Alexander C. Lichtler - US grants

Type 1 collagen is the major structural protein in bone, and it's high level synthesis is an important characteristic of the osteoblast phenotype. We have evidence that transcription of the alpha1 (I) collagen gene is regulated differently in osteoblasts than in other collagen producing cell types. We believe that by investigating the DNA sequences and transcription factors that are involved in regulating the gene in osteoblasts that we will achieve a greater fundamental understanding of osteoblast differentiation, which is critical to our understanding of many bone disorders. The specific aims of this grant are: A. To functionally characterize the DNA sequences and clone the proteins which bind to a 49 bp region between -1719 and -1670 bp in the rat alpha1(1) collagen gene which has been shown to be necessary for significant expression of the gene in bone. This will be carried out by analysis of site specific mutations in transgenic animals, analysis of DNA-protein interactions using gel mobility shifts, cloning transcription factors, and analyzing their functions using expression vectors and antisense nucleic acids. B. To evaluate the possible role of the region between -2297 bp and -3521 bp of the alpha1(1) promoter in transcription during normal bone development using immunohistochemistry and in situ hybridization. If we find that this region plays a role in transcription of the gene during some phase of the osteoblast life cycle, we will analyze the DNA sequences and transcription factors responsible for this regulation by in vitro analysis of DNA-protein interactions, in vitro mutagenesis, transfection into cultured bone cells, and analysis of select mutations in transgenic mice. C. To determine the regions of the alpha1(1) gene necessary to direct high level transcription in bone and other collagen producing cells in transgenic animals and by transfection of cultured cells. D. The second basic goal of this grant and the fourth specific aim is to determine the mechanism of the 1,25 dihydroxyvitamin D (vitamin D) inhibition of the alpha1(1) gene in bone cells. This will be accomplished by testing mutated promoter constructs for vitamin D response in transfected cells and transgenic mice, by analyzing the binding of the vitamin D receptor to fragments of the alpha1(I) promoter, and by evaluating possible interactions of the vitamin D receptor with factors involved in basal transcription of the gene.

DESCRIPTION (provided by applicant): The goal of this grant application is to generate and test transgenic mouse lines containing ligand regulated Cre recombinase driven by 3.6 and 2.3 kb fragments of the rat Collal promoter. Currently available information T2 indicates that a tamoxifen regulated Cre, Cre-ER , is probably the most appropriate for our studies, Unmodified Cre recombinase constitutively catalyzes deletion of DNA between loxP sites, however Cre-ER T2 is inactive in the absence of the estrogen analogue tamoxifen. Constructs containing the 3.6 and 2.3 kb Col I al promoter driving Cre-ER T22 will be produced and used to generate transgenic mice. Testing of these transgenic mouse lines will be carried out by crossing them with a ROSA 26 Cre reporter mouse strain in which expression of B-galactosidase (beta-gal) or green fluorescent protein (GFP) is dependent on Cre activity. It is expected that reporter gene expression will be specific to the cell types that express the Collal promoters, and dependent on injection of tamoxifen. We will characterize the minimum dosage and time course of tamoxifen treatment needed to optimally induce the reporter gene. We will also characterize the effects of long-term tamoxifen treatment at this dosage on bone metabolism. The purpose for developing these lines is to enable tissue specific and temporally regulated inactivation of genes that are expressed in osteoblasts and are believed to play important roles in regulating osteoblast function, but whose role in adult animals is not understood. By allowing normal expression of a gene during early development and inactivating the gene in adult osteoblasts, we can learn about the specific role of the gene in adult bone metabolism.

DESCRIPTION (provided by applicant): The goal of our studies is to contribute to the understanding of the transcriptional regulation of osteoblastic differentiation. This is crucial to our understanding of the mechanisms controlling bone formation. Greater understanding of this process may lead to more rational design of therapies for osteoporosis and other bone diseases. Our studies and those of others have suggested that Dlx5, a homeodomain-containing transcription factor, plays an important role in the induction of osteoblastic differentiation. The Specific Aims of this grant application are: 1. To assess the role of Dlx5 in osteoblast differentiation in vivo by studying the effect of over expression of Dlx5 in osteoblasts off transgenic mice at different stages of osteoblast lineage progression. 2. To study the effect of inactivation of Dlx5 in cultured osteoblasts, and to assess the effect of Dlx5 on osteoblastic gene expression. We will study in vitro differentiation of osteoblasts derived from Dlx5 knockout mice. We will also carry out focused microarray analysis of cells plus and minus Dlx5 to identify genes that are induced or inhibited by Dlx5, and analyze bone development in an osteoblast directed knockout of the Dlx5 gene. This will allow us to separate the effects of Dlx5 expression in osteoblasts from the effect of this gene in other cell types. 3. To assess the potential for other members of the Dlx gene family to contribute to induction of osteoblast differentiation in vivo. We will continue to examine the expression of Dlx1, 2,3, 6 and 7 in osteoblasts. We will continue studies on Dlx3, which we already have detected in differentiating osteoblasts. We will continue studies on the ability of other Dlx genes to induce osteoblastic differentiation in the same in vitro system that we have used to study Dlx5. We will also study an osteoblast-directed knockout of the Dlx3 gene, to evaluate the role of Dlx3 in bone development in vivo. Our proposed studies will contribute to a more complete understand of the role of the Dlx protein family in osteoblast differentiation.

[unreadable] DESCRIPTION (provided by applicant): The specific aim of this proposal is to generate a physically linked multiple-gene-reporter mouse model that is relevant to bone biology. This animal model will have broad research applications in the field of bone biology. It will aid basic scientists in their ability to understand how cells within the bone lineage grow and differentiate. It may also be used by private industry to aid in the identification of novel compounds, which may lead to treatment of bone abnormalities, such as osteoporosis. The generation of linked multi-gene transgenic mice is novel and may influence the future design of transgenic mice. We propose to assemble a multi-gene-reporter DNA construct using Bacteria Artificial Chromosomes (BACs) and recombination technology in bacteria. A distinct feature that separates this transgenic mouse model from currently existing ones, is that the transgenic DNA construct will contain multiple gene reporter elements that are physically linked. The presence of multiple reporter genes in the same mouse has several advantages, including increasing the rate of data acquisition and the ability to identify and isolate distinct cell populations from the same mouse. As a result of linkage of the reporter genes, this mouse model can be easily crossed with other lines of mice without separation of individual reporters. As proof of concept, we have chosen three genes to drive the expression of three spectral variant fluorescent reporters to identify three distinct cell types important to bone biology. This animal model will have broad applications in research and we believe it will contribute to improvements in public health. It will aid basic scientists in their ability to understand how bone cells form, function, and interact with each other. It may also be used by private industry to aid in the identification of new drugs, which may lead to treament of bone diseases, such as osteoporosis. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Craniometaphyseal dysplasia (CMD) is a rare genetic craniotubular bone disorder which begins in childhood with cranial hyperostosis and long bone hypoostosis. Its lifelong progression leads to life-threatening consequences in some patients. There is no treatment other than repetitive surgery as of yet. CMD is heavily under-studied because the pathoetiology of this disorder is complex and bone tissue for research is rarely available. Therefore, we propose to create a source of induced pluripotent stem cells (iPS) from CMD patients and control individuals, which can be differentiated into cells involved in bone remodeling. This exploratory application focuses on the reprograming of skin fibroblasts from CMD patients and normal control individuals into iPS cells. We will use retrovirus vectors and protocols developed by Takahashi et al. to produce iPS cells and apply any useful modifications to the protocol that become available. IPS cell clones will be thoroughly characterized for normal karyotype, gene expression profile, epigenetic profile and will be tested for pluripotency using teratoma assays. We propose to differentiate suitable control and CMD iPS clones into osteoblasts in culture and analyze their potential for differentiation and bone matrix deposition in detail with a broad range of methods. Our goal is to obtain osteoblasts with properties that have the greatest possible similarity to primary osteoblasts. Before initiating studies with iPS cells, we will use NIH-approved human embryonic stem cells (hESC) to evaluate and optimize existing protocols for differentiating hESC into osteoblasts. Currently we expect to concentrate on protocols that initially differentiate the cells into mesenchymal stem cell-like populations, and then differentiate them into osteoblasts using standard conditions. As time and resources allow, we will perform initial phenotypic comparisons of iPS cell-derived osteoblasts from CMD patients and control individuals. This model will be the first to allow extensive studies of CMD (or any other craniotubular disorder) with human cells. PUBLIC HEALTH RELEVANCE: This application is designed to develop human inducible pluripotent stem (IPS) cells from skin biopsies, which are capable to differentiate into typical mature bone forming cells (osteoblasts). The goal of this investigation is to obtain osteoblasts from patients with rare bone disorders for detailed studies of disease mechanisms, which would otherwise not be possible. We chose to create IPS-derived osteoblasts from patients with craniometaphyseal dysplasia (CMD), a debilitating genetic bone disorder, because we have very interesting data from a mouse model for CMD but it is currently impossible to translate our findings to the human system.