect cellular function via the expression variability of two VLDLR subtypes is still unknown.Thus, we explored the expression and function of type II VLDLR during the induction of human gastric adenocarcinoma cell line SGC7901 and in cells treated with two ligands of VLDLR to explore the relationship of type II VLDLR with cell function. To investigate the relationship between the expression variability of two VLDLR subtypes and cellular functions during the induction of SGC7901 cells, we use all-trans retinoic acid (ATRA) to induce SGC7901 differentiation and phorbol-12-myristate-13-acetate (PMA) to induce a change of differentiation to relatively lower. The mRNA expression of Telomerase reverse transcriptase (TERT) acts as a marker to identify the extent of cellular differentiation. The expression of two subtypes of VLDLR after treatment was detected by western blotting. The cells became well differentiated when induced by ATRA, accompanied by decrease in expression of type II VLDLR and gradually attenuated cell proliferation and migration. However, the cells became poorly differentiated when induced by PMA. These cells had increased receptor expression, and enhanced cell proliferation and migration. Our data indicate that the increased expression or the activity of type II VLDLR may be associated with the poor differentiation, and the enhanced proliferation and migration of the cells. Then, in order to further understand the expression of two VLDLR subtypes and the cellular function, we use two VLDLR ligands, uPA-PAI-1 complex and TFPI, which can affect cell proliferation and migration, to incubate with SGC7901 cells. In this study, we showed that cell proliferation and migration were inhibited by TFPI, but promoted by uPA-PAI-1 complex. In addition, we also demonstrated that TFPI treatment caused a decrease, but uPA-PAI-1 complex caused an increase, in the expression of type II VLDLR, suggesting that increasing type II VLDLR activity might be associated with augmenting cell proliferation and migration. In conclusion, the expression of type II VLDLR had a general phenomenon during the differentiation of cancer cells: the expression of type II VLDLR increased in lowly-differentiated cells with high proliferation and migration, but it decreased in highly-differentiated cells with low proliferation and migration. These indicate that the increased expression or the activity of type II VLDLR may be associated with the poor differentiation, and the enhanced proliferation and migration of the cells.It was reported that during the development of nervous tissue, VLDLR mediates Reelin-Dab1 signal pathway, modulates tau phosphorylation through glycogen synthase kinase-3beta cascade and affects tissue remodeling and cell migration. Recent study indicates that VLDLR is a negative regulator of the wnt signaling pathway. These studies suggest that VLDLR may play an important role in wnt signal pathway. In addition VLDLR can bind with different ligands relative with proliferation, regulate different signal pathway, and affect cell function. The binding of VLDLR and urokinase-type plasminogen activator and plasminogen activator inhibitor 1 (uPA-PAI-1) complex can sustain the phosphorylation of extracellular signal-regulates kinase (ERK) to promote cell proliferation and migration. But VLDLR binding with TFPI can inhibit cell proliferation through activating p38 signal pathway. The two ligands appear to have quite different effects on cell function through VLDLR. It was reported that the activation of ERK can induce the expression ofβ-catenin, which promote the transcription of certain target genes inclding matrix metalloproteinase (MMPs). These studies suggest that the effect of VLDLR on cell function may be related with mitogen-activated protein kinases (MAPK) signal pathway and wnt signal. But it was unclear whether the two VLDLR subtypes were regulated differently and affected cell function through dinstinct signal pathway.It was speculated that the role of type II VLDLR may be related with MAPK and wnt signal pathway. So we observe the possible signal pathway involved in the role of type II VLDLR. Our results indicated that the well differentiated cells induced by ATRA and the cells treated with TFPI with a significant decrease in type II VLDLR expression accompanied by a gradually attenuatedβ-catenin and MMP-2 and MMP-9 expression, but in the poorly differentiated cells induced by PMA and in the cells treated with uPA-PAI-1 complex with an increase in type II VLDLR expression showed an increase inβ-catenin and MMP-2 and MMP-9 expression. These suggested that the role of type II VLDLR could be related with the aggregation of intracellularβ-catenin, which promotes some specific target genes transcription.In our study, uPA-PAI-1 complex can rapidly activate the ERK phosphorylation of SGC7901 cells after 5 min incubation and it can sustain for at least 30 min. The phosphorylation of ERK1 can last for 1 h obviously. Thise was agreed with previous study. But TFPI can inhibit the phosphorylation of ERK. Studies about LDLR found that stress-activated p38 MAPK regulates LDL receptor expression via negatively modulation of p42/44 MAPK cascade. So it was speculated that TFPI inhibited ERK through activating p38 MAPK. These results suggest that the ligand promoting cell proliferation and migration can activate ERK through type II VLDLR; but the ligand inhibiting cell proliferation and migration can reduce the phosphorylation of ERK through type I VLDLR. Our studies suggested that the role of type II VLDLR on cell function may be related with the activation of ERK, then induce the aggregation ofβ-catenin, promoting the transcription of some specific down-stream genes to affect cell function. These suggested that type II VLDLR may play an important role in promoting cell proliferation and migration, and inhibiting cell differentiation.In conclusion, type II VLDLR may bind and internalize specific ligand, mediate the relative signal pathway, and affect cell function. Our study revealed the relationship between type II VLDLR and cellular function and the possible related signal pathway, which provided profound views of VLDLR function and new cognition of lipoprotein receptor as a cellular Swiss army knife.