Wiebe Olijve

Comprehensive microarray analysis of bone morphogenetic protein 2-induced osteoblast differentiation resulting in the identification of novel markers for bone development

Osteoblasts are cells responsible for matrix deposition during bone development and although temporal expression of many genes has been related to osteoblast differentiation, a complete description of osteoblast‐specific gene regulation will lead to a better understanding of osteoblast function. In this study, microarray technology was used to analyze gene expression on a broad scale during osteoblast differentiation. Expression analysis of 9596 sequences revealed 342 genes and expressed sequence tags (ESTs) to be modulated differentially during a time course experiment in which murine C2C12 mesenchymal progenitor cells were induced to differentiate into mature osteoblasts by treatment with bone morphogenetic protein 2 (BMP‐2). By means of hierarchical clustering, these genes were grouped by similarities in their expression profiles, resulting in subsets of early, intermediate, and late response genes, which are representative of the distinct stages of osteoblast differentiation. To identify new bone markers, the bone specificity of the late response genes was determined by comparing BMP‐induced expression in C2C12 and MC3T3 osteoblasts with that in NIH3T3 fibroblasts. This resulted in the identification of nine novel genes and ESTs that were induced specifically in osteoblasts, in addition to the well‐known markers ALP and osteocalcin. For at least one of these novel genes, Wnt inhibitory factor 1, and two of the ESTs, expression in developing bone was verified in vivo by in situ hybridization of E16.5 mouse embryos. In conclusion, by a combination of in vitro and in vivo screening approaches, a set of new genes related to osteoblast differentiation and skeletal development has been identified.

Identification of novel regulators associated with early-phase osteoblast differentiation.

Key regulatory components of the BMP‐induced osteoblast differentiation cascade remain to be established. Microarray and subsequent expression analyses in mice identified two transcription factors, Hey1 and Tcf7, with in vitro and in vivo expression characteristics very similar to Cbfa1. Transfection studies suggest that Tcf7 modulates BMP2‐induced osteoblast differentiation. This study contributes to a better definition of the onset of BMP‐induced osteoblast differentiation.

Microarray analysis of bone morphogenetic protein, transforming growth factor β, and activin early response genes during osteoblastic cell differentiation

Bone morphogenetic protein (BMP) 2, a member of the transforming growth factor (TGF) β family, is a potent regulator of osteoblast differentiation. In addition, both TGF‐β and activin A can either induce bone formation or inhibit bone formation depending on cell type and differentiation status. Although much is known about the receptors and intracellular second messengers involved in the action of TGF‐β family members, little is known about how selectivity in the biological response of individual family members is controlled. In this study, we have investigated selective gene induction by BMP‐2, TGF‐β1 and activin A in relation to their ability to control differentiation of mouse mesenchymal precursor cells C2C12 into osteoblastic cells. TGF‐β1 can inhibit BMP‐2‐induced differentiation of these cells, whereas activin A was found to be without morphogenetic effect. Using a gene expression microarray approach covering 8636 sequences, we have identified a total of 57 established genes and expressed sequence tags (ESTs) that were either up‐regulated or down‐regulated 2 h after treatment with at least one of these three stimuli. With respect to the established genes, 15 new target genes for TGF‐β family members thus were identified. Furthermore, a set of transcripts was identified, which was oppositely regulated by TGF‐β1 and BMP‐2. Based on the inverse biological effects of TGF‐β1 and BMP‐2 on C2C12 cells, these genes are important candidates for controlling the process of growth factor‐induced osteoblast differentiation.

Gene Array Analysis of Bone Morphogenetic Protein Type I Receptor-Induced Osteoblast Differentiation†

The genomic response to BMP was investigated by ectopic expression of activated BMP type I receptors in C2C12 myoblast using cDNA microarrays. Novel BMP receptor target genes with possible roles in inhibition of myoblast differentiation and stimulation of osteoblast differentiation were identified.

Human serotonin 5‐HT7 receptor: cloning and pharmacological characterisation of two receptor variants

Two splice variants of the 5‐HT7 receptor were identified in human brain that differ in the lengths of their intracellular carboxy terminal tail. Identification of the variants of this receptor is of particular interest since the 5‐HT7 receptor is known to have a high affinity for a number of antidepressants and is localized in brain regions thought to be implicated in depression. The two isoforms are expressed in roughly equal amounts in various regions of the human brain. When expressed in NIH‐3T3 cells, both variants encode functional 5‐HT7 receptors, positively coupled to adenylyl cyclase. We suggest that both variants are derived from a single gene by alternative mRNA splicing. Furthermore, our results from Southern blot analysis studies suggest that additional 5‐HT7 receptor genes may exist in human.

Genomic organization, coding sequence and functional expression of human 5-HT2 and 5-HT1A receptor genes

The family of serotonin receptors consists of at least eight distinct subtypes, divided into four classes based on their pharmacological and functional characteristics. Here we report the cloning and expression in Swiss 3T3 cells of the human 5-HT2 and 5-HT1A receptor subtypes. Both genes encode functional receptors for 5-HT, that differ considerably in genomic structure, primary amino acid sequence, pharmacology and signal transduction. The 5-HT1A receptor transfectants displayed a single high affinity site for the agonist [3H](+/-)-8-hydroxy-2-(di-n-propylamino)tetralin HBr ([3H]8-OH-DPAT) and a pharmacological profile specific for the 5-HT1A receptor. In these transfectants, 5-HT mediated a dose-dependent inhibition of forskolin-stimulated cAMP levels. Cells expressing the 5-HT2 receptor exhibited high affinity binding for the antagonist [3H]ketanserin with a 5-HT2 receptor specific pharmacological profile. In these cells 5-HT activated phospholipase C in a dose-dependent manner. The 5-HT2 receptor displayed a genomic organization quite different from the 5-HT1A, 5-HT1B and 5-HT1D receptor subtypes. While these receptors are encoded by one single exon, the 5-HT2 receptor is encoded by three exons separated by two introns. The latter finding adds and additional molecular criterion for receptor classification.

Molecular cloning and characterization of a novel orphan receptor (P2P) expressed in human pancreas that shows high structural homology to the P2U purinoceptor

Here we report the cloning of a gene encoding a new member of the superfamily of G protein‐coupled receptors. The gene encodes a protein of 365 amino acids closely resembling two recently cloned nucleotide binding receptors, called P2U and P2Y purinoceptors (71% and 49% sequence identity within the transmembrane domains, respectively). Our studies show that this new putative purinoceptor (designated P2P) is encoded by an intronless single copy gene that is exclusively expressed in pancreas, in contrast to the P2U and the P2Y purinoceptors which are widely distributed throughout the periphery. The identification of a pancreas‐specific human putative P2 purinoceptor makes it attractive to speculate that the reported actions of ADP/ATP analogues in pancreas on insulin secretion are mediated through this receptor.

Genomic organisation and functional expression of the gene encoding the human serotonin 5-HT2C receptor

The 5-HT2C receptor gene is unique among the members of the 5-HT receptor family by virtue of its genomic organisation. The human 5-HT2C receptor gene, unlike many other genes for guanine nucleotide binding (G)-proteins, contains three introns which interrupt the coding sequence into four exons. The first two introns are at equivalent positions as compared to the intervening sequences previously found in the 5-HT2(A) receptor gene, suggesting a close evolutionary relationship between both genes. Southern blot analysis shows that the 5-HT2C receptor gene is a single copy gene. Furthermore, we report the functional expression of a complementary DNA for the 5-HT2C receptor, cloned from hippocampal RNA. Membranes prepared from NIH 3T3 cells stably expressing the 5-HT2C receptor cDNA, displayed a single population of high affinity sites for the antagonist [3H]mesulergine (Kd = 2.9 +/- 0.4 nM, Bmax = 44.3 +/- 7.2 pmol/mg protein) as well as for [3H]5-HT (Kd = 9.9 +/- 0.7 nM, Bmax = 13.6 +/- 1.0 pmol/mg protein). Displacement of [3H]mesulergine and [3H]5HT binding by ligands indicated a pharmacological similarity of these binding sites with porcine and rat choroid plexus 5-HT2C receptors. Furthermore, activation of the 5-HT2C receptor with 5-HT results in an increased phospholipase C activity.

A new approach to the generation of human or murine antibody producing hybridomas.

A new and very efficient method for the generation of human and murine monoclonal antibodies has been developed. The method is based on clonal expansion of single B cells in the presence of human T cell supernatant and irradiated murine thymoma helper cells. Subsequently, the clonally expanded B cells are immortalized by electric field mediated cell fusion. The high efficiency of the method permits the processing of small numbers of lymphocytes, e.g., obtained by preselection of specific B cells, small amounts of human donor material and murine PBL or lymph node cells. The method may be an alternative for the EBV-transformation technique used for the generation of human monoclonal antibodies, which immortalizes only a subset of B cells and frequently yields poorly growing or unstable cell lines. This report describes the generation of murine anti-HIV and human anti-rubella antibodies combining the clonal expansion of B cells and mini-electrofusion.

Immortalizaton of antigen selected B cells

This paper reports the generation of monoclonal antibody producing hybridomas from a small number of antigen-specific B cells selected by panning on antigen-coated dishes and rosetting with antigen-coupled paramagnetic beads. Anti-HIV positive B cells from spleen could be recovered by panning with an efficiency of 5% and a purity of 24%. Immunobead selection of anti-HIV positive B cells from the same mice yielded a recovery of 17% and a purity of 7%. Various experimental conditions with respect to the selection of specific B cells were investigated, leading to an optimized protocol for the isolation of a limited subset of B cells. The selected cells retained their property to produce immunoglobulins and could be clonally expanded in the presence of human T cell supernatant and irradiated murine thymoma helper cells to generate sufficient cells for a mini-electrofusion with NS-1 myeloma cells. Up to 78 specific hybridomas could be generated from one anti-HIV positive B cell. An overall efficiency of specific B cell immortalization of up to 10% was obtained.