Janet M. Kerr

Single‐Colony Derived Strains of Human Marrow Stromal Fibroblasts Form Bone After Transplantation In Vivo

Populations of marrow stromal fibroblasts (MSFs) can differentiate into functional osteoblasts and form bone in vivo. It is not known, however, what proportion of MSF precursor cells, colony forming units‐fibroblast (CFU‐Fs), have osteogenic potential. In the present study, analysis of bone formation in vivo by single‐colony derived strains of human marrow stromal fibroblasts (HMSFs) has been performed for the first time. Each strain originated from an individual CFU‐F and underwent four passages in vitro prior to subcutaneous implantation into immunodeficient mice within vehicles containing hydroxyapatite‐tricalcium phosphate ceramic. Multicolony derived HMSF strains were also transplanted to serve as positive controls. After 8 weeks, abundant bone formation was found in the transplants of all multicolony derived HMSF strains, whereas 20 out of 34 (58.8%) single‐colony derived strains from four donors formed bone. Immunostaining with antibody directed against human osteonectin and in situ hybridization for human‐specific alu sequences demonstrated that cells forming new bone were of human origin and were vital for at least 45 weeks post‐transplantation. Both the incidence of bone‐forming colonies and the extent of bone formation by single‐colony derived HMSF strains were increased by cultivation with dexamethasone and ascorbic acid phosphate. Other factors, including type of transplantation vehicle, morphology, size, and structure of the original HMSF colonies showed no obvious correlation with the incidence or extent of bone formation. Hematopoietic tissue within the newly formed bone was developed in the transplants exhibiting exuberant bone formation. These results provide evidence that individual human CFU‐Fs have osteogenic potential and yet differ from each other with respect to their osteogenic capacity.

cDNA cloning, mRNA distribution and heterogeneity, chromosomal location, and RFLP analysis of human osteopontin (OPN) ☆

A human osteopontin (OP) cDNA was isolated from a library made from primary cultures of human bone cells. The distribution of osteopontin mRNA in human tissues was investigated by Northern analysis and showed that the human message was predominant in cultures of bone cells and in decidua cells isolated at 6-12 weeks of gestation. Immunohistochemical analysis confirmed that OP expression is high in decidua cells as well as in the endometrial glands of a non-pregnant secretory-phase human uterus. Two variants of the OP message were evident on the basis of DNA sequencing and polymerase chain reaction amplification of bone and decidua cell mRNA. The peptides potentially translated by the variant messages differ by the presence (OP1b) or absence (OP1a) of 14 amino acids at residue 58 of the molecule. The deduced human protein sequence shows a conservation between species in the position of the Arg-Gly-Asp (RGD) cell attachment site. Chromosomal mapping of the osteopontin gene (OPN) using human-rodent cell hybrids demonstrated a location on chromosome 4 in the human genome. In situ hybridization of metaphase chromosomes using radiolabeled OP1a as a probe indicated that the gene is located on a region of 4q that is near the centromere. A high-frequency restriction fragment length polymorphism was evident in the DNA from 29 unrelated individuals using the enzyme BglII. Analysis of total genomic DNA by digestion with several restriction enzymes, Southern blotting, and hybridization with the human osteopontin cDNA indicated that the gene is a single copy with an approximate length of 5.4-8.2 kb.

Murine bone sialoprotein (BSP): cDNA cloning, mRNA expression, and genetic mapping.

Bone sialoprotein (BSP) is a small ( -70 ,000 Mr), highly post-translationally modified protein that is an abundant noncollagenous component of the bone matrix (Fisher et al. 1983). With a combination of immunohistochemistry and in situ hybridization, it has been shown that in the human and rat, BSP is produced by bone-forming cells and mature osteoblasts, recently entombed osteocytes, osteoclasts, and hypertrophic chondrocytes within the growth plate (Bianco et al. 1991; Chen et al. 1991). The only nonskeletal source that shows expression of BSP is the mononucleated trophoblast cells and their multinucleated syncytia in the developing placenta. Studies in the rat also showed that BSP is expressed in the odontoblasts of the developing mandibular incisor (Chen et al. 1992b). In an in vitro model of differentiating fetal bovine osteoblasts, BSP mRNA was increased 120-fold at a time point that corresponded to a dramatic formation of mineralizing nodules and trabecular-like structures (Ibaraki et al. 1992). This localization of BSP to sites of new mineral formation implicates BSP in this process. To determine the structure and describe the expression of BSP in the mouse, we have cloned and sequenced mouse BSP cDNA. In this study we report the nucleotide sequence of the murine cDNA, its deduced amino acid sequence, and we report the mapping of the mouse BSP gene (Ibsp) to Chromosome (Chr) 5. Mouse BSP cDNA was cloned from a 16-day mouse embryo cDNA library in a lambda SHlox vector (Novogen Cat #69641-1) with a 1.8-kb insert of rat cDNA (Oldberg et al. 1988a) as a probe under moderate hybridization conditions (Sambrook et al. 1989). One clone contained an insert size of 1.3 kb and was sequenced completely through-

Differential display of human marrow stromal cells reveals unique mRNA expression patterns in response to dexamethasone

Human bone marrow stromal cells (hBMSC) are pluripotent cells that have the ability to differentiate into bone, cartilage, hematopoietic‐supportive stroma, and adipocytes in a process modulated by dexamethasone (DEX). To characterize changes in hBMSC in response to DEX, we carried out differential display experiments using hBMSC cultured for 1 week in the presence or absence of 10−8 M DEX. When RNA from these cells was used for differential display, numerous cDNA bands were identified that were up‐regulated and down‐regulated by DEX. The cDNA bands were reamplified by PCR and directly used to screen an hBMSC cDNA library. Seven clones were isolated and characterized by DNA sequencing and found to encode the following genes: transforming growth factor‐β‐induced gene product (βig‐h3), calphobindin II, cytosolic thyroid‐binding protein, 22‐kDA smooth muscle protein (SM22), and the extracellular matrix proteins osteonectin/SPARC, type III collagen, and fibronectin. To confirm that these genes were regulated by DEX, the cells were treated continuously with this hormone for periods ranging from 2 to 30 days, and steady‐state mRNA levels were measured by Northern blot analysis. All genes showed some level of regulation by DEX. The most profound regulation by DEX was observed in the βig‐h3 gene, which showed a relative 10‐fold decrease in mRNA levels after 6 days of treatment. Interestingly, βig‐h3 expression was not altered by DEX in fibroblasts from other human tissues, including thymus stromal fibroblasts, spleen stromal fibroblasts, and foreskin fibroblasts. In summary, differential display of DEX‐treated hBMSC revealed unique patterns of gene expression and has provided new information about phenotypic changes that accompany the differentiation of hBMSC toward osteogenesis. J. Cell. Biochem. 76:231–243, 1999. Published 1999 Wiley‐Liss, Inc.

The cDNA cloning and RNA distribution of bovine osteopontin

We have isolated and sequenced the bovine cDNA (OPN) counterpart of osteopontin. The cDNA is 1356 nucleotides (nt) in length with an open reading frame of 834 nt, encoding a 278-amino acid (aa) protein. Cell-free transcription and translation of OPN RNA resulted in a major species of approx. 40 kDa in size, in agreement with the predicted size of the deduced aa sequence. Northern analysis of bovine OPN RNA indicated the presence of the message in mineralized, as well as soft tissues. A comparison of the deduced aa sequence among various species indicates both regions of similarity and divergence. One prominent region of dissimilarity in bovine OPN compared to all other species is a 22-aa gap which may represent a loss of a potential Ca(2+)-binding loop. Despite the variability among the species, several regions of conservation are apparent, including a hydrophobic leader sequence, a potential site for Asn-linked glycosylation, a stretch of polyaspartic acid residues, and the cell attachment Arg-Gly-Asp tripeptide. Whether bovine OPN enhances cell attachment is unknown. Furthermore, whether the loss of a potential Ca(2+)-binding loop alters the function of OPN would be interesting to determine.

MOLECULAR AND CELLULAR BIOLOGY OF THE MAJOR NONCOLLAGENOUS PROTEINS IN BONE

Publisher Summary This chapter discusses the molecular and cellular biology of the major non-collagenous proteins in bone. It also presents the structure and expression of a protein called osteonectin. By cDNA cloning and subsequent protein sequencing, it has become apparent that osteonectin is widely distributed in the body and likely to have multiple functions in mineralized and nonmineralized tissues as well. Despite its wide tissue distribution, it is, comparatively, highly expressed in bone. For this reason, the osteonectin gene has been used as a model to study transcriptional control in human- and bovine-derived cultured bone cells. The chapter also describes the two cell attachment bone matrix proteins, osteopontin and bone sialoprotein. While they both share the same small cell attachment motif, they are otherwise distinct in structure and in their extensive posttranslational modifications. The transcriptional control of the two genes also appears to be distinct and varies considerably with direct hormone/vitamin treatment as well as during development and with cellular transformation.

Mapping of the human and mouse bone sialoprotein and osteopontin loci.

~School of Biological Sciences and Departments of Dental Medicine and Surgery, 3.239, Stopford Building, University of Manchester, Manchester, M13 9PT, UK 2Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA 3Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA 4Bone Research Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892, USA

Diverse forms of stress results in changes in cellular levels of osteonectin/SPARC without altering mRNA levels in osteoligament cells

SummaryThe osteonectin/SPARC gene has been shown to possess motifs for a heat shock element and metal responsiveness. Also, the expression of the protein has been associated with culture stress in endothelial cells. In the present study, osteoligament (OL) cells derived from the patellar ligament were subjected to diverse forms of stress that included (a) exposure to sodium arsenite, (b) heat shock, (c) cadmium ion, and (d) the amino acid analog, AZC. Osteonectin/SPARC levels in OL cells were determined by Western blot analyses, and immunoprecipitation using antiosteonectin antibodies. Expression of osteonectin/SPARC mRNA was determined by Northern analysis using a 1.5 kb EcoRI restriction fragment of bovine osteonectin cDNA. These studies reveal that osteonectin/SPARC is produced following diverse forms of stress, however, the levels are lower than observed in unchallenged OL cells. In all instances, the mRNA levels were comparable to control cells. These studies indicate that expression of osteonectin/SPARC mRNA is tightly controlled in OL cells and that the protein may be regulated at the level of protein translation.

The Human Bone Sialoprotein Gene Contains an NF-E1/YY1 Cis-Acting Sequence with Putative Regulatory Activity

Bone sialoprotein (BSP) is a noncollagenous matrix glycoprotein localized predominantly in mineralized tissues but also detected in extraskeletal sites undergoing focal mineralization. We have previously characterized the human BSP gene and have shown that the upstream sequence contains inverted TATA and CCAAT motifs at the expected locations from the transcriptional start site (J. M. Kerr et al. [13]) and a potential YY1 binding motif located within the first 30 bp of intron 1 of the human gene. Deletion analyses of the human BSP promoter/exon 1 sequence fused to a CAT reporter gene indicate that CCAAT enhances basal transcription of BSP in transiently transfected rat UMR106-01 BSP osteosarcoma and rat skin fibroblasts. Though this enhancing activity was lost with inclusion of 68 bp of intron containing a YY1 motif in these constructs, reporter activity in the UMR106-01-BSP cells was elevated four- to seven-fold relative to that of rat fibroblasts. Gel electrophoretic mobility shift, UV-crosslinking, and southwestern experiments indicate that YY1 is present only in the extracts of nuclei isolated from the UMR cells and may contribute to the elevated transcriptional activity of the human BSP promoter construct in UMR106-01-BSP.