Role of LRP1b in cancer and atherosclerosis

Low-density lipoprotein receptor-related protein 1b (LRP1b) is a recently discovered member of the low-density lipoprotein (LDL) receptor family, which consists of structural similar transmembrane proteins with multiple functions including ligand uptake and intracellular signaling. The LRP1b gene is inactivated in several human tumors and LRP1b was therefore postulated as a potential tumor suppressor. LRP1b is a very large (600 kDa) transmembrane protein with a restricted expression in murine (brain, testis, adrenal gland) and human (brain, thyroid gland, skeletal muscle) tissues. Previous studies, including the generation of LRP1b knockout mice and the identification of some intracellular and extracellular ligands, failed to unequivocally assign a specific function to this receptor. In this proposal I suggest four different strategies to increase our understanding of the functions of LRP1b. The study of the receptor is hampered by the lack of cell lines expressing substantial amounts of LRP1b. Therefore, a recombinant full-length LRP1b receptor will be constructed and expressed in immortalized human cell lines. The proliferation and migration characteristics of stably transfected cells will be analyzed compared to mock transfected cells. Alternatively, a recently identified ovarian carcinoma cell line (OVCAR-3) with high endogenous LRP1b expression will be used. Furthermore, using FLAG-tagged versions of LRP1b constructs, transmembrane and intracellular binding partners will be identified. Secondly, novel circulating ligands will be purified from plasma by affinity chromatography using immobilized recombinant LRP1b ectodomains. The binding of identified ligands will then be verified by immunoprecipitation and the affinity of the interactions will be measured. Most previous studies of LRP1b inactivation in tumors have been performed with cultured tumor cell lines. To exclude cell culture artifacts, tumor cells from malignant pleural and ascitic effusions will be tested directly for inactivation of LRP1b using RT-PCR, PCR from somatic DNA and Western blotting. The alterations of the LRP1b gene will be correlated to clinical data and other tumor markers. Like other LDL receptor family members (LDL receptor, LRP1, VLDL receptor), LRP1b has recently been shown to be expressed by smooth muscle cells in the arterial wall, where it may play an important role in the in the formation of atherosclerotic lesions by modifying the proliferation of smooth muscle cells analogous to its postulated role in tumor formation. To investigate a possible role of LRP1b in atherosclerosis, the expression of the receptor in cells of the arterial wall (endothelial cells, smooth muscle cells, and macrophages) will be measured quantitatively by RT-PCR. The binding of lipoproteins (LDL, beta-VLDL, HDL) to LRP1b and the effect of ligands on the proliferation and migration of these cells will be investigated. Together, these experiments are expected to considerably extend our current understanding of the physiological function of LRP1b as well as the role of this receptor in cancer and atherosclerosis, both major health burdens.

Atherosclerosis and cancer are undisputedly the leading causes of death worldwide. The aim of this research project was the characterization of the LRP1b, a protein found on the surface of cells, in these two devastating diseases. LRP1b is a member of a protein family (LDL receptor family) with important roles in the formation of atherosclerotic lesions and was originally discovered during studies of lung cancer cell lines. These experiments showed frequent inactivation of LRP1b in these cell lines and the receptor was therefore postulated as potential tumor suppressor. To investigate the specific functions of LRP1b in a cell culture model, the genetic information coding for LRP1b (cDNA) was planned to be isolated, packed into a vector and transferred into human cells. The main obstacle for this approach was the enormous size of the receptor. LRP1b with its 4600 amino acids encoded by 13800 nucleotides is one of the largest single peptide chain proteins. Nevertheless, we succeeded in constructing an LRP1b expression vector construct and were successful in transferring it into human cells. Initial results showed an inhibition of cellular growth by LRP1b consistent with the proposed tumor suppressor function. The second aim was the identification of soluble ligands of the receptor from human plasma, which was performed using fragments of the extracellular part of LRP1b. These studies led to the identification of clusterin, a protein found on HDL lipoproteins, and fibrinogen, an important factor in blood coagulation, as ligands of LRP1b. The third set of experiments comprised the detection of LRP1b in tumor cells with high sensitivity. This was performed using specific probes to measure the messenger RNA (mRNA) responsible for the synthesis of the receptor. Using this method, tumor cell lines with high, moderate and absent LRP1b expression were identified. Finally, binding of LRP1b to different lipoproteins was investigated. These experiments were performed with fragments of the extracellular part of LRP1b and showed binding of VLDL and HDL lipoproteins to the receptor. In addition, internalization of VLDL into LRP1b carrying cells could be demonstrated. A further important result of the research project was the analysis of LRP1b tissue distribution. Here we found a restricted expression of the receptor confined to few tissues (brain, thyroid gland, skeletal muscle, and testis). In summary, the work performed in this research project has yielded several important insights for our understanding of the role of LRP1b in cancer and atherosclerosis which may help in diagnosis and therapy of these diseases in the future.