ABSTRACT
The nuclear orphan receptor human estrogen receptor-related receptor (ERR)-alpha is implicated in bone metabolism. We studied the effect of ERRalpha silencing in human mesenchymal stem cells (hMSCs) during osteoblastogenesis. We found that ERRalpha silencing led to an increase of bone sialoprotein and a decrease of osteopontin mRNA levels, suggesting enhanced osteoblastic differentiation. This was confirmed by an increased ability of hMSCs to deposit calcium. Concomitantly, knockdown of ERRalpha inhibited adipogenesis, resulting in a decrease in adipocyte number and adipocyte marker gene expression. In line with a negative role of ERRalpha in bone metabolism, we found that adult female and male ERRalpha-deficient mice displayed a moderate increase in femoral cancellous bone volume and density. Osteoblast surface was increased and marrow fat volume decreased in these animals. Furthermore, ERRalpha-deficient osteoblasts displayed increased differentiation properties in vitro in line with our observations in hMSCs. In summary, we identified a role for ERRalpha in bone mass regulation by affecting osteoblastic differentiation.
Subject(s)
Adipocytes/cytology , Bone and Bones/cytology , Cell Differentiation , Mesenchymal Stem Cells/metabolism , Osteoblasts/cytology , Receptors, Estrogen/metabolism , Adipocytes/metabolism , Adipogenesis , Animals , Bone Density , Bone Marrow/anatomy & histology , Cell Line , Cell Lineage , Female , Gene Knockdown Techniques , Gene Silencing , Humans , Lentivirus , Male , Mice , Osteoblasts/metabolism , Phenotype , ERRalpha Estrogen-Related ReceptorABSTRACT
Membrane associated and secreted proteins are translated as precursors containing a signal peptide that allows protein-insertion into the membrane of the endoplasmic reticulum and is co-translationally removed in the lumen. The ability of the signal peptide to direct a polypeptide into the secretory pathway is exploited in methods developed to select cDNAs encoding such proteins. Different strategies are known in which cDNA libraries can be screened for signal peptides by the ability of the latter to rescue the translocation of signal sequence-less proteins. In one method, a cDNA library is tested for interleukin 2 receptor alpha chain translocation to the membrane in COS cells, in another one for invertase secretion from yeast. In this work, we compared the two systems by testing six mouse signal peptides in COS and yeast cells. All of them were functional in the mammalian system, whereas only three of them in yeast. Two other sequences needed the 5' cDNA sequence flanking the ATG codon to be removed in order to enable protein translocation. Although the structure of signal sequences and the functioning of the secretory machinery are well conserved from prokaryotes to eukaryotes, it seems evident that not all signal peptides can be interchanged between different proteins and organisms. In particular, signal peptides that are functional in the mammalian system do not necessarily lead to protein translocation in yeast.
Subject(s)
Glycoside Hydrolases/metabolism , Membrane Proteins/metabolism , Peptides/metabolism , Protein Sorting Signals/physiology , Receptors, Interleukin-2/metabolism , Yeasts/metabolism , Animals , Base Sequence , COS Cells/cytology , Endothelium, Vascular/metabolism , Eukaryotic Cells/metabolism , Gene Library , Mammals , Mice , Molecular Sequence Data , Protein Transport/physiology , Species Specificity , Yeasts/cytology , beta-FructofuranosidaseABSTRACT
Neovascularization is a prerequisite for tumor growth. Thus, selective destruction of the tumor vasculature should prevent tumor expansion. We have established a method to identify proteins that are specifically expressed on the surface of endothelial cells in tumors. CD31-positive endothelial cells were isolated from Lewis lung carcinoma lung metastases as well as from normal lung tissue. cDNAs derived from these cells were subjected to a subtractive hybridization procedure, and cDNAs overrepresented in tumor-derived endothelial cells were isolated; those encoding surface proteins were selected using a signal sequence trap assay. One isolated cDNA encoded H/T-cadherin. In this report, we show that mouse H/T-cadherin is overexpressed on endothelial cells of several tumors, whereas it is expressed only on a subset of endothelial cells in healthy organs. On the basis of the expression of H/T-cadherin in lung metastases of different tumors, we suggest that different tumors can have a differential influence on the expression of endothelial cell surface proteins.
Subject(s)
Cadherins/biosynthesis , Carcinoma, Lewis Lung/blood supply , Endothelium, Vascular/metabolism , Neovascularization, Pathologic/metabolism , Amino Acid Sequence , Animals , Cadherins/genetics , Carcinoma, Lewis Lung/genetics , Carcinoma, Lewis Lung/secondary , DNA, Complementary/genetics , DNA, Complementary/metabolism , DNA, Neoplasm/genetics , DNA, Neoplasm/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Nude , Molecular Sequence Data , Neoplasm Transplantation , Platelet Endothelial Cell Adhesion Molecule-1/biosynthesis , Tumor Cells, CulturedABSTRACT
Monoclonal antibody (Ab) directed against the vascular endothelial growth factor, one of the major inducers of angiogenesis, can inhibit tumor growth in mice. Treatment of cancer patients with monoclonal Ab requires large-scale production of the clean Ab and frequent application of the Ab. This might be improved by using single-chain Ab fragments (scFvs), which can be produced in large quantities in bacteria and are attractive for gene therapeutic approaches. Here we describe anti-vascular endothelial growth factor scFvs derived from a human phage-display library able to block the vascularization of the chorioallantoic membrane of chick embryos and reduce the growth of s.c. tumors in nude mice. This work opens the way to develop gene therapy-based strategies using a scFv to treat angiogenesis-dependent diseases.
